Why do I need a blog on microbiology?

Microbiology is a very fascinating subject. I have been reading it for the last 8 years and I am still not bored. With so much research happening on so many diverse ares, it was hard for me to remember the articles that I read. Whenever, I read an article I generally make a brief summary of it and write it down on the article itself. I would generally start with the introduction, to understand the known facts and the problem. Then, I would summarize the methodologies used and results observed and finally I would jot down the conclusions and future perspectives.
Recently, I realized that most of my previous labor has been either lost or not in very great form at present. The idea of writing a blog on the papers that I read is the brain child of my husband and I am highly grateful to him for that.
Its been one month since I am writing these blogs on various articles and I am really feeling updated. Having a blog on a particular article also gives me the chance to look back, whenever I am in doubt. I must say that I am not a very good writer but I like to write. My blog gives me a platform to my thoughts and my understanding. It is also one of the easiest ways to communicate with my friends and colleagues. I hope that I will continue reading these amazing works and keep writing.

An article on pathology of Psoriasis

My today's blog is a a brief summary of a very interesting article on pathology of psoriasis by Dr Jensen and team, University of Pennsylvania School of Medicine, Philadelphia, USA.

Interleukin-1 regulates keritinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK-1) dependent and independent pathways.

Psoriasis is a noncontagious and chronic skin condition that nearly affects 0.3 to 3% of world population. Prevalence of psoriasis varies widely depending up on the ethnicity. Psoriasis occurs most commonly in Caucasians. The disease is characterized by disfigured red thickened skin covered with white/silvery scales. The management is generally done using anti-inflammatory agents such as corticosteroids and biologics such as TNF-alpha neutralizing Etanercept.
The cause of psoriasis is unknown and there is no cure. Several hypotheses have been proposed to find the cause of psoriasis. Based on these, the cause appears to be a multifactorial process which involves both genes and the environment leading to the dysregulated skin structure and chronic inflammation. Keratinocytes which are the primary constituents of the epidermis, hyper-proliferate and fail to undergo differentiation in affected skin.
Previous studies have revealed that a vast array of genes including cytokines, growth factors and differentiation markers is differentially regulated during the disease. New studies have demonstrated that activation of keratinocytes is sufficient to trigger psoriasis like lesions in transgenic mice. This process is however, dependent up on the presence of T cells demonstrating the interplay between keratinocytes and T cells. Recent reports have also suggested the role of IL-1 in the pathology of psoriasis.

There are two forms of IL-1, IL-1alpha and IL-1beta, which are encoded by separate genes. Both the IL-1alpha and IL-1beta bind to the IL-1 receptor type 1 (IL-1RI). Together with IL-1RI accessory protein (IL-1RIAcP), IL-1RI activates intracellular signaling cascades leading to activation of the transcription factors AP-1 and NF-kappaB. These transcription factors play important roles in signal transduction pathways and they regulate expression of many genes and gene products e.g., complement factors and cytokines.

After binding of the ligand to the IL-1R, IL-1R associated kinase-1 (IRAK1), a serine-threonine kinase, is recruited to the intracellular domain of IL1-RI. After activation, it gets disassociated from the receptors and activates down stream signaling factors, which in turn regulate activation of NF-kappaB. The role of IRAK1 in activation of transcription factors, AP-1 is controversial.

Il-1 has been shown to be expressed in psoriatic skin lesions. Recent studies using functional genomics have suggested that transcriptome profile derived from psoriatic skin lesions were similar to those of IL-1 stimulated keratinocytes. Thus, IL-1 has been implicated in the psoriasis associated hyper-proliferation of keratinocytes. As recent studies have suggested links between activated keratinocytes and T cells and also shown the involvement of IL-1 in psoriasis, the authors searched for potential role of Il-1 in linking keratinocytes to T cell.

The involvement of IRAK1 in IL-1 signaling in keratinocytes and the potential role that elevated IRAK1 levels may have in psoriasis pathology have never been explored. In this study, the authors have demonstrated that IL-1 stimulated keratinocytes express elevated levels of Cys-Cys (CC) and Cys-X-Cys (CXC) motif chemokines, which are involved in T cell recruitment.

The Cys-Cys chemokine (or beta-chemokine) have two adjacent cysteines, near their amino terminus. There are at least 27 distinct members of this subgroup reported for mammals, called CC chemokine ligands (CCL)-1 to -28. In case of , Cys-X-Cys chemokines (or α-chemokines), the amino terminal cysteines are separated by one amino acid, called X. There are 17 different types in mammals.

The authors also observed that while IL-1 regulated CC chemokine production was IRAK1 dependent, IL-1 and IFN-gamma regulated CXC production was IRAK-1 independent.
The major results of the study are as follows:
1. IL-1 alpha and beta stimulated keratinocytes express T cell targeting CC chemokine (CCL5 and CCl20) mRNAs present in psoriatic lesions
2. IL-1 alpha and beta up-regulates CC chemokine secretion (CCL5 and CCl20) by keratinocytes
3. IL-1beta up-regulates CXC chemokine mRNA levels but does not lead to protein secretion
4. IL-1beta amplifies chemokine production induced by INF-γ and TNF-α
5. Elevated IRAK1 levels enhance chemokine expression
6. IL-1 induced CC chemokine expression is dependent upon IRAK1
7. IL-1 effect on IFN-gamma induced CXC chemokine expression is IRAK1 independent

Reference: Sanmiguel JC, Olaru F, Li J, Mohr E, Jensen LE. Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways. Cell Signal. 2009 May;21(5):685-94.

Today, I happened to read a very interesting article from the lab of Dr. Llyod S Miller, Division of Dermatology, UCLA, USA. This article shows that IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. The article is cited as Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, Magorien JE, Blauvelt A, Kolls JK, Cheung AL, Cheng G, Modlin RL, Miller LS. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest. 2010 May 3;120(5):1762-73.

I have tried to prepare a brief synopsis of the article and it is as follows:
Staphylococcus aureus is a gram positive bacteria that is one of the causes of skin and soft tissue infections. In recent years, virulent antibiotic resistant strains, such as methicillin resistant S. aureus (MRSA) have become a major cause of public health concern. Immune based therapy may provide an alternative approach for treatment of these infections. The use of immune based therapy requires a clear understanding of the protective immune response against S. aureus infection in the skin. The neutrophils recruitment has been shown to play an essential role S. aureus infection. Other studies have also provided evidence that in addition to neutrophils, T cells also play important roles in host defense against S. aureus cutaneous infection. However, the mechanisms by which T cells promote protective immunity against S. aureus skin infection are not clear. Thus, in the present study the investigators decided to evaluate the contribution and mechanism by which alpha beta or gamma delta T cells participate in cutaneous host defense. For this they used an in vivo mouse model of cutaneous S. aureus infection.

The study was performed in a stepwise manner and these were as follows:

1. First step was to know which T cells contribute to host defense against S. aureus infection in skin. Thus, wild type mice and mice deficient in either alpha beta or gamma delta T cells were inoculated intradermally with the bioluminescent S. aureus SH100 strain. The lesion size and in vivo bioluminescence of the live actively metabolizing bacteria within the lesions were evaluated. It was observed that gamma delta T cell deficient mice developed skin lesions approximately three fold larger than wild type or alpha beta deficient mice. To determine a possible association of large lesions of gamma delta deficient mice with impaired bacterial clearance the authors anesthesized the mice and determined the bacterial counts within the lesions in real time. It was observed that number of CFUs obtained from 8mm punch biopsies were up to 10 fold higher in gamma delta T cells deficient mice than in WT mice on days 1 and 3 of infection. The authors have also earlier observed that after, S. aureus infection, WT and alpha beta T cell deficient mice had similar bioluminescent signals that decreased over 14 days. In contrast, gamma delta T cells deficient mice showed bioluminescent signals that were up to 20 fold higher than those of WT mice at all time points.
All these data indicate that gamma delta T cells, but not alpha beta T cells are required for mediating host defense and bacterial clearance against cutaneous S. aureus infection.


2. The next step was to know the role of gamma delta T cells in recruitment of neutrophils to site of infection. To investigate this, histology and myeloperoxidase (MPO, an indicator of neutrophils function) activity were evaluated in skin lesions biopsies performed after one day on inoculation in WT and gamma delta T cell deficient mice. The lesions of gamma delta T cell deficient mice had highly decreased number of neutrophils with impaired abscess formation and a good number of S. aureus bacteria as observed by Gram staining in comparison to WT mice. The MPO activity was also found t be less in deficient mice. These data suggest that gamma delta T cell deficient mice show impaired recruitment of neutrophils to the site of infection and thus gamma delta T cells play important role in recruitment of neutrophils. Next step was to further investigate the mechanism of impaired neutrophils recruitment observed in gamma delta T cell deficient mice. This was done by assessing the levels of soluble factors which are known to have direct neutrophils chemotactic activity. These included neutrophils chemokines, KC and MIP2 and granulopoeisis factor GM-CFS. The levels were measured by performing real time PCR and protein array analysis on homogenized lesion biopsies 8 hr after inoculation. The gamma delta T cell deficient mice showed significantly decreased levels of mRNA and proteins. The results suggested the impaired neutrophils recruitment in gamma delta T cell deficient mice was most probably caused by a decrease in production of neutrophils chemotactant.

3. The third step was to investigate the production of T cell derived cytokines which are known to promote neutrophils recruitment. These cytokines are IL-17 and IL-22. These cytokines trigger fibroblasts and epithelial cells to produce neutrophils recruitment factors, e.g., KC, MIP2 and GM-CSF. The authors also evaluated production of IL-21. They found that WT mice showed an early gene expression of IL-17A and IL-17F in response to the bacterial challenge (mRNA levels by real time PCR). In contrast, gamma delta T cell deficient mice did not show this early expression of IL-17A and IL-17F. IL-22 was found to be expressed in both groups of mice. The authors then further studied the source of IL-17 in skin sample lesions from WT mice. It was noted that IL-17Aand IL-17F were highly expressed in the epidermal compartment and there was more or less no expression in the dermis. The authors then positively selected the CD3+ from cell suspensions by using a specific monoclonal antibody directed against the T cell marker. It was observed that after 8 hrs of infection, IL-17A and IL-17F induction was detected exclusively in T cell fraction (CD3+ cells). In contrast, no such induction was observed in T cell fractions or the negative fractions from epidermis of gamma delta T cell deficient mice. The authors further selected gamma delta T cells from cell suspensions using gamma delta specific antibody, GL3. IL17A and IL17F induction was detected exclusively in gamma delta T cell fractions from WT mice. All these results indicate that gamma delta T cells are major source of IL-17 production in response to S. aureus cutaneous challenge.

