RESUMEN
Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.
Asunto(s)
Aurodox , Sistemas de Secreción Tipo III , Sistemas de Secreción Tipo III/metabolismo , Aurodox/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
The first report of transmissible carbapenem resistance encoded by blaIMP-1 was discovered in Pseudomonas aeruginosa GN17203 in 1988, and blaIMP-1 has since been detected in other bacteria, including Enterobacterales. Currently, many variants of blaIMPs exist, and point mutations in the blaIMP promoter have been shown to alter promoter strength. For example, the promoter (Pc) of blaIMP-1, first reported in P. aeruginosa GN17203, was a weak promoter (PcW) with low-level expression intensity. This study investigates whether point mutations in the promoter region have helped to create strong promoters under antimicrobial selection pressure. Using bioinformatic approaches, we retrieved 115 blaIMPs from 14,529 genome data of Pseudomonadota and performed multiple alignment analyses. The results of promoter analysis of the 115 retrieved blaIMPs showed that most of them used the Pc located in class 1 integrons (n = 112, 97.4%). The promoter analysis by year revealed that the blaIMP population with the strong promoter, PcS, was transient. In contrast, the PcW-TG population, which had acquired a TGn-extended -10 motif in PcW and had an intermediate promoter strength, gradually spread throughout the world. An inverse correlation between Pc promoter strength and Intl1 integrase excision efficiency has been reported previously [1]. Because of this trade-off, it is unlikely that blaIMPs with strong promoters will increase rapidly, but the possibility that promoter strength will increase with the use of other integrons cannot be ruled out. Monitoring of the blaIMP genes, including promoter analysis, is necessary for global surveillance of carbapenem-resistant bacteria.
Asunto(s)
Regiones Promotoras Genéticas , Pseudomonas aeruginosa , beta-Lactamasas , beta-Lactamasas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Integrones/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mutación PuntualRESUMEN
The blaNDM-1 gene encodes a carbapenemase, New Delhi metallo-ß-lactamase (NDM-1), and the ability to produce NDM-1 is spread among Enterobacteriaceae via horizontal gene transfer of plasmids. It has been widely accepted that blaNDM-1 is regulated by a hybrid promoter (PISAba125) consisting of a -10 box from the original blaNDM-1 and a -35 box from ISAba125. However, the conservation of this promoter and the vertical transmission of blaNDM genes by chromosomal integration have not been comprehensively analyzed. We retrieved the region containing the ORF of blaNDM-1 (>95% translated protein identity) and a region 120 bp upstream of the blaNDM-1 start codon from the complete sequence data of Enterobacteriaceae plasmids (n = 10,914) and chromosomes (n = 4908) deposited in GenBank, and the 310 extracted blaNDM genes were analyzed by an in-silico approach. The results showed that most blaNDM genes (99.0%) utilized the promoter, PISAba125. Interestingly, two blaNDM-1 genes from the genus Citrobacter utilized the ISCR1-derived outward-oriented promoters POUT (PISCR1). Furthermore, the insertion of ISAba125 and ISCR1 occurred upstream of the CCATATTT sequence, which is located upstream of the -10 box. We also confirmed that most of the blaNDM genes were disseminated by horizontal gene transfer of the plasmid, but 10 cases of the blaNDM genes were integrated into the chromosome via mobile genetic elements such as IS26, IS150, ISCR1, ICE, and Tn7-like elements. Thus, plasmid-mediated transmission of the PISAba125-blaNDM genes is predominant in Enterobacteriaceae. However, the spread of blaNDM genes with new promoters and vertical dissemination via chromosomal integrations may pose additional serious clinical problems.
