Metallo-protease Peptidase M84 from Bacillusaltitudinis induces ROS-dependent apoptosis in ovarian cancer cells by targeting PAR-1.

We have purified Peptidase M84 from Bacillus altitudinis in an effort to isolate anticancer proteases from environmental microbial isolates. This metallo-protease had no discernible impact on normal cell survival, but it specifically induced apoptosis in ovarian cancer cells. PAR-1, a GPCR which is reported to be overexpressed in ovarian cancer cells, was identified as a target of Peptidase M84. We observed that Peptidase M84 induced PAR-1 overexpression along with activating its downstream signaling effectors NF-κB and MAPK to promote excessive reactive oxygen species (ROS) generation. This evoked apoptotic death of the ovarian cancer cells through the intrinsic route. In in vivo set-up, weekly intraperitoneal administration of Peptidase M84 in syngeneic mice significantly diminished ascites accumulation, increasing murine survival rates by 60%. Collectively, our findings suggested that Peptidase M84 triggered PAR-1-mediated oxidative stress to act as an apoptosis inducer. This established Peptidase M84 as a drug candidate for receptor mediated targeted-therapy of ovarian cancer.

Subtilisin from Bacillus amyloliquefaciens induces apoptosis in breast cancer cells through ubiquitin-proteasome-mediated tubulin degradation.

To search for novel proteases from environmental isolates which can induce apoptosis in cancer cells, we have purified subtilisin from Bacillus amyloliquefaciens and studied its anti-cancer properties. Subtilisin induced apoptosis in colon (HT29) and breast (MCF7) cancer cells but showed no effect on mouse peritoneal macrophages and normal breast cells (MCF10A). Western blot analysis showed that Bax, Bcl-2 level remained unchanged but tubulin level decreased significantly. Subtilisin does not induce the intrinsic pathway of apoptosis, rather it induced tubulin degradation in MCF-7 cells, whereas in normal cells (MCF-10A) tubulin degradation was not observed. Subtilisin activates ubiquitination and proteasomal-mediated tubulin degradation which was completely restored in presence of proteasome inhibitor MG-132. We further observed PARKIN, one of the known E3-ligase, is overexpressed and interacts with tubulin in subtilisin treated cells. Knockdown of PARKIN effectively downregulates ubiquitination and inhibits degradation of tubulin. PARKIN activation and tubulin degradation lead to ER-stress which in turn activates caspase-7 and PARP cleavage, thus guiding the subtilisin treated cells towards apoptosis. To our knowledge this is the first report of subtilisin induced apoptosis in cancer cells by proteasomal degradation of tubulin.

Secreted proteases: A new insight in the pathogenesis of extraintestinal pathogenic Escherichia coli.

Bacterial secreted proteases are the key factors that increase the virulence potential of different pathogens. Extraintestinal pathogenic E. coli (ExPEC) is a distinct pathotype that has unique ability to infect various body sites apart from the gastrointestinal tract causing several life-threatening diseases both in human and animals. Thus, understanding of ExPEC pathogenesis is crucial in effective management of disease caused by these pathogens. It is known that ExPEC possesses a broad spectrum of virulence factors including the secreted proteases which elude the host defence system. Recent studies have shown that high prevalence as well as the action of the secreted proteases influence the pathogenesis of ExPEC. However, literature on the secreted proteases present in ExPEC and their role in promoting virulence of ExPEC is rather limited. This review describes the distribution, characterization and the role of serine and metalloproteases secreted by diverse pathotypes of ExPEC, highlighting the significance of secreted proteases of ExPEC in pathogenesis.

SslE (YghJ), a Cell-Associated and Secreted Lipoprotein of Neonatal Septicemic Escherichia coli, Induces Toll-Like Receptor 2-Dependent Macrophage Activation and Proinflammation through NF-κB and MAP Kinase Signaling.

SslE (YghJ), a cell surface-associated and secreted lipoprotein, was identified as a potential vaccine candidate for extraintestinal pathogenic Escherichia coli, providing nearly complete protection from sepsis in a mouse model. We earlier found that SslE from neonatal septicemic E. coli could trigger the secretion of various proinflammatory cytokines in murine macrophages, the signaling pathway of which is still obscure. In this study, we showed that SslE specifically binds to Toll-like receptor 2 (TLR2)/TLR1 heterodimers and recruits downstream adaptors MyD88, TIRAP, and TRAF6. In addition, SslE stimulates nuclear translocation of NF-κB and activates different mitogen-activated protein (MAP) kinase signaling cascades specific to the secretion of each cytokine in murine macrophages, which becomes impaired in TLR2 small interfering RNA (siRNA)-transfected cells and in cells blocked with a monoclonal antibody (MAb) against TLR2, suggesting the involvement of TLR2 in NF-κB and MAP kinase activation and subsequent cytokine secretion. Furthermore, our study is the first to show that SslE can stimulate TLR2-dependent production of other proinflammatory hallmarks, such as reactive nitrogen and oxygen species as well as type 1 chemokines, which contribute to the anti-infection immune response of the host. Also, the overexpression of major histocompatibility complex class II (MHC II) and other costimulatory molecules (CD80 and CD86) in macrophages essentially indicates that SslE promotes macrophage activation and M1 polarization, which are crucial in framing the host's innate immune response to this protein, and hence, SslE could be a potent immunotherapeutic target against E. coli sepsis.

YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop.

YghJ, also known as SslE (Secreted and surface associated lipoprotein) is a cell surface associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the gene is known to be conserved among both pathogenic and commensal Escherichia coli isolates, the expression and secretion of YghJ was found to be higher among diverse E. coli pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and hence facilitates colonization of these enteric pathogens to intestinal epithelial cells. Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study, we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue damage in mouse ileum and is also able to induce significant fluid accumulation in a dose dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal pathogenic E. coli.

Role in proinflammatory response of YghJ, a secreted metalloprotease from neonatal septicemic Escherichia coli.

Neonatal sepsis is the invasion of microbial pathogens into blood stream and is associated with a systemic inflammatory response with production and release of a wide range of inflammatory mediators. The increased serum levels of cytokines were found to correlate with the severity and mortality in course of sepsis. There have been no reports on the role of microbial proteases in stimulation of proinflammatory response in neonatal sepsis. We have identified YghJ, a secreted metalloprotease from a neonatal septicemic Escherichia coli (NSEC) isolate. The protease was partially purified from culture supernatant by successive anion and gel filtration chromatography. MS/MS peptide sequencing of the protease showed homology with YghJ. YghJ was cloned, expressed and purified in pBAD TOPO expression vector. YghJ was found to be proteolytically active against Methoxysuccinyl Ala-Ala-Pro-Met-p-nitroanilide oligopeptide substrate, but not against casein and gelatin. YghJ showed optimal activity at pH 7-8 and at temperatures 37-40°C. YghJ showed clear changes in cellular morphologies of Int407, HT-29 and HEK293 cells. YghJ stimulated the secretion of cytokines IL-1α, IL-1ß and TNF-α in murine macrophages (RAW 264.7) and IL-8 from human intestinal epithelial cells (HT-29). YghJ also down-regulated the production of anti-inflammatory cytokines such as IL-10. YghJ is present in both septicemic (78%) and fecal E. coli isolates (54%). However, expression and secretion of YghJ is significantly higher among the septicemic (89%) than the fecal isolates (33%). This is the first study to show the role of a microbial protease, YghJ in triggering proinflammatory response in NSEC.

Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae.

Proteases in Vibrio cholerae have been shown to play a role in its pathogenesis. V. cholerae secretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization of V. cholerae strains in adult mice. In conclusion, our study shows that V. cholerae OMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.