RESUMEN
Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin-degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG-thiazoline (NGT)-mediated inhibition of a recombinantly expressed GH20 ß-N-acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116. NGT strongly inhibited VhGlcNAcase with an IC50 of 11.9 ± 1.0 µm and Ki 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a Kd of 32 ± 1.2 µm. The observed ΔG°binding of -7.56 kcal·mol-1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy-driven. The structural complex shows that NGT fully occupies the substrate-binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the -1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections. DATABASE: Structural data are available in PDB database under the accession number 6K35.
Asunto(s)
Acetilglucosamina/análogos & derivados , Acetilglucosaminidasa/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Tiazoles/farmacología , Vibrio/enzimología , Acetilglucosamina/química , Acetilglucosamina/farmacología , Acetilglucosaminidasa/química , Acetilglucosaminidasa/metabolismo , Animales , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/prevención & control , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Oligosacáridos/metabolismo , Dominios Proteicos , Especificidad por Sustrato , Termodinámica , Tiazoles/química , Vibrio/efectos de los fármacos , Vibrio/genética , Vibrio/crecimiento & desarrolloRESUMEN
In vitro- and in vivo-polarised absorptive epithelia (enterocytes) are considered to be non-phagocytic towards bacteria with invasive pathogenic strains relying on virulence factors to 'force' entry. Here, we report a serendipitous discovery that questions these beliefs. Thus, we uncover in well-established models of human small (Caco-2; TC-7) and large (T84) intestinal enterocytes a polarization-dependent mechanism that can transfer millions of bacteria from the basal to apical compartment. Antibiotic-protection assays, confocal imaging and drug inhibitor data are consistent with a transcellular route in which internalized, basolateral-membrane enclosed bacteria are trafficked to and across the apical surface. Basal-to-apical transport of non-pathogenic bacteria (and inert beads) challenged the idea of pathogens relying on virulence factors to force entry. Indeed, studies with Salmonella demonstrated that it's entry-forcing virulence factor (SPI-I) was not required to enter via the basolateral surface but to promote another virulence-associated event (intra-enterocyte accumulation).
Asunto(s)
Traslocación Bacteriana , Enterocitos/microbiología , Transporte Biológico , Células CACO-2 , Línea Celular , Polaridad Celular/fisiología , Enterocitos/efectos de los fármacos , Escherichia coli Enteropatógena/patogenicidad , Escherichia coli Enteropatógena/fisiología , Células HeLa , Humanos , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Factores de Virulencia/farmacologíaRESUMEN
Objectives/Hypothesis. To determine if laryngopharyngeal reflux alters mucin gene expression in laryngeal mucosa. Methods. In situ hybridization was employed to study the expression of the 8 well-characterised mucin genes MUC1-4, 5AC, 5B, 6, and 7 in reflux laryngeal mucosa from laryngeal ventricles, posterior commissures, and vocal folds compared to control/normal laryngeal mucosa. Results. MUC1-5 genes are expressed in normal and reflux laryngeal mucosa. MUC1, 3 and 4 are expressed in respiratory and squamous mucosa whereas MUC2 and 5AC are expressed in respiratory mucosa only. MUC3, 4 and 5AC are downregulated in reflux mucosa. MUC5AC expression is significantly reduced in the 3 mucosal sites and when mucosal type was taken into account, this remains significant in combined laryngeal and ventricular mucosa only. Conclusions. MUC3, 4 and 5AC expression is downregulated in laryngopharyngeal reflux. This may be due to laryngeal mucosal metaplasia and/or alteration of mucin gene expression in the preexisting mucosa. Altered mucin gene expression might predispose laryngeal mucosa to the damaging effect of reflux.
