RESUMEN
BACKGROUND & AIMS: Variants at the ABCB4 or MDR2 locus, which encodes a biliary transport protein, are associated with a spectrum of cholestatic liver diseases. Exacerbation of liver disease has been linked to increased hepatic levels of interleukin (IL) 17, yet the mechanisms of this increase are not understood. We studied mice with disruption of Mdr2 to determine how defects in liver and alteration in the microbiota contribute to production of IL17 by intrahepatic γδ T cells. METHODS: We performed studies with Mdr2-/- and littermate FVB/NJ (control) mice. IL17 was measured in serum samples by an enzyme-linked immunosorbent assay. Mice were injected with neutralizing antibodies against the γδ T-cell receptor (TCR; anti-γδ TCR) or mouse IL17A (anti-IL17A). Livers were collected and bacteria were identified in homogenates by culture procedures; TCRγδ+ cells were isolated by flow cytometry. Fecal samples were collected from mice and analyzed by 16S ribosomal DNA sequencing. Cells were stimulated with antibodies or bacteria, and cytokine production was measured. We obtained tissues from 10 patients undergoing liver transplantation for primary sclerosing cholangitis or chronic hepatitis C virus infection. Tissues were analyzed for cytokine production by γδ TCR+ cells. RESULTS: Mdr2-/- mice had collagen deposition around hepatic bile ducts and periportal-bridging fibrosis with influx of inflammatory cells and increased serum levels of IL17 compared with control mice. Administration of anti-IL17A reduced hepatic fibrosis. Livers from Mdr2-/- mice had increased numbers of IL17A+ γδTCR+ cells-particularly of IL17A+ Vγ6Jγ1 γδ TCR+ cells. Fecal samples from Mdr2-/- mice were enriched in Lactobacillus, and liver tissues were enriched in Lactobacillus gasseri compared with control mice. Mdr2-/- mice also had increased intestinal permeability. The γδ TCR+ cells isolated from Mdr2-/- livers produced IL17 in response to heat-killed L gasseri. Intraperitoneal injection of control mice with L gasseri led to increased serum levels of IL17 and liver infiltration by inflammatory cells; injection of these mice with anti-γδ TCR reduced serum level of IL17. Intravenous injections of Mdr2-/- mice with anti-γδ TCR reduced fibrosis; liver levels of IL17, and inflammatory cells; and serum levels of IL17. γδTCR+ cells isolated from livers of patients with primary sclerosing cholangitis, but not hepatitis C virus infection, produced IL17. CONCLUSIONS: In Mdr2-/- mice, we found development of liver fibrosis and inflammation to require hepatic activation of γδ TCR+ cells and production of IL17 mediated by exposure to L gasseri. This pathway appears to contribute to development of cholestatic liver disease in patients.
Asunto(s)
Colestasis/patología , Microbioma Gastrointestinal , Interleucina-17/metabolismo , Linfocitos Intraepiteliales/metabolismo , Cirrosis Hepática/patología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adulto , Anciano , Animales , Conductos Biliares/citología , Conductos Biliares/inmunología , Conductos Biliares/microbiología , Células Cultivadas , Colangitis Esclerosante/microbiología , Colangitis Esclerosante/patología , Colangitis Esclerosante/cirugía , Colestasis/inmunología , Colestasis/microbiología , Colestasis/cirugía , Modelos Animales de Enfermedad , Enfermedad Hepática en Estado Terminal/microbiología , Enfermedad Hepática en Estado Terminal/patología , Enfermedad Hepática en Estado Terminal/cirugía , Femenino , Hepatitis C Crónica/patología , Hepatitis C Crónica/cirugía , Hepatitis C Crónica/virología , Humanos , Interleucina-17/antagonistas & inhibidores , Interleucina-17/sangre , Interleucina-17/inmunología , Lactobacillus gasseri/inmunología , Hígado/citología , Hígado/inmunología , Hígado/microbiología , Hígado/patología , Cirrosis Hepática/inmunología , Cirrosis Hepática/microbiología , Cirrosis Hepática/cirugía , Trasplante de Hígado , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T gamma-delta/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Adulto Joven , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Acinetobacter baumannii strain AB5075 forms two cell types distinguished by their opaque (VIR-O) or translucent (AV-T) colonies. VIR-O cells possess a thicker capsule and are more resistant to a variety of stressors than AV-T cells. However, the direct role of the capsule in these stressors was unknown. This study demonstrates that the capsule is required for resistance to disinfectants, lysozyme, and desiccation in Acinetobacter baumannii In addition, the capsule is required for survival in a mouse lung model of infection.
