RESUMO
Pathogenic strains of Escherichia coli infecting poultry, commonly called avian pathogenic E. coli (APEC) present significant risks, to the health of both poultry and the general public. This systematic review aimed to examine the prevalence of APEC serotypes, sequence types (ST), phylogenetic groups, virulence factors and antibiotic resistance patterns based on 189 research papers sourced from PubMed, Web of Science, and ProQuest. Then, data were extracted from the selected studies and analyzed to assess the global distribution and characteristics of APEC strains. The metaprop codes in the Meta and Metafor packages of R as implemented in RStudio were then used to conduct meta-analysis. Among APEC strains identified from these different research reports serogroup O78 had the highest overall prevalence (16 %), followed by serogroups O2 (10 %), and O117 (8 %). The most common ST profiles were ST117 (20 %), ST140 (15 %), ST95 (12 %), and ST131 (9 %). ST117 and ST140 are known reservoirs for pathogenic E. coli in humans. Moreover, phylogenetic assessment highlighted the prevalence of phylogroups A, A1, F, D, and B2 among APEC strains indicating diversity in phylogenetic origin within poultry populations. The presence of antimicrobial resistance was notable among APEC strains against antibiotics such as tetracyclines, penicillins, and cephalosporins. This resistance may be linked to use of antimicrobials in poultry production in certain regions presenting challenges for both animal health management and human infection control. Analysis of sequences linked to adherence or virulence indicated that genes encoding adhesins (csg, fimC), iron/metal uptake (sitB, sitC, iroD) and cytotoxicity (estB, hlyF), and serum resistance (traT, iss) were highly prevalent. These factors have been reported to contribute to APEC host colonization and virulence in poultry. In summary, this overview of the characteristics of APEC highlights the pressing importance of monitoring and implementing management approaches to reduce antimicrobial resistance considering that a phylogenetic diversity of E. coli strains causes infections in both poultry and humans and represents a risk to both animal and public health. Further, determining the major conserved aspects and predominant mechanisms of virulence of APEC is critical for improving diagnostics and developing preventative measures to reduce the burden of infection caused by pathogenic E. coli in poultry and lower risks associated with foodborne transmission of E. coli to humans through poultry and poultry products.
RESUMO
Urinary tract infections (UTIs) are a common bacterial infectious disease in humans, and strains of uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTIs. During infection, UPEC must cope with a variety of stressful conditions in the urinary tract. Here, we demonstrate that the small RNA (sRNA) RyfA of UPEC strains is required for resistance to oxidative and osmotic stresses. Transcriptomic analysis of the ryfA mutant showed changes in expression of genes associated with general stress responses, metabolism, biofilm formation and genes coding for cell surface proteins. Inactivation of ryfA in UPEC strain CFT073 decreased urinary tract colonization in mice and the ryfA mutant also had reduced production of type 1 and P fimbriae (pili), adhesins which are known to be important for UTI. Furthermore, loss of ryfA also reduced UPEC survival in human macrophages. Thus, ryfA plays a key regulatory role in UPEC adaptation to stress, which contributes to UTI and survival in macrophages.
