RESUMO
Helicobacter pylori plays an essential role in the pathogenesis of gastritis, peptic ulcer disease, and gastric cancer. The serine protease HtrA, an important secreted virulence factor, disrupts the gastric epithelium, which enables H. pylori to transmigrate across the epithelium and inject the oncogenic CagA protein into host cells. The function of periplasmic HtrA for the H. pylori cell is unknown, mainly due to unavailability of the htrA mutants. In fact, htrA has been described as an essential gene in this bacterium. We have screened 100 worldwide H. pylori isolates and show that only in the N6 strain it was possible to delete htrA or mutate the htrA gene to produce proteolytically inactive HtrA. We have sequenced the wild-type and mutant chromosomes and we found that inactivation of htrA is associated with mutations in SecA - a component of the Sec translocon apparatus used to translocate proteins from the cytoplasm into the periplasm. The cooperation of SecA and HtrA has been already suggested in Streptococcus pneumonia, in which these two proteins co-localize. Hence, our results pinpointing a potential functional relationship between HtrA and the Sec translocon in H. pylori possibly indicate for the more general mechanism responsible to maintain bacterial periplasmic homeostasis.
Assuntos
Proteínas de Bactérias/genética , Infecções por Helicobacter/genética , Helicobacter pylori/genética , Proteínas SecA/genética , Serina Proteases/genética , Antígenos de Bactérias/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Interações Hospedeiro-Patógeno/genética , Humanos , MutaçãoRESUMO
The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Caderinas/metabolismo , Helicobacter pylori/enzimologia , Serina Proteases/metabolismo , Motivos de Aminoácidos , Antígenos CD , Proteínas de Bactérias/antagonistas & inibidores , Caderinas/química , Caderinas/genética , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Proteólise , Proteômica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina Proteases/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Ressonância de Plasmônio de SuperfícieRESUMO
Sustained identification of innovative chemical entities is key for the success of chemical biology and drug discovery. We report the fragment-based, computer-assisted deâ novo design of a small molecule inhibiting Helicobacter pylori HtrA protease. Molecular binding of the designed compound to HtrA was confirmed through biophysical methods, supporting its functional activity in vitro. Hit expansion led to the identification of the currently best-in-class HtrA inhibitor. The results obtained reinforce the validity of ligand-based deâ novo design and binding-kinetics-guided optimization for the efficient discovery of pioneering lead structures and prototyping drug-like chemical probes with tailored bioactivity.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Peptídeo Hidrolases/química , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Desenho Assistido por Computador , Descoberta de Drogas , Infecções por Helicobacter/microbiologia , Helicobacter pylori/enzimologia , Humanos , Ligantes , Relação Estrutura-AtividadeRESUMO
Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden. C. jejuni can cross the intestinal epithelial barrier as visualized in biopsies derived from human patients and animal models, however, the underlying molecular mechanisms and associated immunopathology are still not well understood. We have recently shown that the secreted serine protease HtrA (high temperature requirement A) plays a key role in C. jejuni cellular invasion and transmigration across polarized epithelial cells in vitro. In the present in vivo study we investigated the role of HtrA during C. jejuni infection of mice. We used the gnotobiotic IL-10(-/-) mouse model to study campylobacteriosis following peroral infection with the C. jejuni wild-type (WT) strain NCTC11168 and the isogenic, non-polar NCTC11168ΔhtrA deletion mutant. Six days post infection (p.i.) with either strain mice harbored comparable intestinal C. jejuni loads, whereas ulcerative enterocolitis was less pronounced in mice infected with the ΔhtrA mutant strain. Moreover, ΔhtrA mutant infected mice displayed lower apoptotic cell numbers in the large intestinal mucosa, less colonic accumulation of neutrophils, macrophages and monocytes, lower large intestinal nitric oxide, IFN-γ, and IL-6 as well as lower TNF-α and IL-6 serum concentrations as compared to WT strain infected mice at day 6 p.i. Notably, immunopathological responses were not restricted to the intestinal tract given that liver and kidneys exhibited mild histopathological changes 6 days p.i. with either C. jejuni strain. We also found that hepatic and renal nitric oxide levels or renal TNF-α concentrations were lower in the ΔhtrA mutant as compared to WT strain infected mice. In conclusion, we show here that the C. jejuni HtrA protein plays a pivotal role in inducing host cell apoptosis and immunopathology during murine campylobacteriosis in the gut in vivo.