4. The next step was to determine the exact location of gamma delta T cells within the infected skin lesions and the percentage of gamma delta T cells that express the known skin specific Vgamma5 chain. The authors thus examined the sections of lesional biopsy at 8 and 24 hrs after skin inoculation and observed CD3+ cells in epidermal compartment of WT mice. Only rare CD3+ cells were seen in epidermis of gamma delta T cell deficient mice. Further, epidermal cell suspensions of skin biopsies from normal uninfected WT mouse skin were labeled with specific mAbs for CD3, gamma delta T cells and Vgamma5 expressions were analyzed by flow cytometry. 90% of CD3+ T cells from WT mice epidermis were found to express Vgamma5 chain. Purified GL3+ epidermal gamma delta T cells from uninfected mice were left unstimulated or were activated with PMA/ionomycin and cultured in the absence or presence of IL-1beta, IL-23 or both IL-1beta and Il-23. Supernatants were collected after 24 hrs and IL-17 levels were analyzed by ELISA. PMA/ionomycin stimulation resulted in a three fold increase in production of IL-17 compared with unstimulated cells. The addition of exogenous recombinant IL-1beta or IL-23 or combination of both resulted in 2-3.5 fold increase in IL-17 production over PMA/ionamycin alone. All these data indicate that resident epidermal gamma delta T cells expressing Vgamma5 chain are the predominant T cell subset present in mouse skin at baseline and during an early immune response to S. aureus skin infection and they have the capacity to produce IL-17 upon activation.

5. In the next step, the authors determined whether IL-17 production in response to S. aureus infection by gamma delta T cells is dependent up on IL-1, TLR-2 (TLR-2 has been recently shown to be expressed by gamma delta T cells) and IL-23. To determine whether the induction of IL-17A and IL-17F was dependent up on IL-1 and/or TLR-2, the authors inoculated WT mice and mice deficient in IL-1R, TLR-2 or MyD88 and determined IL-17A and Il-17F mRNA production at different time points by real time PCR. At 8 hrs after inoculation, IL-1R-, TLR-2 and MyD88-deficient mice showed impaired expression of IL-17A and IL-17F compared with WT mice. Similarly, to determine the dependence of IL-17A and IL-17F expression on IL-23, the authors inoculated WT mice and mice deficient in p40 (shared subunit for IL-12 and IL-23) and p19 (IL-23 specific subunit) and p35 (Il-12 specific subunit). It was observed that at 8 hrs after challenge p40 and p19 but not p35 deficient mice showed decreased expression of IL-17A or IL-17F in comparison to WT mice. This shows that IL-23 and not IL-12 plays an important role in induction of IL-17A and IL-17F.

6. The further step was to determine whether IL-17 contributes directly to the impaired neutrophils recruitment and host defense against S. aureus cutaneous challenge as seen in gamma delta T cell deficient mice. Thus, WT mice and mice deficient in the IL-17R were inoculated intradermally with S. aureus. It was observed that IL-17R deficient mice developed significantly larger lesions and higher bacterial counts in comparison to WT mice. Thus, these data indicate that IL-17 producing gamma delta T cells are required for host defense against S. aureus skin infections.

7. Given the critical role of gamma delta T cells and IL-17 in host defense against cutaneous S. aureus challenge, the final step was to determine whether the addition of IL-17A (recombinant IL-17) can affect the increased lesion size and bacterial counts in gamma delta T cell deficient mice. Administration of single dose of rIL-17 along with the S. aureus inoculum resulted in significantly reduced lesion sizes and bacterial counts compared with gamma delta T cell deficient mice that were inoculated with S. aureus alone. The lesion size and bacterial counts observed in gamma delta T cell deficient mice that were immunized with rIL-17 were found to be comparable to those observed in wild type mice. These data suggest that IL-17 is the essential mediator by which gamma delta T cells contribute to host defense against S. aureus cutaneous infection.

BBK07 as a potential marker for serodiagnosis of Lyme Disease in humans.

Lyme Disease (LD) or lyme borreliosis, an infectious disease, is caused by three species of bacteria belonging to the genus Borrelia. In USA, Borrelia burgdorferi is the main cause of LD, while Borrelia afzelli and B. garinii have mostly been found in Asia and Europe. The disease is named after a town, Lyme in USA, where a number of cases were identified in 1975. B. burgdorferi was identified as the cause of LD nearly 28 years ago (in 1982). LD is difficult to diagnose as the signs and symptoms of the disease are non-specific. Due to the low pathogenic load of the organisms in clinical samples (CSF or blood), slow growth of B. burgdorferi, and high cost and labor intensive procedures, the use of culture as a diagnostic method of LD is limited. PCR detection has been described but has been observed to be relatively less sensitive in clinical samples. The most commonly method currently used to diagnose is serodiagnosis. Serodiagnosis can be performed by using either whole cell antigens or by using recombinant proteins or peptide fragments. The problem with whole cell antigens is false positivity due to cross reactivity with antibodies against other bacteria. To overcome this problem, several recombinant proteins have been evaluated in immunodetection. These include OspC, BmpA, VlsE, BBK32, L25, P37, and DbpA. The various studies using these proteins have suggested while the use of these r-proteins can increase the specificity of detection by reducing the cross reactivity, they decrease the sensitivity of detection as only selected antigens are used. Bacon et al suggested combined detection of IgM against pepC10 and IgG against C6 to increase the sensitivity. Recently, Barbour et al used synthetic protein arrays to test the immunogenicity of the majority of open reading frames of B. burgdorferi. They identified several novel antigens. These also included BBK07 and BBK12. These proteins are extremely similar in sequence. In the present study, the authors characterize the expression, surface localization and immune response against BBK07 to evaluate its use as a diagnostic marker in the diagnosis of LD. This paper is from the lab of Dr. Coleman and Dr. Pal, University of Maryland, USA and has been published in clinical and vaccine immunology, November, 2009 issue.
The major questions asked and how they were resolved is as follows:

Question 1: Do the BBK07 gene or BBK12 gene, or both are transcribed by B. burgdorferi in vivo and in vitro?
The authors checked the expression of both BBK07 and BBK12 from cultures of bacteria as well as from mice tissues infected with B. burgdorferi. Thus, primers were designed for both the genes. Total RNA was extracted from cultured Borrelia as well as from various mouse tissues (infected with B. burgdorferi). Now, the expression of both the genes was studied by qRT-PCR. It was observed that both the genes were expressed at low levels in vitro (from cultures). Only BBK07 transcripts were present in infected murine skin samples (in vivo) one week after the inoculation. To study the transcriptional profile of the BBK07 gene through out the representative life cycle stages of B. burgdorferi, mice and ticks were infected with the organism and total RNA was extracted from infected tissue samples representing the life cycle stages of B. burgdorferi. It was observed that BBK07 was expressed in all murine tissue samples tested but was undetectable in tick samples. The same RNA samples did not show detectable quantities of BBK12 transcripts in any tissue sample at any point of time.
Answer: The BBK07, but not the paralogous BBK12 gene, is selectively expressed in the mammal during the infection cycle of B. burgdorferi.

Question 2: Whether this protein, BBK007 is surface exposed and immunogenic?
To answer this question, the authors tried to express this protein. Since, expressing the full length protein proved difficult, the authors expressed and purified an amino-terminal fragment. This was referred to as BBK07N. Mice were immunized with this fragment with an adjuvant. Serum samples from these mice were collected at different time points and the immune response to BBK07N and B. burgdorfei lysate was measured by immunoblot. It was observed that BBK07N evoked a robust immune response. BBK07N antiserum recognized both purified BBK07N and native BBK07 from B. burgdorferi lysate. Now, to test whether this protein is surface exposed or not, a proteinase kinase assay was done. In this assay, B. burgdorferi was incubated with proteinase K and without it. These samples were then tested with FlaB, OspA and BBK07N antiserum (i.e., sera raised in mice by inoculating FlaB, OspA and BBK07N, respectively) by immunoblot. The results showed that FlaB was not degraded while OspA and BBK07 were significantly degraded.
Answer: Amino-terminal region of BBK07 is surface exposed and immunogenic.

Question 3: Is the BBK07N specific antibody response is produced during active infection only or it is also produced in hosts immunized with lysed organisms?
Two groups of mice were taken. One group of mice was immunized with B. burgdorfei. Another group of mice was immunized with sonicated B. burgdorferi. Serum samples from these mice were tested against B. burgdorferi lysate or BBK07N at different time points. As a negative control, Lp6.6, which is abundant in vitro but down regulated during murine infection, was also tested by these serum samples. In the group of mice infected with B. burgdorferi, a robust immune response was observed against BBK07N or lyaste but not against lp6.6. The mice immunized with sonicated B. burgdorfei, produced an insignificant immune response against BBK07N protein. In order to check the dependency of antibody response against BBK07 on route of infection, groups of naïve mice were infected with tick bite. Serum samples from these mice were collected and antibody response was checked and it was observed that serum samples showed a similar response to BBK07 and lysate.
Answer: BBK07-specific immune response is pronounced during active borrelial infection but absent in hosts immunized with lysed pathogens.

Question 4: is BBK07N suitable as a marker for diagnosis of LD?
To compare the immunogenecity of BBK07 against other antigens, serum samples from infected and un-infected mice were used to probe purified proteins (BBK07N, VlsE, BmpA, OspC, Lp6.6) or sonicated B. burgdorferi and levels of antibody response were tested by ELISA. Un-infected serum samples showed low reactivity to all antigens. BBK07N showed most robust immune response among all antigens tested.
Answer: BBK07N is a sensitive marker for detection of LD.

Question 5: Is the protein BBK07N suitable as marker for diagnosis of LD in human samples?
Serum samples from patients diagnosed with LD were tested against rBBK07N, BmpA, OspC or B. burgdorfei lysate by ELISA. B. burgdorferi lysate displayed highest sensitivities for the antigen tested. BBK07N showed higher specificity than lysate. Using other antigens lower sensitivities were observed.
Answer: BBK07N can be used and developed into a diagnostic tool for evaluating human LD patients.

Source: Coleman AS, Pal U. BBK07, a dominant in vivo antigen of Borrelia burgdorferi, is a potential marker for serodiagnosis of Lyme disease. Clin Vaccine Immunol. 2009 Nov;16(11):1569-75. Epub 2009 Sep 23. PubMed PMID: 19776192; PubMed Central PMCID: PMC2772389.