RESUMEN
Although there have been great advances in our understanding of the bacterial cytoskeleton, major gaps remain in our knowledge of its importance to virulence. In this study we have explored the contribution of the bacterial cytoskeleton to the ability of Salmonella to express and assemble virulence factors and cause disease. The bacterial actin-like protein MreB polymerises into helical filaments and interacts with other cytoskeletal elements including MreC to control cell-shape. As mreB appears to be an essential gene, we have constructed a viable ΔmreC depletion mutant in Salmonella. Using a broad range of independent biochemical, fluorescence and phenotypic screens we provide evidence that the Salmonella pathogenicity island-1 type three secretion system (SPI1-T3SS) and flagella systems are down-regulated in the absence of MreC. In contrast the SPI-2 T3SS appears to remain functional. The phenotypes have been further validated using a chemical genetic approach to disrupt the functionality of MreB. Although the fitness of ΔmreC is reduced in vivo, we observed that this defect does not completely abrogate the ability of Salmonella to cause disease systemically. By forcing on expression of flagella and SPI-1 T3SS in trans with the master regulators FlhDC and HilA, it is clear that the cytoskeleton is dispensable for the assembly of these structures but essential for their expression. As two-component systems are involved in sensing and adapting to environmental and cell surface signals, we have constructed and screened a panel of such mutants and identified the sensor kinase RcsC as a key phenotypic regulator in ΔmreC. Further genetic analysis revealed the importance of the Rcs two-component system in modulating the expression of these virulence factors. Collectively, these results suggest that expression of virulence genes might be directly coordinated with cytoskeletal integrity, and this regulation is mediated by the two-component system sensor kinase RcsC.
Asunto(s)
Sistemas de Secreción Bacterianos/fisiología , Citoesqueleto/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella/metabolismo , Salmonella/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citoesqueleto/genética , Femenino , Flagelos/genética , Flagelos/metabolismo , Eliminación de Gen , Islas Genómicas/fisiología , Ratones , Salmonella/genética , Infecciones por Salmonella/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Virulencia/genéticaRESUMEN
Salmonella enterica serovar Typhi (S. typhi) causes typhoid fever. We show that exposure of S. typhi to neuroendocrine stress hormones results in haemolysis, which is associated with the release of haemolysin E in membrane vesicles. This effect is attributed to increased expression of the small RNA micA and RNA chaperone Hfq, with concomitant downregulation of outer membrane protein A. Deletion of micA or the two-component signal-transduction system, CpxAR, abolishes the phenotype. The hormone response is inhibited by the ß-blocker propranolol. We provide mechanistic insights into the basis of neuroendocrine hormone-mediated haemolysis by S. typhi, increasing our understanding of inter-kingdom signalling.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Epinefrina/fisiología , Proteínas Hemolisinas/metabolismo , Norepinefrina/fisiología , Salmonella typhi/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación hacia Abajo , Epinefrina/farmacología , Regulación Bacteriana de la Expresión Génica , Proteínas Hemolisinas/genética , Hemólisis , Hormonas/genética , Hormonas/metabolismo , Norepinefrina/farmacología , Propranolol/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Salmonella typhi/genética , Salmonella typhi/fisiología , Transducción de Señal , Estrés FisiológicoRESUMEN
OBJECTIVES/HYPOTHESIS: Exposure of pig laryngeal mucosa to pepsin and acid will have a differential damaging effect depending on the anatomical site, mirroring the effects seen in the human larynx in laryngopharyngeal reflux (LPR). This study aims to quantitate damage caused to laryngeal tissue by acid alone, and acid and pepsin, and also to determine if the extent of this damage depends on the tissue site. STUDY DESIGN: Prospective translational research study. METHODS: An excised porcine laryngeal damage model in a small Ussing chamber was used to measure the effect of pepsin and acid on five sites (ventricles, vocal folds, posterior commissure, supraglottic, and subglottic mucosa). The tissue samples were incubated on the lumenal side for 1 hour with pH 2 and 4 HCl, pH 2 plus 1 mg/mL pepsin, and pH 4 plus 1 mg/mL pepsin. Damage was assessed by changes in absorbance of the bathing solution at optical density (OD) 260 nm and OD 280 nm and by measurement of released DNA compared to tissues bathed in pH 7.4 buffer. Damage was also assessed histologically. RESULTS: Based on histology, all the tissues were resistant to pH 4.0 except the subglottic mucosa. Only the posterior commissure was not damaged by pH 2.0 plus pepsin. Similar patterns were observed with absorbance changes and DNA release. CONCLUSIONS: The subglottic mucosa was the most susceptible to damage and the posterior commissure the least. Laryngeal tissues are essentially resistant to damage at pH 4.0, but are damaged when pepsin is present. This suggests that in LPR, pH 4.0 or above refluxate would only be damaging if it contains pepsin.
