RESUMEN
Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S') transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.
Asunto(s)
Bacteriófago P1 , Escherichia coli , Antígenos O , Shigella flexneri , Transducción Genética , Proteínas de la Cola de los Virus , Escherichia coli/genética , Escherichia coli/virología , Antígenos O/genética , Antígenos O/fisiología , Shigella flexneri/genética , Shigella flexneri/virología , Bacteriófago P1/genética , Bacteriófago P1/fisiología , Proteínas de la Cola de los Virus/genéticaRESUMEN
Bacteriophages are highly abundant molecular machines that have evolved proteins to target the surface of host bacterial cells. Given the ubiquity of lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria, we reasoned that targeting proteins from bacteriophages could be leveraged to target the surface of Gram-negative pathogens for biotechnological applications. To this end, a short tail fiber (GP12) from the T4 bacteriophage, which infects Escherichia coli (E. coli), was isolated and tested for the ability to adhere to whole bacterial cells. We found that, surprisingly, GP12 effectively bound the surface of Pseudomonas aeruginosa cells despite the established preferred host of T4 for E. coli. In efforts to elucidate why this binding pattern was observed, it was determined that the absence of the O-antigen region of LPS on E. coli improved cell surface tagging. This indicated that O-antigens play a significant role in controlling cell adhesion by T4. Probing GP12 and LPS interactions further using deletions of the enzymes involved in the biosynthetic pathway of LPS revealed the inner core oligosaccharide as a possible main target of GP12. Finally, we demonstrated the potential utility of GP12 for biomedical applications by showing that GP12-modified agarose beads resulted in the depletion of pathogenic bacteria from solution.
Asunto(s)
Escherichia coli/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Estructurales Virales/metabolismo , Membrana Externa Bacteriana/metabolismo , Adhesión Celular , Lipopolisacáridos/metabolismo , Antígenos O/fisiología , Oligosacáridos/metabolismoRESUMEN
Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD-mediated pyroptosis via activation of caspase-1, caspase-4 and caspase-8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island-1 (SPI-1) injectisome, HilA-mediated overexpression of the SPI-1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O-antigen length regulator, which induces the production of very long O-antigen chains. Using a ΔfepE mutant we established that the very long O-antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome-mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar-specific inflammasome modulation, which mirrors the pro- and anti-inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome-mediated detection through the elaboration of very long LPS O-polysaccharides.
Asunto(s)
Inflamasomas/metabolismo , Macrófagos/microbiología , Macrófagos/fisiología , Antígenos O/fisiología , Fiebre Paratifoidea/microbiología , Piroptosis , Salmonella paratyphi A/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Caspasas/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/inmunología , Antígenos O/química , Proteínas de Unión a Fosfato/metabolismo , Salmonella paratyphi A/inmunología , Células THP-1 , Sistemas de Secreción Tipo III/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Salmonella Enteritidis is a non-typhoidal serovar of great public health significance worldwide. The RpoE sigma factor and CpxRA two-component system are the major regulators of the extracytoplasmic stress response. In this study, we found that the CpxR has highly significant, but opposite effects on the auto-aggregation and swarming motility of S. Enteritidis. Auto-aggregation was negatively affected in the ∆cpxR mutant, whereas the same mutant significantly out-performed its wild-type counterpart with respect to swarming motility, indicating that the CpxR plays a role in biofilm-associated phenotypes. Indeed, biofilm-related assays showed that the CpxR is of critical importance in biofilm development under both static (microtiter plate) and dynamic (flow cell) media flow conditions. In contrast, the RpoE sigma factor showed no significant role in biofilm development under dynamic conditions. Transcriptomic analysis revealed that the cpxR mutation negatively affected the constitutive expression of the operons critical for biosynthesis of O-antigen and adherence, but positively affected the expression of virulence genes critical for Salmonella-mediated endocytosis. Conversely, CpxR induced the expression of curli csgAB and fimbrial stdAC operons only during biofilm development and flagellar motAB and fliL operons exclusively during the planktonic phase, indicating a responsive biofilm-associated loop of the CpxR regulator.