Asunto(s)
Enterobacteriaceae , beta-Lactamasas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Enterobacteriaceae/genética , Transferencia de Gen Horizontal , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Bordetella bronchiseptica injects virulence proteins called effectors into host cells via a type III secretion system (T3SS) conserved among many Gram-negative bacteria. Small proteins called chaperones are required to stabilize some T3SS components or localize them to the T3SS machinery. In a previous study, we identified a chaperone-like protein named Bcr4 that regulates T3SS activity in B. bronchiseptica. Bcr4 does not show strong sequence similarity to well-studied T3SS proteins of other bacteria, and its function remains to be elucidated. Here, we investigated the mechanism by which Bcr4 controls T3SS activity. A pulldown assay revealed that Bcr4 interacts with BscI, based on its homology to other bacterial proteins, to be an inner rod protein of the T3SS machinery. An additional pulldown assay using truncated Bcr4 derivatives and secretion profiles of B. bronchiseptica producing truncated Bcr4 derivatives showed that the Bcr4 C-terminal region is necessary for the interaction with BscI and activation of the T3SS. Moreover, the deletion of BscI abolished the secretion of type III secreted proteins from B. bronchiseptica and the translocation of a cytotoxic effector into cultured mammalian cells. Finally, we show that BscI is unstable in the absence of Bcr4. These results suggest that Bcr4 supports the construction of the T3SS machinery by stabilizing BscI. This is the first demonstration of a chaperone for the T3SS inner rod protein among the virulence bacteria possessing the T3SS. IMPORTANCE The type III secretion system (T3SS) is a needle-like complex that projects outward from bacterial cells. Bordetella bronchiseptica uses the T3SS to inject virulence proteins into host cells. Our previous study reported that a protein named Bcr4 is essential for the secretion of virulence proteins from B. bronchiseptica bacterial cells and delivery through the T3SS. Because other bacteria lack a Bcr4 homologue, the function of Bcr4 has not been elucidated. In this study, we discovered that Bcr4 interacts with BscI, a component of the T3SS machinery. We show that a B. bronchiseptica BscI-deficient strain was unable to secrete type III secreted proteins. Furthermore, in a B. bronchiseptica strain that overproduces T3SS component proteins, Bcr4 is required to maintain BscI in bacterial cells. These results suggest that Bcr4 stabilizes BscI to allow construction of the T3SS in B. bronchiseptica.
Asunto(s)
Bordetella bronchiseptica , Bordetella , Animales , Sistemas de Secreción Tipo III/metabolismo , Bordetella/metabolismo , Bordetella bronchiseptica/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Mamíferos/metabolismoRESUMEN
Aurodox was originally isolated in 1972 as a linear polyketide compound exhibiting antibacterial activity against Gram-positive bacteria. We have since identified aurodox as a specific inhibitor of the bacterial type III secretion system (T3SS) using our original screening system for inhibition of T3SS-mediated hemolysis in enteropathogenic Escherichia coli (EPEC). In this research, we synthesized 15 derivatives of aurodox and evaluated EPEC T3SS inhibitory activity as well as antibacterial activity against EPEC. One of the derivatives was highly selective for T3SS inhibition, equivalent to that of aurodox, but without exhibiting antibacterial activity (69-fold selectivity). This work revealed the structure-activity relationship for the inhibition of T3SS by aurodox and suggests that the target of T3SS is distinct from the target for antibacterial activity.
