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bioRxiv · 10.1101/2023.03.30.534876

Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis

Mukku, R. P.·Kokavalla, P.·Yadav, S.·Raghunand, T. R.
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Abstract

The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses. The genome of M. tb encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family, which comprise about 10% of its coding potential. The presence of these proteins in pathogenic mycobacteria strongly suggests that they play a role in disease pathogenesis. To understand its role in M. tb physiology we have begun to characterise the PPE50 (Rv3135)-PPE51 (Rv3136) gene cluster, one of nine PPE-PPE clusters in the M. tb genome. We demonstrate that this cluster encodes a co-transcriptional unit and that PPE50 and PPE51 interact both in vitro and in vivo, the first demonstration of PPE-PPE interaction. THP-1 macrophages infected with recombinant M. smegmatis strains expressing PPE50 and PPE51 showed less intracellular viability than the control strain containing the vector alone, the decline in viable counts correlating with an increase in transcript levels of inducible nitric oxide synthase (iNOS2). Macrophages infected with the recombinant strains exhibited an upregulation in levels of the anti-inflammatory cytokine IL-10, indicating an immunomodulatory role for these proteins. Using pull-down assays, we discovered TLR1 to be the cognate receptor for PPE50, with signalling through the receptor being indicated by an increase in IRAK1 phosphorylation. All the phenotypes observed on infection of THP-1 macrophages including the decrease in CFUs, the increase in iNOS2 and IL-10 levels, as well as signalling through the receptor, were reversed on treatment of macrophages with an anti-TLR1 antibody prior to infection, validating the functional outcome of PPE50-TLR1 interaction. Our data points to a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and a concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance.

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Mukku, R. P., Kokavalla, P., Yadav, S., Raghunand, T. R.. 2023-03-30. Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis. https://doi.org/10.1101/2023.03.30.534876

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