RESUMO
Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.
Assuntos
Suplementos Nutricionais , Modelos Animais de Doenças , Pneumopatias/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Virulência/efeitos dos fármacos , Zinco/administração & dosagem , Animais , Feminino , Pneumopatias/tratamento farmacológico , Pneumopatias/microbiologia , Camundongos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.
Assuntos
Proteínas de Bactérias/metabolismo , Colo do Útero/citologia , Células Epiteliais/microbiologia , Gonorreia/metabolismo , L-Lactato Desidrogenase/metabolismo , Viabilidade Microbiana/genética , Neisseria gonorrhoeae/enzimologia , Proteínas de Bactérias/genética , Feminino , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Gonorreia/microbiologia , Humanos , Ferro/metabolismo , L-Lactato Desidrogenase/genética , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/crescimento & desenvolvimento , RNA Viral/genéticaRESUMO
Bacterial pathogens encounter a variety of adverse physiological conditions during infection, including metal starvation, metal overload and oxidative stress. Here, we demonstrate that group A Streptococcus (GAS) utilises Mn(II) import via MtsABC during conditions of hydrogen peroxide stress to optimally metallate the superoxide dismutase, SodA, with Mn. MtsABC expression is controlled by the DtxR family metalloregulator MtsR, which also regulates the expression of Fe uptake systems in GAS. Our results indicate that the SodA in GAS requires Mn for full activity and has lower activity when it contains Fe. As a consequence, under conditions of hydrogen peroxide stress where Fe is elevated, we observed that the PerR-regulated Fe(II) efflux system PmtA was required to reduce intracellular Fe, thus protecting SodA from becoming mismetallated. Our findings demonstrate the co-ordinate action of MtsR-regulated Mn(II) import by MtsABC and PerR-regulated Fe(II) efflux by PmtA to ensure appropriate Mn(II) metallation of SodA for optimal superoxide dismutase function.
Assuntos
Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/farmacologia , Manganês/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Streptococcus pyogenes/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Bactérias/genética , Ferro/metabolismo , Estresse Oxidativo/genética , Streptococcus pyogenes/genética , Superóxido Dismutase/genéticaRESUMO
Streptococcus pyogenes (group A Streptococcus [GAS]) causes a wide range of human infections. The pathogenesis of GAS infections is dependent on the temporal expression of numerous secreted and surface-associated virulence factors that interact with host proteins. Streptococcal pyrogenic exotoxin B (SpeB) is one of the most extensively studied toxins produced by GAS, and the coordinate growth phase-dependent regulation of speB expression is linked to disease severity phenotypes. Here, we identified the endopeptidase PepO as a novel growth phase-dependent regulator of SpeB in the invasive GAS M1 serotype strain 5448. By using transcriptomics followed by quantitative reverse transcriptase PCR and Western blot analyses, we demonstrate through targeted mutagenesis that PepO influences growth phase-dependent induction of speB gene expression. Compared to wild-type and complemented mutant strains, we demonstrate that the 5448ΔpepO mutant strain is more susceptible to killing by human neutrophils and is attenuated in virulence in a murine model of invasive GAS infection. Our results expand the complex regulatory network that is operating in GAS to control SpeB production and suggest that PepO is a virulence requirement during GAS M1T1 strain 5448 infections.IMPORTANCE Despite the continuing susceptibility of S. pyogenes to penicillin, this bacterial pathogen remains a leading infectious cause of global morbidity and mortality. A particular subclone of the M1 serotype (M1T1) has persisted globally for decades as the most frequently isolated serotype from patients with invasive and noninvasive diseases in Western countries. One of the key GAS pathogenicity factors is the potent broad-spectrum cysteine protease SpeB. Although there has been extensive research interest on the regulatory mechanisms that control speB gene expression, its genetic regulation is not fully understood. Here, we identify the endopeptidase PepO as a new regulator of speB gene expression in the globally disseminated M1T1 clone and as being essential for virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/patogenicidade , Animais , Animais Geneticamente Modificados , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Modelos Animais de Doenças , Exotoxinas/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutagênese , Neutrófilos/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Zinc plays an important role in host innate immune function. However, the innate immune system also utilizes zinc starvation ("nutritional immunity") to combat infections. Here, we investigate the role of zinc import and export in the protection of Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive bacterial pathogen responsible for a wide spectrum of human diseases, against challenge from host innate immune defense. In order to determine the role of GAS zinc import and export during infection, we utilized zinc import (ΔadcA ΔadcAII) and export (ΔczcD) deletion mutants in competition with the wild type in both in vitro and in vivo virulence models. We demonstrate that nutritional immunity is deployed extracellularly, while zinc toxicity is utilized upon phagocytosis of GAS by neutrophils. We also show that lysosomes and azurophilic granules in neutrophils contain zinc stores for use against intracellular pathogens.