Molecular detection of viable bacterial pathogens in water by ratiometric pre-RNA analysis

The use of PCR in detecting bacteria in clinical or environmental samples is limited in part by false positive detection (PCR can detect non-viable cells) and free DNA in environmental samples. Today, I happened to read a very interesting article on molecular detection of viable bacterial pathogens in water. The article instantly reminded me of a journal club presentation by my friend, Chhavi on detection of viable cells by PCR. So, I did some searches, and came across many articles on use of novel techniques to selectively detect live bacteria.
First, let me elaborate viability in terms of bacteria. Bacteria were traditionally considered viable when they could be cultured. However, today's viability concept is based on the presence of some form of metabolic activity, responsiveness, RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane (Meschke et al., 2010). The presence of an intact membrane has been the focus of recent approaches to limit detection of intact cells using ethidium monoazide or propidium monoazide. These are DNA intercalating dyes and they can selectively enter those cells that have compromised cell walls and membranes. Within these cells, they can covalently link with the DNA. This linked DNA then can not be amplified by PCR, thus, allowing selective amplification of DNA only from live cells.
Recently, Dr. John S. Meschke and his group (University of Washington, Seattle) have developed a new method for detection of viable bacterial pathogens in water. The article is published in the February, 2010 issue of applied and environmental microbiology.
Here is a brief summary of the article:
One approach to detect viable cells is to detect microbial RNA, which is less stable than DNA and as already quickly degrades after cell death. However, mRNA can be difficult to detect, while mature rRNA is stable within inactivated cells. The authors have suggested that microbial-rRNA precursors (pre-rRNA) could be an alternative RNA target. These pre-rRNA molecules have leader and tail sequences that are enzymatically removed during rRNA maturation. These sequences are also phylogenetically specific, so one can detect them in complex samples. It is assumed that once the growth of bacteria ceases, the rRNA processing in bacterial cells continues, while pre-rRNA synthesis stops and thus pre-rRNA conc. decreases. Therefore, the pre-rRNA concentrations could be indicative of the physiological activity of micro-organisms.
In the present work, the authors exploited the replenishment of pre-rRNA that occurs immediately upon nutritional stimulation of nutrient limited bacterial cells. Thus, species-specific pre-rRNA was measured in environmental samples after brief exposure to culture medium by qPCR. The values obtained were compared with those seen in un-exposed control samples. The values that exceed those of control indicate the presence of viable cells. The authors called this approach ratiometric pre-rRNA analysis (RPA) and tested it on rapidly growing Aeromonas hydrophila and slowly growing Mycobacterium avium.

Methodology and Results:

 To assess the time course of pre-rRNA replenishment upon nutrient stimulation: For this, early stationary phase A. hydrophila ATCC7966 cells were washed, re-suspended in autoclaved tap water (ATW) and incubated for seven days with aeration. Similarly, early stationary phase M. avium strain HMC02 cells were washed, re-suspended in ATW, and then incubated for 14 days. The purpose of these experiments was to create nutritionally deficient cells and to drain pre-rRNA pools in simulated water supply environments. To conduct RPA, these cultures were divided into two parts and centrifuged. One pellet was re-suspended in the culture medium (nutritional stimulation) and other in ATW (control). After various time periods of incubations, these cells were lysed, RNA was extracted and qPCR for pre-rRNA was performed from cDNA. The ratios of RT-qPCR values in stimulated and control samples were calculated following normalization to genomic DNA standard curves. It was observed that pre-rRNA stimulation was very rapid in both organisms. For A. hdrophila, 15 minutes and for M. avium, 4 hrs were adequate for near maximum stimulation.

 To assess the specificity of RPA for viable cells: For these experiments, sodium hypochlorite was used to generate suspension with various ratios of viable and inactivated cells. These cell suspensions were incubated for specific periods, plated on medium and post-treatment percent viability was determined by RPA. In addition, the authors also quantified the DNA concentration of the samples by qPCR with the same primers used for RPA. It was observed that samples with no detectable viable cells (0% viability, 0 CFU/ml) exhibited pre-RNA stimulation ratios of one or less. All other sample showed pre-rRNA stimulation ratios of more than or equal to 2. In contrast, qPCR detection of A. hydrophila genomic DNA was strongly positive in all samples. Also, the pre-rRNA stimulation ratios were significantly lower in samples with no detectable CFUs in comparison to samples with detectable CFUs. However, no such correlation of CFU with no. of genomic DNA copies obtained by qPCR was observed.


 Field test of RPA for A. hydrophila: Three fresh water samples and one salt water sample were collected from various sites in Seattle, WA. These water samples were concentrated by filtration, diluted two fold in nutrient broth (stimulated) and in ATW (control). After incubation, these were tested by RPA. Viable counts were also obtained by plating these samples onto ampicillin-dextrin agar with vancomycin. The freshwater sample yielded vales between 280 to 798 CFU/ml A. hydrophila and the RPA results ranged from 4.8 to 39.8. The salt water sample yielded 6CFU/ml, but no pre-rRNA. Therefore, the authors suggest that in its current form, RPA applied to natural samples has a detection limit between 6 to 280 CFU/ml.
In practical terms, RPA can be conducted by dividing a sample into two aliquots. One aliquot is made nutritionally stimulated while other is re-suspended in water. After stimulation for less than or equal to one generation time, species specific pre-rRNA levels can be determined by RT-qPCR and compared with un-stimulated sample. Ideally a threshold pre-stimulation rRNA ratio of one would indicate the presence of viable cells. At present RPA is not quantitative and optimization and standardization of diluted samples may give quantitative results.

Future applications and conclusions:
In the present work, the authors show that RPA can be used to specifically detect viable cells in environmental samples. The method was compared to traditional PCR and it was observed that it reduced the number of false positives obtained by amplification of dead cells. It may prove highly useful in food and water safety analysis.

Investigation of the Genes Involved in Antigenic Switching at the vlsE Locus in Borrelia burgdorferi: An Essential Role for the RuvAB Branch Migrase

Most of the pathogens adopt the strategy of antigenic variation to evade immune responses by host. These variations occur through targeted genome rearrangements. However, the exact molecular details of the recombination processes that generate diversity in the antigen-expressing genes are not fully known.
Borrelia burgdorferi is a spirochete that causes lyme borreliosis. This disease progresses through three stages, early, disseminated and persistent. The persistent infection requires continual segmental gene conversion at the vlsE locus. This locus encodes a 35KDa membrane lipoprotein and is present on the linear plasmid Ip-28-1. When this locus is deleted or when the plasmid is not present, a productive murine infection occurs, but the organisms are cleared between 8-21days post infection. Adjacent to vlsE (also referred to as vls1), is present a contiguous upstream array of 15 silent cassettes separated from each other by 17 bp direct repeats. It has been suggested that during murine infection, information is transferred uni-directionally from the silent cassettes into the expression site to generate diversity at six regions (VR-1 to VR-6) with in the central region of the vlsE gene. VR-1 to VR-6 regions are believed to be prominently displayed antigenic areas. The antigenic diversity is generated through segmental gene conversion so that information from several silent cassettes can be transferred into the single vlsE locus to generate a mosaic gene. And thus, unique vlsE proteins can be generated. It has been shown that all silent cassettes are used as sequence donors in the gene conversion events at vlsE. The mammalian signal that triggers recombinational switching is unknown. The protein machinery that promotes recombinational switching at vlsE remains unknown. This fascinating work is from the lab of Dr. George Chaconas, Department of Microbiology and infectious disease, university of Calgary, Canada.
In this paper, the authors generated 17 mutants carrying disruptions in known DNA recombination, repair and replication genes to identify proteins involved in recombinational switching at vlsE.
The methodology used and major results obtained are as follows:

 Construction of DNA repair and replication gene disruptions in B. burgdorferi- 21 different DNA replication, repair and recombination genes were disrupted to investigate their role in vlsE recombination. For this, knockout plasmids carrying a one kb gentamicine cassette that replaced the central portion of the target gene, were constructed and used to transform the infectious B. burgdorferi B31 clone 5A4. Following transformation, allelic exchange results in successful disruption. However, recombination can also lead to integrative recombination leading to the formation of merodiploids. Thus, construct verification was done. For this, each gene disruption was subjected to four PCR assays. Firstly, all the samples were subjected to PCR for the presence of gentamicin cassette. Secondly, samples were subjected to PCR using knock out primers to confirm the absence of deleted gene. Thirdly, all the samples were subjected to PCR using target gene primers to confirm the size. Fourthly, combination of target gene primers and primers internal to gentamicin cassette were used to amplify the boundaries. Of the 21 DNA replication, repair and recombination gene knockout attempted, 17 were successful.

 Effect of gene disruption on mouse (C3h/HeN) infection- For each gene disruption, two clones were chosen, which contained the full plasmid complement required for infectivity. These were used to infect C3H/HeN immunocompetent mouse. Cultures were grown from blood samples at day 7 of infectivity. At days 14 and 21, ear biopsies were used for monitoring infection and switching at vlsE. A portion of the vlsE expression site containing the variable regions was amplified from organisms obtained from ear biopsies at day 21. The incorporation of new restriction sites from the cassettes into the variable region of vlsE provides a clear indication of the switching process. Thus, these were further subjected to RFLP analysis to study switching. The results obtained can be used to divide the 17 mutations in two groups. In the first group, >75% of blood cultures were positive at day 7 and same was observed at day 21. All these mutants also displayed switching. In the second group, the mutant strains displayed <75% positive cultures from ear biopsies at 21 days post-infection. In case of sbcD, sbcC and BBG32, switching was observed by RFLP. For the remainder of mutant strains in this group, the infection was allowed till day 35. At that time, mice were sacrificed and the spirochetes were cultivated from heart, bladder, joint and ear. The RFLP experiments demonstrated that switching could be observed in all the strains except those carrying ruvA or ruvB mutations. These genes encode for the two subunits of a Holliday junction branch migrase.

 Effect of B. burgdorferi gene disruptions on SCID C3H/HeN mouse infections: The presence of an effective immune response might exert a selective pressure on antigenic variation, thus to remove this selective pressure the mutant strains of the second group were used to infect SCID mice. This removes the pressure as well as it allows the organisms to persist in the host for longer duration. In a wild type mouse, the strains defective in switching would be cleared by day 21. However, in SCID mice, this will not happen and thus analysis of switching can be done beyond 21 days. It was observed that all the mutant strains tested displayed wild type levels of infectivity and persistence throughout 35 days of infection. This showed that mutant strains that displayed reduced infectivity at 21 days were fully competent for the infection process in mice lacking an acquired immune response.