Asunto(s)
Aurodox , Escherichia coli Enteropatógena , Proteínas de Escherichia coli , Antibacterianos/farmacología , Aurodox/farmacología , Relación Estructura-Actividad , Sistemas de Secreción Tipo IIIRESUMEN
Bordetella pertussis uses a type III secretion system (T3SS) to inject virulence proteins into host cells. Although the B. pertussis T3SS was presumed to be involved in host colonization, efficient secretion of type III secreted proteins from B. pertussis has not been observed. To investigate the roles of type III secreted proteins during infection, we attempted to optimize culture conditions for the production and secretion of a type III secreted protein, BteA, in B. pertussis We observed that B. pertussis efficiently secretes BteA in ascorbic acid-depleted (AsA-) medium. When L2 cells, a rat lung epithelial cell line, were infected with B. pertussis cultured in the AsA- medium, BteA-dependent cytotoxicity was observed. We also performed an immunofluorescence assay of L2 cells infected with B. pertussis Clear fluorescence signals of Bsp22, a needle structure of T3SS, were detected on the bacterial surface of B. pertussis cultured in the AsA- medium. Since ascorbic acid is known as a reducing agent, we cultured B. pertussis in liquid medium containing other reducing agents such as 2-mercaptoethanol and dithioerythritol. Under these reducing conditions, the production of type III secreted proteins was repressed. These results suggest that in B. pertussis, the production and secretion of type III secreted proteins are downregulated under reducing conditions.IMPORTANCE The type III secretion system (T3SS) of Bordetella pertussis forms a needlelike structure that protrudes from the bacterial cell surface. B. pertussis uses a T3SS to translocate virulence proteins called effectors into host cells. The culture conditions for effector production in B. pertussis have not been investigated. We attempted to optimize culture medium compositions for producing and secreting type III secreted proteins. We found that B. pertussis secretes type III secreted proteins in reducing agent-deprived liquid medium and that BteA-secreting B. pertussis provokes cytotoxicity against cultured mammalian cells. These results suggest that redox signaling is involved in the regulation of B. pertussis T3SS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bordetella pertussis/patogenicidad , Regulación Bacteriana de la Expresión Génica , Sistemas de Secreción Tipo III/metabolismo , Tos Ferina/microbiología , Animales , Línea Celular , Medios de Cultivo , Regulación hacia Abajo , Oxidación-Reducción , Ratas , VirulenciaRESUMEN
Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713-deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713.
Asunto(s)
Macrófagos/inmunología , Stenotrophomonas maltophilia/inmunología , Animales , Técnicas de Cocultivo , Femenino , Homeostasis/inmunología , Interleucina-10/deficiencia , Interleucina-10/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCIDRESUMEN
Bordetella species, including B. pertussis, have a type III secretion system that is highly conserved among gram-negative pathogenic bacteria. Genes encoding the component proteins of the type III secretion system are localized at the bsc locus in the Bordetella genome. Here, the function of a hypothetical protein Bcr4 encoded at the bsc locus in the B. bronchiseptica genome was investigated. A Bcr4-deficient mutant was created and the amounts of type III secreted proteins (e.g., BopB, BopN and Bsp22) in both the supernatant fraction and whole-cell lysates of the Bcr4-deficient mutant were determined. It was found that the amounts of these proteins were significantly lower than in the wild-type strain. The amounts of type III secreted proteins in the supernatant fraction and whole-cell lysates were much greater in a Bcr4-overproducing strain than in the wild-type strain. The type III secreted protein BspR reportedly negatively regulates the type III secretion system. Here, it was observed that a Bcr4 + BspR double-knockout mutant did not secrete type III secreted proteins, whereas the amounts of these proteins in whole-cell lysates of this mutant were nearly equal to those in whole-cell lysates of the BspR-deficient mutant. Bcr4 thus appears to play an essential role in the extracellular secretion of type III secreted proteins. Our data also suggest that Bcr4 antagonizes the negative regulatory function of BspR.
Asunto(s)
Proteínas Bacterianas/genética , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/metabolismo , Genes Bacterianos/genética , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Animales , Bordetella pertussis/genética , Proteínas Portadoras/genética , Línea Celular , Regulación Bacteriana de la Expresión Génica , Técnicas de Inactivación de Genes , Peso Molecular , Mutación , Transporte de Proteínas , Ratas , TranscriptomaRESUMEN
Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.
Asunto(s)
Escherichia coli Enteropatógena/patogenicidad , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Organofosfatos/metabolismo , Polímeros/metabolismo , Factores de Virulencia/metabolismo , Animales , Modelos Animales de Enfermedad , Escherichia coli Enteropatógena/genética , Escherichia coli O157 , Proteínas de Escherichia coli/genética , Femenino , Células HeLa , Humanos , Enfermedades Intestinales/microbiología , Ratones , Ratones Endogámicos C57BL , Chaperonas Moleculares/genética , Tirosina/genética , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0148387.].
