RESUMO
Type 17 cytokines have been strongly implicated in mucosal immunity, in part by regulating the production of antimicrobial peptides. Using a mouse model of Citrobacter rodentium infection, which causes colitis, we found that intestinal IL-17RA and IL-17RC were partially required for control of infection in the colon and IL-17 regulates the production of luminal hydrogen peroxide as well as expression of Tnsf13 Reduced Tnfsf13 expression was associated with a profound defect in generating C. rodentium-specific IgA+ Ab-secreting cells. Taken together, intestinal IL-17R signaling plays key roles in controlling invading pathogens, in part by regulating luminal hydrogen peroxide as well as regulating the generation of pathogen-specific IgA+ Ab-secreting cells.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Imunoglobulina A Secretora/imunologia , Mucosa Intestinal/imunologia , Oxirredutases/imunologia , Receptores de Interleucina-17/imunologia , Transdução de Sinais/imunologia , Animais , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/genética , Humanos , Peróxido de Hidrogênio/imunologia , Imunoglobulina A Secretora/genética , Camundongos , Camundongos Knockout , Oxirredutases/genética , Receptores de Interleucina-17/genética , Transdução de Sinais/genéticaRESUMO
Enterotoxigenic Escherichia coli (ETEC) remain a major cause of diarrheal mortality and morbidity in children in low-resource settings. Few studies have explored the consequences of simultaneous intoxication with heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) despite the increased prevalence of wild ETEC isolates expressing both toxins. We therefore used a combination of tissue culture and murine models to explore the impact of simultaneous ST + LT intoxication on epithelial and myeloid cells. We report that LT induces sustained production of interleukin 33 (IL-33) and interleukin 1 receptor antagonist (IL-1Ra) in T84 intestinal epithelial cells via cAMP production and protein kinase A activation. We demonstrate that combined ST + LT intoxication hastens epithelial transcriptional responses induced more slowly by LT alone. ST- and LT-mediated luminal fluid accumulation in vivo correlates with significant increases in IL-33 and IL-1Ra in small intestinal mucosal scrapings. Additionally, IL-33 receptor (IL-33R)-deficient mice are significantly less susceptible to ST-mediated secretion than wildtype mice. In the immune compartment, IL-33 is sensed by myeloid cells, and LT suppresses IL-33-induced tumor necrosis factor α (TNF-α) secretion from macrophages and bone marrow-derived dendritic cells (BMDCs) but amplifies IL-33-mediated induction of IL-6 from BMDCs. In conclusion, our studies suggest that enterotoxin-induced IL-33 and IL-1Ra modulate intestinal inflammation and IL-1 receptor signaling in the intestinal mucosa in response to ETEC enterotoxins.
Assuntos
Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Animais , Toxinas Bacterianas/metabolismo , Linhagem Celular , Citocinas/metabolismo , Enterotoxinas , Proteínas de Escherichia coli/metabolismo , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-33 , CamundongosRESUMO
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.
Assuntos
GMP Cíclico/biossíntese , Escherichia coli Enterotoxigênica/fisiologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/etiologia , Infecções por Escherichia coli/metabolismo , Interleucina-33/biossíntese , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Animais , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade nas Mucosas , Imunização , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , CamundongosRESUMO
BACKGROUND: Biofilms are microbial communities surrounded by a self-produced extracellular matrix which protects them from environmental stress. Bacteria within biofilms are 10- to 1000-fold more resistant to antibiotics, making it challenging but imperative to develop new therapeutics that can disperse biofilms and eradicate infection. Gram-negative bacteria produce outer membrane vesicles (OMV) that play critical roles in communication, genetic exchange, cargo delivery, and pathogenesis. We have previously shown that OMVs derived from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. RESULTS: Here, we examine the antibiofilm activity of Burkholderia thailandensis OMVs against the oral biofilm-forming pathogen Streptococcus mutans. We demonstrate that OMV treatment reduces biofilm biomass, biofilm integrity, and bacterial cell viability. Both heat-labile and heat-stable components, including 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline and long-chain rhamnolipid, contribute to the antibiofilm activity of OMVs. When OMVs are co-administered with gentamicin, the efficacy of the antibiotic against S. mutans biofilms is enhanced. CONCLUSION: These studies indicate that bacterial-derived OMVs are highly effective biological nanoparticles that can inhibit and potentially eradicate biofilms.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vesículas Extracelulares/química , Streptococcus mutans/fisiologia , Membrana Externa Bacteriana/química , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/patogenicidadeRESUMO
