RESUMO
Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen capable of causing illness in humans. In a previous study, our group showed that a STEC isolate belonging to O22:H8 serotype (strain 154) can interfere with STEC O157:H7 colonization both in vitro and in vivo. Using whole-genome sequencing and genomic comparative, we predicted a subset of genes acquired by O22:H8 strain 154 through horizontal gene transfer that might be responsible for the phenotype previously described by our group. Among them were identified genes related to the pathogenesis of non-LEE (locus of enterocyte effacement) STEC, specific metabolic processes, antibiotic resistance and genes encoding for the T6SS-1 that is related to inter-bacterial competition. In addition, we showed that this strain carries stx1c and stx2dact, a mucus-inducible variant. The results obtained in this study provide insights into STEC genomic plasticity and the importance of genomic islands in the adaptation and pathogenesis of this pathogen.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Filogenia , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.
Assuntos
Infecções por Escherichia coli/epidemiologia , Escherichia coli O157/genética , Genoma Bacteriano , Toxina Shiga/genética , Fatores de Virulência/genética , Animais , Argentina/epidemiologia , Bovinos , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Genótipo , Filogenia , Prófagos , Sorogrupo , Toxina Shiga/metabolismo , VirulênciaRESUMO
The zoonotic enterohemorrhagic Escherichia coli (EHEC) O157: H7 bacterium causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) in humans. Cattle are primary reservoirs and EHEC O157: H7; the bacteria predominately inhabit the colon and recto-anal junctions (RAJ). The early innate immune reactions in the infected gut are critical in the pathogenesis of EHEC O157: H7. In this study, calves orally inoculated with EHEC O157: H7 showed infiltration of neutrophils in the lamina propria of ileum and RAJ at 7 and 14 days post-infection. Infected calves had altered mucin layer and mast cell populations across small and large intestines. There were differential transcription expressions of key bovine ß defensins, tracheal antimicrobial peptide (TAP) in the ileum, and lingual antimicrobial peptide (LAP) in RAJ. The main Gram-negative bacterial/LPS signaling Toll-Like receptor 4 (TLR4) was downregulated in RAJ. Intestinal infection with EHEC O157: H7 impacted the gut bacterial communities and influenced the relative abundance of Negativibacillus and Erysipelotrichaceae in mucosa-associated bacteria in the rectum. Thus, innate immunity in the gut of calves showed unique characteristics during infection with EHEC O157: H7, which occurred in the absence of major clinical manifestations but denoted an active immunological niche.
Assuntos
Infecções por Escherichia coli/imunologia , Escherichia coli O157/metabolismo , Microbioma Gastrointestinal/imunologia , Adesinas Bacterianas/imunologia , Animais , Bovinos , Doenças dos Bovinos/imunologia , Diarreia/microbiologia , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/imunologia , Síndrome Hemolítico-Urêmica/microbiologia , Íleo/patologia , Reto/microbiologiaRESUMO
Bacterial pathogens utilize a myriad of mechanisms to invade mammalian hosts, damage tissue sites, and evade the immune system. One essential strategy of Gram-negative bacteria is the secretion of virulence factors through both inner and outer membranes to reach a potential target. Most secretion systems are harbored in mobile elements including transposons, plasmids, pathogenicity islands, and phages, and Escherichia coli is one of the more versatile bacteria adopting this genetic information by horizontal gene transfer. Additionally, E. coli is a bacterial species with members of the commensal intestinal microbiota and pathogens associated with numerous types of infections such as intestinal, urinary, and systemic in humans and other animals. T6SS cluster plasticity suggests evolutionarily divergent systems were acquired horizontally. T6SS is a secretion nanomachine that is extended through the bacterial double membrane; from this apparatus, substrates are conveyed straight from the cytoplasm of the bacterium into a target cell or to the extracellular space. This nanomachine consists of three main complexes: proteins in the inner membrane that are T4SS component-like, the baseplate complex, and the tail complex, which are formed by components evolutionarily related to contractile bacteriophage tails. Advances in the T6SS understanding include the functional and structural characterization of at least 13 subunits (so-called core components), which are thought to comprise the minimal apparatus. So far, the main role of T6SS is on bacterial competition by using it to kill neighboring non-immune bacteria for which antibacterial proteins are secreted directly into the periplasm of the bacterial target after cell-cell contact. Interestingly, a few T6SSs have been associated directly to pathogenesis, e.g., roles in biofilm formation and macrophage survival. Here, we focus on the advances on T6SS from the perspective of E. coli pathotypes with emphasis in the secretion apparatus architecture, the mechanisms of pathogenicity of effector proteins, and the events of lateral gene transfer that led to its spread.
RESUMO
Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.
