RESUMO
Hemolysin from uropathogenic Escherichia coli (UPEC) is a hemolytic and cytotoxic protein active against a broad range of species and cell types. Expression of hemolysin correlates with severity of infection, as up to 78% of UPEC isolates from pyelonephritis cases express hemolysin. Despite decades of research on hemolysin activity, the mechanism of intoxication and the function of hemolysin in UPEC infection remain elusive. Early in vitro research established the role of hemolysin as a lytic protein at high doses. It is hypothesized that hemolysin is secreted at sublytic doses in vivo and recent research has focused on understanding the more subtle effects of hemolysin both in vitro and in elegant infection models in vivo, including inoculation by micropuncture of individual kidney nephrons. As the field continues to evolve, comparisons of hemolysin function in isolates from a range of UTI infections will be important for delineating the role of this toxin. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Assuntos
Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/metabolismo , Animais , Humanos , Escherichia coli Uropatogênica/patogenicidadeRESUMO
Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains. The results show that varying the substitution pattern of the hydrophobic subunit leads to relatively small changes in antibacterial activity but causes significant changes in hemolytic activity. We hypothesize that these differences in biological activity profile arise, at least in part, from variations among the conformational propensities of the hydrophobic subunits. The α,α,ß,ß-tetramethyl unit is optimal among the subunits we have examined, providing copolymers with potent antibacterial activity and excellent prokaryote vs eukaryote selectivity. Bacteria do not readily develop resistance to the new antibacterial nylon-3 copolymers. These findings suggest that variation in subunit conformational properties could be generally valuable in the development of synthetic polymers for biological applications.
Assuntos
Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Nylons/farmacologia , Polímeros/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Salmonella enterica/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Células NIH 3T3 , Nylons/síntese química , Nylons/química , Polímeros/síntese química , Polímeros/química , Relação Estrutura-AtividadeRESUMO
d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238-1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity.
Assuntos
Antibacterianos/farmacologia , Ciclosserina/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Mutação , RNA Interferente Pequeno/metabolismo , Antibacterianos/metabolismo , Meios de Cultura/química , Ciclosserina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Interferente Pequeno/genética , Urina/microbiologiaRESUMO
During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.
Assuntos
GMP Cíclico/análogos & derivados , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/genética , Infecções Urinárias/microbiologia , Sistema Urinário/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulose/genética , Celulose/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , Citoplasma/metabolismo , Citoplasma/microbiologia , Escherichia coli/enzimologia , Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Feminino , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Deleção de Genes , Peróxido de Hidrogênio/metabolismo , Ferro/metabolismo , Camundongos , Fósforo-Oxigênio Liases/metabolismo , Sistema Urinário/metabolismo , Infecções Urinárias/metabolismo , Urina/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismoRESUMO
Expression of virulence genes in pathogenic Escherichia coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters, but how H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA strand sequence called operon polarity suppressor (ops). RfaH-ops interactions establish long-lived RfaH-EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify eight RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination, whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS-binding features, explaining minor-paralog targeting. IMPORTANCE Bacterial pathogens adapt to hosts and host defenses by reprogramming gene expression, including by H-NS counter-silencing. Counter-silencing turns on transcription initiation when regulators bind to promoters and rearrange repressive H-NS nucleoprotein filaments that ordinarily block transcription. The specialized NusG paralog RfaH also reprograms virulence genes but regulates transcription elongation. To understand how elongation regulators might affect genes silenced by H-NS, we mapped H-NS, StpA (an H-NS paralog), RfaH, σ70, and RNA polymerase (RNAP) locations on DNA in the uropathogenic E. coli strain CFT073. Although H-NS-StpA filaments bind only 18% of the CFT073 genome, all loci at which RfaH binds RNAP are also bound by H-NS-StpA and are silenced when RfaH is absent. Thus, RfaH represents a distinct class of counter-silencer that acts on elongating RNAP to enable transcription through repressive nucleoprotein filaments. Our findings define a new mechanism of elongation counter-silencing and explain how RfaH functions as a virulence regulator.