Asunto(s)
Proteínas Bacterianas/fisiología , Biopelículas/crecimiento & desarrollo , Proteínas Fimbrias/fisiología , Antígenos O/fisiología , Salmonella enteritidis/fisiología , Factores de Virulencia/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Perfilación de la Expresión Génica , Antígenos O/genética , Antígenos O/metabolismo , Salmonella enteritidis/genética , Salmonella enteritidis/metabolismo , TranscriptomaRESUMEN
Salmonella enterica serovar Typhimurium, a Gram-negative bacterium, can cause infectious diseases ranging from gastroenteritis to systemic dissemination and infection. However, the molecular mechanisms underlying this bacterial dissemination have yet to be elucidated. A study indicated that using the lipopolysaccharide (LPS) core as a ligand, S Typhimurium was able to bind human dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (hCD209a), an HIV receptor that promotes viral dissemination by hijacking antigen-presenting cells (APCs). In this study, we showed that S Typhimurium interacted with CD209s, leading to the invasion of APCs and potentially the dissemination to regional lymph nodes, spleen, and liver in mice. Shielding of the exposed LPS core through the expression of O-antigen reduces dissemination and infection. Thus, we propose that similar to HIV, S Typhimurium may also utilize APCs via interactions with CD209s as a way to disseminate to the lymph nodes, spleen, and liver to initiate host infection.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Salmonella typhimurium/patogenicidad , Animales , Células Presentadoras de Antígenos/microbiología , Femenino , Interacciones Huésped-Patógeno , Humanos , Lipopolisacáridos/fisiología , Mananos/farmacología , Ratones , Ratones Endogámicos C57BL , Antígenos O/fisiología , Ganglios Linfáticos Agregados/fisiología , Fagocitosis , Células RAW 264.7RESUMEN
Tailed bacteriophages specific for Gram-negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events that orchestrate phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O-antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O-antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O-antigen specific host attachment must not always come along with O-antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O-antigen but does not degrade it, whereas rough strains or strains lacking O-antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host's O-antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O-antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps.
Asunto(s)
Antígenos O/metabolismo , Proteínas de la Cola de los Virus/metabolismo , Bacteriófago P22/metabolismo , Bacteriófagos/fisiología , Lipopolisacáridos/metabolismo , Antígenos O/fisiología , Salmonella typhimurium/metabolismo , Relación Estructura-ActividadRESUMEN
Riptortus pedestris harboring Burkholderia symbiont is a useful symbiosis model to study the molecular interactions between insects and bacteria. We recently reported that the lipopolysaccharide O-antigen is absent in the Burkholderia symbionts isolated from Riptortus guts. Here, we investigated the symbiotic role of O-antigen comprehensively in the Riptortus-Burkholderia model. Firstly, Burkholderia mutant strains deficient of O-antigen biosynthesis genes were generated and confirmed for their different patterns of the lipopolysaccharide by electrophoretic analysis. The O-antigen-deficient mutant strains initially exhibited a reduction of infectivity, having significantly lower level of symbiont population at the second-instar stage. However, both the wild-type and O-antigen mutant symbionts exhibited a similar level of symbiont population from the third-instar stage, indicating that the O-antigen deficiency did not affect the bacterial persistence in the host midgut. Taken together, we showed that the lipopolysaccharide O-antigen of gut symbiont plays an exclusive role in the initial symbiotic association.
Asunto(s)
Burkholderia/fisiología , Heterópteros/microbiología , Antígenos O/fisiología , Animales , Antibacterianos/farmacología , Larva/microbiología , Pruebas de Sensibilidad Microbiana , Polimixina B/farmacología , SimbiosisRESUMEN