 Analysis of switching at the vlsE by DNA sequencing of mutant strains recovered from SCID mouse infection- Since RFLP analysis is not quantitative and because of the previous observations that switching is apparently less frequent in SCID mice, the investigators analyzed switching in a limited set of mutants by sequencing. They chose the two mutants (ruvA and ruvB) that were defective in switching by RFLP analysis and two mutants (recJ and mutL) that were shown to switch by RFLP at 35 days post infection, but displayed no organisms in 21 days. In these experiments, the PCR product used for RFLP and obtained from different tissue (heart, joints, bladder, and ear) was used for cloning and sequencing. Sequencing of 10 clones from each tissue type culture was performed for each mutation. Thus, total 40 clones were sequenced for each mutation. It was observed that for wild type clones, 10 out of the 10 clones showed switching and contained sequences similar to those in silent cassettes in the heart and bladder tissue culture. While 5/10 and 8/10 showed switching in the joint and ear tissue culture, respectively. These results are similar to a previously reported data. In case of ruvA mutants, it was observed that only one of the 40 clones differed from the wild type vlsE (that is one showed switching). In case of ruvB, all 40 clones were similar to the wild type vlsE sequences (that is no switching). These results further confirm the negative switching observed in the ruvA and ruvB strains by RFLP. In case of mutL mutants, 27.5% of clones showed nucleotide changes and thus switching, while in case of recJ mutants 57.5% of mutants displayed switching. The major conclusions of the study are as follows:

a. The investigators successfully disrupted 17 of the 21 genes involved in DNA replication, repair and recombination. It remains to be elucidated whether the reaming four genes are essential for B. burgdorferi or whether the gene disruption mechanisms used in this study are ideal for these loci.
b. By creating these mutants, the authors aimed to study the possible role of genes in question on recombinational switching at the vlsE locus. However, the effect of these 17 mutations on generalized recombination and DNA repair is not known and authors are studying it.
c. It was observed by RFLP that ruvA/ruvB disruption leads to no switching. The results were further confirmed by sequencing.
d. Several of the mutants exhibited an altered infectivity phenotype in C3H/HeN mice that was not attributable to the defect in vlsE switching. When these mutants were inoculated into SCID C3H/HeN mice, their infectivity was restored and was comparable to wild type strains. The exact mechanism for such changes is not known.
e. recA is not required for recombinational switching.
f. A role of ruvAB encoded branch migarse in recombinational switching at vlsE
g. A possible role for mutL in recombinational switching at vlsE- No clear-cut involvement of mutL in recombination switching of vlsE was observed and this needs to be studied further. It may be possible that mutL is actually required for switching at vlsE but some other B. burgdorferi protein can substitute for mutL because of functional redundancy.
h. Role of recJ in switching?- Similarly, the possible role of recJ in switching is not clear and needs to be studied further.
i. Comparison with other antigenic variation systems: Information about proteins involved in gene conversion events is available only from the studies on N. gonorrhoeae and Trypanosoma brucei. However, the process of antigenic variation by recombinational switching in B. burgdorferi at the vlsE locus differs dramatically from that of N. gonorrhoeae and further studies are required.

The Influence of IS1301 in the Capsule Biosynthesis Locus on Meningococcal Carriage and Disease

Continuing on their previous finding that the insertion of IS1301 in the intergenic region (IGR) between siaA and ctrA operons in the MenC isolates from Spain was responsible for resistance to serum bactericidal antibodies, the authors in the present work have investigated whether isolates with IS1301 in IGR are emerging among vaccine failure strains in UK.

1. IS1301 insertion in the IGR was not associated with MenC conjugate vaccine failure in UK: MenC isolates from individuals from UK with meningococcal disease who had completed a course of the MenC conjugate vaccine were screened. 79% of the isolates were ST-11. IS1301 was not present in the IGR of any of the 33 MenC isolates, even though most of these had IS1301 insertions elsewhere in the genome. The investigators also examined MenC isolates in the UK before the introduction of conjugate vaccine. Insertion of IS1301 in the IGR region was observed in only one out of 104 isolates. In this isolate IS1301 was inserted in the same location but in opposite direction to isolates from Spain. Thus, the results suggest that IS1301 insertion in IGR is not responsible for resistance to immunity induced by vaccine, in UK.

2. Presence of IS1301 in the IGR of UK meningococcal disease and carriage isolates: The authors further examined 741 carriage and 345 disease isolates of different serogroups, collected from 1998 to 2000 in the South East of England. Insertion of IS1301 in the IGR was observed to be significantly more frequent in the disease isolates than in the carriage isolates. In the study, insertion of IS1301 was most frequently observed in the MenB isolates belonging to clonal complex ST-269 (cc269). In the collection cc269 was more commonly found among the disease isolates compared to the carriage isolates.


3. IGRs with IS1301 in MenB isolates are associated with polymorphisms: Of the 44 isolates in which IS1301 insertion was observed in the IGR in the UK, one isolate showed exactly same insertion to that of Spanish isolates. Most of the UK isolates showed 8bp deletion upstream of siaA in the IS1301. In other cases IGR contained inverted IS1301 with an internal 69 bp duplications.

4. Polymorphic sia/ctr IGRs with IS1301 lead to increased transcription of the genes for capsule transport but not capsule biosynthesis: To investigate the effect of insertion of IS1301 with associated polymorphisms in the IGR, the investigators constructed isogenic strains by introducing two common types of IGR with IS1301 with IS-8 and IS1301 with iIS+69 into a MenB cc269 strain. Transformants were selected with and without changes in IGR. That is those containing IS1301 with IS-8+ and iIS+69+ and those with IS1301 with IS-8- and IS+69-. Both IS-8+ and iIS+69+ led to a two fold increase in the expression of ctrA, which was comparable to that observed in MenC isolates. However, no upregulation in siaA expression was observed.


5. Increased transcription of capsule transport genes does not change the amount of capsules or resistance against complement mediated killing: The previous study conducted in Spain showed that increased transcription of siaA and ctrA leads to increase capsule expression but does the increased transcription of only ctrA has the same effect on capsule expression? To understand this, FACS analysis of the amount of capsule expressed by IS-8+, IS-8-, IS-69-, IS-69+ and D-157 (MenB cc269 disease isolate without insertion in the IGR) was done. It was observed that polymorphic IGRs with IS1301 do not express increased amounts of capsules. The SBA titres were also same for strains with and without the polymorphic IGRs with IS1301.

6. Presence of IS1301 in MenB cc269 diseases and carriage isolates: The investigators further examined a large number of MenB cc269 isolates (241 carriage and 421 disease isolates). Consistent with the earlier finding the insertion of IS1301 was more frequent in disease versus carriage isolates. All the insertions were polymorphic and were either IS-8 or iIS+69. Next, the authors studied genetic relatedness between STs within cc269. It was observed that the STs that are distant from the ancestral ST-269 and share only four out of seven MLST loci with ST-269, were clustered together around the subgroup founder ST-275. IS1301 insertion was observed in only one of these 217 isolates. These were significantly more prevalent in the carriage isolates. In contrast, 50% of the isolates that shared 5 or more loci with ST269 had IS1301 in the IGR. These isolates showed a similar prevalence in both the disease and carriage isolates.


Conclusions and Further investigations:
1. MenC isolates from people immunized with the MenC vaccine in the UK had no evidence of IS1301 in the IGR.
2. In UK isolates, IS1301 insertion inn IGR was restricted to MenB isolates (cc269).
3. There was a significant association of IS1301 in IGR in disease compared to carriage isolates.
4. Novel polymorphisms were observed in the IS1301 in IGR,
5. The mechanisms by which insertion of IS1301 leads to upregulation of sia (in MenC) and ctr (in MenB and MenC isolates) is not known.
6. Sia expression was not upregulated in MenB isolates in either the presence of IS-8 or iIS+69. The reason is not known.
7. Studies are being carried out to determine virulence factors by comparing the frequency of genetic traits between disease and carriage isolates.

A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies

Neisseria meningitidis is a gram negative diplococcus and it is one of the leading causes of bacterial meningitis and septicemia. It has been shown that individuals with complement deficiency are sensitive to meningococcal infection, thus indicating the complement system is crucial for immunity against this pathogen. Furthermore, it has been shown that serum of individuals infected with N. meningitidis contains bactericidal antibodies. These antibodies initiate the activation of classical complement fixation pathway, which ultimately leads to insertion of membrane attack complex into the outer membrane of the organism and subsequently bacterial lysis.

Recently, a new vaccine has been introduced into countries across Europe and North America. This vaccine is N. meningitidis serogroup C conjugate (MCC) vaccine, as there have been rising incidences of N. meningitidis serogroup C (MenC) belonging to sequence type 11 (ST11). The MCC vaccine consists of alpha 2-9 linked polysialic capsular polysaccharide coupled to a carrier protein. The protection is offered by bactericidal antibodies directed against the capsule. This vaccine has been quite successful and dramatic decrease in the incidence of MenC disease has been observed. However, the continued use of this vaccine might lead to emergence of ST-11 strains expressing other capsular types or other strains of N. meningitidis might fill the ecological niche vacated by MenC. But, till now such concerns are largely speculative.

In the present study, the authors have described the identification and characterization of three MenC strains with enhanced resistance against bactericidal antibodies elicited by MCC vaccine. N. meningitidis contains an intergenic region (IGR) between the sia and ctr operons. These operons are involved in the capsule biosynthesis and export. The authors observed that an insertion sequence, IS1301 in this IGR was responsible for the resistance. This insertion results in increase transcription of these two operons, which leads to an increase in the amount of capsular polysaccharides and impairment of the alternative pathway of complement activation.

Due to this single genetic change the bacteria can evade killing by bactericidal antibodies. Given the widespread distribution of IS1301 in N. meningitidis genome and bacterium’s competence for DNA uptake, this genetic change is very important.