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/genética , Nucleoproteínas/genética , Fatores de Alongamento de Peptídeos/genética , Transativadores/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Vibrio cholerae is a human diarrhoeal pathogen that is a major cause of gastrointestinal disease and death worldwide. Pathogenic V. cholerae strains are characterized by the presence of a Vibrio pathogenicity island (VPI) that encodes virulence factors, including the toxin co-regulated pilus (TCP). TagA is encoded within the VPI and is positively co-regulated with cholera toxin and TCP. TagA is a sequelogue of the StcE mucinase of Escherichia coli O157â:âH7. We investigated whether this sequence homology reflected a conserved enzymic substrate profile. TagA exhibited metalloprotease activity toward crude purified mucins, salivary mucin and LS174T goblet cell surface mucin. Like StcE, TagA did not cleave general protease substrates, but unlike StcE, TagA did not cleave the mucin-like serpin C1 esterase inhibitor. Both proteins cleaved the immune cell surface mucin CD43, but TagA demonstrated reduced enzymic efficiency relative to StcE. TagA was expressed and secreted by V. cholerae under ToxR-dependent conditions. A tagA-deficient V. cholerae strain showed no defect in a model of in vitro attachment to the HEp-2 cell line; however, overexpression of a proteolytically inactive mutant, TagA(E433D), caused a significant increase in attachment. The increased attachment was reduced by pretreatment of epithelial monolayers with active TagA. Our results indicate that TagA is a mucinase and suggest that TagA may directly modify host cell surface molecules during V. cholerae infection.
Assuntos
Proteínas de Bactérias/metabolismo , Metaloendopeptidases/metabolismo , Mucinas/metabolismo , Vibrio cholerae/genética , Aderência Bacteriana , Proteínas de Bactérias/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Humanos , Metaloendopeptidases/genética , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Saliva/química , Fatores de Transcrição/metabolismo , Vibrio cholerae/enzimologiaRESUMO
Escherichia coli O157:H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157:H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157:H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation.
Assuntos
Escherichia coli O157/enzimologia , Proteínas de Escherichia coli/metabolismo , Metaloendopeptidases/metabolismo , Neutrófilos/fisiologia , Animais , Adesão Celular , Células Cultivadas , Quimiotaxia de Leucócito , Embrião não Mamífero/imunologia , Embrião não Mamífero/metabolismo , Proteínas de Escherichia coli/genética , Humanos , Inflamação , Antígenos Comuns de Leucócito/metabolismo , Leucossialina/metabolismo , Metaloendopeptidases/genética , Microscopia de Fluorescência , Ativação de Neutrófilo , Neutrófilos/citologia , Neutrófilos/imunologia , Explosão Respiratória , Peixe-Zebra/embriologia , Peixe-Zebra/imunologia , Peixe-Zebra/metabolismoRESUMO
The complement system is an essential component of host defense against pathogens. Previous research in our laboratory identified StcE, a metalloprotease secreted by Escherichia coli O157:H7 that cleaves the serpin C1 esterase inhibitor (C1-INH), a major regulator of the classical complement cascade. Analyses of StcE-treated C1-INH activity revealed that surprisingly, StcE enhanced the ability of C1-INH to inhibit the classical complement-mediated lysis of sheep erythrocytes. StcE directly interacts with both cells and C1-INH, thereby binding C1-INH to the cell surface. This suggests that the augmented activity of StcE-treated C1-INH is due to the increased concentration of C1-INH at the sites of potential lytic complex formation. Indeed, removal of StcE abolishes the ability of C1-INH to bind erythrocyte surfaces, whereas the proteolysis of C1-INH is unnecessary to potentiate its inhibitory activity. Physical analyses showed that StcE interacts with C1-INH within its aminoterminal domain, allowing the unaffected serpin domain to interact with its targets. In addition, StcE-treated C1-INH provides significantly increased serum resistance to E. coli K-12 over native C1-INH. These data suggest that by recruiting C1-INH to cell surfaces, StcE may protect both E. coli O157:H7 and the host cells to which the bacterium adheres from complement-mediated lysis and potentially damaging inflammatory events.
