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Peptidyl prolyl isomerases (PPIases) are well‐conserved protein‐folding enzymes that moonlight as regulators of bacterial virulence. Peptidyl prolyl isomerase A, PPiA (Rv0009) is a secretory protein of Mycobacterium tuberculosis that possesses sequence and structural similarity to eukaryotic cyclophilins. In this study, we validated the interaction of PPiA with stimulator of interferon genes (STING) using both, Escherichia coli‐based and mammalian in vitro expression systems. In vitro pull‐down assays confirmed that the cytosolic domain of STING interacts with PPiA, and moreover, we found that PPiA could induce dimerization of STING in macrophages. In silico docking analyses suggested that the PXXP (PDP) motif of PPiA is crucial for interaction with STING, and concordantly, mutations in the PDP domain (PPiA MUT‐II) abrogated this interaction, as well as the ability of PPiA to facilitate STING dimerization. In agreement with these observations, fluorescence microscopy demonstrated that STING and wild‐type PPiA, but not PPiA MUT‐II, could colocalize when expressed in HEK293 cells. Highlighting the importance of the PDP domain further, PPiA, but not PPiA MUT‐II could activate Tank binding kinase 1 (TBK1)‐interferon regulatory factor 3 (IRF3) signaling to promote the release of interferon‐beta (IFNβ). PPiA, but not PPiA MUT‐II expressed in Mycobacterium smegmatis induced IFNβ release and facilitated bacterial survival in macrophages in a STING‐dependent manner. The PPiA‐induced release of IFNβ was c‐GAS independent. We conclude that PPiA is a previously undescribed mycobacterial regulator of STING‐dependent type I interferon production from macrophages. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Here, a macrophage infection model was used to unravel the role of the histone deacetylase sirtuin 6 (SIRT6) in Mtb‐triggered regulation of the innate immune response. Mtb infection downregulated microRNA‐26a and upregulated its target SIRT6. SIRT6 suppressed glycolysis and expression of HIF‐1α‐dependent glycolytic genes during infection. In addition, SIRT6 regulated the levels of intracellular succinate which controls stabilization of HIF‐1α, as well as the release of interleukin (IL)‐1β. Furthermore, SIRT6 inhibited inducible nitric oxide synthase (iNOS) and proinflammatory IL‐6 but augmented anti‐inflammatory arginase expression. The miR‐26a/SIRT6/HIF‐1α axis therefore regulates glycolysis and macrophage immune responses during Mtb infection. Our findings link SIRT6 to rewiring of macrophage signaling pathways facilitating dampening of the antibacterial immune response. [ABSTRACT FROM AUTHOR]

Ionic homeostasis is essential for the survival and replication of Mycobacterium tuberculosis within its host. Low potassium ion concentrations trigger a transition of M. tuberculosis into dormancy. Our current knowledge of the transcriptional regulation mechanisms governing genes involved in potassium homeostasis remains limited. Potassium transport is regulated by the constitutive Trk system and the inducible Kdp system in M. tuberculosis. The two‐component system KdpDE (also known as KdpD/KdpE) activates expression of the kdpFABC operon, encoding the four protein subunits of the Kdp potassium uptake system (KdpFABC). We show that, under potassium deficiency, expression of the two‐component system senX3/regX3 is upregulated, and bacterial survival is compromised in a regX3‐inactivated mutant, ΔregX3. Electrophoretic mobility shift assays (EMSAs), promoter reporter assays and chromatin immunoprecipitation (ChIP) show that RegX3 binds to the kdpDE promoter and activates it under potassium deficiency, whereas RegX3 (K204A), a DNA binding‐deficient mutant, fails to bind to the promoter. Mutation of the RegX3 binding motifs on the kdpDE promoter abrogates RegX3 binding. In addition, EMSAs and ChIP assays show that RegX3 represses Rv0500A, a repressor of kdpFABC, by binding to consensus RegX3 binding motifs on the rv0500A promoter. Our findings provide important insight into two converging pathways regulated by RegX3; one in which it activates an activator of kdpFABC, and the other in which it represses a repressor of kdpFABC, during potassium insufficiency. This culminates in increased expression of the potassium uptake system encoded by kdpFABC, enabling bacterial survival. These results further expand the growing transcriptional network in which RegX3 serves as a central node to enable bacterial survival under stress. [ABSTRACT FROM AUTHOR]

A cluster of genes encoded by ORFs Rv0014c-Rv0018c in Mycobacterium tuberculosis encodes candidate cell division proteins RodA and PBPA, a pair of serine/threonine kinases (STPKs), PknA and PknB, and a phosphatase, PstP. The organization of genes encompassing this region is conserved in a large number of mycobacterial species. This study demonstrates that recombinant PBPA of M. tuberculosis binds benzylpenicillin. Knockout of its counterpart in M. smegmatis resulted in hindered growth and defective cell septation. The phenotype of the knockout (PBPA-KO) could be restored to that of the wild-type upon expression of PBPA of M. tuberculosis. PBPA localized to the division site along with newly synthesized peptidoglycan, between segregated nucleoids. In vivo coexpression of PBPA and PknB, in vitro kinase assays and site-specific mutagenesis substantiated the view that PknB phosphorylates PBPA on T362 and T437. A T437A mutant could not complement PBPA-KO. These studies demonstrate for the first time that PBPA, which belongs to a subclass of class B high-molecular-mass PBPs, plays an important role in cell division and cell shape maintenance. Signal transduction mediated by PknB and PstP likely regulates the positioning of this PBP at the septum, thereby regulating septal peptidoglycan biosynthesis.

The dual multimodular class A penicillin-binding proteins (PBP) in Mycobacterium leprae (M. leprae)was compared with the PBP in Escherichia coli (E. coli). The PBP has been produced in Escherichia coli by extracting the membranes with 3-[(3-cholamidoporyl)-dimenthylammonio]-1-propane-sulfunate and purified by Ni(sup 2+)-nitrilotriacetic acid-agarose chromatography. Results show that the the modular design of the M. leprae PBP1 is very similar with the E. coli PBP1a and E. coli PBP1b. However, they differ in terms of penicillin sensitivity, thermostability and reactions to detergents.

Western blotting and immunoprecipitation analyses help characterize the penicillin-binding proteins (PBPs) of Shigella dysenteriae. A comparative study reveals the close similarity between the PBP type of the membranes of S. dysenteriae and that of Escherichia coli membranes. Standard procedures facilitate the purification of a 38 kDa PBP, an important target for the oxacephem moxalactam, the penem SCH34343 and the cephamycin cefoxitin. Stationary-phase cells of S. dysenteriae have significant levels of PBP.