Enterotoxigenic Escherichia coli (ETEC) is a major cause of infectious diarrhea in children, travelers, and deployed military personnel. As such, development of a vaccine would be advantageous for public health. One strategy is to use subunits of colonization factors combined with antigen/adjuvant toxoids as an ETEC vaccine. Here, we investigated the intradermal (i.d.) or sublingual (s.l.) delivery of CFA/I fimbrial antigens, including CfaEB and a CfaE-heat-labile toxin B subunit (LTB) chimera admixed with double mutant heat-labile toxin (LT) LT-R192G/L211A (dmLT). In addition, we compared dmLT with other LT proteins to better understand the generation of adjuvanted fimbrial and toxoid immunity as well as the influence on any local skin reactogenicity. We demonstrate that immunization with dmLT admixed with CfaEB induces robust serum and fecal antibody responses to CFA/I fimbriae and LT but that i.d. formulations are not optimal for s.l. delivery. Improved s.l. vaccination outcomes were observed when higher doses of dmLT (1 to 5 µg) were admixed with CfaEB or, even better, when a CfaE-LTB chimera antigen was used instead. Serum anti-CFA/I total antibodies, detected by enzyme-linked immunosorbent assay, were the best predictor of functional antibodies, based on the inhibition of red blood cell agglutination by ETEC. Immunization with other LT proteins or formulations with altered B-subunit binding during i.d. immunization (e.g., by addition of 5% lactose, LTA1, or LT-G33D) minimally altered the development of antibody responses and cytokine recall responses but reduced skin reactogenicity at the injection site. These results reveal how formulations and delivery parameters shape the adaptive immune responses to a toxoid and fimbria-derived subunit vaccine against ETEC.
Assuntos
Anticorpos Antibacterianos/sangue , Escherichia coli Enterotoxigênica/imunologia , Enterotoxinas/imunologia , Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/química , Antígenos de Bactérias , Toxinas Bacterianas/imunologia , Fezes/química , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologiaAssuntos
Fibrose Cística , Humanos , Fibrose Cística/genética , Interleucina-33 , Sistema Respiratório , InflamaçãoRESUMO
Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation (P < 0.01). Double and triple mutants with deficiency in brpA and/or psr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically (P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans, indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Biofilmes , Parede Celular/metabolismo , Streptococcus mutans/enzimologia , Streptococcus mutans/fisiologia , Fatores de Transcrição/metabolismo , Transferases/metabolismo , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Divisão Celular , Parede Celular/genética , Meios de Cultura/química , Meios de Cultura/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptococcus mutans/citologia , Streptococcus mutans/genética , Fatores de Transcrição/genética , Transferases/genéticaRESUMO
Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.
Assuntos
Biofilmes/crescimento & desenvolvimento , Membrana Celular/fisiologia , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Biossíntese de Proteínas/fisiologia , Streptococcus mutans/metabolismo , DNA Bacteriano/genética , Streptococcus mutans/genética , Streptococcus mutans/fisiologia , Streptococcus mutans/ultraestrutura , Regulação para CimaRESUMO
Streptococcus mutans, the primary aetiological agent of dental caries, possesses an YjeE-like protein that is encoded by locus SMU.409, herein designated brpB. In this study, a BrpB-deficient mutant, JB409, and a double mutant deficient of BrpB and BrpA (a paralogue of the LytR-CpsA-Psr family of cell wall-associated proteins), JB819, were constructed and characterized using function assays and microscopy analysis. Both JB409 and JB819 displayed extended lag phases and drastically slowed growth rates during growth in brain heart infusion medium as compared to the wild-type, UA159. Relative to UA159, JB409 and JB819 were more than 60- and 10-fold more susceptible to acid killing at pH 2.8, and more than 1 and 2 logs more susceptible to hydrogen peroxide, respectively. Complementation of the deficient mutants with a wild-type copy of the respective gene(s) partly restored the acid and oxidative stress responses to a level similar to the wild-type. As compared to UA159, biofilm formation by JB409 and JB819 was drastically reduced (P<0.001), especially during growth in medium containing sucrose. Under a scanning electron microscope, JB409 had significantly more giant cells with an elongated, rod-like morphology, and JB819 formed marble-like super cells with apparent defects in cell division. As revealed by transmission electron microscopy analysis, BrpB deficiency in both JB409 and JB819 resulted in the development of low electron density patches and formation of a loose nucleoid structure. Taken together, these results suggest that BrpB likely functions together with BrpA in regulating cell envelope biogenesis/homeostasis in Strep. mutans. Further studies are under way to elucidate the mechanism that underlies the BrpA- and BrpB-mediated regulation.
Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Divisão Celular , Regulação Bacteriana da Expressão Gênica , Streptococcus mutans/fisiologia , Streptococcus mutans/ultraestrutura , Estresse Fisiológico , Ácidos/toxicidade , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Técnicas de Inativação de Genes , Teste de Complementação Genética , Peróxido de Hidrogênio/toxicidade , Concentração de Íons de Hidrogênio , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/genéticaRESUMO
Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/homeostasis and affects stress responses and biofilm formation by Strep. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically compared with the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. Real-time-PCR and Western blot analyses revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by Strep. mutans. Transmission electron microscopy analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in Strep. mutans.
Assuntos
Proteínas de Bactérias/metabolismo , Glucanos/metabolismo , Proteínas Repressoras/metabolismo , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Ácidos/toxicidade , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Western Blotting , Parede Celular/ultraestrutura , Deleção de Genes , Perfilação da Expressão Gênica , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/fisiologiaRESUMO
Enterotoxigenic E. coli (ETEC) are endemic in low-resource settings and cause robust secretory diarrheal disease in children less than five years of age. ETEC cause secretory diarrhea by producing the heat-stable (ST) and/or heat-labile (LT) enterotoxins. Recent studies have shown that ETEC can be carried asymptomatically in children and adults, but how ETEC subvert mucosal immunity to establish intestinal residency remains unclear. Macrophages are innate immune cells that can be exploited by enteric pathogens to evade mucosal immunity, so we interrogated the ability of ETEC and other E. coli pathovars to survive within macrophages. Using gentamicin protection assays, we show that ETEC H10407 is phagocytosed more readily than other ETEC and non-ETEC isolates. Furthermore, we demonstrate that ETEC H10407, at high bacterial burdens, causes nitrite accumulation in macrophages, which is indicative of a proinflammatory macrophage nitric oxide killing response. However, at low bacterial burdens, ETEC H10407 remains viable within macrophages for an extended period without nitrite accumulation. We demonstrate that LT, but not ST, intoxication decreases the number of ETEC phagocytosed by macrophages. Furthermore, we now show that macrophages exposed simultaneously to LPS and LT produce IL-33, which is a cytokine implicated in promoting macrophage alternative activation, iron recycling, and intestinal repair. Lastly, iron restriction using deferoxamine induces IL-33 receptor (IL-33R) expression and allows ETEC to escape macrophages. Altogether, these data demonstrate that LT provides ETEC with the ability to decrease the perceived ETEC burden and suppresses the initiation of inflammation. Furthermore, these data suggest that host IL-33/IL-33R signaling may augment pathways that promote iron restriction to facilitate ETEC escape from macrophages. These data could help explain novel mechanisms of immune subversion that may contribute to asymptomatic ETEC carriage.
RESUMO
Enterotoxigenic Escherichia coli (ETEC) are a significant cause of childhood diarrhea in low-resource settings. ETEC are defined by the production of heat-stable enterotoxin (ST) and/or heat-labile enterotoxin (LT), which alter intracellular cyclic nucleotide signaling and cause the secretion of water and electrolytes into the intestinal lumen. ETEC take cues from chemicals (e.g., glycans, bile salts, and solutes) that may be liberated following enterotoxin activity to recognize entrance into the host. ETEC then alter the expression of surface adhesins called colonization factors (CFs) to attach to the intestinal epithelium, proliferate, and cause disease. Here, we used an in vivo model of oral ST intoxication to determine its impact on luminal ion concentrations via ICP-MS. We also used functional assays, including Western blots, qPCR, and toxin activity assays, to assess the impact of luminal ion flux on CF and toxin expression. Finally, we assessed ETEC strains with CFs CFA/I or CS6 in a streptomycin mouse model of ETEC colonization. ST causes rapid and significant increases in luminal chloride but significant decreases in luminal magnesium and iron. We confirmed that increased sodium chloride suppresses CFA/I production in ETEC H10407 but does not affect CS6 production in ETEC 214-4. CFA/I production in ETEC H10407 is increased when magnesium becomes limiting, although it does not affect CS6 production in ETEC 214-4. Iron restriction via deferoxamine induces CFA/I expression in ETEC H10407 but not CS6 expression in ETEC 214-4. We demonstrate that ST production is suppressed via iron restriction in H10407, 214-4, and over 50 other ETEC clinical isolates. Lastly, we demonstrate that the iron restriction of mice using oral deferoxamine pre-treatment extends the duration of ETEC H10407 (CFA/I+) fecal shedding while accelerating ETEC 214-4 (CS6+) fecal shedding. Combined, these data suggest that enterotoxins modulate luminal ion flux to influence ETEC virulence including toxin and CF production.
