Tuesday, October 12, 2010

ESCRT factors restrict mycobacterial growth

Mycobacterium tuberculosis is considered as one of the most successful pathogen. Some of the reasons that are responsible for its success include its ability to survive within the macrophages. But how does the bacteria survive intra-cellularly is not known. In the present paper, the authors have tried to address this question.

Source: Philips JA, Porto MC, Wang H, Rubin EJ, Perrimon N. ESCRT factors 

Restrict mycobacterial growth. Proc Natl Acad Sci U S A. 2008 Feb 26;105

(8):3070-5. Epub 2008 Feb 19. PubMed PMID: 18287038; 

PubMed Central PMCID: PMC2268586.


 

It is known that virulent mycobacterial are able to alter the maturation of phagosome inside the macrophages. They reside in a cellular compartment that resembles an early endosome. This endosome retains early endosome markers such as Rab5 and fails to recruit mature enodosomal markers such as Rab7.

In an earlier study, the authors performed a functional genomic screening to identify host factors, which can influence the uptake and growth of Mycobacteria. The authors used Drosophilla S2 cells, a macrophage like cell line that is open to RNAi and M. fortuitum, which like M. tuberculosis restricts phagosome fusion with lysosomes. M. fortuitum was able to induce the expression macrophage activated promoters (maps), which are the bacterial genes that are specifically induced when the bacteria grows intra-cellularly. Thus, by visualizing GFP (Green fluorescent proteins, proteins that produce free fluorescence when exposed to blue light) produced under the control of the map24 promoter, the authors were able to monitor the bacterial response to the intracellular environment. In another previous study, the authors identified 86 dsRNAs, which were able to diminish bacterial GFP production. As production of GFP reflects both the bacterial response to the phagosomal environment and bacterial growth, these dsRNAs can affect the infection by altering bacterial uptake, intra-cellular bacterial growth or induction from the map24 promoter.

In the present study, the authors further characterized three host cell activities Rab7, CG8743, and the ESCRT machinery. First to check the affect of dsRNAs, they used M. smegmatis, as it is difficult to measure intra-cellular growth of M. fortuitum. Thus, they examined two possibilities, if dsRNA treatment results in less intra-cellular growth of M. fortuitum and then it should have little effect on M. stegmatis because they do not grow in S2 cells. The other possibility was if the dsRNA altered phagosome environment and caused diminished map induction, then treatment with dsRNA should cause increase in permissiveness of phagosome for bacterial growth and M. stegmatis should grow. They treated S2 cells with dsRNA and then infected them with M. stegmatis. After two days, cells were examined by microscopy. They observed that four dsRNAs out of 86 strongly increased the percentage of heavily infected cells. These dsRNAs targeted Rab7, dVps28, CG8055 and CG8743. To confirm that bacterial growth was occurring intra-cellularly, they further blocked the bacterial uptake by using a dsRNA which targets Pes, a receptor required for entry of bacteria into the cell. The investigators observed that blocking did not cause any increase in bacterial growth and thus the results indicated that growth must be occurring intra-cellularly. Based on these results, the authors suggested that Rab7, dVps28, CG8055 and CG8743 might be playing important roles in restricting the intracellular growth of M. stegmatis.

Rab7 is a GTP binding protein and a marker for late enodosome. But the role of dVps28, CG8055 and CG8743 are not known. dVps28 and CG8055 are members of ESCRT machinery, which is made up of three proteins ESCRTI, ESCRTII and ESCRTIII. The authors thus, tested the effect of other dsRNAs that target additional ESCRT components on M. stegmatis growth. The authors observed that all dsRNAs, which robustly disrupt ESCRT machinery, caused increased growth of M. stegmatis and also altered the phagosome environment resulting in map24 and map49 induction by M. fortuitum and increased growth of M. stegmatis.

It is known that the ESCRT machinery acts on the membrane of the endosome. its function is to deliver ubiquitinated receptors into intraluminal vesicles of multivesicular bodies. These bodies are finally delivered to the lysosome for degradation. In Drosophila, a lack of ESCRT functioning causes accumulation of enlarged endosomes with accumulation of ubiquitin.

The authors next sought to determine whether ESCRT machinery acts directly on mycobacterial phagosome. They examined the localization of bacteria in ESCRT-depleted cells. S2 cells, which were depleted of dTsg101 and Vps28 (part of ESCRTI) were infected with M. stegmatis. After three hrs of infection, bacteria were found within the heavily ubiquinated vesicular compartments. These results showed that bacteria reside in the compartment on which ESCRT machinery acts. These results indicate that ESCRT machinery directly acts on the bacterial phagosome.

To test whether ESCRT machinery affects mycobacterial growth infection in mammalian macrophages, the authors depleted dTsg101 and Vps28 from murine macrophages RAW264.7 using SiRNA. These cells were then infected with M. fortuitum map24:GFP. The depletion caused less GFP production when cells were infected with M. fortuitum map24:GFP in comparison to controls. To further determine whether phagosome in ESCRT depleted cells was also less restrictive for mycobacterial growth, Raw264.7 cells were depleted of dTsg101 and Vps28 and infected with M. smegmatis hsp60::GFP. In control cells there was little growth 24 hrs post infection. In contrast, substantial growth was observed in depleted cells. Thus, the authors concluded that ESCRT machinery is required to restrict the growth of M. stegmatis in mammalian macrophages.

The diminished GFP production in cells depleted of ESCRT and infected with M. fortuitum map24:GFP is due to diminished expression and not due to decreased bacterial growth. To validate this, the authors used an M. fortuitum strain that expresses both red fluorescent protein (dsRed2) and GFP under control of two separate promoters. dsRed2 is under the control of msp12 promoter and is constitutively expressed. GFP is expressed under the control of map49, which is induced during intracellular growth. When ESCRTI depleted cells were infected with this bacteria, the authors observed diminished GFP expression as early as 3 hrs post infection. Examination of red fluorescence however, revealed that bacterial uptake was similar in both depleted and control cells and bacterial growth was similar or slightly increased in controls. These results suggested the phagosomal environment in ESCRT depleted cells was different from the non-depleted cells. The effect of this difference is such that the induction from the map24 and map49 promoters was decreased and bacterial growth is not efficiently restricted.

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