Assuntos
Streptococcus pyogenes/metabolismo , Zinco/metabolismo , Zinco/toxicidade , Animais , DNA Bacteriano , Deleção de Genes , Regulação da Expressão Gênica/imunologia , Humanos , Complexo Antígeno L1 Leucocitário/metabolismo , Lisossomos , Camundongos , Camundongos Transgênicos , Neutrófilos/fisiologia , Plasminogênio/genética , Plasminogênio/metabolismo , Pele/citologia , Pele/metabolismo , Pele/microbiologia , Dermatopatias Bacterianas/metabolismo , Dermatopatias Bacterianas/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/patogenicidade , VirulênciaRESUMO
Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estresse Oxidativo , Streptococcus pyogenes/enzimologia , Adenosina Trifosfatases/genética , Animais , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Homeostase , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Transgênicos , Mutação , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/genética , Estreptonigrina/farmacologiaRESUMO
We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , Salmonella typhimurium/fisiologia , Salmonella/fisiologia , Receptores Toll-Like/metabolismo , Zinco/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Células Cultivadas , Cobre , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Toll-Like/genéticaRESUMO
Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.
Assuntos
Infecções Bacterianas/imunologia , Cobre/fisiologia , Imunidade Inata , Zinco/fisiologia , Animais , Infecções Bacterianas/microbiologia , Transporte Biológico , Interações Hospedeiro-Patógeno , HumanosRESUMO
BACKGROUND: Zinc plays an important role in human immunity, and it is known that zinc deficiency in the host is linked to increased susceptibility to bacterial infection. In this study, we investigate the role of zinc efflux in the pathogenesis of Streptococcus pyogenes (group A Streptococcus [GAS]), a human pathogen responsible for superficial infections, such as pharyngitis and impetigo, and severe invasive infections. METHODS: The clinically important M1T1 wild-type strain was used in this study, and isogenic mutants were constructed with deletions in the czcD gene (Spy0653; which encodes a putative zinc efflux pump) and adjacent gczA gene (Spy0654; which encodes a putative zinc-dependent activator of czcD). Wild-type, isogenic mutants and complemented strains were tested for resistance against zinc stress, intracellular zinc accumulation, and virulence. RESULTS: Both czcD and gczA mutants exhibited increased sensitivity to zinc. Transcriptional analyses indicate that GczA upregulates czcD in response to zinc. Both mutants displayed increased susceptibility to human neutrophil killing and reduced virulence in a murine infection model. Furthermore, we showed that neutrophils mobilize zinc in response to GAS. CONCLUSIONS: These data indicate that the innate immune system may use zinc as an antimicrobial agent and that zinc efflux is an important contributor to GAS pathogenesis.
Assuntos
Imunidade Inata/fisiologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes , Zinco/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica/imunologia , Humanos , Streptococcus pyogenes/genéticaRESUMO
Lactate is an abundant metabolite, produced by host tissues and commensal organisms, and it represents an important potential carbon source for bacterial pathogens. In the case of Neisseria spp., the importance of the lactate permease in colonization of the host has been demonstrated, but there have been few studies of lactate metabolism in pathogenic Neisseria in the postgenomic era. We describe herein the characterization of genome-annotated, respiratory, and substrate-level lactate dehydrogenases (LDHs) from the obligate human pathogen Neisseria gonorrhoeae. Biochemical assays using N. gonorrhoeae 1291 wild type and isogenic mutant strains showed that cytoplasmic LdhA (NAD(+)-dependent D-lactate dehydrogenase) and the membrane-bound respiratory enzymes, LdhD (D-lactate dehydrogenase) and LldD (L-lactate dehydrogenase) are correctly annotated. Mutants lacking LdhA and LdhD showed greatly reduced survival in neutrophils compared with wild type cells, highlighting the importance of D-lactate metabolism in gonococcal survival. Furthermore, an assay of host colonization using the well-established human primary cervical epithelial cell model revealed that the two respiratory enzymes make a significant contribution to colonization of and survival within the microaerobic environment of the host. Taken together, these data suggest that host-derived lactate is critical for the growth and survival of N. gonorrhoeae in human cells.
