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1.
Nat Immunol ; 17(2): 150-8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26595890

RESUMO

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.


Assuntos
Imunidade Inata , Interferons/metabolismo , Mucosa/imunologia , Mucosa/metabolismo , Viroses/imunologia , Viroses/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL10/biossíntese , Modelos Animais de Doenças , Feminino , Expressão Gênica , Glicosilação , Herpes Simples/genética , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 2/imunologia , Humanos , Interferons/genética , Ligantes , Camundongos , Camundongos Knockout , Mucosa/virologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/imunologia , Receptores CXCR3/deficiência , Receptores CXCR3/metabolismo , Vagina/imunologia , Vagina/metabolismo , Vagina/virologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Carga Viral , Viroses/virologia
2.
PLoS Pathog ; 16(8): e1008776, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845938

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a diarrheagenic pathotype associated with traveler's diarrhea, foodborne outbreaks and sporadic diarrhea in industrialized and developing countries. Regulation of virulence in EAEC is mediated by AggR and its negative regulator Aar. Together, they control the expression of at least 210 genes. On the other hand, we observed that about one third of Aar-regulated genes are related to metabolism and transport. In this study we show the AggR/Aar duo controls the metabolism of lipids. Accordingly, we show that AatD, encoded in the AggR-regulated aat operon (aatPABCD) is an N-acyltransferase structurally similar to the essential Apolipoprotein N-acyltransferase Lnt and is required for the acylation of Aap (anti-aggregation protein). Deletion of aatD impairs post-translational modification of Aap and causes its accumulation in the bacterial periplasm. trans-complementation of 042aatD mutant with the AatD homolog of ETEC or with the N-acyltransferase Lnt reestablished translocation of Aap. Site-directed mutagenesis of the E207 residue in the putative acyltransferase catalytic triad disrupted the activity of AatD and caused accumulation of Aap in the periplasm due to reduced translocation of Aap at the bacterial surface. Furthermore, Mass spectroscopy revealed that Aap is acylated in a putative lipobox at the N-terminal of the mature protein, implying that Aap is a lipoprotein. Lastly, deletion of aatD impairs bacterial colonization of the streptomycin-treated mouse model. Our findings unveiled a novel N-acyltransferase family associated with bacterial virulence, and that is tightly regulated by AraC/XylS regulators in the order Enterobacterales.


Assuntos
Acetiltransferases/metabolismo , Fator de Transcrição AraC/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Acetiltransferases/genética , Acilação , Animais , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Óperon , Filogenia , Conformação Proteica , Virulência
3.
Infect Immun ; 88(9)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32631917

RESUMO

Symptomatic and asymptomatic infection with the diarrheal pathogen enteroaggregative Escherichia coli (EAEC) is associated with growth faltering in children in developing settings. The mechanism of this association is unknown, emphasizing a need for better understanding of the interactions between EAEC and the human gastrointestinal mucosa. In this study, we investigated the role of the aggregative adherence fimbriae II (AAF/II) in EAEC adherence and pathogenesis using human colonoids and duodenal enteroids. We found that a null mutant in aafA, the major subunit of AAF/II, adhered significantly less than wild-type (WT) EAEC strain 042, and adherence was restored in a complemented strain. Immunofluorescence confocal microscopy of differentiated colonoids, which produce an intact mucus layer comprised of the secreted mucin MUC2, revealed bacteria at the epithelial surface and within the MUC2 layer. The WT strain adhered to the epithelial surface, whereas the aafA deletion strain remained within the MUC2 layer, suggesting that the presence or absence of AAF/II determines both the abundance and location of EAEC adherence. In order to determine the consequences of EAEC adherence on epithelial barrier integrity, colonoid monolayers were exposed to EAEC constructs expressing or lacking aafA Colonoids infected with WT EAEC had significantly decreased epithelial resistance, an effect that required AAF/II, suggesting that binding of EAEC to the epithelium is necessary to impair barrier function. In summary, we show that production of AAF/II is critical for adherence and barrier disruption in human colonoids, suggesting a role for this virulence factor in EAEC colonization of the gastrointestinal mucosa.