Assuntos
Toxinas Bacterianas , Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Animais , Camundongos , Enterotoxinas , Escherichia coli Enterotoxigênica/metabolismo , Toxinas Bacterianas/metabolismo , Virulência , Ferro/metabolismo , Desferroxamina/metabolismo , Temperatura Alta , Magnésio/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismoRESUMO
A human homologue of the iron-sulfur cluster assembly protein IscA (hIscA1) has been cloned and expressed in Escherichia coli cells. The UV-visible absorption and EPR (electron paramagnetic resonance) measurements reveal that hIscA1 purified from E. coli cells contains a mononuclear iron centre and that the iron binding in hIscA1 expressed in E. coli cells can be further modulated by the iron content in the cell growth medium. Additional studies show that purified hIscA1 binds iron with an iron association constant of approx. 2x1019 M-1, and that the iron-bound hIscA1 is able to provide the iron for the iron-sulfur cluster assembly in a proposed scaffold protein, IscU of E. coli, in vitro. The complementation experiments indicate that hIscA1 can partially substitute for IscA in restoring the cell growth of E. coli in the M9 minimal medium under aerobic conditions. The results suggest that hIscA1, like E. coli IscA, is an iron-binding protein that may act as an iron chaperone for biogenesis of iron-sulfur clusters.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Humanos , Proteínas Ferro-Enxofre/química , CinéticaRESUMO
Endogenous peptides and structurally similar bacterial heat-stable enterotoxins (ST) bind guanylate cyclase-C (GC-C), resulting in fluid homeostasis or diarrhea, respectively. In this issue of Cell Host & Microbe, Carey et al., show how bats have evolutionarily maintained homeostatic signaling while avoiding pathogenic effects of ST.
Assuntos
Toxinas Bacterianas/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/metabolismo , Guanilato Ciclase/metabolismo , Animais , Quirópteros , GMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/microbiologia , Diarreia/patologia , Enterócitos/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Guanilato Ciclase/genética , Ligação Proteica , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Streptococcus mutans appears to possess a sole iron-sulfur (Fe-S) cluster biosynthesis system encoded by the sufCDSUB cluster. This study was designed to examine the role of sufCDSUB in S. mutans physiology. Allelic exchange mutants deficient of the whole sufCDSUB cluster and in individual genes were constructed. Compared to the wild-type, UA159, the sufCDSUB-deficient mutant, Δsuf::kan r , had a significantly reduced growth rate, especially in medium with the absence of isoleucine, leucine or glutamate/glutamine, amino acids that require Fe-S clusters for biosynthesis and when grown with medium adjusted to pH 6.0 and under oxidative and nitrosative stress conditions. Relative to UA159, Δsuf::kan r had major defects in stress tolerance responses with reduced survival rate of > 2-logs following incubation at low pH environment or after hydrogen peroxide challenge. When compared to UA159, Δsuf::kan r tended to form aggregates in broth medium and accumulated significantly less biofilm. As shown by luciferase reporter fusion assays, the expression of sufCDSUB was elevated by > 5.4-fold when the reporter strain was transferred from iron sufficient medium to iron-limiting medium. Oxidative stress induced by methyl viologen increased sufCDSUB expression by > 2-fold, and incubation in a low pH environment led to reduction of sufCDSUB expression by > 7-fold. These results suggest that lacking of SufCDSUB in S. mutans causes major defects in various cellular processes of the deficient mutant, including growth, stress tolerance responses and biofilm formation. In addition, the viability of the deficient mutant also suggests that SUF, the sole Fe-S cluster machinery identified is non-essential in S. mutans, which is not known in any other bacterium lacking the NIF and/or ISC system. However, how the bacterium compensates the Fe-S deficiency and if any novel Fe-S assembly systems exist in this bacterium await further investigation.
