RESUMEN
Bacterial cell envelopes play a critical role in host-pathogen interactions. Macromolecular components of these structures have been closely linked to the virulence of pathogens. Piscirickettsia salmonis is a relevant salmonid pathogen with a worldwide distribution. This bacterium is the etiological agent of piscirickettsiosis, a septicemic disease that causes a high economic burden, especially for the Chilean salmon farming industry. Although P. salmonis has been discovered long ago, its pathogenicity and virulence mechanisms are not completely understood. In this work, we present a genetic approach for producing in-frame deletion mutants on genes related to the biosynthesis of membrane-associated polysaccharides. We provide a detailed in vitro phenotype description of knock-out mutants on wzx and wcaJ genes, which encode predicted lipopolysaccharide (LPS) flippase and undecaprenyl-phosphate glucose phosphotransferase enzymes, respectively. We exhibit evidence that the wzx mutant strain carries a defect in the probably most external LPS moiety, while the wcaJ mutant proved to be highly susceptible to the bactericidal action of serum but retained the ability of biofilm production. Beyond that, we demonstrate that the deletion of wzx, but not wcaJ, impairs the virulence of P. salmonis in an intraperitoneally infected Atlantic salmon, Salmo salar, model of piscirickettsiosis. Our findings support a role for LPS in the virulence of P. salmonis during the onset of piscirickettsiosis.
Asunto(s)
Enfermedades de los Peces , Salmo salar , Animales , Enfermedades de los Peces/microbiología , Lipopolisacáridos , Piscirickettsia , VirulenciaRESUMEN
Few studies have addressed the impact of the upwelling oceanographical conditions on biological processes, such as growth, using a molecular and physiological approach. Upwelling conditions are characterized by low temperature seawater and high nutrient availability, which represents an ideal opportunity to understand how habitat modulates animal performance at different levels of biological complexity. We aimed to assess intraspecific variations in weight, oxygen consumption, protein content, and key signaling pathways involved in muscle-growth (protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)) under experimental trials considering high/low seawater temperatures with full/restricted food rations. For this purpose, we studied Girella laevifrons, one of the most abundant fish species inhabiting rocky intertidal zones along the Eastern South Pacific coasts. Using fish obtained from upwelling (U) and non-upwelling (NU) zones, we reported that U animals displayed higher growth performance during both contrasting trials, with a weight gain (~3â¯g), lower oxygen consumption (~12%), and higher protein contents (~20%). Only ERK showed significant differences during the trials (~2-fold downregulation between NU and U fish). We also found that U fish increased protein ubiquitination in high water temperature and restricted food ration in contrast to NU fish. Our results help to elucidate how upwelling conditions may influence fish growth at physiological and molecular levels. Still, future analyses are necessary to improve the information regarding the impact of U and NU condition on animals, as well as the possible applications of this data in the aquaculture industry.