Bacterial surface components have a major role in the development of biofilms. In the present study, the effect of Escherichia coli O8-antigen on biofilms was investigated using two E. coli K-12 derived strains that differed only in the O8-antigen biosynthesis. In the presence of O8-antigen both bacterial adhesion and biofilm formation slightly decreased under static conditions whereas a substantial increase in adhesion and biofilm formation was observed under agitated conditions. It was noted that, irrespective of the O8-antigen status, the hydrophobic interactions played an important role in bacterial adhesion under both static and agitated conditions. However, under agitated conditions, the extent of bacterial adhesion in the O8-antigen bearing strain was predominantly determined by the electrostatic interactions. Results showed that the presence of O8-antigen decreases the surface hydrophobicity and surface charge. Moreover, O8-antigen facilitates adhesion on hydrophilic and hydrophobic surfaces as revealed through tests with modified substrata. Our results indicate that O8-antigen, which appears dispensable for biofilm formation under static conditions, actually enhances E. coli biofilm formation under agitated conditions.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Escherichia coli K12/fisiología , Antígenos O/fisiología , Adhesión Bacteriana , Escherichia coli K12/genética , Escherichia coli K12/inmunología , Escherichia coli K12/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Lipopolisacáridos/fisiología , Antígenos O/química , Antígenos O/genética , Propiedades de SuperficieRESUMEN
We previously reported that lipopolysaccharide (LPS) -related sugars are associated with the glycosylation of the collagen adhesin EmaA, a virulence determinant of Aggregatibacter actinomycetemcomitans. In this study, the role of LPS in the secretion of other virulence factors was investigated. The secretion of the epithelial adhesin Aae, the immunoglobulin Fc receptor Omp34 and leukotoxin were examined in a mutant strain with inactivated TDP-4-keto-6-deoxy-d-glucose 3,5-epimerase (rmlC), which resulted in altered O-antigen polysaccharides (O-PS) of LPS. The secretion of Aae and Omp34 was not affected. However, the leukotoxin secretion, which is mediated by the TolC-dependent type I secretion system, was altered in the rmlC mutant. The amount of secreted leukotoxin in the bacterial growth medium was reduced nine-fold, with a concurrent four-fold increase of the membrane-bound toxin in the mutant compared with the wild-type strain. The altered leukotoxin secretion pattern was restored to the wild-type by complementation of the rmlC gene in trans. Examination of the ltxA mRNA levels indicated that the leukotoxin secretion was post-transcriptionally regulated in the modified O-PS containing strain. The mutant strain also showed increased resistance to vancomycin, an antibiotic dependent on TolC for internalization, indicating that TolC was affected. Overexpression of TolC in the rmlC mutant resulted in an increased TolC level in the outer membrane but did not restore the leukotoxin secretion profile to the wild-type phenotype. The data suggest that O-PS mediate leukotoxin secretion in A. actinomycetemcomitans.
Asunto(s)
Aggregatibacter actinomycetemcomitans/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Carbohidrato Epimerasas/genética , Exotoxinas/biosíntesis , Lipopolisacáridos/fisiología , Proteínas de Transporte de Membrana/genética , Factores de Virulencia/biosíntesis , Aggregatibacter actinomycetemcomitans/genética , Proteínas de la Membrana Bacteriana Externa/genética , Carbohidrato Epimerasas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Técnicas de Inactivación de Genes , Lipopolisacáridos/química , Mutación , Antígenos O/fisiología , Resistencia a la Vancomicina/genéticaRESUMEN
Human brucellosis is caused mainly by Brucella melitensis, which is often acquired by ingesting contaminated goat or sheep milk and cheese. Bacterial factors required for food-borne infection of humans by B. melitensis are poorly understood. In this study, a mouse model of oral infection was characterized to assess the roles of urease, the VirB type IV secretion system, and lipopolysaccharide for establishing infection through the digestive tract. B. melitensis strain 16M was consistently recovered from the mesenteric lymph node (MLN), spleen, and liver beginning at 3 or 7 day postinfection (dpi). In the gut, persistence of the inoculum was observed up to 21 dpi. No inflammatory lesions were observed in the ileum or colon during infection. Mutant strains lacking the ureABC genes of the ure1 operon, virB2, or pmm encoding phosphomannomutase were constructed and compared to the wild-type strain for infectivity through the digestive tract. Mutants lacking the virB2 and pmm genes were attenuated in the spleen (P < 0.05) and MLN (P < 0.001), respectively. The wild-type and mutant strains had similar levels of resistance to low pH and 5 or 10% bile, suggesting that the reduced colonization of mutants was not the result of reduced resistance to acid pH or bile salts. In an in vitro lymphoepithelial cell (M-cell) model, B. melitensis transited rapidly through polarized enterocyte monolayers containing M-like cells; however, transit through monolayers containing only enterocytes was reduced or absent. These results indicate that B. melitensis is able to spread systemically from the digestive tract after infection, most likely through M cells of the mucosa-associated lymphoid tissue.