Assuntos
Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Pequeno RNA não Traduzido/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Adaptação Fisiológica , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Animais , Fímbrias Bacterianas/metabolismo , Perfilação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Camundongos , Osmorregulação , Estresse Oxidativo , RNA Bacteriano/genética , Deleção de Sequência , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Escherichia coli Uropatogênica/fisiologia , VirulênciaRESUMO
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen producing Shiga toxins (Stx1 and Stx2), which can cause hemorrhagic diarrhea and life-threatening infections. O157:H7 strain EDL933 carries prophages CP-933V and BP-933W, which encode Shiga toxin genes (stx1 and stx2, respectively). The aim of this work was to investigate the mechanisms of adaptive resistance of EHEC strain EDL933 to a typically lethal dose of gamma irradiation (1.5 kGy). Adaptive selection through six passages of exposure to 1.5 kGy resulted in the loss of CP-933V and BP-933W prophages from the genome and mutations within three genes: wrbA, rpoA, and Wt_02639 (molY). Three selected EHEC clones that became irradiation adapted to the 1.5-kGy dose (C1, C2, and C3) demonstrated increased resistance to oxidative stress, sensitivity to acid pH, and decreased cytotoxicity to Vero cells. To confirm that loss of prophages plays a role in increased radioresistance, clones C1 and C2 were exposed to bacteriophage-containing lysates. Although phage BP-933W could lysogenize C1, C2, and E. coli K-12 strain MG1655, it was not found to have integrated into the bacterial chromosome in C1-Φ and C2-Φ lysogens. Interestingly, for the E. coli K-12 lysogen (K-12-Φ), BP-933W DNA had integrated at the wrbA gene (K-12-Φ). Both C1-Φ and C2-Φ lysogens regained sensitivity to oxidative stress, were more effectively killed by a 1.5-kGy gamma irradiation dose, and had regained cytotoxicity and acid resistance phenotypes. Further, the K-12-Φ lysogen became cytotoxic, more sensitive to gamma irradiation and oxidative stress, and slightly more acid resistant. IMPORTANCE Gamma irradiation of food products can provide an effective means of eliminating bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, a significant foodborne pathogen that can cause severe disease due to the production of Stx. To decipher the mechanisms of adaptive resistance of the O157:H7 strain EDL933, we evolved clones of this bacterium resistant to a lethal dose of gamma irradiation by repeatedly exposing bacterial cells to irradiation following a growth restoration over six successive passages. Our findings provide evidence that adaptive selection involved modifications in the bacterial genome, including deletion of the CP-933V and BP-933W prophages. These mutations in EHEC O157:H7 resulted in loss of stx1 and stx2, loss of cytotoxicity to epithelial cells, and decreased resistance to acidity, critical virulence determinants of EHEC, concomitant with increased resistance to lethal irradiation and oxidative stress. These findings demonstrate that the potential adaptation of EHEC to high doses of radiation would involve elimination of the Stx-encoding phages and likely lead to a substantial attenuation of virulence.
Assuntos
Bacteriófagos , Escherichia coli Êntero-Hemorrágica , Escherichia coli O157 , Proteínas de Escherichia coli , Animais , Chlorocebus aethiops , Toxina Shiga/genética , Prófagos/genética , Células Vero , Toxinas Shiga/farmacologia , Bacteriófagos/genética , Genômica , Proteínas Repressoras/farmacologiaRESUMO
Fimbrial adhesins promote bacterial adherence and biofilm formation. Sequencing of avian pathogenic Escherichia coli (APEC) strain QT598 identified new fimbriae belonging to the π group, which we named PL (P-like) fimbriae since the genetic organization and sequence are similar to those of P and related fimbriae. Genes encoding PL fimbriae located on IncF plasmids are present in diverse E. coli isolates from poultry, human systemic infections, and other sources. As with P fimbriae, PL fimbriae exhibit divergence in adhesin-encoding genes and could be divided into 5 classes based on sequence differences in the PlfG adhesin. plf genes from two predominant PlfG adhesin classes, PlfG class I (PlfGI) and PlfGII, were cloned. PL fimbriae were visualized by electron microscopy, associated with increased biofilm, demonstrated distinct hemagglutination profiles, and promoted adherence to human bladder and kidney epithelial cells. The genes encoding hybrid fimbriae were comprised of genes from plfQT598, wherein plfG was replaced by papG; the adhesin-encoding genes were also functional and mediated adherence to epithelial cells, demonstrating compatibility between the components of these two types of fimbriae. Deletion of plf genes did not reduce colonization of the mouse urinary tract in a single-strain infection model. In contrast, loss of plf genes significantly reduced competitive colonization in the mouse kidneys. Furthermore, plf gene expression was increased over 40-fold in the bladder compared to during in vitro culture. Overall, PL fimbriae represent a new group of fimbriae demonstrating both functional differences from and similarities to P fimbriae, which mediated adherence to host cells and improved competitive colonization of the mouse kidney. IMPORTANCE Fimbriae are important colonization factors in many bacterial species. The identification of a new type of fimbriae encoded on some IncF plasmids in E. coli was investigated. Genomic sequences demonstrated these fimbrial gene clusters have genetic diversity, particularly in the adhesin-encoding plfG gene. Functional studies demonstrated differences in hemagglutination specificity, although both types of Plf adhesin under study mediated adherence to human urinary epithelial cells. A plf mutant also showed decreased colonization of the kidneys in a mouse competitive infection model. PL fimbriae may represent previously unrecognized adhesins that could contribute to host specificity and tissue tropism of some E. coli strains.