Asunto(s)
Peces/crecimiento & desarrollo , Músculo Esquelético/citología , Músculo Esquelético/crecimiento & desarrollo , Agua de Mar/química , Transducción de Señal , Animales , Peso Corporal , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Peces/metabolismo , Músculo Esquelético/metabolismo , Consumo de OxígenoRESUMEN
In order to establish infection, bacterial pathogens modulate host cellular processes by using virulence factors, which are delivered from the bacteria to the host cell leading to cellular reprogramming. In this context, several pathogens regulate the ubiquitin proteasome system in order to regulate the cellular effectors required for their successful colonization and persistance. In this study, we investigated how Helicobacter pylori affect the ubiquitination of the host proteins to achieve the adherence to the cells, using AGS gastric epithelial cells cultured with H. pylori strains, H. pylori 26695 and two isogenic mutants H. pylori cag::cat and vacA::apha3, to characterize the ability of H. pylori to reprogram the ubiquitin proteasome systems. The infection assays suggest that the ubiquitination of the total proteins does not change when cells were co-culture with H. pylori. We also found that the proteasome activity is necessary for H. pylori adhesion to AGS cells and the adherence increases when the level of KCTD5, an adaptor of Cullin-3, decrease. Moreover, we found that KCTD5 is ubiquitinated and degraded by the proteasome system and that CagA and VacA played no role on reducing KCTD5 levels. Furthermore, H. pylori impaired KCTD5 ubiquitination and did not increase global proteasome function. These results suggest that H. pylori affect the ubiquitin-proteasome system (UPS) to facilitate the adhesion of this microorganism to establish stable colonization in the gastric epithelium and improve our understanding of how H. pylori hijack host systems to establish the adherence.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/patogenicidad , Canales de Potasio/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Línea Celular , Técnicas de Cocultivo , Proteínas Cullin/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/fisiología , Interacciones Huésped-Patógeno/fisiología , Humanos , Lisosomas , Factores de Virulencia/metabolismoRESUMEN
Growth is one of the main biological processes in aquatic organisms that is affected by environmental fluctuations such as upwelling (characterized by food-rich waters). In fish, growth is directly related with skeletal muscle increase; which represents the largest tissue of body mass. However, the effects of upwelling on growth, at the physiological and molecular level, are unknown. This study used Girella laevifrons (one of the most abundant intertidal fish in Eastern South Pacific) as a biological model, considering animals from upwelling (U) and non-upwelling (NU) areas. Here, we evaluated the effect of nutritional composition and food availability on growth performance and expression of key growth-related genes (insulin-kike growth factor 1 (igf1) and myosin heavy-chain (myhc)) and atrophy-related genes (muscle ring-finger 1 (murf1), F-box only protein 32 (atrogin-1) and BCL2/adenovirus E1B 19kDa-interacting protein 3 (bnip3)). We reported that, among zones, U fish displayed higher growth performance in response to nutritional composition, specifically between protein- and fiber-rich diets (~1g). We also found in NU fish that atrophy-related genes were upregulated with fiber-rich diet and during fasting (~2-fold at minimum respect U). In conclusion, our results suggest that the growth potential of upwelling fish may be a consequence of differential muscle gene expression. Our data provide a preliminary approach contributing on how upwelling influence fish growth at the physiological and molecular levels. Future studies are required to gain further knowledge about molecular differences between U and NU animals, as well as the possible applications of this knowledge in the aquaculture industry.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Perciformes/crecimiento & desarrollo , Perciformes/genética , Animales , Dieta de Carga de Carbohidratos , Ecosistema , Cadena Alimentaria , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/crecimiento & desarrollo , Perciformes/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Ríos/química , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Agua de Mar/química , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismoRESUMEN
Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection. [Int Microbiol 2016; 49-55].
Asunto(s)
Proteínas Bacterianas/metabolismo , Piscirickettsia/metabolismo , Proteínas Represoras/metabolismo , Animales , Proteínas Bacterianas/genética , Enfermedades de los Peces/microbiología , Peces/microbiología , Genes Bacterianos , Hierro/metabolismo , Proteínas Represoras/genéticaRESUMEN