Asunto(s)
Proteínas Bacterianas/fisiología , Brucella melitensis/patogenicidad , Tracto Gastrointestinal/microbiología , Lipopolisacáridos/fisiología , Proteínas de Transporte de Membrana/fisiología , Antígenos O/fisiología , Ureasa/fisiología , Factores de Virulencia/fisiología , Animales , Proteínas Bacterianas/genética , Línea Celular , Colon/patología , Recuento de Colonia Microbiana , Enterocitos/microbiología , Femenino , Eliminación de Gen , Íleon/patología , Lipopolisacáridos/genética , Hígado/microbiología , Ganglios Linfáticos/microbiología , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Antígenos O/genética , Bazo/microbiología , Ureasa/genética , Factores de Virulencia/genéticaRESUMEN
Brucella abortus es una bacteria que causa abortos e infertilidad en el ganado y fiebre ondulante en el hombre. Se multiplica en el citoplasma celular evadiendo los mecanismos de muerte intracelular. El óxido nítrico (NO) es importante en la regulación de la respuesta inmune. En el presente trabajo estudiamos la habilidad de tres cepas de B. abortus para sobrevivir intracelularmente en dos líneas celulares de macrófagos. La multiplicación de bacterias en ambas líneas celulares fue determinada a distintos tiempos en número de UFC/ml, también fue observada al microscopio de campo claro y de fluorescencia utilizando Giemsa y naranja de acridina, respectivamente. La tinción de ambas líneas celulares inoculadas con B. abortus mostró un resultado concordante con el encontrado en la determinación del número de UFC. Fue confirmada la presencia de B. abortus por microscopía electrónica. Para medir la producción de NO se utilizó el reactivo de Griess. La multiplicación de la cepa rugosa RB51 disminuyó en ambas líneas celulares y los niveles de NO fueron mayores en células inoculadas con dicha cepa que cuando fueron inoculadas con las cepas lisas (S19 y 2308). Estos resultados sugieren que probablemente la ausencia de cadena O en el lipopolisacárido afecta el crecimiento intracelular de B. abortus.
Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably has affects the intracellular growth of B. abortus.
Asunto(s)
Animales , Bovinos , Ratones , Cápsulas Bacterianas/fisiología , Brucella abortus/crecimiento & desarrollo , Macrófagos/microbiología , Óxido Nítrico/biosíntesis , Cápsulas Bacterianas/química , Brucella abortus/clasificación , Brucella abortus/metabolismo , Brucella abortus/ultraestructura , División Celular , Línea Celular/metabolismo , Línea Celular/microbiología , Microscopía Electrónica , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Macrófagos/metabolismo , Antígenos O/fisiología , Especificidad de la EspecieRESUMEN
Burkholderia pseudomallei is a facultative intracellular gram-negative bacterium that can survive and multiply inside macrophages. One of the mechanisms by which B. pseudomallei escapes macrophage killing is by interfering with the expression of inducible nitric oxide synthase (iNOS). However, the bacterial components that modulate antimicrobial activity of the macrophage have not been fully elucidated. In the present study, we demonstrated that B. pseudomallei strain SRM117, a lipopolysaccharide (LPS) mutant that lacks the O-antigenic polysaccharide moiety, was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain (1026b). Unlike the wild type, the LPS mutant could readily stimulate Y701-STAT-1 phosphorylation (pY701-STAT-1) and interferon-regulatory factor 1 (IRF-1) expression, both of which are essential transcription factors of iNOS. Neutralizing antibody against beta interferon was able to inhibit the phosphorylation of Y701-STAT-1 and the expression of IRF-1 and iNOS, all of which resulted in an increased rate of intracellular replication. These data suggest that the O-antigenic polysaccharide moiety of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophages.
Asunto(s)
Burkholderia pseudomallei/crecimiento & desarrollo , Burkholderia pseudomallei/genética , Líquido Intracelular/microbiología , Lipopolisacáridos/metabolismo , Macrófagos/microbiología , Antígenos O/fisiología , Polisacáridos Bacterianos/fisiología , Animales , Burkholderia pseudomallei/metabolismo , Línea Celular , Líquido Intracelular/metabolismo , Macrófagos/metabolismo , Ratones , Mutación , Antígenos O/genética , Antígenos O/metabolismo , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismoRESUMEN
In this article, different aspects of virulence factors of Proteus bacilii (P. mirabilis, P. vulgaris, P. penneri i P. hauseri) are presented. These are opportunistic pathogens that cause different kinds of infections, most frequently of the urinary tract. These bacteria have developed several virulence factors, such as adherence due to the presence of fimbriae or afimbrial adhesins, invasiveness, swarming phenomenon, hemolytic activity, urea hydrolysis, proteolysis, and endotoxicity. Below we focus on data concerning the molecular basis of the pathogenicity of Proteus bacilli.