RESUMEN
Bordetella bronchiseptica infects a wide variety of mammals, the type III secretion system (T3SS) being involved in long-term colonization by Bordetella of the trachea and lung. T3SS translocates virulence factors (commonly referred to as effectors) into host cells, leading to alterations in the host's physiological function. The Bordetella effectors BopN and BteA are known to have roles in up-regulation of IL-10 and cytotoxicity, respectively. Nevertheless, the mechanism by which BopN is translocated into host cells has not been examined in sufficient detail. Therefore, to determine the precise mechanisms of translocation of BopN into host cells, truncated derivatives of BopN were built and the derivatives' ability to translocate into host cells evaluated by adenylate cyclase-mediated translocation assay. It was found that N-terminal amino acid (aa) residues 1-200 of BopN are sufficient for its translocation into host cells. Interestingly, BopN translocation was completely blocked by deletion of the N-terminal aa residues 6-50, indicating that the N-terminal region is critical for BopN translocation. Furthermore, BopN appears to play an auxiliary role in BteA-mediated cytotoxicity. Thus, BopN can apparently translocate into host cells and may facilitate activity of BteA.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Bordetella/microbiología , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/metabolismo , Transporte de Proteínas , Adenilil Ciclasas , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos , Proteínas Bacterianas/inmunología , Línea Celular , Citotoxicidad Inmunológica , ADN Bacteriano , Escherichia coli/genética , Regulación de la Expresión Génica , Genes Bacterianos , Vectores Genéticos , Interacciones Huésped-Patógeno , Interleucina-10 , Transporte de Proteínas/fisiología , Ratas , Eliminación de Secuencia , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Regulación hacia Arriba , Factores de Virulencia/metabolismoRESUMEN
BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Sistemas de Secreción Bacterianos , Bordetella bronchiseptica/fisiología , Macrófagos/metabolismo , Macrófagos/microbiología , Fagocitosis , Transducción de Señal , Citoesqueleto de Actina/efectos de los fármacos , Aminoácidos/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/efectos de los fármacos , Células COS , Forma de la Célula/efectos de los fármacos , Chlorocebus aethiops , Citocalasina B/farmacología , Endocitosis/efectos de los fármacos , Gentamicinas/farmacología , L-Lactato Deshidrogenasa/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Proteínas Mutantes/metabolismo , Necrosis , Fagocitos/metabolismo , Fagocitos/microbiología , Fagocitosis/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Ratas , Transducción de Señal/efectos de los fármacos , Imagen de Lapso de TiempoRESUMEN
Bordetella bronchiseptica is genetically related to B. pertussis and B. parapertussis, which cause respiratory tract infections in humans. These pathogens possess a large number of virulence factors, including the type III secretion system (T3SS), which is required for the delivery of effectors into the host cells. In a previous study, we identified a transcriptional regulator, BspR, that is involved in the regulation of the T3SS-related genes in response to iron-starved conditions. A unique feature of BspR is that this regulator is secreted into the extracellular milieu via the T3SS. To further characterize the role of BspR in extracellular localization, we constructed various truncated derivatives of BspR and investigated their translocation into the host cells using conventional translocation assays. In this study, the effector translocation was evaluated by the T3SS of enteropathogenic E. coli (EPEC), since the exogenous expression of BspR triggers severe repression of the Bordetella T3SS expression. The results of the translocation assays using the EPEC T3SS showed that the N-terminal 150 amino acid (aa) residues of BspR are sufficient for translocation into the host cells in a T3SS-dependent manner. In addition, exogenous expression of BspR in HeLa cells demonstrated that the N-terminal 100 aa residues are involved in the nuclear localization. In contrast, the N-terminal 54 aa residues are sufficient for the extracellular secretion into the bacterial culture supernatant via the EPEC T3SS. Thus, BspR is not only a transcriptional regulator in bacteria cytosol, but also functions as an effector that translocates into the nuclei of infected host cells.