RESUMO
Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus that causes the disease melioidosis. B. pseudomallei expresses a number of proteins that contribute to its intracellular survival in the mammalian host. We previously demonstrated that immunization with OMVs derived from B. pseudomallei grown in nutrient-rich media protects mice against lethal disease. Here, we evaluated if OMVs derived from B. pseudomallei grown under macrophage-mimicking growth conditions could be enriched with intracellular-stage proteins in order to improve the vaccine. We show that OMVs produced in this manner (M9 OMVs) contain proteins associated with intracellular survival yet are non-toxic to living cells. Immunization of mice provides significant protection against pulmonary infection similar to that achieved with a live attenuated vaccine and is associated with increased IgG, CD4+, and CD8+ T cells. OMVs possess inherent adjuvanticity and drive DC activation and maturation. These results indicate that M9 OMVs constitute a new promising vaccine against melioidosis.
RESUMO
IscA/SufA paralogues are the members of the iron-sulfur cluster assembly machinery in Escherichia coli. Whereas deletion of either IscA or SufA has only a mild effect on cell growth, deletion of both IscA and SufA results in a null-growth phenotype in minimal medium under aerobic growth conditions. Here we report that cell growth of the iscA/sufA double mutant (E. coli strain in which both iscA and sufA had been in-frame-deleted) can be partially restored by supplementing with BCAAs (branched-chain amino acids) and thiamin. We further demonstrate that deletion of IscA/SufA paralogues blocks the [4Fe-4S] cluster assembly in IlvD (dihydroxyacid dehydratase) of the BCAA biosynthetic pathway in E. coli cells under aerobic conditions and that addition of the iron-bound IscA/SufA efficiently promotes the [4Fe-4S] cluster assembly in IlvD and restores the enzyme activity in vitro, suggesting that IscA/SufA may act as an iron donor for the [4Fe-4S] cluster assembly under aerobic conditions. Additional studies reveal that IscA/SufA are also required for the [4Fe-4S] cluster assembly in enzyme ThiC of the thiamin-biosynthetic pathway, aconitase B of the citrate acid cycle and endonuclease III of the DNA-base-excision-repair pathway in E. coli under aerobic conditions. Nevertheless, deletion of IscA/SufA does not significantly affect the [2Fe-2S] cluster assembly in the redox transcription factor SoxR, ferredoxin and the siderophore-iron reductase FhuF. The results suggest that the biogenesis of the [4Fe-4S] clusters and the [2Fe-2S] clusters may have distinct pathways and that IscA/SufA paralogues are essential for the [4Fe-4S] cluster assembly, but are dispensable for the [2Fe-2S] cluster assembly in E. coli under aerobic conditions.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Hidroliases/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Aerobiose , Aminoácidos de Cadeia Ramificada/biossíntese , Aminoácidos de Cadeia Ramificada/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Divisão Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Hidroliases/genética , Ferro/metabolismo , Proteínas Ferro-Enxofre/classificação , Proteínas Ferro-Enxofre/genética , Mutação , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies have identified heat-stable enterotoxin (ST)-producing ETEC as one of the major diarrhea-causing pathogens in children younger than five years. In this study, we examined iron and zinc binding by both human and porcine ST variants and determined how host metallothionein could detoxify ST. We found that ST purified from ETEC culture supernatants eluted as a doublet during C18 reverse-phase chromatography. Leading edge fractions of the ST doublet were found to be devoid of iron, while trailing edge fractions of the ST doublet were found to contain measurable iron. Next, we found that purified ST could be reconstituted with iron under reducing and anaerobic conditions, and iron-bound ST attenuated the induction of cGMP in T84 epithelial cells. Moreover, we demonstrated that supernatants of ETEC 214-4 grown under increasing iron concentrations were only able to induce cGMP at iron concentrations greater than 5 µM. In vitro studies also demonstrated that ST binds zinc, and once bound, zinc removal from ST required denaturing conditions. Zinc-bound ST also failed to induce cGMP. We found that ST contributes disulfide bonds to the perceived oxidized glutathione pool, increases the rate of zinc release from metallothionein, and can be detoxified by metallothionein. Lastly, we showed ST induces transcriptional changes in genes previously shown to be regulated by deferoxamine. These studies demonstrate ST ETEC pathogenesis may be tied intimately to host mucosal metal status.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries, deployed military personnel, and travelers to regions of endemicity. The heat-stable toxin (ST) is a small nonimmunogenic secreted peptide with 3 disulfide bonds. It has been appreciated that dietary disulfides modulate intestinal redox potential and that ST could be detoxified using exogenous reductants. Using biochemical and spectroscopic approaches, we demonstrated that ST can separately bind iron and zinc under reducing conditions, thereby reducing ST toxicity. Moreover, we demonstrated that ST modulates the glutathione (GSH)/oxidized glutathione (GSSG) ratio and that ST should be considered a toxin oxidant. ST can be detoxified by oxidizing zinc-loaded metallothionine, causing free zinc to be released. These studies help lay a foundation to understand how diarrheal pathogens modulate intestinal redox potential and may impact how we design therapeutics and/or vaccines for the pathogens that produce them.