Asunto(s)
Infecciones por Proteus/microbiología , Proteus/fisiología , Proteus/patogenicidad , Factores de Virulencia/química , Factores de Virulencia/fisiología , Animales , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/fisiología , Biopelículas/crecimiento & desarrollo , Secuencia de Carbohidratos/fisiología , Catéteres de Permanencia/microbiología , Fimbrias Bacterianas/fisiología , Proteínas Hemolisinas/fisiología , Humanos , Ratones , Antígenos O/química , Antígenos O/fisiología , Proteus/química , Proteus/clasificación , Infecciones por Proteus/fisiopatología , Conejos , Especificidad de la Especie , Infecciones Urinarias/microbiologíaRESUMEN
Predation from intestinal amoebae may provide selective pressure for the maintenance of high genetic diversity at the Salmonella enterica rfb locus, whereby serovars better escape predators in particular environments depending on the O-antigens they express. Here, the hypothesis that amoebae from a particular intestinal environment collectively prefer one serovar over another is tested. Collections of Acanthamoeba, Tetramitus, Naegleria and Hartmannella were isolated from the intestinal tracts of several vertebrate hosts, including bullfrog tadpoles, goldfish, turtles and bearded dragons, and their feeding preferences were determined. Congeneric amoebae from the same environment had significantly similar feeding preferences. Strikingly, even unrelated amoebae - such as Naegleria and Tetramitus from goldfish - also had significantly similar feeding preferences. Yet amoebae isolated from different environments showed no similarity in prey choice. Thus, feeding preferences of amoebae appear to reflect their environment, not their taxonomic relationships. A mechanism mediating this phenotypic convergence is discussed.
Asunto(s)
Amoeba/crecimiento & desarrollo , Intestinos/microbiología , Intestinos/parasitología , Viabilidad Microbiana , Salmonella enterica/crecimiento & desarrollo , Acanthamoeba/crecimiento & desarrollo , Acanthamoeba/aislamiento & purificación , Amoeba/clasificación , Amoeba/genética , Amoeba/aislamiento & purificación , Animales , ADN Protozoario/química , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Carpa Dorada/microbiología , Carpa Dorada/parasitología , Hartmannella/crecimiento & desarrollo , Hartmannella/aislamiento & purificación , Datos de Secuencia Molecular , Naegleria/crecimiento & desarrollo , Naegleria/aislamiento & purificación , Antígenos O/fisiología , Filogenia , ARN Ribosómico 18S/genética , Rana catesbeiana/microbiología , Rana catesbeiana/parasitología , Análisis de Secuencia de ADN , Tortugas/microbiología , Tortugas/parasitologíaRESUMEN
Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus.
Asunto(s)
Cápsulas Bacterianas/fisiología , Brucella abortus/crecimiento & desarrollo , Macrófagos/microbiología , Óxido Nítrico/biosíntesis , Animales , Cápsulas Bacterianas/química , Brucella abortus/clasificación , Brucella abortus/metabolismo , Brucella abortus/ultraestructura , Bovinos , División Celular , Línea Celular/metabolismo , Línea Celular/microbiología , Macrófagos/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Ratones , Microscopía Electrónica , Antígenos O/fisiología , Especificidad de la EspecieRESUMEN
We show that the expression of a Yersinia enterocolitica O:8 pYV-encoded type III secretion system was altered in a rough mutant (YeO8-R) due to elevated levels of FlhDC. H-NS might underlie flhDC upregulation in YeO8-R, and the data suggest a relationship between the absence of O antigen and the expression of H-NS.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Antígenos O/fisiología , Yersinia enterocolitica/genética , Yersinia enterocolitica/patogenicidad , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Células HeLa , Humanos , Mutación , Antígenos O/biosíntesis , Antígenos O/genética , Plásmidos/genética , Plásmidos/metabolismo , Virulencia/genética , Yersinia enterocolitica/inmunologíaRESUMEN
Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.