Asunto(s)
Proteínas Bacterianas/genética , Bordetella bronchiseptica/genética , Regulación Bacteriana de la Expresión Génica , Sistemas de Secreción Tipo III/metabolismo , Factores de Virulencia/genética , Secuencias de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bordetella bronchiseptica/metabolismo , Bordetella bronchiseptica/patogenicidad , Células COS , Núcleo Celular/química , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citosol/química , Citosol/metabolismo , Escherichia coli Enteropatógena/genética , Escherichia coli Enteropatógena/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Sistemas de Secreción Tipo III/genética , Factores de Virulencia/química , Factores de Virulencia/metabolismoRESUMEN
Bordetella bronchiseptica colonizes the respiratory tracts of a wide variety of mammals and causes a range of diseases, from lethal pneumonia to asymptomatic chronic infection. We report the complete genome sequence of Bordetella bronchiseptica strain S798, isolated from a pig with atrophic rhinitis in Japan.
RESUMEN
Bordetella bronchiseptica is closely related with B. pertussis and B. parapertussis, the causative agents of whooping cough. These pathogenic species share a number of virulence genes, including the gene locus for the type III secretion system (T3SS) that delivers effector proteins. To identify unknown type III effectors in Bordetella, secreted proteins in the bacterial culture supernatants of wild-type B. bronchiseptica and an isogenic T3SS-deficient mutant were compared with iTRAQ-based, quantitative proteomic analysis method. BB1639, annotated as a hypothetical protein, was identified as a novel type III secreted protein and was designated BspR (Bordetella secreted protein regulator). The virulence of a BspR mutant (ΔbspR) in B. bronchiseptica was significantly attenuated in a mouse infection model. BspR was also highly conserved in B. pertussis and B. parapertussis, suggesting that BspR is an essential virulence factor in these three Bordetella species. Interestingly, the BspR-deficient strain showed hyper-secretion of T3SS-related proteins. Furthermore, T3SS-dependent host cell cytotoxicity and hemolytic activity were also enhanced in the absence of BspR. By contrast, the expression of filamentous hemagglutinin, pertactin, and adenylate cyclase toxin was completely abolished in the BspR-deficient strain. Finally, we demonstrated that BspR is involved in the iron-responsive regulation of T3SS. Thus, Bordetella virulence factors are coordinately but inversely controlled by BspR, which functions as a regulator in response to iron starvation.
Asunto(s)
Sistemas de Secreción Bacterianos/genética , Bordetella bronchiseptica/genética , Regulación Bacteriana de la Expresión Génica/genética , Factores de Virulencia de Bordetella/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bordetella bronchiseptica/patogenicidad , Hemaglutininas/metabolismo , Ratones , Proteómica , Conejos , Ratas , Virulencia , Factores de Virulencia de Bordetella/genéticaRESUMEN
The type III secretion system (T3SS) plays a key role in the exertion of full virulence by Bordetella bronchiseptica. However, little is known about the environmental stimuli that induce expression of T3SS genes. Here, it is reported that iron starvation is a signal for T3SS gene expression in B. bronchiseptica. It was found that, when B. bronchiseptica is cultured under iron-depleted conditions, secretion of type III secreted proteins is greater than that in bacteria grown under iron-replete conditions. Furthermore, it was confirmed that induction of T3SS-dependent host cell cytotoxicity and hemolytic activity is greatly enhanced by infection with iron-depleted Bordetella. In contrast, production of filamentous hemagglutinin is reduced in iron-depleted Bordetella. Thus, B. bronchiseptica controls the expression of virulence genes in response to iron starvation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Bordetella bronchiseptica/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Animales , Toxinas Bacterianas/metabolismo , Bordetella bronchiseptica/genética , Células Cultivadas , Eritrocitos/efectos de los fármacos , Hemólisis , Ratones , Conejos , Factores de Virulencia/metabolismoRESUMEN
The type III secretion system (T3SS) is a sophisticated protein secretion machinery that delivers bacterial virulence proteins into host cells. A needle-tip protein, Bsp22 , is one of the secreted substrates of the T3SS and plays an essential role in the full function of the T3SS in Bordetella bronchiseptica. In this study, we found that BB1618 functions as a chaperone for Bsp22 . The deletion of BB1618 resulted in a dramatic impairment of Bsp22 secretion into the culture supernatants and Bsp22 stability in the bacterial cytosol. In contrast, the secretion of other type III secreted proteins was not affected by the BB1618 mutation. Furthermore, the BB1618 mutant strain could not induce cytotoxicity and displayed the same phenotypes as the Bsp22 mutant strain. An immunoprecipitation assay demonstrated that BB1618 interacts with Bsp22 , but not with BopB and BopD . Thus, we identified BB1618 as a specific type III chaperone for Bsp22 . Therefore, we propose that BB1618 be renamed Btc22 for the Bordetella type III chaperone for Bsp22 .