Assuntos
Infecções por Escherichia coli , Escherichia coli Extraintestinal Patogênica , Proteínas de Fímbrias , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Animais , Aderência Bacteriana/fisiologia , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Humanos , CamundongosRESUMO
Macrophages represent one of the first lines of defense during infections and are essential for resolution of inflammation following pathogen clearance. Rapid activation or suppression of protein synthesis via changes in translational efficiency allows cells of the immune system, including macrophages, to quickly respond to external triggers or cues without de novo mRNA synthesis. The translational repressors eIF4E-binding proteins 4E-BP1 and 4E-BP2 (4E-BP1/2) are central regulators of proinflammatory cytokine synthesis during viral and parasitic infections. However, it remains to be established whether 4E-BP1/2 play a role in translational control of anti-inflammatory responses. By comparing translational efficiencies of immune-related transcripts in macrophages from wild-type and 4E-BP1/2 double-knockout mice, we found that translation of mRNAs encoding two major regulators of inflammation, IL-10 and PG-endoperoxide synthase 2/cyclooxygenase-2, is controlled by 4E-BP1/2. Genetic deletion of 4E-BP1/2 in macrophages increased endogenous IL-10 and PGE2 protein synthesis in response to TLR4 stimulation and reduced their bactericidal capacity. The molecular mechanism involves enhanced anti-inflammatory gene expression (sIl1ra, Nfil3, Arg1, Serpinb2) owing to upregulation of IL-10-STAT3 and PGE2-C/EBPß signaling. These data provide evidence that 4E-BP1/2 limit anti-inflammatory responses in macrophages and suggest that dysregulated activity of 4E-BP1/2 might be involved in reprogramming of the translational and downstream transcriptional landscape of macrophages during pathological conditions, such as infections and cancer.
Assuntos
Proteínas de Transporte/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Inflamação/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular , Dinoprostona/metabolismo , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Regulação para Cima/fisiologiaRESUMO
TagB, TagC (tandem autotransporter genes B and C), and Sha (Serine-protease hemagglutinin autotransporter) are recently described members of the SPATE (serine protease autotransporters of Enterobacteriaceae) family. These SPATEs can cause cytopathic effects on bladder cells and contribute to urinary tract infection in a mouse model. Bladder epithelial cells form an important barrier in the urinary tract. Some SPATEs produced by pathogenic E. coli are known to breach the bladder epithelium. The capacity of these newly described SPATEs to alter bladder epithelial cells and the role of the serine protease active site were investigated. All three SPATE proteins were internalized by bladder epithelial cells and altered the distribution of actin cytoskeleton. Sha and TagC were also shown to degrade mucin and gelatin respectively. Inactivation of the serine catalytic site in each of these SPATEs did not affect secretion of the SPATEs from bacterial cells, but abrogated entry into epithelial cells, cytotoxicity, and proteolytic activity. Thus, our results show that the serine catalytic triad of these proteins is required for internalization in host cells, actin disruption, and degradation of host substrates such as mucin and gelatin.
Assuntos
Citoesqueleto de Actina/metabolismo , Escherichia coli Extraintestinal Patogênica/enzimologia , Mutação , Serina Endopeptidases/metabolismo , Bexiga Urinária/citologia , Domínio Catalítico , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli Extraintestinal Patogênica/genética , Gelatina/metabolismo , Humanos , Mucinas/metabolismo , Proteólise , Serina Endopeptidases/química , Serina Endopeptidases/genética , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologiaRESUMO
The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC (adrA in Salmonella) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA, affecting at the same time the inversion of the fim promoter switch (fimS). In the pst mutant, inactivation of yaiC restored fim-dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC, which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model.IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the expression of type 1 fimbriae and attenuates UPEC virulence. Herein, we identified that alteration of the phosphate metabolism increases production of the signaling molecule c-di-GMP, which in turn decreases the expression of type 1 fimbriae. We also determine the regulatory cascade leading to the accumulation of c-di-GMP and identify the Pho regulon as new players in c-di-GMP-mediated cell signaling. By understanding the molecular mechanisms leading to the expression of virulence factors, we will be in a better position to develop new therapeutics.