Assuntos
Toxinas Bacterianas/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferro/metabolismo , Metalotioneína/metabolismo , Zinco/metabolismo , Animais , Linhagem Celular , Células Epiteliais/microbiologia , Infecções por Escherichia coli/microbiologia , Glutationa/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Peptídeos/metabolismo , Ligação Proteica , SuínosRESUMO
Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.
Assuntos
Antibacterianos/metabolismo , Antibiose/fisiologia , Burkholderia/metabolismo , Vesículas Extracelulares/metabolismo , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Antibacterianos/farmacologia , Membrana Externa Bacteriana/metabolismo , Biofilmes/crescimento & desenvolvimento , Glicolipídeos/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Quinolinas/metabolismoRESUMO
Enterotoxigenic Escherichia coli (ETEC) is a significant cause of childhood diarrhea and is a leading cause of traveler's diarrhea. ETEC strains encoding the heat-stable enterotoxin (ST) are more often associated with childhood diarrhea than ETEC strains that encode only the heat-labile enterotoxin (LT). Colonization factors (CFs) also have a demonstrated role in ETEC virulence, and two of the most prevalent CFs among ETEC that have caused diarrhea are colonization factor antigen I (CFA/I) and CS6. In the current report, we describe the genomes of 269 CS6- or CFA/I-encoding ST-only ETEC isolates that were associated with human diarrhea. While the CS6 and CFA/I ETEC were identified in at least 13 different ETEC genomic lineages, a majority (85%; 229/269) were identified in only six lineages. Complete genome sequencing of selected isolates demonstrated that a conserved plasmid contributed to the dissemination of CFA/I whereas at least five distinct plasmids were involved in the dissemination of ST and/or CS6. Additionally, there were differences in gene content between CFA/I and CS6 ETEC at the phylogroup and lineage levels and in association with their geographic location of isolation as well as lineage-related differences in ST production. Thus, we demonstrate that genomically diverse E. coli strains have acquired ST, as well as CFA/I or CS6, via one or more plasmids and that, in some cases, isolates of a particular lineage or geographic location have undergone additional modifications to their genome content. These findings will aid investigations of virulence and the development of improved diagnostics and vaccines against this important human diarrheal pathogen. IMPORTANCE Comparative genomics and functional characterization were used to analyze a global collection of CFA/I and CS6 ST-only ETEC isolates associated with human diarrhea, demonstrating differences in the genomic content of CFA/I and CS6 isolates related to CF type, lineage, and geographic location of isolation and also lineage-related differences in ST production. Complete genome sequencing of selected CFA/I and CS6 isolates enabled descriptions of a highly conserved ST-positive (ST+) CFA/I plasmid and of at least five diverse ST and/or CS6 plasmids among the CS6 ETEC isolates. There is currently no approved vaccine for ST-only ETEC, or for any ETEC for that matter, and as such, the current report provides functional verification of ST and CF production and antimicrobial susceptibility testing and an in-depth genomic characterization of a collection of isolates that could serve as representatives of CFA/I- or CS6-encoding ST-only ETEC strains for future studies of ETEC pathogenesis, vaccine studies, and/or clinical trials.