Vol. 4 No. 2 October 2005

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Afeyan Interview

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Student Research

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The BioTECH Quarterly

Student Research Spotlight

Exploring the Role of the Pst System in Pathogenesis of Mycobacterium Tuberculosis
Gateways to the Laboratory Program, Weill Cornell/Rockefeller/Sloan-Kettering, Tri-Institutional MD-PhD

Sophia Kamran is a current sophomore at MIT planning to be a biology or a BE major. She spent her summer in the Weill Cornell/Rockefeller/Sloan-Kettering Gateways to the Laboratory program researching the genes responsible for the counter-immune defense of tuberculosis.

By Sophia Kamran í08

   Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, persists within the human host despite a vigorous host immune response. The bacterium uses specific counter-immune strategies to persist in host tissues, specifically macrophages.

   Despite the fact that drug therapy against TB has been available since the early 1950ís, full understanding of Mtb pathogenesis and worldwide control of TB still elude scientists and physicians today.

   By studying Mtb, we can learn more about the environment and host defenses Mtb must overcome in order to survive within a hostís macrophage. It is clear that Mtb has evolved specific counterimmune (cim) strategies to resist the immune system.

   Using signature-tagged transposon mutagenesis (STM) as a screening method, several M. tuberculosis genes have been identified that are involved in counter immune responses. My summer research focused on one gene, pstA1, that came out of the screen.

   PstA1 is part of a system known as the Pst system that is activated to take up phosphate in limiting phosphate conditions. Only few virulent microorganisms have this Pst system.

   The Mtb genome has two pstA domains as well as two pstC membrane spanning domains and three pstS substrate binding domains, all encoding different parts of the Pst system. For our experiments we used the pstA1 transposon mutant as well as an unmarked in-frame deletion of pstS3.

   Inorganic phosphate is an essential nutrient and may be limiting in the macrophage phagosome as a part of the host defense.

   In order to demonstrate the importance of the Pst system to M. tuberculosis virulence, we examined the replication of pstA1 and pstS3 compared to the wildtype (WT) strain in mice.

   The pstA1and pstS3 mutants demonstrated reduced virulence as compared to the wildtype strain (Fig 1), concluding that the Pst system and our mutant genes, pstA1 and pstS3, are essential components to Mtb virulence.

   We conducted two different experiments to further explore the mechanisms behind the importance of the Pst system, growing the WT and mutant strains in low phosphate media to maximally induce Pst activity.

    In a radioactive phosphate uptake experiment, the results have shown that while pstA1 takes up phosphate less quickly than the WT strain, pstS3 has no defect. PstS3 is highly homologous to pstS2 and we hypothesize that pstS2 is able to support phosphate uptake in the absence of pstS3.

   In contrast, pstA1 and pstA2 share limited homology and may not have the same degree of redundancy in uptake capacity. In previous studies, it has been demonstrated that pstS2 mutants cannot take up phosphate at low phosphate concentrations. This suggests that pstS2 plays an important role in phosphate uptake, and that pstS1 or pstS3 mutants canít take up phosphate in low Pi concentrations without pstS2.

   Using RT-QPCR, we observed that the pstS2 gene is expressed more than the other genes in both high phosphate and limiting phosphate conditions (Fig. 2).

   The genes appear to be upregulated in limiting phosphate conditions.

   It has been hypothesized that pstS2 plays the central role in phosphate uptake for Mtb. These two experiments support this hypothesis. Further experiments are needed, and are currently in progress, to examine and understand the role pstS2 plays in phosphate uptake and Mtb virulence.

   These experiments can help us to understand the importance of phosphate uptake in similar pathogens. The Pst system could be a potential drug target for the treatment of TB.


Fig. 1. The pstA1 and pstS3 mutant strains have decreased growth as compared to WT bacteria in mice infections.


Fig. 2 Expression of 5 targeted pst genes in both phosphate and no-phosphate conditions using quantitative RT-PCR. It seems that the pstS2 gene has more expression in both conditions than the other 4 genes.

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