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Escherichia coli Uropatogênica/genética , Transportadores de Cassetes de Ligação de ATP/genética , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/genética , GMP Cíclico/metabolismo , Infecções por Escherichia coli/microbiologia , Fímbrias Bacterianas/genética , Humanos , Camundongos , Família Multigênica , Mutação , Óperon , Fosfatos/metabolismo , Recombinases/genética , Regulon , Fatores de Transcrição/genética , Bexiga Urinária/citologia , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/enzimologia , Escherichia coli Uropatogênica/metabolismo , VirulênciaRESUMO
Escherichia coli can cause extraintestinal infections in humans and animals. The hlyF gene is epidemiologically associated with virulent strains of avian pathogenic E. coli and human neonatal meningitis-associated E. coli. We demonstrated that culture supernatants of E. coli expressing HlyF induced autophagy in eukaryotic cells. This phenotype coincided with an enhanced production of outer membrane vesicles (OMVs) by bacteria expressing HlyF. The HlyF protein displays a predicted catalytic domain of the short-chain dehydrogenase/reductase superfamily. This conserved domain was involved the ability of HlyF to promote the production of OMVs. The increased production of OMVs was associated with the release of toxins. hlyF was shown to be expressed during extraintestinal infection and to play a role in the virulence of extraintestinal pathogenic E. coli in a chicken model of colibacillosis. This is the first evidence that pathogenic bacteria produce a virulence factor directly involved in the production of OMVs.
Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica/fisiologia , Fatores de Hemolisina/metabolismo , Fatores de Virulência/metabolismo , Animais , Autofagia , Membrana Celular/genética , Galinhas , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Humanos , Mutagênese Sítio-Dirigida , Filogenia , Doenças das Aves Domésticas/microbiologia , Vacúolos , Virulência , Fatores de Virulência/genéticaRESUMO
The genotoxin colibactin, synthesized by Escherichia coli, is a secondary metabolite belonging to the chemical family of hybrid polyketide/nonribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the high-pathogenicity island, encoding the siderophore yersiniabactin, which belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90, which is involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mutagênicos/metabolismo , Peptídeos/metabolismo , Fenóis/metabolismo , Policetídeos/metabolismo , Sideróforos/metabolismo , Tiazóis/metabolismo , Animais , Modelos Animais de Doenças , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Feminino , Deleção de Genes , Proteínas de Choque Térmico HSP90/genética , Camundongos Endogâmicos C57BL , Mapeamento de Interação de Proteínas , Ratos Wistar , VirulênciaRESUMO
Campylobacter jejuni is the leading cause of bacterial gastroenteritis. It relies on several virulence factors for host colonization, including glycosylated flagella. C. jejuni NCTC 11168 modifies its flagellins with pseudaminic acid derivatives. It is also presumed to modify these proteins with legionaminic acid, although no glycopeptide evidence was available at the onset of this study. The enzyme encoded by cj1319 can be used to make legionaminic acid in vitro, but the pathway for legionaminic acid synthesis partially inferred by knockout mutagenesis in Campylobacter coli VC167 excludes Cj1319. To address this contradiction, we examined the presence of legionaminic acid in flagellin glycopeptides of wild-type (WT) C. jejuni NCTC 11168 and of a cj1319 knockout mutant. We used high-energy collision-induced dissociation to obtain amino acid sequences while also visualizing signature sugar oxonium ions. Data analysis was performed with PEAKS software, and spectra were manually inspected for glycopeptide determination and verification. We showed that legionaminic acid is present on the flagellins of C. jejuni NCTC 11168 and that flagellin glycosylation is highly heterogeneous, with up to six different sugars singly present at a given site. We found that the cj1319 mutant produces more legionaminic acid than WT, thus excluding the requirement for Cj1319 for legionaminic acid synthesis. We also showed that this mutant has enhanced chicken colonization compared with WT, which may in part be attributed to the high content of legionaminic acid on its flagella.
