RESUMO
Protein secretion plays a central role in modulating interactions of the human pathogen Listeria monocytogenes with its environment. Recently, secretion of RNA has emerged as an important strategy used by the pathogen to manipulate the host cell response to its advantage. In general, the Sec-dependent translocation pathway is a major route for protein secretion in L. monocytogenes, but mechanistic insights into the secretion of RNA by these pathways are lacking. Apart from the classical SecA1 secretion pathway, L. monocytogenes also encodes for a SecA paralogue (SecA2) which targets the export of a specific subset of proteins, some of which are involved in virulence. Here, we demonstrated that SecA2 co-sediments with translating ribosomes and provided evidence that it associates with a subset of secreted small non-coding RNAs (sRNAs) that induce high levels of IFN-ß response in host cells. We found that enolase, which is translocated by a SecA2-dependent mechanism, binds to several sRNAs, suggesting a pathway by which sRNAs are targeted to the supernatant of L. monocytogenes.
Assuntos
Listeria monocytogenes , Proteínas de Membrana Transportadoras , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , RNA/metabolismoRESUMO
Mice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , NF-kappa B/imunologia , Proteínas Proto-Oncogênicas c-rel/imunologia , Transcrição Gênica/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , CamundongosRESUMO
Introduction: Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. Methods: We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Results: Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation.
Assuntos
Peróxido de Hidrogênio , Streptococcus pneumoniae , Streptococcus pneumoniae/imunologia , Peróxido de Hidrogênio/metabolismo , Humanos , Fosforilação , Interações Hospedeiro-Patógeno/imunologia , Linhagem Celular , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Transdução de SinaisRESUMO
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.
Assuntos
Peróxido de Hidrogênio , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolismo , Peróxido de Hidrogênio/metabolismo , Cisteína/metabolismo , Estreptolisinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
The NF-κB transcription factor c-Rel plays a crucial role in promoting and regulating immune responses and inflammation. However, the function of c-Rel in modulating the mucosal immune system is poorly understood. T follicular helper (Tfh) cells and IgA production in gut-associated lymphoid tissues (GALT) such as Peyer's patches (PPs) are important for maintaining the intestinal homeostasis. Here, c-Rel was identified as an essential factor regulating intestinal IgA generation and function of Tfh cells. Genetic deletion of c-Rel resulted in the aberrant formation of germinal centers (GCs) in PPs, significantly reduced IgA generation and defective Tfh cell differentiation. Supporting these findings, the Ag-specific IgA response to Citrobacter rodentium was strongly impaired in c-Rel-deficient mice. Interestingly, an excessive expansion of segmented filamentous bacteria (SFB) was observed in the small intestine of animals lacking c-Rel. Yet, the production of IL-17A, IgA, and IL-21, which are induced by SFB, was impaired due to the lack of transcriptional control by c-Rel. Collectively, the transcriptional activity of c-Rel regulates Tfh cell function and IgA production in the gut, thus preserving the intestinal homeostasis.