Asunto(s)
Ácido Gástrico/metabolismo , Mucosa Laríngea/patología , Reflujo Laringofaríngeo/patología , Pepsina A/efectos adversos , Animales , Modelos Animales de Enfermedad , Concentración de Iones de Hidrógeno , Mucosa Laríngea/efectos de los fármacos , Mucosa Laríngea/metabolismo , Reflujo Laringofaríngeo/etiología , Reflujo Laringofaríngeo/metabolismo , Laringe/metabolismo , Laringe/patología , Laringe/cirugía , Pepsina A/metabolismo , Estudios Prospectivos , PorcinosRESUMEN
Live Salmonella vaccines are limited in use by the inherent toxicity of the lipopolysaccharide. The waaN gene encodes a myristyl transferase required for the secondary acylation of lipid A in lipopolysaccharide. A waaN mutant exhibits reduced induction of the inflammatory cytokines associated with lipopolysaccharide toxicity. Here the characteristics of a Salmonella enterica serovar Typhimurium aroA waaN mutant (SK100) in vitro and in vivo compared with its parent aroA strain (SL3261) were described. Phenotypic analysis of purified lipopolysaccharide obtained from SK100 confirmed that the physical and biological activities of the lipopolysaccharide had been altered. Nevertheless both strains had similar patterns of colonization and persistence in mice and significantly the aroA waaN mutant was equally as effective as the parent at protecting against challenge with wild-type S. Typhimurium. Furthermore, a SK100 strain was constructed expressing both tetanus toxin fragment C and the circumsporozoite protein of a malaria parasite. In marked contrast to its isogenic parent, the new attenuated strain induces significantly enhanced immune responses against the circumsporozoite protein. The waaN mutation enhances the ability of this strain to elicit immune responses towards guest antigens. This study provides important insights into the development of safe and effective multivalent Salmonella vaccines.
Asunto(s)
Vacunas contra la Malaria/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/genética , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antiprotozoarios/sangre , Proteínas Bacterianas/genética , Línea Celular , Recuento de Colonia Microbiana , Femenino , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/toxicidad , Hígado/microbiología , Macrófagos/microbiología , Vacunas contra la Malaria/genética , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Polisacáridos Bacterianos/aislamiento & purificación , Polisacáridos Bacterianos/toxicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/prevención & control , Vacunas contra la Salmonella/genética , Salmonella typhimurium/inmunología , Salmonella typhimurium/patogenicidad , Bazo/microbiología , Toxina Tetánica/genética , Toxina Tetánica/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Vacunas Tifoides-Paratifoides/genética , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunologíaRESUMEN
Pathogenic bacteria employ a variety of mechanisms to resist a barrage of stresses they encounter during active growth in or outside the host as well as during growth stasis. An in silico screen of the Salmonella genome sequence revealed that Salmonella typhimurium LT2 possesses a homologue belonging to the universal stress protein A (UspA) family. We assessed the transcriptional profile of uspA in S. typhimurium C5 by constructing a lacZ fusion revealing that uspA is induced by metabolic, oxidative, and temperature stresses. The highest transcriptional levels occurred in cells entering stationary phase, an observation consistent with expression patterns in Escherichia coli. The protein was purified as a fusion with GST (UspA(F)) and antibodies raised against UspA(F) revealed elevated protein levels in stressed and growth-arrested cells. Inactivation of uspA in S. typhimurium C5, lead to increased susceptibility to stress conditions. Furthermore, UspA makes an important contribution to the in vivo virulence of Salmonella in mice thus highlighting the importance of stress resistance regulation in pathogenicity and survival within the host.