Assuntos
Flagelina/metabolismo , Glicopeptídeos/metabolismo , Ácidos Siálicos/metabolismo , Açúcares Ácidos/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidade , Flagelina/química , Flagelina/genética , Glicopeptídeos/química , Glicosilação , Interações Hospedeiro-Patógeno/genética , Humanos , Mutagênese , Ácidos Siálicos/química , Açúcares Ácidos/químicaRESUMO
Salmochelins are glucosylated forms of enterobactin (enterochelin) and contribute to the virulence of Salmonella enterica and some extra-intestinal pathogenic Escherichia coli (ExPEC). Fes, IroD and IroE esterases degrade salmochelins and enterobactin to release iron. We investigated the apparently redundant role of these esterases in virulence and in salmochelin production and utilization of the ExPEC strain χ7122. The ΔiroD, ΔfesΔiroD and ΔfesΔiroDΔiroE mutants displayed attenuated virulence phenotypes in an avian systemic infection model. Growth of ΔfesΔiroD and ΔfesΔiroDΔiroE mutants was severely reduced in the presence of conalbumin, and although enterobactin was produced, no salmochelins were detected in the culture supernatants of these mutants. Elimination of catecholate synthesis via an entA deletion in a ΔfesΔiroDΔiroE restored growth in the presence of conalbumin, but only partially restored the virulence of the strain. Salmochelin production was reestablished by reintroducing active esterases. Intracellular accumulation of cyclic mono-glucosylated enterobactin was observed in the triple mutant ΔfesΔiroDΔiroE, and deletion of fepC, required for catecholate import into the cytoplasm, restored salmochelin detection in supernatants. These results suggest that in the absence of esterases, cyclic salmochelins are synthesized and secreted, but remain cell-bound after internalization indicating that esterase-mediated degradation is required for re-secretion of catecholate siderophore molecules following their utilization.
Assuntos
Enterobactina/análogos & derivados , Escherichia coli/patogenicidade , Esterases/metabolismo , Sideróforos/metabolismo , Animais , Transporte Biológico , Galinhas , Enterobactina/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Compostos Férricos/metabolismo , Glucosídeos/metabolismo , Salmonella enterica/metabolismo , VirulênciaRESUMO
The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.
Assuntos
Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/patogenicidade , Heptoses/metabolismo , Polissacarídeos Bacterianos/metabolismo , Animais , Cápsulas Bacterianas/genética , Ácidos e Sais Biliares/metabolismo , Células CACO-2 , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Sequência de Carboidratos , Galinhas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Trato Gastrointestinal/microbiologia , Técnicas de Inativação de Genes , Heptoses/genética , Humanos , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Células RAW 264.7 , VirulênciaRESUMO
tcf (Typhi colonization factor) is one of the 12 putative chaperone/usher fimbrial clusters present in the Salmonella enterica serovar Typhi genome. We investigated the production, expression and regulation of tcf as well as its role during interaction with human cells. The tcf gene cluster was cloned and induced in Escherichia coli and S. Typhi, and the production of intertwined fibres similar to the Cbl (cable) pili of Burkholderia cepacia was observed on the bacterial surface by electron microscopy. In S. Typhi, tcf was expressed more after growth in M63 minimal medium than in standard Luria-Bertani medium. Analysis of the promoter region identified putative binding sites for the global regulators RcsB, ArgR and Fur. The expression of tcf was measured in isogenic strains lacking these global regulators. Under the conditions tested, the results showed that tcf expression was higher in the fur mutant and was regulated by iron concentration. Fur may regulate these fimbriae indirectly via the small RNAs RyhB1 and RyhB2. An isogenic mutant harbouring a deletion of the tcf cluster did not demonstrate any defect in adhesion or invasion of human epithelial cells, or in phagocytosis or survival in macrophages, when compared to the WT serovar Typhi strain. However, the tcf cluster contributed to adherence to human epithelial cells when introduced into E. coli. Thus, tcf genes encode functional fimbriae that can act as an adhesin and may contribute to colonization during typhoid fever.