Assuntos
Adesinas de Escherichia coli/imunologia , Células Epiteliais/microbiologia , Escherichia coli/imunologia , Fímbrias Bacterianas/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Organoides/microbiologia , Adesinas de Escherichia coli/genética , Aderência Bacteriana , Colo/imunologia , Colo/metabolismo , Colo/microbiologia , Contagem de Colônia Microbiana , Duodeno/imunologia , Duodeno/metabolismo , Duodeno/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Fímbrias Bacterianas/genética , Deleção de Genes , Regulação da Expressão Gênica , Teste de Complementação Genética , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucina-2/genética , Mucina-2/imunologia , Organoides/imunologia , Organoides/metabolismo , Transdução de Sinais
4.
PLoS Pathog ; 13(8): e1006545, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28806780

RESUMO

The AraC Negative Regulators (ANR) comprise a large family of virulence regulators distributed among diverse clinically important Gram-negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., and pathogenic E. coli strains. We have previously reported broad effects of the ANR members on regulators of the AraC/XylS family. Here, we interrogate possible broader effects of the ANR members on the bacterial transcriptome. Our studies focused on Aar (AggR-activated regulator), an ANR family archetype in enteroaggregative E. coli (EAEC) isolate 042. Transcriptome analysis of EAEC strain 042, 042aar and 042aar(pAar) identified more than 200 genes that were differentially expressed (+/- 1.5 fold, p<0.05). Most of those genes are located on the bacterial chromosome (195 genes, 92.85%), and are associated with regulation, transport, metabolism, and pathogenesis, based on the predicted annotation; a considerable number of Aar-regulated genes encoded for hypothetical proteins (46 genes, 21.9%) and regulatory proteins (25, 11.9%). Notably, the transcriptional expression of three histone-like regulators, H-NS (orf1292), H-NS homolog (orf2834) and StpA, was down-regulated in the absence of aar and may explain some of the effects of Aar on gene expression. By employing a bacterial two-hybrid system, LacZ reporter assays, pull-down and electrophoretic mobility shift assay (EMSA) analysis, we demonstrated that Aar binds directly to H-NS and modulates H-NS-induced gene silencing. Importantly, Aar was highly expressed in the mouse intestinal tract and was found to be necessary for maximal H-NS expression. In conclusion, this work further extends our knowledge of genes under the control of Aar and its biological relevance in vivo.


Assuntos
Fator de Transcrição AraC/metabolismo , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Virulência/fisiologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/metabolismo , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase
5.
Mol Microbiol ; 101(2): 314-32, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27038276

RESUMO

AraC Negative Regulators (ANR) suppress virulence genes by directly down-regulating AraC/XylS members in Gram-negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR-activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC-like member AggR. ANR-AggR binding disrupted AggR dimerization and prevented AggR-DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α-helices. Site-directed mutagenesis studies suggest that at least predicted α-helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.


Assuntos
Fator de Transcrição AraC/genética , Genes araC/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genes araC/fisiologia , Bactérias Gram-Negativas/genética , Mutagênese Sítio-Dirigida , Filogenia , Relação Estrutura-Atividade , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Virulência/genética
6.
PLoS Pathog ; 10(5): e1004153, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24875828

RESUMO

We have reported that transcription of a hypothetical small open reading frame (orf60) in enteroaggregative E. coli (EAEC) strain 042 is impaired after mutation of aggR, which encodes a global virulence activator. We have also reported that the cryptic orf60 locus was linked to protection against EAEC diarrhea in two epidemiologic studies. Here, we report that the orf60 product acts as a negative regulator of aggR itself. The orf60 protein product lacks homology to known repressors, but displays 44-100% similarity to at least fifty previously undescribed small (<10 kDa) hypothetical proteins found in many gram negative pathogen genomes. Expression of orf60 homologs from enterotoxigenic E. coli (ETEC) repressed the expression of the AraC-transcriptional ETEC regulator CfaD/Rns and its regulon in ETEC strain H10407. Complementation in trans of EAEC 042orf60 by orf60 homologs from ETEC and the mouse pathogen Citrobacter rodentium resulted in dramatic suppression of aggR. A C. rodentium orf60 homolog mutant showed increased levels of activator RegA and increased colonization of the adult mouse. We propose the name Aar (AggR-activated regulator) for the clinically and epidemiologically important orf60 product in EAEC, and postulate the existence of a large family of homologs among pathogenic Enterobacteriaceae and Pasteurellaceae. We propose the name ANR (AraC Negative Regulators) for this family.


