Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

PE12 interaction with TLR4 promotes intracellular survival of Mycobacterium tuberculosis by suppressing inflammatory response.

Macrophages serve as the primary immune cells responsible for the innate immune defense against Mycobacterium tuberculosis (MTB) infection within the host. Specifically, NLRP3, a member of the NLRs family, plays a significant role in conferring resistance against MTB infection. Conversely, MTB evades innate immune killing by impeding the activation of the NLRP3 inflammasome, although the precise mechanism remains uncertain. In this study, we have identified PE12 (Rv1172c), a member of the PE/PPE family proteins, as an extracellular protein of MTB. PE12 interacts with Toll like receptor 4 (TLR4) in macrophages, forming the PE12-TLR4 complex which subsequently inhibits the transcription and expression of NLRP3. As a result, the transcription and secretion of IL-1ß are reduced through the PE12-TLR4-NLRP3-IL-1ß immune pathway. In vitro and in vivo experiments using a PE12-deficient strain (H37RvΔPE12) demonstrate a weakening of the suppression of the inflammatory response to MTB infection. Our findings highlight the role of the PE12 protein in not only inhibiting the transcription and release of inflammatory cytokines but also mediating the killing of MTB escape macrophages through TLR4 and inducing lung injury in MTB-infected mice. These results provide evidence that PE12 plays a significant role in the inhibition of the host immune response by MTB.

Extracellular DNase MAP3916c attacks the neutrophil extracellular traps and is needed for Mycobacterium avium subsp. paratuberculosis virulence.

Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.