Assuntos
Proteínas Inativadoras do Complemento 1/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Metaloendopeptidases/administração & dosagem , Animais , Células COS , Proteínas Inativadoras do Complemento 1/metabolismo , Via Clássica do Complemento/efeitos dos fármacos , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Escherichia coli O157/enzimologia , Hemólise/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Técnicas In Vitro , Modelos Biológicos , OvinosRESUMO
Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin.IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.
Assuntos
Proteínas de Bactérias/genética , Ferro/metabolismo , Regulon , Proteínas Repressoras/genética , Escherichia coli Uropatogênica/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismo , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/genéticaRESUMO
The repeats-in-toxin (RTX) family represents a unique class of bacterial exoproteins. The first family members described were toxins from Gram-negative bacterial pathogens; however, additional members included exoproteins with diverse functions. Our review focuses on well-characterized RTX family toxins from Aggregatibacteractinomycetemcomitans (LtxA), Mannheimiahaemolytica (LktA), Bordetella pertussis (CyaA), uropathogenic Escherichia coli (HlyA), and Actinobacillus pleuropneumoniae (ApxIIIA), as well as the studies that have honed in on a single host cell receptor for RTX toxin interactions, the ß2 integrins. The ß2 integrin family is composed of heterodimeric members with four unique alpha subunits and a single beta subunit. ß2 integrins are only found on leukocytes, including neutrophils and monocytes, the first responders to inflammation following bacterial infection. The LtxA, LktA, HlyA, and ApxIIIA toxins target the shared beta subunit, thereby targeting all types of leukocytes. Specific ß2 integrin family domains are required for the RTX toxin's cytotoxic activity and are summarized here. Research examining the domains of the RTX toxins required for cytotoxic and hemolytic activity is also summarized. RTX toxins attack and kill phagocytic immune cells expressing a single integrin family, providing an obvious advantage to the pathogen. The critical question that remains, can the specificity of the RTX-ß2 integrin interaction be therapeutically targeted?
Assuntos
Proteínas de Bactérias , Toxinas Bacterianas , Citotoxinas , Exotoxinas , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Citotoxinas/química , Citotoxinas/toxicidade , Exotoxinas/química , Exotoxinas/toxicidade , Interações Hospedeiro-Patógeno , Humanos , Monócitos/imunologia , Neutrófilos/imunologia , Domínios Proteicos , Receptores de Superfície Celular/imunologiaRESUMO
The Escherichia coli hemolysin (HlyA) is a pore-forming exotoxin associated with severe complications of human urinary tract infections. HlyA is the prototype of the repeats-in-toxin (RTX) family, which includes LtxA from Aggregatibacter actinomycetemcomitans, a periodontal pathogen. The existence and requirement for a host cell receptor for these toxins are controversial. We performed an unbiased forward genetic selection in a mutant library of human monocytic cells, U-937, for host factors involved in HlyA cytotoxicity. The top candidate was the ß2 integrin ß subunit. Δß2 cell lines are approximately 100-fold more resistant than wild-type U-937 cells to HlyA, but remain sensitive to HlyA at high concentrations. Similarly, Δß2 cells are more resistant than wild-type U-937 cells to LtxA, as Δß2 cells remain LtxA resistant even at >1,000-fold-higher concentrations of the toxin. Loss of any single ß2 integrin α subunit, or even all four α subunits together, does not confer resistance to HlyA. HlyA and LtxA bind to the ß2 subunit, but not to αL, αM, or αX in far-Western blots. Genetic complementation of Δß2 cells with either ß2 or ß2 with a cytoplasmic tail deletion restores HlyA and LtxA sensitivity, suggesting that ß2 integrin signaling is not required for cytotoxicity. Finally, ß2 mutations do not alter sensitivity to unrelated pore-forming toxins, as wild-type or Δß2 cells are equally sensitive to Staphylococcus aureus α-toxin and Proteus mirabilis HpmA. Our studies show two RTX toxins use the ß2 integrin ß subunit alone to facilitate