Assuntos
Proteínas de Bactérias/metabolismo , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Escherichia coli/patogenicidade , Transativadores/metabolismo , Animais , Aderência Bacteriana , Citrobacter rodentium/genética , Diarreia/microbiologia , Infecções por Enterobacteriaceae/genética , Regulação Bacteriana da Expressão Gênica/imunologia , Camundongos , Virulência/genética
7.
PLoS Pathog ; 10(9): e1004404, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25232738

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a leading cause of acute and persistent diarrhea worldwide. A recently emerged Shiga-toxin-producing strain of EAEC resulted in significant mortality and morbidity due to progressive development of hemolytic-uremic syndrome. The attachment of EAEC to the human intestinal mucosa is mediated by aggregative adherence fimbria (AAF). Using X-ray crystallography and NMR structures, we present new atomic resolution insight into the structure of AAF variant I from the strain that caused the deadly outbreak in Germany in 2011, and AAF variant II from archetype strain 042, and propose a mechanism for AAF-mediated adhesion and biofilm formation. Our work shows that major subunits of AAF assemble into linear polymers by donor strand complementation where a single minor subunit is inserted at the tip of the polymer by accepting the donor strand from the terminal major subunit. Whereas the minor subunits of AAF have a distinct conserved structure, AAF major subunits display large structural differences, affecting the overall pilus architecture. These structures suggest a mechanism for AAF-mediated adhesion and biofilm formation. Binding experiments using wild type and mutant subunits (NMR and SPR) and bacteria (ELISA) revealed that despite the structural differences AAF recognize a common receptor, fibronectin, by employing clusters of basic residues at the junction between subunits in the pilus. We show that AAF-fibronectin attachment is based primarily on electrostatic interactions, a mechanism not reported previously for bacterial adhesion to biotic surfaces.


Assuntos
Adesinas de Escherichia coli/imunologia , Aderência Bacteriana/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Escherichia coli/patogenicidade , Fímbrias Bacterianas/química , Interações Hospedeiro-Patógeno/imunologia , Adesinas de Escherichia coli/genética , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fibronectinas/metabolismo , Humanos , Immunoblotting , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos
8.
Infect Immun ; 83(5): 1893-903, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25712927

RESUMO

Enterotoxigenic Escherichia coli (ETEC), a leading cause of acute diarrhea, colonizes the intestine by means of adhesins. However, 15 to 50% of clinical isolates are negative for known adhesins, making it difficult to identify antigens for broad-coverage vaccines. The ETEC strain 1766a, obtained from a child with watery diarrhea in Chile, harbors the colonization factor CS23 but is negative for other known adhesins. One clone, derived from an ETEC 1766a genomic library (clone G10), did not produce CS23 yet was capable of adhering to Caco-2 cells. The goal of this study was to identify the gene responsible for this capacity. Random transposon-based mutagenesis allowed the identification of a 4,110-bp gene that codes for a homologue of the temperature-sensitive hemagglutinin (Tsh) autotransporter described in avian E. coli strains (97% identity, 90% coverage) and that is called TleA (Tsh-like ETEC autotransporter) herein. An isogenic ETEC 1766a strain with a tleA mutation showed an adhesion level similar to that of the wild-type strain, suggesting that the gene does not direct attachment to Caco-2 cells. However, expression of tleA conferred the capacity for adherence to nonadherent E. coli HB101. This effect coincided with the detection of TleA on the surface of nonpermeabilized bacteria, while, conversely, ETEC 1766a seems to secrete most of the produced autotransporter to the medium. On the other hand, TleA was capable of degrading bovine submaxillary mucin and leukocyte surface glycoproteins CD45 and P-selectin glycoprotein ligand 1 (PSGL-1). These results suggest that TleA promotes colonization of the intestinal epithelium and that it may modulate the host immune response.