Here is the summary of major results:
1. Identification of MenC strains that are resistant to sertum bactericidal antibodies elicited by MCC vaccine: To identify the resistant strains, the sera from three patients who had been immunized with MCC vaccine and who had high level serum SBA titres against C11, a MenC strain were chosen. These were used to screen 109 MenC isolates from patients with meningococcal diseases. It was observed that three strains (patient isolates out of 109) were identified, against which the serum bactericidal antibodies (SBA) titres of all three immunized patients was <8. A SBA titre>8 is a proven correlate of protection against MenC disease and a titre less than that shows resistance. None of these three patients (from which R1, R2 and R3 were isolated) had received the meningococcal vaccine. These strains were sequence typed and they were observed to be C:2a:P1.5 and ST-11, subgroup ET-15 by MLST and fumC sequencing.
2. Strains are not resistant because of changes in the LPS sialylation or acetylation of capsular polysaccharides: To understand the basis of the increased resistance of R strains against immune sera, the authors first examined the degree of LPS sialylation of strains. This was done because this modification can contribute to the avoidance of complement mediated lysis. For these studies, five fully sensitive C:2a:P1.5 and ST-11/ET-15 strains were selected from the reference lab as controls. This analysis was done by using Western blot using mAb 3F11, which binds to unsialylated LPS. No consistent difference in the degree of LPS sialylation was observed between R strains and S strains. The authors next examined the role of alteration in acetylation status of the capsule in resistance. Acetylation of capsule is mediated by a transferase encoded by oatC, which is located in the capsule biosynthesis locus (cps). The oatC contains homopolymeric tracts that can mediate ON:OFF switching of acetylation after alteration in the tract length during DNA replication. The nucleotide sequence of oatC in the R and S strains indicated that the gene is predicted to be expressed in all the strains. Furthermore, FACS analysis revealed that capsules of all strains were acetylated.
3. Insertion of IS1301 in the capsule biosynthesis locus is responsible for increased resistance: To investigate the role of capsule itself in the resistance to SBA, the authors generated the capsule negative strains of R3 and S3 by inactivating siaD. SiaD encodes capsule specific polysialyl transferase. Now, the SBA titres of vaccinees sera were measured against these capsule deficient R3 and S3 strains. It was observed that the titres were unchanged against the S3 strain, while titres were increased (from <8 to >512) for the capsule deficient R3 strain. Thus, capsule deficient R3 strain became more susceptible to killing and thus, the expression of capsule is necessary for enhanced resistance of R3.
This observation suggests that R strain might harbor changes in the cps. To test this hypothesis the genomic DNA from strain R3:oatC was used to transform S3 and the transformants were analyzed for their sensitivity to bactericidal antibodies. The authors were able to identify transformants that had become resistant to killing (S3:T1). While others transformants remain fully sensitive (S3:T2 and S3:T3). To identify the particular regions of cps that might be responsible for the resistance, the overlapping fragments of the cps were PCR amplified from the transformants (S3:T1, S3:T2 abd S3:T3). One region of the cps yielded a significantly larger product from the transformant with higher resistance (S3:T1) compared with the sensitive transformants. To define the precise location of this polymorphic region, further PCRs were done. A nearly 1.1kb fragment was amplified from resistant strains, while a 300 bp product was obtained from sensitive strains. These results showed a direct correlation between the size of this region of cps and the ability of the strain to show resistance to SBAs. The authors further performed sequence analysis of this region and found out the presence of an insertion sequence IS1301 in the 134bp IGR between siaA and ctrA in all R strains and S3:T1 strains. IS1301 is an 844 bp mobile element that is present in multiple copies in the genomes of some meningococcal strains, including ST-11 isolates belonging to ET-15.

4. Insertion of IS1301 in the IGR increases capsule expression but does not affect its structure: To determine whether the insertion of IS1301 affected the antigenic properties of the capsular polysaccharide, immune sera were raised against S3, S3:R and R3 strains. Sera raised against S3 mediated killing of the homologous strain S3 but not S3:R or R3. Sera raised against S3:R and R3 were also able to elicit complement-mediated lysis of S3. This showed that the capsular polysaccharide from the R strains retain its immunogenecity. Even high concentration of same sear failed to induce killing of S3:R or R3. This suggested that the increase in resistance of the strain was not due to an alteration in the nature of antigens expressed. Furthermore, the authors studied the composition of the capsular polysaccharides of the strains by NMR spectroscopy. The spectrum of purified capsule from S3 was similar to that of S3:R and R3. To examine whether the insertion of IS1301 in the IGR affected the amount of capsule expressed, the authors performed quantitative real time PCR on siaA and ctrA, and compared the results with the transcripts levels of a house keeping gene, gdh. R3 and S3R strains showed a two to three fold increase in mRNA levels of both siaA and ctrA compared with S3. No significant difference was observed in the mRNA levels of gdh between the sensitive and resistant strains. The authors also determined levels of SiaA protein expressed by all the strains using Western blotting. It demonstrated that R3 and S3:R strains produced higher levels of SiaA protein than S3 strains. The authors also examined the strains by cyto-TEM after labeling live bacteria with the electron dense, cationic stain ferritin. This stain binds to negatively charged bacterial capsules. By using TEM, capsular polysaccharides were clearly visualized around strain S3. In contrast, there was much more capsule surrounding the R3 and S3:R strain.

5. Activation of the alternative complement pathway is reduced on the strains with enhanced resistance: To understand the underlying mechanisms for the resistance, the investigators examined the deposition of complements on the surface of bacteria. For this, the bacterial strains were incubated in immune serum, and the deposition of complements was examined. Both R3 and S3:R showed less deposition of C3 and MAC on their surfaces. Furthermore, the AP activity on the surface of strains was examined by blocking the CP and lectin pathways with magnesium and EGTA. This resulted in overall decrease in levels of complement factors deposited on the surface of bacteria. However, the levels of C3 and MAC present on the surface of R3 and S3:R were still less than S3 even in the absence of the CP and LP. This showed that strains with enhanced resistance had reduced activity of the AP on their surface.

The pubmed citation of this article is: Uria MJ, Zhang Q, Li Y, Chan A, Exley RM, Gollan B, Chan H, Feavers I, Yarwood A, Abad R, Borrow R, Fleck RA, Mulloy B, Vazquez JA, Tang CM. A generic mechanism in Neisseria meningitidis for enhanced resistance against bactericidal antibodies. J Exp Med. 2008 Jun 9;205(6):1423-34.

The Discovery of Endogenous Retro Viruses Part 2

Murine Leukemia Virus (MLV) and mammalian gamma retro viruses
Aaronson et al in 1969 observed spontaneous release of MLV from uninfected murine cell cultures. At the same time when Dr. Weiss found the induction of virus production from chick embryo cells, similar experiments were reported for MLV activation by halogenated pyrimidines. The genetic mapping and analysis of gene expression of endogenous MLV was studied in great detail in 1970s and 1980s. As observed in case of endogenous ALVs, many of the genomes of endogenous MLV were defective, while others contained ORFs or complete, potentially infectious genome.
With this discovery of endogenous ALV, many investigators in the 1970s began to examine cells from other species for similar viruses. Many mammalian species including non-human primates were found to harbor MLV related gamma retro viruses.

Murine Mammary Tumor Virus (MMTV)
Earlier, it was thought that the susceptibility to breast cancer in mice was genetic. However, J.J. Bittner made an important observation in 1936. He observed that foster nursing of a low-incidence strain of new born mice on a high-incidence mother, caused the females to develop breast cancer as adult. Further, in 1949, Dr. Dmochowski observed a filterable oncogenic agent in the milk, which led to the identification of MMTV.
The finding that MMTV is endogenous was made at the same time when endogenous ALV was described. As with ALV and MLV, mice carry numerous MMTV ERVs in their chromosomes.

Xenotropism and Xenotransplantation
It has been observed that many endogenous retroviruses do not readily re-infect their own host cells but can infect other species in vitro or in vivo. Thus the endogenous ALV of chickens infects cells of quail, pheasants and turkey more readily than the
chicken. Jay Levy coined the term ‘xenotropic’ for viruses that only infect foreign species. The host has a selective advantage for being insusceptible to re-infection by a potentially pathogenic ERV. This ERV can not be amplified to reach a high viral load in its host. Resistance mechanisms for re-infection include mutation of receptors, blocking of receptors by endogenous Env expression, and intracellular restriction factors. Murine hybridomas can also release xenotropic MLV, so it is important to ensure that biologic medicines such as therapeutic monoclonal antibodies are not contaminated by retroviruses.

Evolutionary Perspectives

There are many questions on these ERVs. Do they represent junk DNA or do they play important roles in genetic regulation of the host? Do retroviruses serve as vectors for horizontal gene exchange? Do ERVs always become defective over time?

There are evidences that suggest that MLV-related gamma-retroviruses may reside for million of years in the germ line of its hosts and yet remain replication competent. Maintenance of functional genomes with ORFs probably requires retrotranspositions. This might be the reason that the ERVs with the complete genomes are recently recycled ones. Colonization of a new host presumably goes via an infectious phase before insertions occur in its germ-line.
Retroviruses could serve as a horizontal means of exchange of genetic information. However, other than transporting themselves, ERV do not appear to be a career of genes.
At the end I would like to quote the last paragraph from this review which talks about the lacunae in information and the future directions.
“ Finally, one may ask why DNA viruses that have a capacity to integrate into host DNA have not been detected in the germ line. Although integration is not an obligate step in their replication cycles, polyoma viruses, papilloma viruses, hepadnaviruses, adenoviruses and parvoviruses could each have gained a free ride to the next host generation, provided they were able to infect primordial germ cells or early embryo cells before segregation of the germ line. Adeno-associated virus has a preferred integration site on human chromosome 19 but has apparently not become inherited at this locus. Like MLV, the polyoma virus, SV40, can infect embryonal stem cells in vitro, and become latent in them. This would be a good way to endogenize yet there is little evidence that it has happened. I am aware of only one example of a Mendelian DNA virus, that of human herpesvirus 6, and this is not universal in the human population. It will be fascinating to work out why HHV-6 but not other herpesviruses
endogenize, and whether other non-retroviral endogenous genomes will be discovered.”

Discovery of Endogenous Retro Viruses Part 1

My today’s blog is on a very interesting review article by Dr. RA Weiss on the discovery of endogenous retroviruses (ERVs). These were discovered in late 1960s and early 1970s. Three types of ERVs were discovered around the same time. These were avian leucosis virus in the domestic fowl, murine leukaemia virus and murine mammary tumor virus in the laboratory mice. Retroviruses in general can be classified into two types: those having simple genome and those with complex genome. Only the simple retroviruses have become endogenous in their hosts with the exception of spumaviruses (have complex genome).

Retroviruses and the proviral hypothesis-
Retroviral diseases were recognized very early in 20th century, although the name retrovirus was coined only in 1974. In 1904, Vallée and Carré showed that equine anemia was infectiously transmitted by a filtrate and we now know that the etiologic agent is a lentivirus. In 1961, it was shown that Rous Sarcoma virus (RSV) particles contain RNA. Howard Temin observed that cells transformed by RSV maintained stable properties through many mitoses. Based on this inheritability of virus transformed phenotypes even in the absence of viral replication, he postulated that RSV genome makes a DNA copy in the infected cell. This DNA copy is then integrated into the host chromosomal DNA. He called this concept as DNA proviral hypothesis. Dr. Andre Lwoff, who had won a noble prize for the discovery of the prophage and lysogeny, has suggested integration of DNA virus, polyoma virus, into the host genome. The concept of integration of DNA tumor viruses genomes in transformed somatic cells was debated and demonstrated in 1968 by Sambrook et al. In spite of all these advances in the understanding of these concepts, the idea of integration of DNA genomes of RNA tumor viruses into the germ-line of healthy animals was not considered.