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Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/fisiología , Desinfectantes/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/patogenicidad , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desecación , Farmacorresistencia Bacteriana/fisiología , Prueba de Complementación Genética , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/fisiología , Muramidasa/farmacología , MutaciónRESUMEN
Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
Asunto(s)
Proteínas Bacterianas/inmunología , Farmacorresistencia Bacteriana/inmunología , Francisella/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Evasión Inmune/inmunología , Inflamasomas/inmunología , Lipoproteínas/inmunología , Animales , Membrana Celular/genética , Membrana Celular/inmunología , Farmacorresistencia Bacteriana/genética , Francisella/genética , Infecciones por Bacterias Gramnegativas/genética , Evasión Inmune/genética , Inflamasomas/genética , Secuencias Invertidas Repetidas/inmunología , Lipoproteínas/genética , Ratones , Ratones NoqueadosRESUMEN
Burkholderia pseudomallei is a Gram-negative soil bacterium that infects both humans and animals. Although cell culture studies have revealed significant insights into factors contributing to virulence and host defense, the interactions between this pathogen and its intact host remain to be elucidated. To gain insights into the host defense responses to B. pseudomallei infection within an intact host, we analyzed the genome-wide transcriptome of infected Caenorhabditis elegans and identified â¼6% of the nematode genes that were significantly altered over a 12-h course of infection. An unexpected feature of the transcriptional response to B. pseudomallei was a progressive increase in the proportion of down-regulated genes, of which ELT-2 transcriptional targets were significantly enriched. ELT-2 is an intestinal GATA transcription factor with a conserved role in immune responses. We demonstrate that B. pseudomallei down-regulation of ELT-2 targets is associated with degradation of ELT-2 protein by the host ubiquitin-proteasome system. Degradation of ELT-2 requires the B. pseudomallei type III secretion system. Together, our studies using an intact host provide evidence for pathogen-mediated host immune suppression through the destruction of a host transcription factor.
Asunto(s)
Burkholderia pseudomallei/patogenicidad , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/inmunología , Caenorhabditis elegans/microbiología , Factores de Transcripción GATA/metabolismo , Animales , Animales Modificados Genéticamente , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/inmunología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Regulación hacia Abajo , Factores de Transcripción GATA/genética , Interacciones Huésped-Patógeno/inmunología , Procesamiento Postranscripcional del ARN , ARN de Helminto/genética , ARN de Helminto/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Virulencia/inmunologíaRESUMEN
BACKGROUND: Chronic bacterial infections occur as a result of the infecting pathogen's ability to live within a biofilm, hence escaping the detrimental effects of antibiotics and the immune defense system. Burkholderia pseudomallei, a gram-negative facultative pathogen, is distinctive in its ability to survive within phagocytic and non-phagocytic cells, to persist in vivo for many years and subsequently leading to relapse as well as the development of chronic disease. The capacity to persist has been attributed to the pathogen's ability to form biofilm. However, the underlying biology of B. pseudomallei biofilm development remains unresolved. RESULTS: We utilised RNA-Sequencing to identify genes that contribute to B. pseudomallei biofilm phenotype. Transcriptome analysis of a high and low biofilm producer identified 563 differentially regulated genes, implying that expression of ~9.5% of the total B. pseudomallei gene content was altered during biofilm formation. Genes involved in surface-associated motility, surface composition and cell wall biogenesis were over-expressed and probably play a role in the initial attachment of biofilms. Up-regulation of genes related to two component signal transduction systems and a denitrification enzyme pathway suggest that the B. pseudomallei high biofilm producer is able to sense the surrounding environmental conditions and regulate the production of extracellular polymeric substance matrix, a hallmark of microbial biofilm formation. CONCLUSIONS: The transcriptome profile described here provides the first comprehensive view of genes that contribute to the biofilm phenotype in B. pseudomallei.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/genética , Transcripción Genética/genética , Virulencia/genética , Animales , Pared Celular/genética , Femenino , Perfilación de la Expresión Génica/métodos , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Regulación hacia Arriba/genéticaRESUMEN
Emerging resistance to "last-resort" polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance.