Assuntos
Adesinas Bacterianas/genética , Adesinas de Escherichia coli/genética , Aderência Bacteriana , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/fisiologia , Células Epiteliais/microbiologia , Proteínas de Escherichia coli/genética , Adesinas Bacterianas/metabolismo , Adesinas de Escherichia coli/metabolismo , Animais , Células CACO-2 , Pré-Escolar , Chile , Elementos de DNA Transponíveis , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Humanos , Lactente , Recém-Nascido , Mutagênese Insercional
9.
Cell Mol Life Sci ; 71(5): 745-70, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23689588

RESUMO

Serine proteases exist in eukaryotic and prokaryotic organisms and have emerged during evolution as the most abundant and functionally diverse group. In Gram-negative bacteria, there is a growing family of high molecular weight serine proteases secreted to the external milieu by a fascinating and widely employed bacterial secretion mechanism, known as the autotransporter pathway. They were initially found in Neisseria, Shigella, and pathogenic Escherichia coli, but have now also been identified in Citrobacter rodentium, Salmonella, and Edwardsiella species. Here, we focus on proteins belonging to the serine protease autotransporter of Enterobacteriaceae (SPATEs) family. Recent findings regarding the predilection of serine proteases to host intracellular or extracellular protein-substrates involved in numerous biological functions, such as those implicated in cytoskeleton stability, autophagy or innate and adaptive immunity, have helped provide a better understanding of SPATEs' contributions in pathogenesis. Here, we discuss their classification, substrate specificity, and potential roles in pathogenesis.


Assuntos
Sistemas de Secreção Bacterianos/genética , Enterobacteriaceae/enzimologia , Evasão da Resposta Imune/fisiologia , Modelos Moleculares , Conformação Proteica , Serina Proteases/biossíntese , Serina Proteases/química , Serina Proteases/metabolismo , Sequência de Aminoácidos , Sistemas de Secreção Bacterianos/fisiologia , Enterobacteriaceae/patogenicidade , Evolução Molecular , Variação Genética , Evasão da Resposta Imune/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Serina Proteases/classificação , Especificidade da Espécie , Especificidade por Substrato , Virulência
10.
Infect Immun ; 82(4): 1719-24, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24516112

RESUMO

Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.


Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Escherichia coli/fisiologia , Fímbrias Bacterianas/fisiologia , Queratina-8/fisiologia , Laminina/fisiologia , Adesinas de Escherichia coli , Linhagem Celular , Células Epiteliais/fisiologia , Fibronectinas/imunologia , Humanos , Mucosa Intestinal/citologia , Queratina-8/metabolismo , Laminina/imunologia , Proteínas de Membrana
11.
Infect Immun ; 82(6): 2626-36, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24711562

RESUMO

A growing family of virulence factors called serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by Shigella, Salmonella, and Escherichia coli pathotypes. SPATEs are subdivided into class 1 and class 2 based on structural features and phylogenetics. Class 1 SPATEs induce cytopathic effects in numerous epithelial cell lines, and several have been shown to cleave the cytoskeletal protein spectrin in vitro. However, to date the in vivo role of class 1 SPATEs in enteric pathogenesis is unknown. Citrobacter rodentium, a natural mouse pathogen, has recently been shown to harbor class 1 and class 2 SPATEs. To better understand the contribution of class 1 SPATEs in enteric infection, we constructed a class 1 SPATE null mutant (Δcrc1) in C. rodentium. Upon infection of C57BL/6 mice, the Δcrc1 mutant exhibited a hypervirulent, hyperinflammatory phenotype compared with its parent, accompanied by greater weight loss and a trend toward increased mortality in young mice; the effect was reversed when the crc1 gene was restored. Using flow cytometry, we observed increased infiltration of T cells, B cells, and neutrophils into the lamina propria of the distal colon in mice fed the Δcrc1 mutant, starting as early as 5 days after infection. No significant difference in epithelial cytotoxicity was observed. Reverse transcription-PCR (RT-PCR) analysis of distal colonic tissue on day 10 postinfection showed significant increases in mRNA encoding cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-1ß, and inducible nitric oxide synthase (iNOS) but not in mRNA encoding IL-17, IL-4, or IL-10 in the Δcrc1 mutant-infected mice. Our data suggest a previously unsuspected role for class 1 SPATEs in enteric infection.