Assuntos
Aderência Bacteriana/genética , Células Epiteliais/microbiologia , Fímbrias Bacterianas/genética , Macrófagos/microbiologia , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Fatores de Virulência/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Macrófagos/imunologia , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Fatores de Virulência/biossínteseRESUMO
In Escherichia coli, the small regulatory noncoding RNA (sRNA) RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the roles of these regulators in iron homeostasis have been well studied in a nonpathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown for a pathogenic E. coli strain. We thus investigated the roles of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of urinary tract infection (UTI), deletion of fur alone did not attenuate virulence, whereas a ΔryhB mutant and a Δfur ΔryhB double mutant showed significantly reduced bladder colonization. The Δfur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins, and aerobactin than the wild-type strain. In contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhB mutant produced lower levels of siderophores. This decrease was correlated with the downregulation of shiA (encoding a transporter of shikimate, a precursor of enterobactin and salmochelin biosynthesis) and iucD (involved in aerobactin biosynthesis) in this mutant grown in minimal medium or in human urine. iucD was also downregulated in bladders infected with the ΔryhB mutant compared to those infected with the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.
Assuntos
Regulação Bacteriana da Expressão Gênica , Pequeno RNA não Traduzido/metabolismo , Sideróforos/biossíntese , Escherichia coli Uropatogênica/fisiologia , Animais , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Feminino , Deleção de Genes , Camundongos Endogâmicos CBA , Pequeno RNA não Traduzido/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo , VirulênciaRESUMO
Avian pathogenic Escherichia coli (APEC) strains harbor a number of virulence genes and cause extraintestinal diseases, such as septicemia, swollen-head syndrome, salpingitis, and omphalitis in poultry. APEC strains are not known to cause intestinal diseases. Herein, for the first time, it is reported that APEC strains were able to induce an enterotoxigenic-like effect in rabbit ligated ileal loops. Strain SEPT362 caused cell detachment of the intestinal villi, which also showed a flattened and wilted appearance, but the integrity of the tight junctions was maintained. Additionally, this strain did not adhere to enterocytes in vivo, although adhesin encoding genes ( fimH, csgA, lpfA2-3, and ECP) were present while other lpfA types, sfa, afa, papC, and ral genes were not. This enterotoxigenic-like activity was conserved after thermal treatment of the supernatant at 65°C but not at 100°C. Moreover, experiments based on filtering with different molecular weight cut-off (MWCO) pore sizes demonstrated that the component associated with the observed biological effect has a molecular weight >100 kDa. Blast search and polymerase chain reaction assays for known E. coli virulence factors showed that strain SEPT362 harbors the gene encoding for the toxin EAST-1 and the serine protease autotransporter (SPATE) Tsh, but is negative for genes encoding for the toxins LT-I, STh, STp, Stx1, Stx2, CNF-1, CNF-2, CDT and the SPATEs Sat, Pic, Vat, SigA, SepA, EatA, EspP, or EspC. A cloned copy of the tsh gene in E. coli K-12 was also tested and was shown to have an enterotoxic effect. These results suggest that APEC might induce fluid accumulation in the rabbit gut. The Tsh autotransporter seems to be one of the factors associated with this phenotype.