Asunto(s)
Acinetobacter baumannii/genética , Amidohidrolasas/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Lípido A/metabolismo , Factores de Transcripción/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/aislamiento & purificación , Amidohidrolasas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Polimixina B/farmacología , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of disease are diverse, ranging from chronic infection to acute septicaemia. The current gold standard of diagnosis involves bacterial culture and identification which is time consuming and often too late for early medical intervention. Hence, rapid diagnosis of melioidosis is crucial for the successful management of melioidosis. METHODS: The study evaluated 4 purified B. pseudomallei recombinant proteins (TssD-5, Omp3, smBpF4 and Omp85) as potential diagnostic agents for melioidosis. A total of 68 sera samples from Malaysian melioidosis patients were screened for the presence of specific antibodies towards these proteins using enzyme-linked immunosorbent assay (ELISA). Sera from patients with various bacterial and viral infections but negative for B. pseudomallei, as well as sera from healthy individuals, were also included as non-melioidosis controls. The Mann Whitney test was performed to compare the statistical differences between melioidosis patients and the non-melioidosis controls. RESULTS: TssD-5 demonstrated the highest sensitivity of 71% followed by Omp3 (59%), smBpF4 (41%) and Omp85 (19%). All 4 antigens showed equally high specificity (89-96%). A cocktail of all 4 antigens resulted in slightly reduced sensitivity of 65% but improved specificity (99%). Multiple-antigen ELISA provided improved sensitivity of 88.2% whilst retaining good specificity (96%). There was minimal reactivity with sera from healthy individuals proposing the utility of these antigens to demarcate diseased from non-symptomatic individuals in an endemic country. CONCLUSIONS: TssD-5 demonstrated high detection sensitivity and specificity and the results were obtained within a few hours compared to time consuming culture and IFAT methods commonly used in a clinical setting. The use of multiple-antigens resulted in improved sensitivity (88.2%) whilst maintaining superior specificity. These data highlight the use of TssD-5 and other recombinant antigens tested in this study as potential serodiagnostic agents for melioidosis.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos , Burkholderia pseudomallei/inmunología , Melioidosis/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes , Sensibilidad y Especificidad , Pruebas Serológicas/métodosRESUMEN
The gut-brain axis, a bidirectional signaling network between the intestine and the central nervous system, is crucial to the regulation of host physiology and inflammation. Recent advances suggest a strong correlation between gut dysbiosis and neurological diseases, however, relatively little is known about how gut bacteria impact the brain. Here, we reveal that gut commensal bacteria can translocate directly to the brain when mice are fed an altered diet that causes dysbiosis and intestinal permeability, and that this also occurs without diet alteration in distinct murine models of neurological disease. The bacteria were not found in other systemic sites or the blood, but were detected in the vagus nerve. Unilateral cervical vagotomy significantly reduced the number of bacteria in the brain, implicating the vagus nerve as a conduit for translocation. The presence of bacteria in the brain correlated with microglial activation, a marker of neuroinflammation, and with neural protein aggregation, a hallmark of several neurodegenerative diseases. In at least one model, the presence of bacteria in the brain was reversible as a switch from high-fat to standard diet resulted in amelioration of intestinal permeability, led to a gradual loss of detectable bacteria in the brain, and reduced the number of neural protein aggregates. Further, in murine models of Alzheimer's disease, Parkinson's disease, and autism spectrum disorder, we observed gut dysbiosis, gut leakiness, bacterial translocation to the brain, and microglial activation. These data reveal a commensal bacterial translocation axis to the brain in models of diverse neurological diseases.
RESUMEN
Diabetes mellitus is a predisposing factor of melioidosis, contributing to higher mortality rates in diabetics infected with Burkholderia pseudomallei. To investigate how diabetes alters the inflammatory response, we established a streptozotocin (STZ) -induced diabetic murine acute-phase melioidosis model. Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42-hr course of infection but the hyperglycaemic environment did not increase the bacterial burden. However, after 24 hr, granulocyte counts increased in response to infection, whereas blood glucose concentrations decreased over the course of infection. A genome-wide expression analysis of the STZ-diabetic murine acute melioidosis liver identified ~1000 genes whose expression was altered in the STZ-diabetic mice. The STZ-diabetic host transcriptional response was compared with the normoglycaemic host transcriptional response recently reported by our group. The microarray data suggest that the presence of elevated glucose levels impairs the host innate immune system by delaying the identification and recognition of B. pseudomallei surface structures. Consequently, the host is unable to activate the appropriate innate immune response over time, which may explain the increased susceptibility to melioidosis in the STZ-diabetic host. Nevertheless, a general 'alarm signal' of infection as well as defence programmes are still triggered by the STZ-diabetic host, although only 24 hr after infection. In summary, this study demonstrates that in the face of a B. pseudomallei acute infection, poor glycaemic control impaired innate responses during the early stages of B. pseudomallei infection, contributing to the increased susceptibility of STZ-induced diabetics to this fatal disease.