Assuntos
Proteínas de Bactérias/fisiologia , Citrobacter rodentium/fisiologia , Colite/microbiologia , Serina Proteases/fisiologia , Análise de Variância , Animais , Linfócitos B/citologia , Toxinas Bacterianas/metabolismo , Citrobacter rodentium/genética , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Colite/imunologia , Colo/citologia , Colo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/citologia , Transporte Proteico/fisiologia , RNA Mensageiro/metabolismo , Linfócitos T/citologia
12.
Proc Natl Acad Sci U S A ; 108(31): 12881-6, 2011 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-21768350

RESUMO

The serine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted by pathogenic Gram-negative bacteria through the autotransporter pathway. We previously classified SPATE proteins into two classes: cytotoxic (class 1) and noncytotoxic (class 2). Here, we show that Pic, a class 2 SPATE protein produced by Shigella flexneri 2a, uropathogenic and enteroaggregative Escherichia coli strains, targets a broad range of human leukocyte adhesion proteins. Substrate specificity was restricted to glycoproteins rich in O-linked glycans, including CD43, CD44, CD45, CD93, CD162 (PSGL-1; P-selectin glycoprotein ligand 1), and the surface-attached chemokine fractalkine, all implicated in leukocyte trafficking, migration, and inflammation. N-terminal sequencing of proteolytic products revealed Pic (protease involved in colonization) cleavage sites to occur before Thr or Ser residues. The purified carbohydrate sLewis-X implied in inflammation and malignancy inhibited cleavage of PSGL-1 by Pic. Exposure of human leukocytes to purified Pic resulted in polymorphonuclear cell activation, but impaired chemotaxis and transmigration; Pic-treated T cells underwent programmed cell death. We also show that the Pic-related protease Tsh/Hbp, implicated in extraintestinal infections, exhibited a spectrum of substrates similar to those cleaved by Pic. In the guinea pig keratoconjunctivitis model, a Shigella pic mutant induced greater inflammation than its parent strain. We suggest that the class-2 SPATEs represent unique immune-modulating bacterial virulence factors.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Glicoproteínas/metabolismo , Leucócitos/metabolismo , Serina Proteases/metabolismo , Shigella flexneri/enzimologia , Animais , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Movimento Celular , Quimiocina CX3CL1/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/fisiologia , Citometria de Fluxo , Cobaias , Interações Hospedeiro-Patógeno , Humanos , Receptores de Hialuronatos/metabolismo , Ceratoconjuntivite/microbiologia , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/microbiologia , Leucossialina/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação , Ativação de Neutrófilo , Filogenia , Receptores de Complemento/metabolismo , Serina Proteases/classificação , Serina Proteases/genética , Shigella flexneri/fisiologia , Especificidade por Substrato
13.
Sci Rep ; 13(1): 7024, 2023 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-37120613

RESUMO

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein-protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.


Assuntos
Escherichia coli Enteropatogênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Humanos , Animais , Camundongos , Virulência/genética , Escherichia coli Enteropatogênica/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição
14.
Infect Immun ; 80(8): 2791-801, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22645287

RESUMO

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.


Assuntos
Adesinas Bacterianas/metabolismo , Antígenos de Bactérias/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Aderência Bacteriana/fisiologia , Sequência de Bases , Células CACO-2 , Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Mutação , Filogenia
15.
Mol Microbiol ; 81(1): 179-91, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21542864

RESUMO

Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well-characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.


Assuntos
Toxinas Bacterianas/biossíntese , Proteína Receptora de AMP Cíclico/metabolismo , Enterotoxinas/biossíntese , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fator Proteico para Inversão de Estimulação/metabolismo , Regulação Bacteriana da Expressão Gênica , Serina Endopeptidases/biossíntese , Transcrição Gênica , Sequência de Bases , Elementos de DNA Transponíveis , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos
16.
Sci Rep ; 10(1): 10533, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601325

RESUMO

EAEC is a common cause of diarrheal illness worldwide. Pathogenesis is believed to occur in the ileum and colon, where the bacteria adhere and form a robust aggregating biofilm. Among the multiple virulence factors produced by EAEC, the Pic serine protease has been implicated in bacterial colonization by virtue of its mucinolytic activity. Hence, a potential role of Pic in mucus barrier disruption during EAEC infection has been long postulated. In this study, we used human colonoids comprising goblet cells and a thick mucin barrier as an intestinal model to investigate Pic's roles during infection with EAEC. We demonstrated the ability of purified Pic, but not a protease defective Pic mutant to degrade MUC2. Western blot and confocal microscopy analysis revealed degradation of the MUC2 layer in colonoids infected with EAEC, but not with its isogenic EAECpic mutant. Wild-type and MUC2-knockdown colonoids infected with EAEC strains exposed a differential biofilm distribution, greater penetration of the mucus layer and increased colonization of the colonic epithelium by Wild-type EAEC than its isogenic Pic mutant. Higher secretion of pro-inflammatory cytokines was seen in colonoids infected with EAEC than EAECpic. Although commensal E. coli expressing Pic degraded MUC2, it did not show improved mucus layer penetration or colonization of the colonic epithelium. Our study demonstrates a role of Pic in MUC2 barrier disruption in the human intestine and shows that colonoids are a reliable system to study the interaction of pathogens with the mucus layer.