cytotoxicity, but downstream integrin signaling is dispensable.IMPORTANCE Urinary tract infections are one of the most common bacterial infections worldwide. Uropathogenic Escherichia coli strains are responsible for more than 80% of community-acquired urinary tract infections. Although we have known for nearly a century that severe infections stemming from urinary tract infections, including kidney or bloodstream infections are associated with expression of a toxin, hemolysin, from uropathogenic Escherichia coli, how hemolysin functions to enhance virulence is unknown. Our research defines the interaction of hemolysin with the ß2 integrin, a human white cell adhesion molecule, as a potential therapeutic target during urinary tract infections. The E. coli hemolysin is the prototype for a toxin family (RTX family) produced by a wide array of human and animal pathogens. Our work extends to the identification and characterization of the receptor for an additional member of the RTX family, suggesting that this interaction may be broadly conserved throughout the RTX toxin family.
Assuntos
Aggregatibacter actinomycetemcomitans/química , Antígenos CD18/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Exotoxinas/química , Proteínas Hemolisinas/química , Aggregatibacter actinomycetemcomitans/genética , Toxinas Bacterianas/química , Antígenos CD18/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/genética , Teste de Complementação Genética , Humanos , Monócitos/microbiologia , Monócitos/patologia , Mutação , Ligação Proteica , Células U937RESUMO
Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections. Nearly half of all UPEC strains secrete hemolysin, a cytotoxic pore-forming toxin. Here, we show that the prevalence of the hemolysin toxin gene (hlyA) is highly variable among the most common 83 E. coli sequence types (STs) represented on the EnteroBase genome database. To explore this diversity in the context of a defined monophyletic lineage, we contextualized sequence variation of the hlyCABD operon within the genealogy of the globally disseminated multidrug-resistant ST131 clone. We show that sequence changes in hlyCABD and its newly defined 1.616-kb-long leader sequence correspond to phylogenetic designation, and that ST131 strains with the strongest hemolytic activity belong to the most extensive multidrug-resistant sublineage (clade C2). To define the set of genes involved in hemolysin production, the clade C2 strain S65EC was completely sequenced and subjected to a genome-wide screen by combining saturated transposon mutagenesis and transposon-directed insertion site sequencing with the capacity to lyse red blood cells. Using this approach, and subsequent targeted mutagenesis and complementation, 13 genes were confirmed to be specifically required for production of active hemolysin. New hemolysin-controlling elements included discrete sets of genes involved in lipopolysaccharide (LPS) inner core biosynthesis (waaC, waaF, waaG, and rfaE) and cytoplasmic chaperone activity (dnaK and dnaJ), and we show these are required for hemolysin secretion. Overall, this work provides a unique description of hemolysin sequence diversity in a single clonal lineage and describes a complex multilevel system of regulatory control for this important toxin.IMPORTANCE Uropathogenic E. coli (UPEC) is the major cause of urinary tract infections and a frequent cause of sepsis. Nearly half of all UPEC strains produce the potent cytotoxin hemolysin, and its expression is associated with enhanced virulence. In this study, we explored hemolysin variation within the globally dominant UPEC ST131 clone, finding that strains from the ST131 sublineage with the greatest multidrug resistance also possess the strongest hemolytic activity. We also employed an innovative forward genetic screen to define the set of genes required for hemolysin production. Using this approach, and subsequent targeted mutagenesis and complementation, we identified new hemolysin-controlling elements involved in LPS inner core biosynthesis and cytoplasmic chaperone activity, and we show that mechanistically they are required for hemolysin secretion. These original discoveries substantially enhance our understanding of hemolysin regulation, secretion and function.
Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas Hemolisinas/biossíntese , Escherichia coli Uropatogênica/metabolismo , Células Cultivadas , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Proteínas Hemolisinas/genética , Humanos , Mutagênese , Óperon , Especificidade da Espécie , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/genética , Sequenciamento do ExomaRESUMO
In vivo accumulation of D-serine by Escherichia coli CFT073 leads to elevated expression of PAP fimbriae and hemolysin by an unknown mechanism. Loss of D-serine catabolism by CFT073 leads to a competitive advantage during murine urinary tract infection (UTI), but loss of both D- and L-serine catabolism results in attenuation. Serine is the first amino acid to be consumed in closed tryptone broth cultures and precedes the production of acetyl phosphate, a high-energy molecule involved in intracellular signaling, and the eventual secretion of acetate. We propose that the colonization defect associated with the loss of serine catabolism is due to perturbations of acetate metabolism. CFT073 grows more rapidly on acetogenic substrates than does E. coli K-12 isolate MG1655. As shown by transcription microarray results, D-serine is catabolized into acetate via the phosphotransacetylase (pta) and acetate kinase (ackA) genes while downregulating expression of acetyl coenzyme A synthase (acs). CFT073 acs, which is unable to reclaim secreted acetate, colonized mouse bladders and kidneys in the murine model of UTI indistinguishably from the wild type. Both pta and ackA are involved in the maintenance of intracellular acetyl phosphate. CFT073 pta and ackA mutants were screened to investigate the role of acetyl phosphate in UTI pathogenesis. Both single mutants are at a competitive disadvantage relative to the wild type in the kidneys but normally colonize the bladder. CFT073 ackA pta was attenuated in both the bladder and the kidneys. Thus, we demonstrate that CFT073 is adapted to acetate metabolism as a result of requiring a proper cycling of the acetyl phosphate pathway for colonization of the upper urinary tract.
Assuntos
Acetatos/metabolismo , Infecções por Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Infecções Urinárias/microbiologia , Acetato Quinase/genética , Acetato Quinase/metabolismo , Animais , Escherichia coli/genética , Infecções por Escherichia coli/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Organofosfatos/metabolismo , Fosfato Acetiltransferase/genética , Fosfato Acetiltransferase/metabolismo , Infecções Urinárias/genética , Infecções Urinárias/metabolismo , Virulência/genéticaRESUMO
Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1ß processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death. Our approach revealed that the hemolysin A (HlyA) toxin is essential for triggering both cell death and NLRP3 inflammasome-mediated IL-1ß release in human macrophages. Random transposon mutagenesis also identified the cof gene, which encodes a poorly characterized phosphatase, as a novel hemolysin regulator; a CFT073 mutant deleted for the cof gene secreted significantly reduced levels of HlyA, had diminished hemolytic activity, and was impaired in its capacity to trigger human macrophage cell death and IL-1ß release. Together, our findings reveal that Cof fine-tunes production of hemolysin, an important determinant of both UPEC-mediated inflammasome activation and human macrophage cell death.
Assuntos
Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Macrófagos/microbiologia , Monoéster Fosfórico Hidrolases/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/metabolismo , Animais , Apoptose , Linhagem Celular , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/genética , Proteínas Hemolisinas/genética , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/citologia , Monoéster Fosfórico Hidrolases/genética , Infecções Urinárias/fisiopatologia , Escherichia coli Uropatogênica/enzimologia , Escherichia coli Uropatogênica/genéticaRESUMO
Escherichia coli are a common cause of infectious disease outside of the gastrointestinal tract. Several independently evolved E. coli clades are common causes of urinary tract and bloodstream infections. There is ample epidemiological and in vitro evidence that several different protein toxins common to many, but not all, of these strains are likely to aid the colonization and immune-evasion ability of these bacteria. This review discusses our current knowledge and areas of ignorance concerning the contribution of the hemolysin; cytotoxic-necrotizing factor-1; and the autotransporters, Sat, Pic, and Vat, to extraintestinal human disease.