Asunto(s)
Burkholderia pseudomallei/patogenicidad , Diabetes Mellitus Experimental/inmunología , Inmunidad Innata/inmunología , Melioidosis/mortalidad , Transcriptoma/inmunología , Animales , Diabetes Mellitus Experimental/complicaciones , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Humanos , Hiperglucemia/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/microbiología , Melioidosis/inmunología , Melioidosis/microbiología , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de Oligonucleótidos , Bazo/inmunología , Bazo/metabolismo , Bazo/microbiología , Factores de TiempoRESUMEN
The ability of enteric pathogens to perceive and adapt to distinct environments within the metazoan intestinal tract is critical for pathogenesis; however, the preponderance of interactions between microbe- and host-derived factors remain to be fully understood. Salmonella enterica serovar Typhimurium is a medically important enteric bacterium that colonizes, proliferates and persists in the intestinal lumen of the nematode Caenorhabditis elegans. Several Salmonella virulence factors important in murine and tissue culture models also contribute to worm mortality and intestinal persistence. For example, PhoP and the virulence plasmid pSLT are virulence factors required for resistance to the C. elegans antimicrobial peptide SPP-1. To uncover additional determinants required for Salmonella typhimurium pathogenesis in vivo, we devised a genetic screen to identify bacterial mutants defective in establishing a persistent infection in the intestine of C. elegans. Here we report on identification of 14 loci required for persistence in the C. elegans intestine and characterization of KdpD, a sensor kinase of a two-component system in S. typhimurium pathogenesis. We show that kdpD mutants are profoundly attenuated in intestinal persistence in the nematode and in macrophage survival. These findings may be attributed to the essential role KdpD plays in promoting resistance to osmotic, oxidative and antimicrobial stresses.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caenorhabditis elegans/microbiología , Macrófagos/microbiología , Proteínas Quinasas/metabolismo , Salmonella typhimurium/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Línea Celular , Tracto Gastrointestinal/microbiología , Técnicas de Inactivación de Genes , Pruebas Genéticas/métodos , Ratones , Viabilidad Microbiana , Proteínas Quinasas/genética , Salmonella typhimurium/genética , VirulenciaRESUMEN
Acinetobacter baumannii is a multidrug-resistant, Gram-negative nosocomial pathogen that exhibits phenotypic heterogeneity resulting in virulent opaque (VIR-O) and avirulent translucent (AV-T) colony variants. Each variant has a distinct gene expression profile resulting in multiple phenotypic differences. Cells interconvert between the VIR-O and AV-T variants at high frequency under laboratory conditions, suggesting that the genetic mechanism underlying the phenotypic switch could be manipulated to attenuate virulence. Therefore, our group has focused on identifying and characterizing genes that regulate this switch, which led to the investigation of ABUW_1132 (1132), a highly conserved gene predicted to encode a LysR-type transcriptional regulator. ABUW_1132 was shown to be a global regulator as the expression of 74 genes was altered ≥ 2-fold in an 1132 deletion mutant. The 1132 deletion also resulted in a 16-fold decrease in VIR-O to AV-T switching, loss of 3-OH-C12-HSL secretion, and reduced surface-associated motility. Further, the deletion of 1132 in the AV-T background caused elevated capsule production, which increased colony opacity and altered the typical avirulent phenotype of translucent cells. These findings distinguish 1132 as a global regulatory gene and advance our understanding of A. baumannii's opacity-virulence switch.
Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Acinetobacter baumannii/genética , Humanos , Fenotipo , Virulencia/genéticaRESUMEN
The outer membrane protects Gram-negative bacteria from the host environment. Lipopolysaccharide (LPS), a major outer membrane constituent, has distinct components (lipid A, core, O-antigen) generated by specialized pathways. In this study, we describe the surprising convergence of these pathways through FlmX, an uncharacterized protein in the intracellular pathogen Francisella. FlmX is in the flippase family, which includes proteins that traffic lipid-linked envelope components across membranes. flmX deficiency causes defects in lipid A modification, core remodeling, and O-antigen addition. We find that an F. tularensis mutant lacking flmX is >1,000,000-fold attenuated. Furthermore, FlmX is required to resist the innate antimicrobial LL-37 and the antibiotic polymyxin. Given FlmX's central role in LPS modification and its conservation in intracellular pathogens Brucella, Coxiella, and Legionella, FlmX may represent a novel drug target whose inhibition could cripple bacterial virulence and sensitize bacteria to innate antimicrobials and antibiotics.