Assuntos
Colo/microbiologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Mucosa Intestinal/microbiologia , Serina Endopeptidases/metabolismo , Colo/metabolismo , Células Caliciformes/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucinas/metabolismo
17.
PLoS Negl Trop Dis ; 14(5): e0008274, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32357189

RESUMO

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.


Assuntos
Adesinas de Escherichia coli/genética , Bacteriemia/microbiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/classificação , Fímbrias Bacterianas/genética , Genótipo , Transativadores/genética , Adolescente , Bacteriemia/epidemiologia , Criança , Pré-Escolar , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Moçambique/epidemiologia , Reação em Cadeia da Polimerase , Sorogrupo , Sequenciamento Completo do Genoma
18.
J Bacteriol ; 191(21): 6571-83, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19734313

RESUMO

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a beta-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-beta or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in approximately 80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker "autotransporter."


Assuntos
Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Chaperonas Moleculares/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Chaperonas Moleculares/genética , Mutação , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Ligação Proteica , Conformação Proteica , Serina Endopeptidases/genética
19.
Infect Immun ; 77(6): 2465-73, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19349428

RESUMO

Enteroaggregative Escherichia coli (EAEC) is increasingly being recognized as a cause of diarrheal disease in diverse populations. No small animal model is currently available to study this pathogen. We report here that conventional mice orally inoculated with prototype EAEC strain 042 generally became colonized, though the abundance of organisms cultured from their stool varied substantially among individual animals. In contrast, mice whose water contained 5 g/liter streptomycin consistently became colonized at high levels (ca. 10(8) CFU/g of stool). Neither conventional nor streptomycin-treated mice developed clinical signs or histopathologic abnormalities. Using specific mutants in competition with the wild-type strain, we evaluated the contribution of several putative EAEC virulence factors to colonization of streptomycin-treated mice. Our data suggest that the dispersin surface protein and Pic, a serine protease autotransporter secreted by EAEC and Shigella flexneri, promote colonization of the mouse. In contrast, we found no role for the aggregative adherence fimbriae, the transcriptional activator AggR, or the surface factor termed Air (enteroaggregative immunoglobulin repeat protein). To study Pic further, we constructed a single nucleotide mutation in strain 042 which altered only the Pic catalytic serine (strain 042PicS258A). Fractionation of the tissue at 24 h and 3 days demonstrated an approximate 3-log(10) difference between 042 and 042PicS258A in the lumen and mucus layer and adherent to tissue. Strains 042 and 042PicS258A adhered similarly to mouse tissue ex vivo. While no growth differences were observed in a continuous-flow anaerobic intestinal simulator system, the wild-type strain exhibited a growth advantage over 042PicS258A in a culture of cecal mucus and in cecal contents in vitro; this difference was manifest only after 6 h of growth. Moreover, enhanced growth of the wild type was observed in comparison with that of the mutant in minimal medium containing mucin but not in the absence of mucin. The data suggest a novel metabolic role for the Pic mucinase in EAEC colonization.


Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Mucinas/metabolismo , Polissacarídeo-Liases/metabolismo , Serina Endopeptidases/fisiologia , Fatores de Virulência/fisiologia , Substituição de Aminoácidos/genética , Animais , Domínio Catalítico , Ceco/microbiologia , Criança , Contagem de Colônia Microbiana , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Feminino , Deleção de Genes , Humanos , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Polissacarídeo-Liases/genética , Serina Endopeptidases/genética , Shigella flexneri/genética , Virulência , Fatores de Virulência/genética
20.
Front Microbiol ; 10: 1965, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31543869

RESUMO

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.

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