Assuntos
Colo do Útero/citologia , Células Epiteliais/microbiologia , Lactato Desidrogenases/metabolismo , Neisseria gonorrhoeae/enzimologia , Neutrófilos/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Lactato Desidrogenases/genética , MutaçãoRESUMO
Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that is carried asymptomatically in the nasopharynx by up to 70% of the human population. Translocation of the bacteria into internal sites can cause a range of diseases, such as pneumonia, otitis media, meningitis, and bacteremia. This transition from nasopharynx to growth at systemic sites means that the pneumococcus needs to adjust to a variety of environmental conditions, including transition metal ion availability. Although it is an important nutrient, iron potentiates oxidative stress, and it is established that in S. pneumoniae, expression of iron transport systems and proteins that protect against oxidative stress are regulated by an orphan response regulator, RitR. In this study, we investigated the effect of iron and manganese ion availability on the growth of a ritR mutant. Deletion of ritR led to impaired growth of bacteria in high-iron medium, but this phenotype could be suppressed with the addition of manganese. Measurement of metal ion accumulation indicated that manganese prevents iron accumulation. Furthermore, the addition of manganese also led to a reduction in the amount of hydrogen peroxide produced by bacterial cells. Studies of virulence in a murine model of infection indicated that RitR was not essential for pneumococcal survival and suggested that derepression of iron uptake systems may enhance the survival of pneumococci in some niches.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Infecções Pneumocócicas/metabolismo , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Transporte de Íons/genética , Camundongos , Mutação , Estresse Oxidativo/genética , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Virulência/genéticaRESUMO
The resurgence of invasive disease caused by Streptococcus pyogenes (group A Streptococcus [GAS]) in the past 30 years has paralleled the emergence and global dissemination of the highly virulent M1T1 clone. The GAS M1T1 clone has diverged from the ancestral M1 serotype by horizontal acquisition of two unique bacteriophages, encoding the potent DNase Sda1/SdaD2 and the superantigen SpeA, respectively. The phage-encoded DNase promotes escape from neutrophil extracellular traps and is linked to enhanced virulence of the M1T1 clone. In this study, we successfully used in vitro lysogenic conversion to transfer the Sda1-encoding phage from the M1T1 clonal strain 5448 to the nonclonal M1 isolate SF370 and determined the impact of this horizontal gene transfer event on virulence. Although Sda1 was expressed in SF370 lysogens, no capacity of the phage-converted strain to survive human neutrophil killing, switch to a hyperinvasive covRS mutant form, or cause invasive lethal infection in a humanized plasminogen mouse model was observed. This work suggests that the hypervirulence of the M1T1 clone is due to the unique synergic effect of the M1T1 clone bacteriophage-specific virulence factor Sda1 acting in concert with the M1T1 clone-specific genetic scaffold.
Assuntos
Desoxirribonuclease I/metabolismo , Fagos de Streptococcus/metabolismo , Streptococcus pyogenes/patogenicidade , Streptococcus pyogenes/virologia , Alelos , Animais , Sequência de Bases , DNA Bacteriano/genética , Desoxirribonuclease I/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fagos de Streptococcus/genética , VirulênciaRESUMO
A scarlet fever outbreak occurred in Hong Kong in 2011. The majority of cases resulted in the isolation of Streptococcus pyogenes emm12 with multiple antibiotic resistances. Phylogenetic analysis of 22 emm12 scarlet fever outbreak isolates, 7 temporally and geographically matched emm12 non-scarlet fever isolates, and 18 emm12 strains isolated during 2005-2010 indicated the outbreak was multiclonal. Genome sequencing of 2 nonclonal scarlet fever isolates (HKU16 and HKU30), coupled with diagnostic polymerase chain reaction assays, identified 2 mobile genetic elements distributed across the major lineages: a 64.9-kb integrative and conjugative element encoding tetracycline and macrolide resistance and a 46.4-kb prophage encoding superantigens SSA and SpeC and the DNase Spd1. Phenotypic comparison of HKU16 and HKU30 with the S. pyogenes M1T1 strain 5448 revealed that HKU16 displays increased adherence to HEp-2 human epithelial cells, whereas HKU16, HKU30, and 5448 exhibit equivalent resistance to neutrophils and virulence in a humanized plasminogen murine model. However, in contrast to M1T1, the virulence of HKU16 and HKU30 was not associated with covRS mutation. The multiclonal nature of the emm12 scarlet fever isolates suggests that factors such as mobile genetic elements, environmental factors, and host immune status may have contributed to the 2011 scarlet fever outbreak.