Asunto(s)
Proteínas Bacterianas/metabolismo , Francisella/metabolismo , Francisella/patogenicidad , Lipopolisacáridos/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Elementos Transponibles de ADN/genética , Escherichia coli/metabolismo , Femenino , Francisella/genética , Galactosamina/metabolismo , Regulación Bacteriana de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Antígenos O/metabolismo , Polimixina B/farmacología , Virulencia/genéticaRESUMEN
BACKGROUND: At present, very little is known about how Burkholderia pseudomallei (B. pseudomallei) interacts with its host to elicit melioidosis symptoms. We established a murine acute-phase melioidosis model and used DNA microarray technology to investigate the global host/pathogen interaction. We compared the transcriptome of infected liver and spleen with uninfected tissues over an infection period of 42 hr to identify genes whose expression is altered in response to an acute infection. RESULTS: Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42 hr course of infection. Microarray analysis of the liver and spleen over this time course demonstrated that genes involved in immune response, stress response, cell cycle regulation, proteasomal degradation, cellular metabolism and signal transduction pathways were differentially regulated. Up regulation of toll-like receptor 2 (TLR2) gene expression suggested that a TLR2-mediated signalling pathway is responsible for recognition and initiation of an inflammatory response to the acute B. pseudomallei infection. Most of the highly elevated inflammatory genes are a cohort of "core host immune response" genes commonly seen in general inflammation infections. Concomitant to this initial inflammatory response, we observed an increase in transcripts associated with cell-death, caspase activation and peptidoglysis that ultimately promote tissue injury in the host. The complement system responsible for restoring host cellular homeostasis and eliminating intracellular bacteria was activated only after 24 hr post-infection. However, at this time point, diverse host nutrient metabolic and cellular pathways including glycolysis, fatty acid metabolism and tricarboxylic acid (TCA) cycle were repressed. CONCLUSIONS: This detailed picture of the host transcriptional response during acute melioidosis highlights a broad range of innate immune mechanisms that are activated in the host within 24 hrs, including the core immune response commonly seen in general inflammatory infections. Nevertheless, this activation is suppressed at 42 hr post-infection and in addition, suboptimal activation and function of the downstream complement system promotes uncontrolled spread of the bacteria.
Asunto(s)
Burkholderia pseudomallei/inmunología , Perfilación de la Expresión Génica , Genoma/genética , Interacciones Huésped-Patógeno/genética , Inmunidad Innata/genética , Melioidosis/genética , Melioidosis/microbiología , Enfermedad Aguda , Reacción de Fase Aguda/complicaciones , Reacción de Fase Aguda/genética , Reacción de Fase Aguda/inmunología , Animales , Caspasas/metabolismo , Muerte Celular/genética , Activación de Complemento/genética , Modelos Animales de Enfermedad , Activación Enzimática , Homeostasis/inmunología , Hígado/inmunología , Hígado/microbiología , Hígado/patología , Melioidosis/complicaciones , Melioidosis/inmunología , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/genética , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismoRESUMEN
We describe a novel genetic mechanism in which tandem amplification of a plasmid-borne integron regulates virulence, opacity variation, and global gene expression by altering levels of a putative small RNA (sRNA) in Acinetobacter baumannii AB5075. Copy number of this amplified locus correlated with the rate of switching between virulent opaque (VIR-O) and avirulent translucent (AV-T) cells. We found that prototypical VIR-O colonies, which exhibit high levels of switching and visible sectoring with AV-T cells by 24 h of growth, harbor two copies of this locus. However, a subset of opaque colonies that did not form AV-T sectors within 24 h were found to harbor only one copy. The colonies with decreased sectoring to AV-T were designated low-switching opaque (LSO) variants and were found to exhibit a 3-log decrease in switching relative to that of the VIR-O. Overexpression studies revealed that the element regulating switching was localized to the 5' end of the aadB gene within the amplified locus. Northern blotting indicated that an sRNA of approximately 300 nucleotides (nt) is encoded in this region and is likely responsible for regulating switching to AV-T. Copy number of the â¼300-nt sRNA was also found to affect virulence, as the LSO variant exhibited decreased virulence during murine lung infections. Global transcriptional profiling revealed that >100 genes were differentially expressed between VIR-O and LSO variants, suggesting that the â¼300-nt sRNA may act as a global regulator. Several virulence genes exhibited decreased expression in LSO cells, potentially explaining their decreased virulence.IMPORTANCEAcinetobacter baumannii remains a leading cause of hospital-acquired infections. Widespread multidrug resistance in this species has prompted the WHO to name carbapenem-resistant A. baumannii as its top priority for research and development of new antibiotics. Many strains of A. baumannii undergo a high-frequency virulence switch, which is an attractive target for new therapeutics targeting this pathogen. This study reports a novel mechanism controlling the frequency of switching in strain AB5075. The rate of switching from the virulent opaque (VIR-O) to the avirulent translucent (AV-T) variant is positively influenced by the copy number of an antibiotic resistance locus encoded on a plasmid-borne composite integron. Our data suggest that this locus encodes a small RNA that regulates opacity switching. Low-switching opaque variants, which harbor a single copy of this locus, also exhibit decreased virulence. This study increases our understanding of this critical phenotypic switch, while also identifying potential targets for virulence-based A. baumannii treatments.