Assuntos
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/metabolismo , Animais , Interações Hospedeiro-Patógeno , HumanosRESUMO
The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic Escherichia coli commonly possess the dsdCXA genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a dsdA mutant outcompetes wild type in the murine model of urinary tract infection. This "hypercolonization" was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of dsdA does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in rpoS and that some D-serine specific effects described in other studies may be affected by the rpoS status of the strains used. Inactivation of dsdA in the original clinical isolate of CFT073 gives CFT073 ΔdsdA a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 ΔdsdA is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic E. coli to colonize the murine urinary tract.
Assuntos
Infecções por Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/patologia , Proteínas de Escherichia coli/genética , Feminino , Humanos , Camundongos , Serina/genética , Serina/metabolismo , Infecções Urinárias/genética , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/genéticaRESUMO
Activation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1-inhibitor deficiency. Heterozygous individuals with <50% levels of active C1-inhibitor are susceptible to angioedema attacks indicating a critical need for C1-inhibitor to be present at maximum levels to prevent unwanted prekallikrein activation. Studies with purified proteins do not adequately explain this observation. Therefore to investigate why reduction of C1-inhibitor to levels seen in angioedema patients results in excessive kallikrein generation we examined the effect of endothelial cells on the inhibition of kallikrein by C1-inhibitor. Surprisingly, it was found that a C1-inhibitor concentration of greater than 1 microM was needed to inhibit 3 nM kallikrein. We propose that this apparent protection from inhibition was mediated by kallikrein binding to the cells via the heavy chain in a high molecular weight kininogen and zinc independent manner. Protection of kallikrein from inhibition was not observed when C1-inhibitor truncated in the amino-terminal domain by the StcE metalloproteinase was used, which suggests a novel function for this unique domain. The requirement for high concentrations of C1-inhibitor to fully inhibit kallikrein is consistent with the fact that reduced levels of C1-inhibitor result in the kallikrein activation seen in angioedema.
Assuntos
Proteínas Inativadoras do Complemento 1/biossíntese , Proteínas Inativadoras do Complemento 1/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Calicreínas/antagonistas & inibidores , Calicreínas/sangue , Angioedema/sangue , Células Cultivadas , Cloro/metabolismo , Coagulantes/farmacologia , Proteína Inibidora do Complemento C1 , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Heterozigoto , Humanos , Calicreínas/metabolismo , Cinética , Metaloendopeptidases/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
UNLABELLED: RpoS (σ(S)), the general stress response sigma factor, directs the expression of genes under a variety of stressful conditions. Control of the cellular σ(S) concentration is critical for appropriately scaled σ(S)-dependent gene expression. One way to maintain appropriate levels of σ(S) is to regulate its stability. Indeed, σ(S) degradation is catalyzed by the ClpXP protease and the recognition of σ(S) by ClpXP depends on the adaptor protein RssB. Three anti-adaptors (IraD, IraM, and IraP) exist in Escherichia coli K-12; each interacts with RssB and inhibits RssB activity under different stress conditions, thereby stabilizing σ(S). Unlike K-12, some E. coli isolates, including uropathogenic E. coli strain CFT073, show comparable cellular levels of σ(S) during the logarithmic and stationary growth phases, suggesting that there are differences in the regulation of σ(S) levels among E. coli strains. Here, we describe IraL, an RssB anti-adaptor that stabilizes σ(S) during logarithmic phase growth in CFT073 and other E. coli and Shigella strains. By immunoblot analyses, we show that IraL affects the levels and stability of σ(S) during logarithmic phase growth. By computational and PCR-based analyses, we reveal that iraL is found in many E. coli pathotypes but not in laboratory-adapted strains. Finally, by bacterial two-hybrid and copurification analyses, we demonstrate that IraL interacts with RssB by a mechanism distinct from that used by other characterized anti-adaptors. We introduce a fourth RssB anti-adaptor found in E. coli species