Assuntos
Surtos de Doenças , Escarlatina/epidemiologia , Escarlatina/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Adolescente , Adulto , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Genômica , Hong Kong/epidemiologia , Humanos , Lactente , Sequências Repetitivas Dispersas , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Fenótipo , Filogenia , Streptococcus pyogenes/efeitos dos fármacosRESUMO
The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]). To establish infection, GAS must survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit. IMPORTANCE During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.
Assuntos
Infecções Estreptocócicas , Streptococcus pyogenes , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Glutationa/metabolismo , Humanos , Evasão da Resposta Imune , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Fatores de Virulência/metabolismoRESUMO
Gram-positive bacteria do not produce lipopolysaccharide as a cell wall component. As such, the polymyxin class of antibiotics, which exert bactericidal activity against Gram-negative pathogens, are ineffective against Gram-positive bacteria. The safe-for-human-use hydroxyquinoline analog ionophore PBT2 has been previously shown to break polymyxin resistance in Gram-negative bacteria, independent of the lipopolysaccharide modification pathways that confer polymyxin resistance. Here, in combination with zinc, PBT2 was shown to break intrinsic polymyxin resistance in Streptococcus pyogenes (Group A Streptococcus; GAS), Staphylococcus aureus (including methicillin-resistant S. aureus), and vancomycin-resistant Enterococcus faecium. Using the globally disseminated M1T1 GAS strain 5448 as a proof of principle model, colistin in the presence of PBT2 + zinc was shown to be bactericidal in activity. Any resistance that did arise imposed a substantial fitness cost. PBT2 + zinc dysregulated GAS metal ion homeostasis, notably decreasing the cellular manganese content. Using a murine model of wound infection, PBT2 in combination with zinc and colistin proved an efficacious treatment against streptococcal skin infection. These findings provide a foundation from which to investigate the utility of PBT2 and next-generation polymyxin antibiotics for the treatment of Gram-positive bacterial infections.
RESUMO
Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli being responsible for >80% of all cases. Asymptomatic bacteriuria (ABU) occurs when bacteria colonize the urinary tract without causing clinical symptoms and can affect both catheterized patients (catheter-associated ABU [CA-ABU]) and noncatheterized patients. Here, we compared the virulence properties of a collection of ABU and CA-ABU nosocomial E. coli isolates in terms of antibiotic resistance, phylogenetic grouping, specific UTI-associated virulence genes, hemagglutination characteristics, and biofilm formation. CA-ABU isolates were similar to ABU isolates with regard to the majority of these characteristics; exceptions were that CA-ABU isolates had a higher prevalence of the polysaccharide capsule marker genes kpsMT II and kpsMT K1, while more ABU strains were capable of mannose-resistant hemagglutination. To examine biofilm growth in detail, we performed a global gene expression analysis with two CA-ABU strains that formed a strong biofilm and that possessed a limited adhesin repertoire. The gene expression profile of the CA-ABU strains during biofilm growth showed considerable overlap with that previously described for the prototype ABU E. coli strain, 83972. This is the first global gene expression analysis of E. coli CA-ABU strains. Overall, our data suggest that nosocomial ABU and CA-ABU E. coli isolates possess similar virulence profiles.
Assuntos
Bacteriúria/microbiologia , Escherichia coli , Cateterismo Urinário , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biofilmes , Cateteres de Demora/microbiologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/patogenicidade , Feminino , Perfilação da Expressão Gênica , Genes Bacterianos , Humanos , Ferro/metabolismo , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Fatores de Virulência/genética , Resistência beta-Lactâmica/genéticaRESUMO
BACKGROUND: Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. RESULTS: Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. CONCLUSIONS: The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.