Assuntos
Adesinas de Escherichia coli/metabolismo , Enterite/microbiologia , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Infecções por Escherichia coli/microbiologia , Íleo/microbiologia , Mucosa Intestinal/microbiologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/toxicidade , Animais , Aderência Bacteriana , Galinhas/microbiologia , Enterite/patologia , Enterite/fisiopatologia , Escherichia coli Enterotoxigênica/crescimento & desenvolvimento , Escherichia coli Enterotoxigênica/isolamento & purificação , Enterotoxinas/genética , Enterotoxinas/toxicidade , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/fisiopatologia , Infecções por Escherichia coli/veterinária , Temperatura Alta , Íleo/metabolismo , Íleo/ultraestrutura , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Fígado/microbiologia , Masculino , Doenças das Aves Domésticas/microbiologia , Coelhos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Sepse/microbiologia , Sepse/veterinária , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/toxicidade , Desequilíbrio Hidroeletrolítico/etiologiaRESUMO
Developing a vaccine against Clostridioides difficile is a key strategy to protect the elderly. Two candidate vaccines using a traditional approach of intramuscular (IM) delivery of recombinant antigens targeting C. difficile toxins A (TcdA) and B (TcdB) failed to meet their primary endpoints in large phase 3 trials. To elicit a mucosal response against C. difficile, we repurposed an attenuated strain of Salmonella Typhimurium (YS1646) to deliver the receptor binding domains (rbd) of TcdA and TcdB to the gut-associated lymphoid tissues, to elicit a mucosal response against C. difficile. In this study, YS1646 candidates with either rbdA or rbdB expression cassettes integrated into the bacterial chromosome at the attTn7 site were generated and used in a short-course multimodal vaccination strategy that combined oral delivery of the YS1646 candidate(s) on days 0, 2, and 4 and IM delivery of recombinant antigen(s) on day 0. Five weeks after vaccination, mice had high serum IgG titers and increased intestinal antigen-specific IgA titers. Multimodal vaccination increased the IgG avidity compared to the IM-only control. In the mesenteric lymph nodes, we observed increased IL-5 secretion and increased IgA+ plasma cells. Oral vaccination skewed the IgG response toward IgG2c dominance (vs IgG1 dominance in the IM-only group). Both oral alone and multimodal vaccination against TcdA protected mice from lethal C. difficile challenge (100% survival vs 30% in controls). Given the established safety profile of YS1646, we hope to move this vaccine candidate forward into a phase I clinical trial.IMPORTANCEClostridioides difficile remains a major public health threat, and new approaches are needed to develop an effective vaccine. To date, the industry has focused on intramuscular vaccination targeting the C. difficile toxins. Multiple disappointing results in phase III trials have largely confirmed that this may not be the best strategy. As C. difficile is a pathogen that remains in the intestine, we believe that targeting mucosal immune responses in the gut will be a more successful strategy. We have repurposed a highly attenuated Salmonella Typhimurium (YS1646), originally pursued as a cancer therapeutic, as a vaccine vector. Using a multimodal vaccination strategy (both recombinant protein delivered intramuscularly and YS1646 expressing antigen delivered orally), we elicited both systemic and local immune responses. Oral vaccination alone completely protected mice from lethal challenge. Given the established safety profile of YS1646, we hope to move these vaccine candidates forward into a phase I clinical trial.
Assuntos
Toxinas Bacterianas , Compostos de Boro , Clostridioides difficile , Humanos , Animais , Camundongos , Idoso , Toxinas Bacterianas/genética , Salmonella typhimurium/genética , Clostridioides difficile/genética , Vacinas Bacterianas , Vacinas Sintéticas , Vacinação , Imunoglobulina G , Imunoglobulina ARESUMO
Infectious bronchitis virus (IBV) is a highly contagious Gammacoronavirus causing moderate to severe respiratory infection in chickens. Understanding the initial antiviral response in the respiratory mucosa is crucial for controlling viral spread. We aimed to characterize the impact of IBV Delmarva (DMV)/1639 and IBV Massachusetts (Mass) 41 at the primary site of infection, namely, in chicken tracheal epithelial cells (cTECs) in vitro and the trachea in vivo. We hypothesized that some elements of the induced antiviral responses are distinct in both infection models. We inoculated cTECs and infected young specific pathogen-free (SPF) chickens with IBV DMV/1639 or IBV Mass41, along with mock-inoculated controls, and studied the transcriptome using RNA-sequencing (RNA-seq) at 3 and 18 h post-infection (hpi) for cTECs and at 4 and 11 days post-infection (dpi) in the trachea. We showed that IBV DMV/1639 and IBV Mass41 replicate in cTECs in vitro and the trachea in vivo, inducing host mRNA expression profiles that are strain- and time-dependent. We demonstrated the different gene expression patterns between in vitro and in vivo tracheal IBV infection. Ultimately, characterizing host-pathogen interactions with various IBV strains reveals potential mechanisms for inducing and modulating the immune response during IBV infection in the chicken trachea.
