RESUMO
An essential feature of the pathogenesis of the Salmonella enterica serovar Enteritidis wild type (WT) is its ability to survive under diverse microenvironmental stress conditions, such as encountering antimicrobial peptides (AMPs) or glucose and micronutrient starvation. These stress factors trigger virulence genes carried on Salmonella pathogenicity islands (SPIs) and determine the efficiency of enteric infection. Although the oligosaccharide/oligonucleotide binding-fold (OB-fold) family of proteins has been identified as an important stress response and virulence determinant, functional information on members of this family is currently limited. In this study, we decipher the role of YdeI, which belongs to OB-fold family of proteins, in stress response and virulence of S Enteritidis. When ydeI was deleted, the ΔydeI mutant showed reduced survival during exposure to AMPs or glucose and Mg2+ starvation stress compared to the WT. Green fluorescent protein (GFP) reporter and quantitative real-time PCR (qRT-PCR) assays showed ydeI was transcriptionally regulated by PhoP, which is a major regulator of stress and virulence. Furthermore, the ΔydeI mutant displayed â¼89% reduced invasion into HCT116 cells, â¼15-fold-reduced intramacrophage survival, and downregulation of several SPI-1 and SPI-2 genes encoding the type 3 secretion system apparatus and effector proteins. The mutant showed attenuated virulence compared to the WT, confirmed by its reduced bacterial counts in feces, mesenteric lymph node (mLN), spleen, and liver of C57BL/6 mice. qRT-PCR analyses of the ΔydeI mutant displayed differential expression of 45 PhoP-regulated genes, which were majorly involved in metabolism, transport, membrane remodeling, and drug resistance under different stress conditions. YdeI is, therefore, an important protein that modulates S Enteritidis virulence and adaptation to stress during infection.IMPORTANCES Enteritidis during its life cycle encounters diverse stress factors inside the host. These intracellular conditions allow activation of specialized secretion systems to cause infection. We report a conserved membrane protein, YdeI, and elucidate its role in protection against various intracellular stress conditions. A key aspect of the study of a pathogen's stress response mechanism is its clinical relevance during host-pathogen interaction. Bacterial adaptation to stress plays a vital role in evolution of a pathogen's resistance to therapeutic agents. Therefore, investigation of the role of YdeI is vital for understanding the molecular basis of regulation of Salmonella pathogenesis. In conclusion, our findings may contribute to finding potential targets to develop new intervention strategies for treatment and prevention of enteric diseases.
Assuntos
Proteínas de Bactérias/fisiologia , Infecções por Salmonella/microbiologia , Salmonella enteritidis/fisiologia , Animais , Proteínas de Bactérias/química , Humanos , Camundongos Endogâmicos C57BL , Conformação Proteica , Salmonella enteritidis/patogenicidade , Estresse Fisiológico , VirulênciaRESUMO
Salmonella enterica serovar Enteritidis (S. Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ -1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S. Enteritidis to stress factors during infection.
Assuntos
Proteínas de Bactérias/genética , Salmonella enteritidis/genética , Salmonella enteritidis/patogenicidade , Fator sigma/genética , Estresse Fisiológico , Fatores de Virulência/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes Bacterianos , Ilhas Genômicas , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fagocitose , Células RAW 264.7 , Salmonelose Animal/microbiologia , Organismos Livres de Patógenos EspecíficosRESUMO
Salmonella, an Enterobacteria is a therapeutically important pathogen for the host. The advancement of genome sequencing of S. enterica serovar Enteritidis have identified a distinct ROD9 pathogenic island, imparting virulence. The occurrence of 17 ROD9 hypothetical proteins, necessitates subsequent bioinformatics approach for structural and functional aspects of protein-protein relations or networks in different pathogenic phenotypes express. A collective analysis using predictive bioinformatics tools that includes NCBI-BLASTp and BLAST2GO annotated the motif patterns and functional significance. The VFDB identified 10 virulence proteins at both genomic and metagenomic level. Phylogenetic analysis revealed a divergent and convergent relationship between 17 ROD9 and 41 SP-1 proteins. Here, combining a comprehensive approach from sequence based, motif recognitions, domain identification, virulence ability to structural modelling provides a precise function to ROD9 proteins biological network, for which no experimental information is available.
Assuntos
Proteínas de Bactérias/análise , Anotação de Sequência Molecular , Salmonella enterica/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Fenótipo , Salmonella enterica/metabolismoRESUMO
Even though surgical experience with wide spectrum of double outlet right ventricle (DORV) is available, the experience with extreme form of complete DORV where both great arteries completely arise from the right ventricle is limited. We present our experience with this unique subset where the systemic outflow is far removed from the interventricular foramen and hence, the systemic ventricle. In this situation, biventricular repair can be technically demanding and challenging. Between June 2002 and February 2006, 12 patients underwent biventricular repair of this subset. The VSD was subaortic in all; eight patients had infundibular and valvar obstruction. Aorta was anterior and to the right, with the pulmonary artery far posterior and to the left. The interventricular foramen was patched with a long Gore-Tex patch to route the LV flow to the aorta. Eight patients had infundibular resection and right ventricular outflow tract (RVOT) enlargement with an autologous monocusp pericardial patch. No patient required a valved conduit. There was no operative mortality. In one patient, there was a small residual VSD that was not of haemodynamic significance. There was no RVOT and left ventricular outflow tract obstruction and no RV inflow obstruction. The early and mid-term results are good.