Endogenous Avian leucosis virus (ALV)
ALV is an alpha retrovirus and it replicates in chick embryo fibroblasts but does not transform them. It is closely related to RSV. However, RSV carries the src oncogene and transforms fibroblasts. The genome organization of these viruses is:

ALV: 5' LTR-gag-pol-env-LTR 3'

RSV (Bryan): 5' LTR-gag-pol-src-LTR 3'

RSV (Prague): 5' LTR-gag-pol-env-src-LTR 3'

The Bryan strain of RSV is defective for replication as src gene is substituted for env gene. Defective Bryan strain can be rescued by ALV which supplies the missing env glycoproteins. Thus, ALV was called a helper virus.

In 1960s, avian leucosis had become a problem in egg-lying hens and thus to screen for leucosis a serological test was devised for group specific antigen (gag) which was common to all ALV serotypes. This test was based on complement fixation and was called COFAL test. It was later observed that the test was not sufficiently specific as some uninfected chickens gave positive results. Furthermore, virus like particles and gag like antigens were detected from ALV-free chicken tissue. Then Payne and Chubb demonstrated that gag-related antigen was inherited as a dominant Mendalian gene in crosses between gag positive and gag negative chicken. Thus, the question arose that whether the endogenous antigen was encoded by a latent retroviral genome or whether it represented a normal host protein with a cross reacting epitope.

Dr. Weiss had observed during his PhD thesis that fibroblast cultures of some chick embryos but not the others, allowed release of infectious Bryan RSV in the absence of “helper” ALV. Dr. Weiss also observed that the envelope of helper free RSV was novel in its receptor specificity and neutralization properties. Similar results were also produced by other labs. Thus, it became evident that some normal chick cells could provide the missing env protein to Bryan RSV. Based on all these observations, Dr. RA Weiss published his work and suggested existence of an integrated retrovirus in normal embryo cells.
Two lines of evidence namely, Mendelian inheritance of gag-related antigen and complementation of an env defective strain of RSV provided a clue that something related to a retrovirus existed in the normal chick embryo cells. So the next step was to determine whether Env complementation and Gag expression were inherited concomitantly. Using inbred chickens, F1 hybrids and back-crosses, the researchers found that both phenotypes were indeed inherited according to Mendel's first law and that they segregated together as a single locus. In 1970, Dr Weiss and Dr. Peter Vogt provided evidence that treatment of normal chicken cells with a variety of activating agents such as ionizing radiations or carcinogens, stimulated release of virus. The authors further used reverse transcriptase activity to measure release of virus particles. Later, based on southern hybridization many proviral copies were found to be present in most chicken breeds. These individual proviral loci were characterized and mapped, many represented incomplete or defective genome.
Dr. Weiss was further interested to know if the chicken ERV was a recent introduction into domestic fowl, or whether it was present in the ancestor species, the red jungle fowl. In 1970, he made a field trip to Malaysia and lived with tribesmen (orang asli) in the Pahang jungle who knew how to trap these birds, in order to take blood samples and to collect eggs for cell culture. The red jungle fowl carried endogenous ALV. He and his team later found that the three other extant species of the same genus, Gallus, did not possess endogenous ALV. Apparently this ERV colonized the chicken germ-line after speciation but before domestication.

Astrin et al identified a rooster that lacked any integrated provirus and a line of chickens was eventually bred from this bird. This showed that viral genomes were not essential for host functions. However, these chickens do carry a second family of ERV called endogenous avian virus (EAV) although they are not infectious. EAV sequences are present in DNA of all species of Gallus and therefore have a more ancient origin.

A third group of avian retroviruses include reticuloendotheliosis virus of turkey.

Sustained desensitization to bacterial Toll-like receptor ligands after resolution of respiratory influenza infection

Reading good articles on host pathogen interactions gives me one of the greatest joys. Today, I will discuss one such article. This is a brief report published in Journal of Experimental Medicine and is from the lab of Dr. Tracy Hussell, Imperial College, London.
In recent years, it has been observed by various studies that one respiratory tract infection may alter immunology and pathology to a second unrelated pathogen, even long after resolution of first pathogen and in the absence of cross-reactive immunity. The outcome of such successive infections may be beneficial to the host or it may be harmful for the host. One of the most common examples is life threatening bacterial pneumonia observed in patients infected with influenza virus. These data suggest that lung undergoes some form of maturation that alters the way it responds to subsequent infections. Many mechanisms have been proposed to explain enhance bacteria at the time of seasonal and pandemic influenza or RSV. These include a disruption of epithelial integrity, up regulation of bacterial adhesion molecules, and/or alteration in antibacterial peptides. This study proposes an additional mechanism, TLR desensitization.
It is generally assumed that innate immune system returns to its normal preinfection state after multiple waves of inflammation. However, it has been observed that the number of dendritic cells and their ability to prime T cells in the murine lung remain elevated even after the resolution of RSV or influenza virus infection. In the present study, evidence was provided that TLR responsiveness is reset after first wave of inflammation. The authors show a long term desensitization of alveolar macrophages to TLR ligands (LPS, flagellin, and lipotechoic acid). Ligation of TLR ligands lead to activation of signaling pathways, resulting in cytokine and chemokine release and finally in pathogen clearance. In this study, the authors show that AMs isolated from lungs of infected animal after resolution of influenza, have impaired NF kappa B translocation in response to TLR ligation. This results in reduced subsequent inflammation and cell recruitment, which further correlates with a higher and prolonged respiratory bacterial load.
Here is a summary of major results and methods used:
1. Impairment in cell recruitment in the post influenza airway in response to TLR ligation: Bacterial TLR ligands (LPS, flagellin and LTA) were introduced in the lungs of post influenza mice. The percentage and total number of macrophages and neutrophils in the lungs and airway was monitored by flow cytometry after 4 weeks. Administration of TLR5 ligand flagellin caused reduced neutrophils migration into the airway compared to the control mice. A similar impairment in the neutrophils number was observed on induction with TLR4 ligand, LPS. At 48hr post induction reduction in macrophage number was also observed. Similar results were observed on TLR-2 ligation.
2. Impairment in neutrophils recruitment on inoculation with Pseudomonas aeruginosa and enhancement in bacterial load.
3. Impairment in neutrophils recruitment on inoculation with group B Streptococcus and enhancement in bacterial load.
4. Reduced neutrophilia to a second respiratory stimulus is due to a reduction in chemotactic signals to draw them there- To prove that reduced neutrophilia to a second respiratory signal is not due to enhanced apoptosis within the lungs, the proportion of apoptic neutrophils was measures and it was observed that proportion of apoptic neutrophils was low and comparable between post influenza and control mice. To further show that reduced neutrophilia was not due to impaired neutrophils transmigration, post influenza and control mice were inoculated with neutrophils chemoattractants. It was observed that equivalent number of neutrophils was recruited in post influenza and control mice. Altogether, these data indicate that the local induction of the TLR signaling pathway is altered by prior viral lung exposure.
5. AMs are desensitized to TLR-mediated signals-The authors further studied which cell types were desensitized to TLR-mediated signals. On systemic administration of flagellin, which targets lung endothelial cells, no impairment in neutrophils infiltration in the lung parenchyma or peripheral sites was observed. This suggested that alteration in TLR signaling is involved when flagellin is administered intra-nasally. To study the extent at which AMs are effected, AMs were isolated 1hr after flagellin challenge and tested ex vivo. They displayed reduced levels of mRNA transcripts for KC, MIP2α and TNF-α in post influenza compared with controls.
6. To determine whether transcriptional regulations occurs independently in AMs or as result of co-operation with other cells e.g., alveolar epithelial cells (AECs), both populations of cells were individually isolated from post influenza mice. These cells were treated with flagellin and NF-kappa B activation was determined in vitro. The authors observed that nuclear translocation of the p65 subunit of NF kappa B in response to flagellin was inhibited in AMs from post influenza mice. No such inhibition was observed in AECs. Thus, the authors proposed that sustained reduction of NF kappa B activation in AMs after the initial viral infection leads to reduced inflammatory response and neutrophils recruitment. To support this, the authors conditionally depleted CD11c+ cells, including AMs, from post influenza mice. This caused nearly 90% reduction in AMs and lung macrophages. The pools of macrophages were regained in 3 weeks. The new macrophages were isolated and induced with flagellin. The reduction in neutrophils recruitment was not observed. These data suggest that AMs are instrumental in the long lived altered response in the post influenza lung.
Things still not known:
1. It remains to be determined whether AMs are directly affected by infection or their phenotypes are altered by interaction with recruited inflammatory cells.
2. In addition, whether the pool of lung macrophages that give rise to AMs is also affected by previous infection warrants further investigation.
3. The molecular mechanisms responsible for long term TLR desensitization remain to be resolved