Assuntos
Galinhas , Infecções por Coronavirus , Perfilação da Expressão Gênica , Vírus da Bronquite Infecciosa , Doenças das Aves Domésticas , Traqueia , Animais , Traqueia/virologia , Traqueia/imunologia , Galinhas/virologia , Vírus da Bronquite Infecciosa/fisiologia , Vírus da Bronquite Infecciosa/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/genética , Células Epiteliais/virologia , Células Epiteliais/imunologia , Transcriptoma , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/genética , Replicação Viral , Organismos Livres de Patógenos EspecíficosRESUMO
In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.
Assuntos
Fímbrias Bacterianas , Osmorregulação , Trealose , Bexiga Urinária , Infecções Urinárias , Animais , Trealose/metabolismo , Camundongos , Bexiga Urinária/microbiologia , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/genética , Infecções Urinárias/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Modelos Animais de Doenças , Feminino , Pressão Osmótica , Escherichia coli Extraintestinal Patogênica/metabolismo , Escherichia coli Extraintestinal Patogênica/genética , Ureia/metabolismo , Trealase/metabolismo , Trealase/genética , Deleção de Genes , Glucose/metabolismoRESUMO
The Escherichia coli Adhesin Involved in Diffuse Adherence (AIDA-I) is a multifunctional protein that belongs to the family of monomeric autotransporters. This adhesin can be glycosylated by the AIDA-associated heptosyltransferase (Aah). Glycosylation appears to be restricted to the extracellular domain of AIDA-I, which comprises imperfect repeats of a 19-amino-acid consensus sequence and is predicted to form a ß-helix. Here, we show that Aah homologues can be found in many Gram-negative bacteria, including Citrobacter rodentium. We demonstrated that an AIDA-like protein is glycosylated in this species by the Aah homologue. We then investigated the substrate recognition mechanism of the E. coli Aah heptosyltransferase. We found that a peptide corresponding to one repeat of the 19-amino-acid consensus is sufficient for recognition and glycosylation by Aah. Mutagenesis studies suggested that, unexpectedly, Aah recognizes a structural motif typical of ß-helices, but not a specific sequence. In agreement with this finding, we observed that the extracellular domain of the Bordetella pertussis pertactin, a ß-helical polypeptide lacking the 19-amino-acid consensus sequence, could be glycosylated by Aah. Overall, our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif.
Assuntos
Adesinas de Escherichia coli/química , Motivos de Aminoácidos , Proteínas de Bactérias/química , Escherichia coli/enzimologia , Glicosiltransferases/química , Bordetella pertussis/enzimologia , Citrobacter rodentium/enzimologia , Sequência Consenso , Glicosilação , Filogenia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Iron is an essential element but can be toxic at high concentrations. Therefore, its acquisition and storage require tight control. Salmonella encodes the global regulator Fur (ferric uptake regulator) and the small regulatory non-coding RNAs (sRNAs) RfrA and RfrB, homologues of RyhB. The role of these iron homeostasis regulators was investigated in Salmonella enterica serovar Typhi (S. Typhi). Strains containing either single or combined deletions of these regulators were obtained. The mutants were tested for growth in low and high iron conditions, resistance to oxidative stress, expression and production of siderophores, and during interaction with host cells. The fur mutant showed a growth defect and was sensitive to hydrogen peroxide. The expression of the sRNAs was responsible for these defects. Siderophore expression by S. Typhi and both sRNAs were regulated by iron and by Fur. Fur contributed to invasion of epithelial cells, and was shown for the first time to play a role in phagocytosis and intracellular survival of S. Typhi in human macrophages. The sRNAs RfrA and RfrB were not required for interaction with epithelial cells, but both sRNAs were important for optimal intracellular replication in macrophages. In S. Typhi, Fur is a repressor of both sRNAs, and loss of either RfrA or RfrB resulted in distinct phenotypes, suggesting a non-redundant role for these regulatory RNAs.