Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth

Abstract

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis

“…The ABC transporter Rv1747 is a virulence factor that has been implicated in transporting intermediates of the cell wall biosynthesis pathway across the Mtb membrane (14). Deletion of Rv1747 leads to Mtb growth defects in macrophages and infected mice.…”

“…Dephosphorylation by PstP reverses this effect. Phase separationmediated clustering could potentially increase transport efficiency due to allosteric activity regulation, amplification of signals by selectively filtering substrates, and/or formation of a scaffold for interactions with additional components of the cell wall biosynthesis pathways (14). In this model, STPKs/PstP allow rapid and reversible activation/deactivation of Rv1747 on initial exposure to challenging environments in infected macrophages.…”

“…Rv1747 , a construct spanning the regulatory module, is capable of intramolecular and intermolecular FHA/pThr interactions that lead to the formation of a variety of higher-order oligomers, depending on the linker phosphorylation state (13). Although implicated in the export of cell wall biosynthesis intermediates, the physiological cargo of Rv1747 is unknown (14). Nevertheless, Rv1747 contributes to Mtb virulence in a manner…”

Underestimated Manipulative Roles of Mycobacterium tuberculosis Cell Envelope Glycolipids During Infection

“…To date, the role and metabolism of PIMs during M. tuberculosis infection and pathogenesis is still unclear, as different PIMs are recognized by different receptors triggering different host responses and outcomes. PIMs are downregulated in stationary phase (162), but it has been observed that the production of PIMs by M. tuberculosis is enhanced during infection in primary human macrophages (163). The latter result indicates that M. tuberculosis can adapt to the environment during infection by modulating its cell envelope composition (164)(165)(166)(167)(168).…”

“…The latter result indicates that M. tuberculosis can adapt to the environment during infection by modulating its cell envelope composition (164)(165)(166)(167)(168). That PIM expression levels during early stages of infection may be in part directly or indirectly involved in bacterial growth, virulence, and entry into persistence is still unclear; however, studies show that a PIM-hyperproducing M. tuberculosis strain ( Rv2623) is hypervirulent in vivo being unable to establish a chronic infection (163), while a PIMhypoproducing M. tuberculosis strain ( Rv1747) is attenuated in vivo in mice (169) ( Table 1).…”