Identification of small RNAs in Mycobacterium tuberculosis

Mycobacterium tuberculosis: I have always been fascinated by this one of the most successful human pathogens. Whenever I happen to see any research work on this pathogen, I try to read it. While doing some random search on pubmed, I happened to come across this very beautiful article. This article is about identification of small RNAs in M. tuberculosis and the authors are Dr. Kristine B. Arnving and Dr. Douglas B. Young, Division of Mycobacterial Research, London UK.
In many bacterial species, it has been shown that the mechanisms of transcription control are complemented by a post transcriptional regulatory network dependent on small regulatory RNA (sRNA) molecules. These sRNA molecules can enhance or suppress translation of mRNA targets by base pairing with the 5’ end of mRNA molecules at different locations relative to the ribosome binding site and start codon. They can also alter stability of mRNA by generating duplex molecules which act as substrate for RNase III or RNase E. It is observed that these sRNAs play important roles in regulation of stress response, bacterial cell cycle and also in bacterial pathogenesis.
The present study was conducted to investigate the occurrence of sRNAs in M. tuberculosis.
Two general approaches have been used for identification of sRNAs in bacteria. The first is bioinformatics prediction by sequence alignment of intergenic regions with known sRNAs, together with identification of appropriately positioned signals for transcriptional initiation and termination. Other approach is direct analysis of low molecular weight RNA molecules from culture of bacteria. In this paper authors used the second approach due to relatively poor definition of transcriptional signals in Mycobacteria. The authors have described a set of nine putative sRNAs identified by this approach and characterized by Northern Blotting and transcript mapping by 5’ and 3’ RACE analysis.
The major results of the study are as follows:
1. Cloning of small RNAs from M. tuberculosis- The initial experiment was aimed for determining the actual presence and abundance of small transcripts, regarded as putative sRNAs. For this, M. tuberculosis was cultured and total RNA was extracted from exponential and stationary growth phases. This total RNA was depleted of rRNA (16SrRNA and 23SrRNA) by microbeExpress kit. After the depletion, it was labeled with 32P-pCp and RNA ligase. The RNA was separated on a denaturing acrylamide gel and visualized by phosphorimaging. Multiple abundant and well defined small transcripts were observed. The pattern of expression was different between the two growth phases. Now, two independent cDNA libraries were constructed from stationary and exponential phase cultures. For preparation of cDNA library, total RNA was size fractionated and transcripts between 20-75 nucleotides were eluted from the gel. The eluted RNA was tailed with CTP and an RNA linker was attached to the 5’end. The RNA was then converted to cDNA, PCR amplified, cloned and sequenced. A total of 192 clones were sequenced and they fell into 6 categories. They were:
a. mRNA fragments with open reading frames (ORF): 11 clones
b. rRNA spacer fragments (5 clones)
c. tRNA fragments (1 clone)
d. unknown RNA encoded in intergenic regions (trans candidates, 23 clones representing six unique regions and 19 repeat clones).
e. Unknown RNA encoded antisense to annotated ORFs
f. Fragments that could not be assigned to a single region due to small size (<17bp) or chimeric sequences.
In order to determine the likelihood of each unknown to be a genuine sRNA or to be a part of an adjacent gene, the genomic positions and context of unknown RNAs were identified. If an intergenic cDNA clone was encoded on the opposite strand of an adjacent gene or more than 100 base pairs from an adjacent gene on the same strand, it was considered to be a valid sRNA candidate. When this criterion was applied, most of the intergenic clones appeared to be independent transcripts and not 5’ or 3’ UTRs.
2. Northern Blots verify the presence of sRNAs in M. tuberculosis- The cDNA libraries identified nine sRNA candidate. To further verify them, northern blotting with riboprobes complementary to the original cDNA clones was performed. The results of Northern blotting demonstrated signals corresponding to small transcripts from each of the candidates. Judging from the signals on the Northern blots, trans encoded sRNAs were expressed at significantly higher levels than the cis encoded sRNAs. All transcripts were larger than the cloned fragments, suggesting that the cloned fragments were probably degradation products.
3. Mapping of transcripts: sRNA transcripts were further characterized by RNA ligase mediated rapid amplification of 5’ and 3’ complementary DNA ends (RLM-RACE). The 5′ ends were mapped by comparing the RACE products obtained with and without prior treatment with tobacco acid pyrophosphatase (TAP), which facilitates the differentiation of transcription start sites from processed 5′ ends. The results suggested several putative transcription start sites. The authors also performed 5’ RACE on RNA from stationary phase.
4. Transcriptional coupling and sequence conservation: Excluding G2, all of the trans encoded sRNA candidates were encoded on the same strand as one of the adjacent protein-encoding genes. To test whether these sRNAs were co-transcribed with the particular upstream or downstream gene, RT PCR was performed by using primers that spanned the sRNA and the adjacent gene. It was observed that B55 was co-transcribed with the upstream Rv0609A. This suggested the possibility that B55 is part of 3’ UTR of the Rv0609A mRNA rather than being an sRNA. C8 was found to be co-transcribed with Rv3722c. On searching the Rfam database with the C8 sequence revealed that this RNA was in fact 4.5SRNA. F6 was co-transcribed with upstream fadA2. No RT PCR product was obtained using primers spanning B11 and Rv3660c. This indicated that B11 is a bonafide sRNA.
5. Prediction of secondary structure of sRNAs: All sRNA candidates except B55 and C8 had a C:G ratio>1.
6. Expression of M. tuberculosis sRNAs during stress: To study this, cultures of M. tuberculosis were subjected to oxidative stress (induced by H2O2), DNA damage (induced by Mitomycin C) and acid stress. It was observed that stress induced expression varied significantly between sRNAs.
7. Overexpression of M. tuberculosis sRNAs: Trans encoded sRNAs, B11, F6 and G2 were cloned in plasmid vectors under the control of the strong rrnB promoter of M. smegmatis and transformed into M. tuberculosis and M. smegmatis mc2155. The constructs expressing B11 and G2 both proved lethal in M. tuberculosis, while the expression of F6 resulted in extremely slow growth with pin prick colonies visible after 3-4 weeks.

In summary, the present study provides first evidence that M. tuberculosis expresses sRNA molecules and that these play important role in bacterial physiology. The authors anticipate that further functional studies in combination with sequence-based RNomics will provide novel insights into the fundamental biology of tuberculosis with the potential to inform development of improved strategies for disease control.

The immune control of HTLV-1 infection: selection forces and dynamics

This is a very interesting review article on immune response to human T cell lymphotrophic virus. The review is by Dr. Charles R M Bangham and his colleagues from Imperial College, London. I would like to highlight some major points from the review.

1. Risk of HAM/TSP depends on HTLV-1 proviral load: Studies have shown a strong and reproducible association between a high proviral load and a high risk of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is a remarkable difference in the stability of proviral load of HTLV-1 in each infected patient over time. There is also variation in the proviral load among patients. As HTLV-1 does not vary much in sequence either within or between hosts, it appears that variation in proviral load among hosts is caused by differences in the host rather in the virus.

2. HTLV-1 proviral load is correlated with the HTLV-1 specific CTL activity: CTL are abundant in the blood of both HAM/TSP patients and asymptomatic carriers. CTLs kill autologous cells that express viral antigens (one of the chief antigens is Tax) and by suppressing viral replications by secreting IFN gamma. Because of very high frequency of infected cells and CTLs in the peripheral blood, it is possible to determine the rate of this autologous CTL mediated lysis in fresh peripheral venous blood. This is done by measuring the effect of varying the frequency of CD8+ cells on the rate of disappearance of HTLV-1-expressing cells. These studies showed that in both HAM/TSP patients and in asymptomatic careers, there was a strong negative correlation between the proportion of HTLV-1 Tax+ cells eliminated per day and the proviral load in vivo. These findings may imply that a high rate of CTL mediated killing suppresses the proviral load in vivo. Based on various other studies it has been hypothesized that positive selection of the virus is caused by the HTLV-1 specific CTL response, these CTLs in turn limit the viral replication in vivo.

3. HTLV-1 infection accelerates the turnover of both CD4+ and CD8+ T cells in vivo- In a recently conducted experiment, the lymphocytes were labeled with denatured glucose administered by intravenous infusion. The results showed that there was a strong increase in the turnover rate of both CD8+ cells and of HTLV-1 infected CD4+ cells. The life span of Tax+ cells as well as HTLV-1 specific CTLs in circulation was decreased. Since this study was conducted in vivo, it provides one of the most direct evidence that HTLV-1 infection is associated with a persistently high rate of lymphocyte proliferation.

4. The rate of HTLV-1 expression varies among hosts and among T cell clones within one host- No association has been observed in the sequence of the virus and either the proviral load or the rate of proviral expression. However, studies have shown significant heterogeneity among HTLV-1 infected hosts in the rate of expression of HTLV-1. Dr. Bangham and others have shown that the level of spontaneous proviral expression in PBMCs ex vivo, is systematically greater in patients with HAM/TSP than in careers, at a given proviral load. It means both groups having same proviral load can have different expression. Studies have also shown that the rate of HTLV-1 virus expression varies among individual T cell clones in a single host. These observations lead to the hypothesis that each provirus containing T cell clone has a characteristic rate of onset of spontaneous proviral expression and this characteristic rate is maintained in the daughter cells of that clone.

Antibody based vaccines for Viruses

As viruses replicate and reside inside a cell, antibodies are of not much use in eliminating a viral infection, as they can not penetrate inside the cells. Some viruses have to exit the cell, to infect neighboring cells. In such cases antibodies may help to prevent spread of infection. But, some viruses can directly spread to neighboring cells with out being exposed to antibodies. So once a virus has entered a host cell, cell mediated responses play important roles in eliminating infection. This is about eliminating infection but antibodies can play important roles in blocking infection. Before entering inside a host cell a virus has to attach to the cell and thus is exposed to the extracellular components at least briefly. Antibodies thus might not be of much help in eliminating already established infection, but they can be very good in protecting against new infection. Due to this reason, most of the antiviral vaccines work by inducing strong and specific antibody responses. In such cases, killed vaccine can also be used, which are safer than attenuated, live vaccines.

What keeps a virus from being blocked by antibodies? There could be many possible reasons but one could be the variable nature of surface antigens on these viruses. One of the most famous example is HIV. The glycoproteins on the surface of HIV are extremely variable. Thus, an antibody that can block one virus strain might not be effective against another. This problem can be solved by targeting those antigens which are not variable. The approach is to find out epitopes on the surface of viruses which are conserved. If a antibody can be designed against such targets, then it might prove useful in protecting against many strains of viruses.

A recent review published in annual reviews of medicine, has summarized progress in understanding the host antibody response to HIV-1 and current strategies for applying this information to develop an effective vaccine.
I would like to quote the abstract here:
Developing an HIV-1 vaccine that can elicit antibodies to prevent infection has been a formidable challenge. Although no single immunogen has generated antibodies that can neutralize diverse isolates, progress has been made in understanding (a) the structure of the HIV-1 envelope glycoprotein, which is targeted by neutralizing antibodies, (b) how HIV-1 evades antibodies made by an infected host, and (c) how rare monoclonal antibodies can exhibit broadly neutralizing activity. Advances in structural and molecular biology coupled with new approaches to isolate neutralizing antibodies from HIV-1-infected individuals are enhancing our understanding of what humoral immune responses will be required for a vaccine.