Assuntos
Citrobacter freundii/metabolismo , Citrobacter koseri/metabolismo , Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Klebsiella oxytoca/metabolismo , Klebsiella pneumoniae/metabolismo , Citrobacter freundii/genética , Citrobacter koseri/genética , Escherichia coli/genética , Proteínas de Fímbrias/classificação , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Klebsiella oxytoca/genética , Klebsiella pneumoniae/genética , FilogeniaRESUMO
Copper (Cu) is an essential metal for bacterial physiology but in excess it is bacteriotoxic. To limit Cu levels in the cytoplasm, most bacteria possess a transcriptionally responsive system for Cu export. In the Gram-positive human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]), this system is encoded by the copYAZ operon. This study demonstrates that although the site of GAS infection represents a Cu-rich environment, inactivation of the copA Cu efflux gene does not reduce virulence in a mouse model of invasive disease. In vitro, Cu treatment leads to multiple observable phenotypes, including defects in growth and viability, decreased fermentation, inhibition of glyceraldehyde-3-phosphate dehydrogenase (GapA) activity, and misregulation of metal homeostasis, likely as a consequence of mismetalation of noncognate metal-binding sites by Cu. Surprisingly, the onset of these effects is delayed by â¼4 h even though expression of copZ is upregulated immediately upon exposure to Cu. Further biochemical investigations show that the onset of all phenotypes coincides with depletion of intracellular glutathione (GSH). Supplementation with extracellular GSH replenishes the intracellular pool of this thiol and suppresses all the observable effects of Cu treatment. These results indicate that GSH buffers excess intracellular Cu when the transcriptionally responsive Cu export system is overwhelmed. Thus, while the copYAZ operon is responsible for Cu homeostasis, GSH has a role in Cu tolerance and allows bacteria to maintain metabolism even in the presence of an excess of this metal ion.IMPORTANCE The control of intracellular metal availability is fundamental to bacterial physiology. In the case of copper (Cu), it has been established that rising intracellular Cu levels eventually fill the metal-sensing site of the endogenous Cu-sensing transcriptional regulator, which in turn induces transcription of a copper export pump. This response caps intracellular Cu availability below a well-defined threshold and prevents Cu toxicity. Glutathione, abundant in many bacteria, is known to bind Cu and has long been assumed to contribute to bacterial Cu handling. However, there is some ambiguity since neither its biosynthesis nor uptake is Cu-regulated. Furthermore, there is little experimental support for this physiological role of glutathione beyond measuring growth of glutathione-deficient mutants in the presence of Cu. Our work with group A Streptococcus provides new evidence that glutathione increases the threshold of intracellular Cu availability that can be tolerated by bacteria and thus advances fundamental understanding of bacterial Cu handling.
Assuntos
Cobre/metabolismo , Glutationa/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico , Cobre/farmacologia , Citoplasma/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Homeostase , Camundongos , Mutação , Streptococcus pyogenes/efeitos dos fármacos , Estresse Fisiológico , VirulênciaRESUMO
In asymptomatic bacteriuria (ABU), bacteria colonize the urinary tract without provoking symptoms. Here, we compared the virulence properties of a collection of ABU Escherichia coli strains to cystitis and pyelonephritis strains. Specific urinary tract infection (UTI)-associated virulence genes, hemagglutination characteristics, siderophore production, hemolysis, biofilm formation, and the ability of strains to adhere to and induce cytokine responses in epithelial cells were analyzed. ABU strains were phylogenetically related to strains that cause symptomatic UTI. However, the virulence properties of the ABU strains were variable and dependent on a combination of genotypic and phenotypic factors. Most ABU strains adhered poorly to epithelial cells; however, we also identified a subgroup of strongly adherent strains that were unable to stimulate an epithelial cell IL-6 cytokine response. Poor immune activation may represent one mechanism whereby ABU E. coli evade immune detection after the establishment of bacteriuria.
Assuntos
Bacteriúria/microbiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/patogenicidade , Infecções Urinárias/microbiologia , Fatores de Virulência/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aderência Bacteriana , Biofilmes/crescimento & desenvolvimento , Cistite/microbiologia , Citocinas/metabolismo , Células Epiteliais/microbiologia , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Feminino , Hemaglutinação , Hemólise , Humanos , Masculino , Pessoa de Meia-Idade , Pielonefrite/microbiologia , Sideróforos/biossíntese , Fatores de Virulência/genéticaRESUMO
A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.