Asunto(s)
Bacteriófago P1/crecimiento & desarrollo , Escherichia coli O157/patogenicidad , Escherichia coli O157/virología , Proteínas de Escherichia coli/genética , Intestinos/microbiología , UDPglucosa 4-Epimerasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacteriólisis , Proteínas Sanguíneas/farmacología , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/genética , Proteínas de Escherichia coli/fisiología , Eliminación de Gen , Proteínas de la Membrana/farmacología , Mutagénesis Insercional , Antígenos O/genética , Antígenos O/fisiología , Conejos , Transducción Genética , UDPglucosa 4-Epimerasa/fisiología , UTP-Glucosa-1-Fosfato Uridililtransferasa/fisiología , Factores de Virulencia/genética , Factores de Virulencia/fisiologíaRESUMEN
Plesiomonas shigelloides is a Gram-negative bacterium associated with waterborne infections, which is common in tropical and subtropical habitats. Contrary to the unified antigenic classification of P. shigelloides, data concerning the structure and activity of their lipopolysaccharides (LPS and endotoxin) are limited. This study completes the structural investigation of phenol- and water-soluble fractions of P. shigelloides O74 (strain CNCTC 144/92) LPS with the emphasis on lipid A heterogeneity, describing the entire molecule and some of its biological in vitro activities. Structures of the lipid A and the affinity-purified decasaccharide obtained by de-N,O-acylation of P. shigelloides O74 LPS were elucidated by chemical analysis combined with electrospray ionization multiple-stage mass spectrometry (ESI-MS(n)), MALDI-TOF MS, and NMR spectroscopy. Lipid A of P. shigelloides O74 is heterogeneous, and three major forms have been identified. They all were asymmetric, phosphorylated, and hexaacylated, showing different acylation patterns. The beta-GlcpN4P-(1-->6)-alpha-GlcpN1P disaccharide was substituted with the primary fatty acids: (R)-3-hydroxytetradecanoic acid [14:0(3-OH)] at N-2 and N-2' and (R)-3-hydroxydodecanoic acid [12:0(3-OH)] at O-3 and O-3'. The heterogeneity among the three forms (I-III) of P. shigelloides O74 lipid A was attributed to the substitution of the acyl residues at N-2' and O-3' with the secondary acyls: (I) cis-9-hexadecenoic acid (9c-16:1) at N-2' and 12:0 at O-3', (II) 14:0 at N-2' and 12:0 at O-3', and (III) 12:0 at N-2' and 12:0 at O-3'. The pro-inflammatory cytokine-inducing activities of P. shigelloides O74 LPS were similar to those of Escherichia coli O55 LPS.
Asunto(s)
Lípido A/química , Lípido A/fisiología , Antígenos O/química , Antígenos O/fisiología , Plesiomonas/química , Secuencia de Carbohidratos , Lípido A/aislamiento & purificación , Espectroscopía de Resonancia Magnética , Modelos Químicos , Datos de Secuencia Molecular , Antígenos O/aislamiento & purificación , Oligosacáridos/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Francisella tularensis live vaccine strain (LVS) produces two colony types when grown on solid media, often referred to as blue variants (BV) and grey variants (GV). Whereas blue variant bacteria possessed a lipopolysaccharide O-side chain, grey variant bacteria lacked O-side chains. Grey variant bacteria appeared in stationary phase bacterial cultures and could be identified using a novel FACS-based assay. Compared to blue variant bacteria, grey variants showed a reduced ability to infect and survive in macrophages. The immunisation of mice with blue variant bacteria, but not grey variant bacteria, induced protective immunity towards fully virulent F. tularensis.
Asunto(s)
Vacunas Bacterianas/inmunología , Francisella tularensis/inmunología , Macrófagos/microbiología , Antígenos O/fisiología , Animales , Línea Celular , Citometría de Flujo , Francisella tularensis/clasificación , Francisella tularensis/aislamiento & purificación , Francisella tularensis/fisiología , Ratones , Ratones Endogámicos BALB CRESUMEN
Yersinia pestis, the cause of bubonic plague, blocks feeding by its vector, the flea. Recent evidence indicates that blockage is mediated by an in vivo biofilm. Y. pestis and the closely related Yersinia pseudotuberculosis also make biofilms on the cuticle of the nematode Caenorhabditis elegans, which block this laboratory animal's feeding. Random screening of Y. pseudotuberculosis transposon insertion mutants with a C. elegans biofilm assay identified gmhA as a gene required for normal biofilms. gmhA encodes phosphoheptose isomerase, an enzyme required for synthesis of heptose, a conserved component of lipopolysaccharide and lipooligosaccharide. A Y. pestis gmhA mutant was constructed and was severely defective for C. elegans biofilm formation and for flea blockage but only moderately defective in an in vitro biofilm assay. These results validate use of the C. elegans biofilm system to identify genes and pathways involved in Y. pestis flea blockage.