and suggest that differences in the regulation of σ(S) levels may contribute to host and niche specificity in pathogenic and nonpathogenic E. coli strains. IMPORTANCE: Bacteria must cope with a variety of environmental conditions in order to survive. RpoS (σ(S)), the general stress response sigma factor, directs the expression of many genes under stressful conditions in both pathogenic and nonpathogenic Escherichia coli strains. The regulation of σ(S) levels and activity allows appropriately scaled σ(S)-dependent gene expression. Here, we describe IraL, an RssB anti-adaptor that, unlike previously described anti-adaptors, stabilizes σ(S) during the logarithmic growth phase in the absence of additional stress. We also demonstrate that iraL is found in a large number of E. coli and Shigella isolates. These data suggest that strains containing iraL are able to initiate σ(S)-dependent gene expression under conditions under which strains without iraL cannot. Therefore, IraL-mediated σ(S) stabilization may contribute to host and niche specificity in E. coli.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Shigella/crescimento & desenvolvimento , Shigella/metabolismo , Fator sigma/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Escherichia coli/genética , Ordem dos Genes , Loci Gênicos , Dados de Sequência Molecular , Fases de Leitura Aberta , Ligação Proteica , Estabilidade Proteica , Alinhamento de Sequência , Shigella/genética , Fator sigma/genética , Sítio de Iniciação de TranscriçãoRESUMO
UNLABELLED: Uropathogenic Escherichia coli (UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (σ(S)), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study of rpoS in UPEC strain CFT073 began after we discovered an rpoS-frameshift mutation in one of our laboratory stocks of "wild-type" CFT073. We demonstrate that an rpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073 rpoS in urine. This indicates that rpoS is needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations in E. coli K-12, CFT073 rpoS is more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073 rpoS in bladder colonization is lost. These results demonstrate that σ(S) is important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though σ(S) affects the pathogenesis of other bacterial species, this is the first work that directly implicates σ(S) as important for UPEC pathogenesis. IMPORTANCE: UPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (σ(S)), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adapted E. coli K-12. Here, we show that σ(S) is needed by the model UPEC strain CFT073 to cope with oxidative stress provided by phagocytes during infection. These findings represent the first report that implicates σ(S) in the fitness of UPEC during infection and support the idea of the need for a better understanding of the effects of this global regulator of gene expression during UTI.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/microbiologia , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Oxirredutases/metabolismo , Fator sigma/metabolismo , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Estresse Oxidativo , Oxirredutases/imunologia , Fagócitos/enzimologia , Fagócitos/imunologia , Fagócitos/microbiologia , Fator sigma/genética , Estresse Fisiológico , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/genéticaRESUMO
Community-acquired urinary tract infections (UTIs) are commonly caused by uropathogenic Escherichia coli (UPEC). We hypothesize that chemotaxis toward ligands present in urine could direct UPEC into and up the urinary tract. Wild-type E. coli CFT073 and chemoreceptor mutants with tsr, tar, or aer deletions were tested for chemotaxis toward human urine in the capillary tube assay. Wild-type CFT073 was attracted toward urine, and Tsr and Tar were the chemoreceptors mainly responsible for mediating this response. The individual components of urine including L-amino acids, D-amino acids and various organic compounds were also tested in the capillary assay with wild-type CFT073. Our results indicate that CFT073 is attracted toward some L- amino acids and possibly toward some D-amino acids but not other common compounds found in urine such as urea, creatinine and glucuronic acid. In the murine model of UTI, the loss of any two chemoreceptors did not affect the ability of the bacteria to compete with the wild-type strain. Our data suggest that the presence of any strong attractant and its associated chemoreceptor might be sufficient for colonization of the urinary tract and that amino acids are the main chemoattractants for E. coli strain CFT073 in this niche.