Asunto(s)
Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidad , Farmacorresistencia Bacteriana/genética , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Integrones , Acinetobacter baumannii/efectos de los fármacos , Animales , Antibacterianos/farmacología , Femenino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Virulencia/genéticaRESUMEN
Antibiotic-resistant infections lead to 700,000 deaths per year worldwide 1 . The roles of phenotypically diverse subpopulations of clonal bacteria in the progression of diseases are unclear. We found that the increasingly pathogenic and antibiotic-resistant pathogen Acinetobacter baumannii harbours a highly virulent subpopulation of cells responsible for disease. This virulent subpopulation possesses a thicker capsule and is resistant to host antimicrobials, which drive its enrichment during infection. Importantly, bacteria harvested from the bloodstream of human patients belong exclusively to this virulent subpopulation. Furthermore, the virulent form exhibits increased resistance to hospital disinfectants and desiccation, indicating a role in environmental persistence and the epidemic spread of disease. We identified a transcriptional 'master regulator' of the switch between avirulent and virulent cells, the overexpression of which abrogates virulence. Furthermore, the overexpression strain is capable of vaccinating mice against lethal challenge. This work highlights a phenotypic subpopulation of bacteria that drastically alters the outcome of infection, and illustrates how knowledge of the regulatory mechanisms controlling such phenotypic switches can be harnessed to attenuate bacteria and develop translational interventions.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/patogenicidad , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/prevención & control , Acinetobacter baumannii/genética , Animales , Línea Celular , Desecación , Modelos Animales de Enfermedad , Desinfectantes/farmacología , Variación Genética , Humanos , Ratones , Fenotipo , Vacunación , VirulenciaRESUMEN
UNLABELLED: The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. IMPORTANCE: Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data demonstrate that the atypical occurrence of two biotin protein ligases in F. novicida is linked to distinct roles in virulence and biotin metabolism.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Francisella/enzimología , Factores de Virulencia/metabolismo , Animales , Recuento de Colonia Microbiana , Modelos Animales de Enfermedad , Prueba de Complementación Genética , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/patología , Macrófagos/microbiología , Ratones , Piel/microbiología , VirulenciaRESUMEN
Burkholderia pseudomallei is resistant to a diverse group of antimicrobials including third generation cephalosporins whilst quinolones and aminoglycosides have no reliable effect. As therapeutic options are limited, development of more effective forms of immunotherapy is vital to avoid a fatal outcome. In an earlier study, we reported on the B. pseudomallei serine MprA protease, which is relatively stable over a wide pH and temperature range and digests physiological proteins. The present study was carried out to evaluate the immunogenicity and protective efficacy of the MprA as a potential vaccine candidate. In BALB/c mice immunized with recombinant MprA protease (smBpF4), a significantly high IgG titer was detectable. Isotyping studies revealed that the smBpF4-specific antibodies produced were predominantly IgG(1), proposing that immunization with smBpF4 triggered a Th2 immune response. Mice were immunized with smBpF4 and subsequently challenged with B. pseudomallei via the intraperitoneal route. Whilst control mice succumbed to the infection by day 9, smBpF4-immunized mice were protected against the lethal challenge and survived beyond 25 days post-infection. In conclusion, MprA is immunogenic in melioidosis patients whilst also eliciting a strong immune response upon bacterial challenge in mice and presents itself as a potential vaccine candidate for the treatment of melioidosis.