Assuntos
Antibacterianos/farmacologia , Escherichia coli Enteropatogênica/efeitos dos fármacos , Escherichia coli Enteropatogênica/metabolismo , Peptídeos/farmacologia , Sistemas de Secreção Tipo III/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Dicroísmo Circular , Escherichia coli Enteropatogênica/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , TermodinâmicaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) are attaching and effacing (A/E) pathogens, which translocate effector proteins to intestinal enterocytes through a type III secretion system (T3SS). T3SS and most of its effector proteins are encoded in a pathogenicity island called LEE. Recently, new effectors have been located outside the LEE. This study aimed to characterize EspY3, a novel non-LEE encoded T3SS effector of EHEC. EspY3 shares homology with SopD and PipB2 effector proteins of Salmonella's T3SS-1 and T3SS-2, respectively. The presence of recombinant EspY3 in the supernatant samples demonstrated that EspY3 was secreted by the T3SS of EHEC and EPEC. Through infection assays, we demonstrated the translocation of EspY3 into Caco-2 cells by T3SS of EPEC. The subcellular localization of EspY3 was determined in the pedestal region, where its presence generates a significant increase in the size of the pedestals area. The EspY3 effector induced the elongation of polymerized actin pedestals in infected Caco-2 by EPEC. This study confirmed that EspY3 is part of the repertoire of T3SS effectors of EHEC O157:H7, and that it participates in modeling cellular actin during the infection.
RESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgun proteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified, which correspond approximately 47% of the predicted proteome of this pathogen. Normalized spectrum abundance factor analysis was performed to estimate the protein abundance. According this analysis, 50 proteins were detected as the most abundant of EHEC O157:H7 proteome. COG analysis showed that the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7.
Assuntos
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/análise , Proteômica , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Escherichia coli O157/classificação , Escherichia coli O157/isolamento & purificação , Humanos , Redes e Vias Metabólicas/genética , Proteoma/análise , Espectrometria de Massas em TandemRESUMO
Cellular immune response was evaluated in lymph nodes and lung with different granulomatous lesions from cattle naturally infected with Mycobacterium bovis. For this purpose, we assessed pro-inflammatory and anti-inflammatory cytokines by immunohistochemical assays. Immunoreaction was observed for all the cytokines analyzed. Fourteen animals displayed advanced stage IV granulomas, with intense immunoreactivity to IFN-γ and TGF-ß in areas of caseous necrosis, macrophages and lymphocytes. Seven animals showed stage III granuloma, with high immunoreactivity to IFN-γ (average of 44.5% immunoreactive cells) and moderate to TNF-α and to the anti-inflammatory cytokines IL-10 and TGF-ß, in relation to the proliferation of fibroblasts in granuloma periphery We found satellite stage I granulomas in 4 bovines and stage II granulomas in 2 bovines, which exhibited low immunostaining response (-13%). Cytokine expression in stage III and IV granulomas was significant, with predominance of immunoreactivity to IFN-γ, thus suggesting a strong, longstanding local immune response mediated by macrophages and epithelioid cells. In addition, these two stages displayed lower reactivity to IL-10; which suggests a deficit of anti-inflammatory cytokines, suppressed immunity and persistence of the infection. High expression of TGF-ß could indicate a chronic process with greater tissue damage and fibrosis. Numerous bacilli observed in necrotic areas in stage III and IV granulomas with low expression of IL-1ß suggest failure in the immune response with bacterial multiplication. In this study, evidence of in situ presence of cytokines demonstrates these cytokines are involved in the development and evolution of bovine tuberculosis granulomas.
Assuntos
Doenças dos Bovinos/imunologia , Citocinas/genética , Granuloma/veterinária , Imunidade Celular , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Citocinas/metabolismo , Feminino , Granuloma/imunologia , Granuloma/microbiologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Tuberculose Bovina/microbiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Escherichia coli O157:H7 is responsible for severe diarrhea and hemolytic uremic syndrome (HUS), and predominantly affects children under 5 years. The major virulence traits are Shiga toxins, necessary to develop HUS and the Type III Secretion System (T3SS) through which bacteria translocate effector proteins directly into the host cell. By SNPs typing, E. coli O157:H7 was separated into nine different clades. Clade 8 and clade 6 strains were more frequently associated with severe disease and HUS. In this study, we aimed to identify differentially expressed proteins in two strains of E. coli O157:H7 (clade 8 and clade 6), obtained from cattle and compared them with the well characterized reference EDL933 strain (clade 3). Clade 8 and clade 6 strains show enhanced pathogenicity in a mouse model and virulence-related properties. Proteins were extracted and analyzed using the TMT-6plex labeling strategy associated with two dimensional liquid chromatography and mass spectrometry in tandem. We detected 2241 proteins in the cell extract and 1787 proteins in the culture supernatants. Attention was focused on the proteins related to virulence, overexpressed in clade 6 and 8 strains compared to EDL933 strain. The proteins relevant overexpressed in clade 8 strain were the curli protein CsgC, a transcriptional activator (PchE), phage proteins, Stx2, FlgM and FlgD, a dienelactone hydrolase, CheW and CheY, and the SPATE protease EspP. For clade 6 strain, a high overexpression of phage proteins was detected, mostly from Stx2 encoding phage, including Stx2, flagellin and the protease TagA, EDL933_p0016, dienelactone hydrolase, and Haemolysin A, amongst others with unknown function. Some of these proteins were analyzed by RT-qPCR to corroborate the proteomic data. Clade 6 and clade 8 strains showed enhanced transcription of 10 out of 12 genes compared to EDL933. These results may provide new insights in E. coli O157:H7 mechanisms of pathogenesis.
Assuntos
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Virulência , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Doenças dos Bovinos/microbiologia , Cromatografia Líquida de Alta Pressão , Bases de Dados Genéticas , Modelos Animais de Doenças , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/genética , Camundongos , Polimorfismo de Nucleotídeo Único , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Espectrometria de Massas em TandemRESUMO
The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.