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/fisiología , Bordetella bronchiseptica/metabolismo , Bordetella bronchiseptica/patogenicidad , Chaperonas Moleculares/metabolismo , Animales , Proteínas Bacterianas/genética , Bordetella bronchiseptica/crecimiento & desarrollo , Línea Celular , Eritrocitos/microbiología , Células HeLa , Hemólisis , Humanos , Inmunoprecipitación , Chaperonas Moleculares/genética , Transporte de Proteínas , Conejos , VirulenciaRESUMEN
Genetic evidence indicates that only the ß-anomer of galactose is transported to Kluyveromyces lactis cells by galactose/glucose transporter Hgt1p, and that aldose-1-epimerase encoded by GAL10 is a prerequisite for growth on galactose. Minor aldose-1-epimerases other than Gal10p also exist in K. lactis. Using a mutant defective in both aldose-1-epimerases, we show by solid-state nuclear magnetic resonance spectroscopy that only ß-anomer is transported in the cell and stays without or with a slow rate of conversion to α-anomer. Signals due to intracellular ß-galactose appeared at two positions, both of which were shifted towards higher magnetic fields than that of ß-galactose in aqueous solution, suggesting that incorporated galactose binds to cellular components, probably proteins.
Asunto(s)
Galactosa/metabolismo , Kluyveromyces/química , Kluyveromyces/metabolismo , Transporte Biológico , Carbohidrato Epimerasas/genética , Galactosa/química , Eliminación de Gen , Isomerismo , Kluyveromyces/enzimología , Kluyveromyces/genética , Espectroscopía de Resonancia MagnéticaRESUMEN
BACKGROUND: Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift. METHODOLOGY/PRINCIPAL FINDING: Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5' untranslated region of bteA, -136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion. CONCLUSION/SIGNIFICANCE: The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis.
Asunto(s)
Proteínas Bacterianas/genética , Bordetella pertussis/genética , Mutagénesis Insercional/genética , Regiones no Traducidas 5'/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Bordetella pertussis/inmunología , Regulación Bacteriana de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutación/genética , Vacuna contra la Tos Ferina/inmunología , Mapeo Físico de Cromosoma , Regiones Promotoras Genéticas/genética , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
The type III secretion system (T3SS) is highly conserved in many Gram-negative pathogenic bacteria and functions as an injector of bacterial proteins (effectors) into host cells. T3SSs are involved in establishing disease processes, but this machinery is not essential for bacterial growth or homeostasis. Thus, T3SS is expected to be a candidate therapeutic target, and inhibitors of T3SSs could potentially reduce virulence without causing bacterial death, thereby avoiding any subsequent development of resistance. We identified a linear polyketide compound, aurodox, as a specific T3SS inhibitor from the culture broth of Streptomyces sp. using a screening system for the T3SS-mediated hemolysis of enteropathogenic Escherichia coli (EPEC) established by our group. Aurodox strongly inhibited T3SS-mediated hemolysis with an IC(50) value of 1.5 µg ml(-1) without affecting bacterial growth in liquid media. We also demonstrated that aurodox specifically inhibits the secretion of type III-secreted proteins such as EspB, EspF and Map, without affecting the expression of the housekeeping protein GroEL. Furthermore, an in vivo infection study using mice clearly indicated that the administration of aurodox allowed the mice to survive a lethal dose of Citrobactor rodentium, a model bacterium for human pathogens such as EPEC. Thus, our in vivo study directly demonstrated for the first time that this putative T3SS inhibitor can be applied as a novel class of anti-infective agents.