Latest in Pneumocystis research

Pneumocystis jirovecii is an opportunistic fungal agent that causes Pneumocystis pneomonia (PCP) in immunocompromised individuals. Although the incidences of PCP have decreased in HIV seropositive individuals after the introduction of highly active anti-retroviral therapy (HAART), it remains one of the most common opportunistic infections in such patients. A higher morbidity and mortality has been observed due to PCP in HIV negative immunocompromised individuals, in comparison to HIV positive individuals. One possible reason for this difference among the two groups of patients could be the difference in ability to mount an inflammatory response. It has been shown that non HIV infected patients have a more robust inflammatory response against the organism in comparison to HIV infected individuals. This exuberant immune response towards the organism has been shown to be more harmful to the host than the organism itself.
A lot of research has been going on Pneumocystis. Today, I read an interesting article entitled, "Pneumocystis cell wall beta D glucan stimulates calcium dependent signaling of IL-8 secretion by human airway epithelial cells". This work has been performed in the lab of Dr. Andrew H. Limper, USA. Pneumocystis organisms are present within the alveolus in lungs in two different forms, i.e. trophic and cyst forms. The cyst forms have a thick cell wall which is rich in beta glucan. Recent studies have shown that this beta glucan is a major initiator of lung inflammation during Pneumocystis infection. However, the mechanisms by which beta glucans induce this exaggerated inflammatory response is not fully clear.
The authors of this paper have earlier demonstrated that beta glucan in the wall of Pneumocystis induce NFkappaB translocation and TNF-alpha production in macrophages. The authors have also demonstrated that Pneumocystis beta glucans (PCBG) stimulate rat airway epithelial cells to secrete macrophage inhibitory protein-2 (MIP-2) through NFkappaB dependent mechanisms. However, the events by which PCBG activates airway epithelial cells are unclear. It has been shown that several bacterial pathogens e.g. Pseudomonas and Salmonella activate airway epithelial cells by increasing intracellular concentration of calcium. Based on this knowledge, the authors have hypothesized that binding of PCBG stimulates airway epithelial cells. These cells induce changes in cytosolic calcium influx. The change in intracellular calcium subsequently activates signal transduction pathways. The activation of these pathways finally leads to cytokine secretion by airway epithelial cells.
Adhesion of the fungus to host cells is an important step to establish infection. Several receptors have been proposed to bind Pneumocystis particles. Lactosylceramide is one such receptor. This group has earlier observed that lactosylceramide is responsible for MIP-2 production. Thus, this study also evaluated the role of glycosphingolipids in cytokine signaling by airway epithelial cells activated with PCBG.


Major Results:
1. PCBG induces IL-8 secretion from airway epithelial cells: Human airway epithelial cells 1HAEo were cultured and challenged with PCBG or S. cerevisiae derived beta glucan or LPS and IL-8 secretions were measured by ELISA. SCBG and PCBG challenged cells secreted IL-8 in a dose dependent manner compared with LPS-challenged cells.

2. IL-8 secretion by airway epithelial cells stimulated with PCBG is calcium dependent- HAEo cells were loaded with Fura-2AM, a calcium binding dye, and incubated with either PCBG or a positive control peptide for indicated time and transient intracellular calcium release was measured. To further show the importance of intracellular calcium mobilization, HAEo cells were pretreated with various extracellular and intracellular Ca chelating agents before stimulating with PCBG. Then the secretion of IL-8 was measured. It was observed that in cells preincubated with intracellular chelating agents, a significant decrease in IL-8 production was observed. In contrast, cells pretreated with extracellular Ca chelating agents did not show any decrease.

3. IL-8 secretion by airway epithelial cells is mediated by NFkappaB and AP-1- IL-8 promoter contains a variety of transcrption factors binding sites (including those of NF and AP-1). To investigate the importance of NF and AP-1 in IL-8 production induced by PCBG, HAEo cells were transfected with IL-8 luciferase reporter construct (promoter) or with IL-8 luciferase reporter construct (promoter) that had targeted mutations in NF or AP-1 binding sites. These cells were than challenged with PCBG. After certain incubation the cells were harvested and luciferase activity was measured. The results showed that PCBG failed to activate IL-8 transcription in cells transfected with either the mutant NF or mutant AP-1 promoter. IL-8 transcription was normal in cells transfected with wild type promoter.

4. IL-8 secretion by PCBG stimulated airway epithelial cells is mediated by MAP kinases- HAEo cells were pretreated with a inhibitor of ERK (PD98059) and then stimulated with PCBG. A dose dependent decrease in IL-8 production was observed. To further understand the kinetics of MAPK/ERK activation, phosphorylation of ERK was determined by western blotting after stimulation of cells for different periods of time. Phosphorylation of ERK p44/42 was detected within five minutes of stimulation and remained slightly elevated as long as two hrs after the initial challenge.
Next, the authors tried to evaluate whether p38, an independent MAPKs pathway, participated in beta glucan mediated IL-8 secretion from airway epithelial cells in response to PCBG. For this, cells were treated with a p38 inhibitor, SB202190 and then stimulated with PCBG. A dose dependent decrease in IL-8 production was observed indicating the role of p38 in release of IL-8. Furthermore the kinetics of p38 was followed by western blot studies. Phosphorylation of p38 was detected at 15 minutes post stimulation and reached a peak at 30 minutes. After 1 hr, phosphorylation of p38 returned to a baseline level. These data show different kinetics of these two pathways (ERK p44/42 and p38). Similar experiments were repeated for JNK. A JNK inhibitor, was used prior to treatment with PCBG. No inhibition in production of IL-8 was observed. These results indicated that JNK does not participate in IL-8 response.

5. MAPK activation in PCGB stimulated HAEo cells stimulates downstream NF expression: To determine whether MAPK activation following beta glucan stimulation also results in downstream NF dependent activation in HAEo cells, HAEo cells transiently infected with NF promoter plasmid were pretreated with inhitor for ERK p44/42 or p38 or JNK and then stumulated with PCBG. Pretreatment of cells with ERK p44/42 and p38 inhibitors led to decrease in transcritional activity of NF. Treatment with JNK inhibitor showed no change.

6. Inhibition of glycophospholipids synthesis further impairs IL-8 secretion by airway epithelial cells stimulated with BCPG- IL-8 secretion in PCBG stimulated cells was assessed in the presence of a potent glycophospholipd synthesis inhibitor. Il-8 production was decreased.

Conclusions: Thus, the present study demonstrated that human airway epithelial cells secrete significant amounts of IL-8 in response to Pneumocystis beta D Glucan. Also, airway epithelial cells mobilize intracellular Ca within seconds after PCBG stimulation. This intra calcium flux initiates the activation of two major MAPk pathways, ERK p44/42 and p38 and subsequent activation of NF and AP-1, which results in release of IL-8.

Future Studies: Better knowledge of the molecular mechanisms regulating chemokine generation will be essential to understand the recruitment of inflammatory cells to the lung during Pneumocystis infection and to design therapeutic strategies for exaggerated lung inflammation.

A study on Leukocyte Ig like receptors in the context of infection

Leukocyte Ig like receptors (LILR or ILT or ILR) are a family of innate immune receptors. These are predominantly expressed on the cells of the myelomonocytic lineage. This family includes 11 proteins that can be further subdivided into activating (subfamily A) or inhibitory (subfamily B) receptors on the basis of their transmembrane or cytoplasmic domains. A lot of research has been going on their role in immune dysregulation and following transplantation. However, not much is known on their role during infection. LILBR4 and LILBR2 are two such important receptors. They have the potential to inhibit T cell proliferation and thus they have been extensively studied in the context of allograft tolerance in case of transplantation and in cancer. The aim of the present work was to better understand the inhibitory potential of LILBR2 and LILBR4 during infection. Salmonella typhimurium is an obligate intracellular pathogen that infects macrophages. In the present study, the modulation of expression of these receptors on antigen presenting cells infected with S. typhimurium was determined.

Cell surface phenotype of LILRB4 ligated dendritic cells= To study this, dendritic cells were cultured in the presence of an isotype control antibody (control), LILBR4 specific antibody with protein G. LPS matured dendritic cells were also cultured in the presence of an isotype control antibody (control) and LILBR4 specific antibody. On these cells flow cytometric analysis of cell surface markers (CD80, CD86, CD83, CD1a) was performed. Ligation of LILBR4 led to an upregulation of costimulatory molecule CD86 on both immature and LPS matured dendritic cells. No significant effects on expression of CD83, CD80 and Cd1a was observed.

To test the effect of ligation of LILBR4 on dendritic cells on T cell stimulation, the immatured and matured dendridic cells were cultured in the presence of isotype control and LILBR4 specific antibody as before. To these cells naive CD4 cells from an allogenic donor were added in various stimulator: responder ratios and cultured for 6 days. On day 6 of culture, triatiated thymidine was added, cells were incubated and analysed using a scintillation plate counter. These experiments are called mixed leukocyte reactions (MLR). The results of such five MLR experiments showed no increase or decrease in T cell proliferation in response to ligation of LILBR4 on dendritic cells.

To study the effect of ligation of LILBR4 on cytokine secretion profile of macrophages, these cells were cultured in the presence of isotype control, LILBR4 specific antibody. Same was done for LPS matured APCs. The concentration of IL-8 appeared to decrease while concentration of IL-10 increased by ligation of LILBR4 in both mature and immature cells. The results were not significant though.

TTo study the effect of S. typhimurium infection on the expression of LILBR4 and LILBR2- monocyte derived macrophages were infected with S. typhimurium (transformed with pFVP25.1-GFP plasmid). Infection of macrophages was confirmed by GFP expression study by flowcytometry. To study the expression of LILBR2 and LILBR4, RNA was extracted from these macrophages and real time PCR was performed. Similar experiments were done on macrophages infected with heat killed bacteria. An upregulation of LILBR2 and LILBR4 was observed in both the cases.

To study the effect of bacterial components (LPS, TLR4 ligand and flagellin TLR-5 ligand) on LILBR2 and LILBR4 expression, macrophages were treated with S. typhimurium LPS, and flagellin. It was observed that treatment of macrophages with LPS led to an increase in upregulation of both LILBR2 and LILBR4 as measured by real time PCR assay. Upregulation was less pronounced for cells treated with flagellin.

The major conclusions of this study are:
1. Continuous ligation of LILBR4 on dendritic cells leads to increased upregulation of CD86. Previous studies have shown that ligation of LILBR2 results in failure to upregulate CD86.
2. The LILBR4 cross linking did not result in any changes in surface phenotype of APCs that could inhibit T cell proliferation. These results complement previous works which show that a soluble form of LILBR4 is sufficient to inhibit T cell stimulation.
3. The LILBR4 ligation showed an increased production of IL-10 (anti-inflammatory factor) and reduced production of IL-8 a potent inflammatory factor.
4. Infection of macrophages with Salmonella led to an increased expression of LILBR2 and LILBR4. Similar upregulation of expression of LILBR2 and LILBR4 was observed when cells were treated with Salmonella LPS or heat killed Salmonella. These results show that the upregulation was due to LPS.

Future studies that need to be done:
1. To determine whether culture of T cells with LILBR4-crosslinked APCs lead to alterations in T cell functions or alterations in the ration of regulatory and suppressor cells.
2. The effect of LILR and TLR signalling on each others expression and function.