Type IV secretion system in Helicobacter pylori: Cag3 function

Source: Pinto-Santini DM, Salama NR. Cag3 is a novel essential component of the Helicobacter pylori Cag type IV secretion system outer membrane subcomplex. J Bacteriol. 2009 Dec;191(23):7343-52. Epub 2009 Oct 2. PubMed PMID: 19801411; PubMed Central PMCID: PMC2786551.

 

Bacteria translocate or transfer effector molecules, for examples proteins, toxins, DNA and enzymes, from the interior to the exterior through secretion. The cell wall of bacteria plays an important role in the secretion. The structure of cell wall of Gram negative bacteria (GNB) is more complex than Gram positive bacteria. Thus, secretion is also more complex in GNBs. According to the literature there are at least six specialized secretion systems in GNBs. These are type I secretion system (T1SS), type II secretion system (T2SS), type III secretion system (T3SS), type IV secretion system (T4SS), type V secretion system (T5SS) and twin arginine translocation.

T4SSs are known to be involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in uptake or release of DNA from/into the extracellular environment, in the secretion of toxins and in the secretion of virulence factors into host cells. It is similar to conjugation system of bacteria and can transfer both proteins and DNA. A lot of research has been going on to define the structure of these T4SS, to identify the molecules which are translocated and effect and mechanism of action of these effector molecules on the host cells. This information is essential to understand the pathogenecity of these bacteria and to design strategies to combat bacterial infections.

Helicobacter pylori is a GNB and it infects 50% of the world population. The infection can lead to chronic gastric and peptic ulcer disease, gastric carcinoma and mucosa associated lymphoid tissue lymphoma. However, only a small percentage of infected people suffer from H. pylori associated illnesses. Earlier studies have established that infection with strains harboring the cag pathogenecity island (PAI) leads to a much higher risk for development of severe illnesses. Cag PAI is a 40kb stretch of DNA that encodes 27 putative proteins. The T4SS VirB/D4 of plant pathogen Agrobacterium tumefaciens has been studied in great detail. Several of the proteins encoded by cag PAI share sequence similarity with components of A. tumefaciens VirB/D4 T4SS. The activity of cag PAI encoded T4SS is responsible for translocation of effector molecule CagA into the host cells and subsequent induction of proinflammatory cytokines.

In this paper, the authors have characterized the biochemical role of a H. pylori specific gene, HP0522/cag3, in Cag T4SS. A previous study has shown that Cag3 could interact with HpVirB8, HpVirB7, CagM and CagG. To understand the molecular basis of Cag3 function in T4SS, the authors investigated the subcellular localization of Cag3 protein as well as the interaction that this protein established with other proteins in bacterial cells.

A brief summary of major results obtained is as follows:

Results:

• cag3 is essential for Cag T4SS function: The CagT4SS is responsible for phosphorylation of CagA and induction of IL-8 secretion. Thus, to evaluate whether cag3 is essential for Cag T4SS function, the authors used a null cag3 mutant, a null cag3 mutant complemented by expression of gene at an unrelated locus rdxA and incubated the mutants as well as the wild strains with AGS cells and determined CagA phosphorylation and secretion of IL-8. The mutant strain was unable to translocate CagA as there was lack of phosphorylation of CagA and was unable to induce IL-8 secretion. However, in case of strains complemented with expression of cag3 at rdxA locus, both the CagA phosphorylation and induction of Il-8 secretion were observed.
  
• Cag3 is a membrane associated protein: To determine the location of Cag3, the investigators fractionated H. pylori cells and performed immunoblotting of each fraction with antibodies to Cag3. Cag3 was found enriched in the membrane associated fractions.
  
• Cag3 co-purifies with predicted T4S compartments: To identify the proteins that interact with Cag3, the authors used affinity purification followed by mass spectrometry. Total extracts from wild type and mutant strains were loaded onto anti-Cag3 affinity columns. The eluates were evaluated for their protein profiles. A number of proteins co-purified specifically with Cag3, i.e. they were absent in eluates of mutant cag3 extracts. Mass spectrometry analysis identified HpVirB7, HpVirD4, CagD and Cag1 as the most specific interactors. Other proteins identified were HpVirB4, HpVirB11, hpVirB10, HpVirB9, HpVirB2 and CagM.
  
• Cag3 interacts with predicted lipoprotein HpVirB7 independent of CagM: Previous studies have shown that HpVirB7 is a part of outer membrane subcomplex and previous studies have also suggested that Cag3 and HpVirB7 can interact. Thus, the authors further looked at the interaction of Cag3 and HpVirB7. For this, whole cell lysates from cag3 mutant strains and wild type strains (expressing HpVirB7-3XFLAG) were immuno-precipitated with anti-FLAG and anti-Cag3 antibodies. The authors observed that anti-FLAG antibodies were able to co-immuno-precipitate Cag3 in a wild type strain but not in the mutant strain. Similarly, anti-Cag3 antibodies were able to co-immuno-precipitate HpVirB7-3XFLAG from the wild type strain and not from the cag3 mutant strain. These observations confirm that Cag3 interacts with HpVirB7. The authors next sought to determine whether the Cag3 and HpVirB7 interaction was CagM dependent. The authors observed that anti-Cag3 antibodies were able to co-immuno-precipitate HpVirB7-3XFLAG in a cagM mutant. These observations indicate that Cag3 can interact with HpVirB7 independently of CagM.
  
• Cag3 and HpVirB7 fractionate in a high molecular weight complex: To further determine the size of Cag3 complexes in the cell, the authors fractionated whole cell wild type extracts and determined the presence of Cag3 and HpVirB7-3XFLAG in each fraction by immunoblotting with anti-Cag3 or anti-FLAG antibodies. Cag3 was precipitated in two peaks; one peak overlapped with the void volume while other peak corresponded to a 150KDa peak. The size predicted for Cag3 monomer is 55KDa. Thus, these results indicate that Cag3 exists in cell as part of two pools. The lower molecular mass form may represent the soluble form, while the higher molecular mass form may represent the Cag3 membrane associated complex.
  
• Protein cross linking reveals HpVirB7-CagM and Cag3-CagM interactions: H. pylori cells (wild type expressing HpVirB7-3XFLAG, mutant HpVirB7, mutant cag3, mutant cag3 complemented with expression of gene at rdxA, and mutant cagM) were cross-lined in vivo with formaldehyde. These cross linked whole cell extracts were fractionated and by SDS-PAGE and immunoblotted with ant-Cag3 or anti-FLAG antibodies. On immunoblotting with anti-Cag3 antibody, monomer sized band as well as several high molecular mass Cag3 containing complexes were observed. These complexes were present even in the HpVirB7 mutants, indicating that HpVirB7 was not present in these complexes. Surprisingly, one complexe was absent in cagM mutant indicating that this complex which was composed of Cag3 and CagM. In contrast, on immunoblotting with anti-FLAG antibody, only two bands were observed. One band corresponded to HpVirB7-3XFLAG while other band was absent in cagM mutant strains, indicating that a dimer containing HpVirB7 and CagM.
  
• Cag3 and HpVirB7 promote each other's stability: The mutant and wild type strains were metabolically labeled with Trans35S-label cys-Met during liquid culture growth for a 30 minutes pulse. After the pulse, the two aliquots from each culture were immuno-precipitated with anti-FLAG or anti-CagA antibody. HpVirB7-3XFLAG was less stable in the cag3 mutant strain background compared to the wild type. Cag3 appeared less stable in the absence of HpVirB7, HpVirB9 or HpVirB10.