The genus Edwardsiella consists of bacteria with an intrinsic resistance to cyclic cationic antimicrobial peptides (CAMPs). Edwardsiella ictaluri, a pathogen of the catfish (Ictalurus punctatus) and the causative agent of a systemic infection, is highly resistant to CAMPs. Previously, we determined that the oligo-polysaccharide (O-PS) of the lipopolysaccharide (LPS) does not play a role in the E. ictaluri CAMP resistance and an intact core-lipid A structure is necessary for CAMPs resistance. Here, we evaluated the influence of the outer-core in the CAMPs resistance and fish virulence. E. ictaluri wabG, a gene that encodes for the UDP-glucuronic acid transferase that links the lipid A-inner-core to the outer-core-oligopolysaccharides, was deleted. Deletion of ΔwabG caused a pleiotropic effect, influencing LPS synthesis, CAMPs resistance, growth, and biofilm formation. E. ictaluri ΔwabG was attenuated in zebrafish indicating the important role of LPS during fish pathogenesis. Also, we evaluated the inflammatory effects of wabG LPS in catfish ligated loop model, showing a decreased inflammatory effect at the gut level respects to the E. ictaluri wild type. We conclude that E. ictaluri CAMPs resistance is related to the molecules present in the LPS outer-core and that fish gut inflammation triggered by E. ictaluri is LPS dependent, reinforcing the hypothesis that fish gut recognizes LPS in an O-PS dependent fashion.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Edwardsiella ictaluri/metabolismo , Edwardsiella ictaluri/patogenicidad , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/microbiología , Lipopolisacáridos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Edwardsiella ictaluri/efectos de los fármacos , Edwardsiella ictaluri/genética , Infecciones por Enterobacteriaceae/microbiología , Ictaluridae , Datos de Secuencia Molecular , Alineación de Secuencia , Virulencia , Pez CebraRESUMEN
Cellular migration and contractility are fundamental processes that are regulated by a variety of concerted mechanisms such as cytoskeleton rearrangements, focal adhesion turnover, and Ca2+ oscillations. TRPM4 is a Ca2+-activated non-selective cationic channel (Ca2+-NSCC) that conducts monovalent but not divalent cations. Here, we used a mass spectrometry-based proteomics approach to identify putative TRPM4-associated proteins. Interestingly, the largest group of these proteins has actin cytoskeleton-related functions, and among these nine are specifically annotated as focal adhesion-related proteins. Consistent with these results, we found that TRPM4 localizes to focal adhesions in cells from different cellular lineages. We show that suppression of TRPM4 in MEFs impacts turnover of focal adhesions, serum-induced Ca2+ influx, focal adhesion kinase (FAK) and Rac activities, and results in reduced cellular spreading, migration and contractile behavior. Finally, we demonstrate that the inhibition of TRPM4 activity alters cellular contractility in vivo, affecting cutaneous wound healing. Together, these findings provide the first evidence, to our knowledge, for a TRP channel specifically localized to focal adhesions, where it performs a central role in modulating cellular migration and contractility.
Asunto(s)
Actinas/metabolismo , Adhesiones Focales/metabolismo , Contracción Muscular/genética , Proteómica , Canales Catiónicos TRPM/metabolismo , Calcio/metabolismo , Linaje de la Célula , Movimiento Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/genética , Humanos , Fosforilación , Canales Catiónicos TRPM/genéticaRESUMEN
BACKGROUND: Bacterial genomes are compacted by association with histone-like proteins to form a complex known as bacterial chromatin. The histone-like protein HU is capable of binding and bending the DNA molecule, a function related to compaction, protection, and regulation of gene expression. In Helicobacter pylori, HU is the only histone-like protein described so far. Proteomic analysis from our laboratory showed that this protein is overexpressed under acidic stress. MATERIALS AND METHODS: We used a purified recombinant wild-type protein and two mutant proteins with the amino acid substitutions K3A/S27D and K62R/V63N/P64A to characterize the function of the N-terminal domain and the flexible arm of HU. RESULTS: In vitro assays for DNA protection, bending, and compaction were performed. We also designed a H. pylori hup::cat mutant strain to study the role of HU in the acid stress response. HUwt protein binds DNA and promotes its bending and compaction. Compared with the wild-type protein, both mutant proteins have less affinity for DNA and an impaired bending and compaction ability. By using qRT-PCR, we confirmed overexpression of two genes related to acid stress response (ureA and speA). Such overexpression was abolished in the hup::cat strain, which shows an acid-sensitive phenotype. CONCLUSIONS: Altogether, we have shown that HUwt -DNA complex formation is favored under acidic pH and that the complex protects DNA from endonucleolytic cleavage and oxidative stress damage. We also showed that the amino-terminal domain of HU is relevant to DNA-protein complex formation and that the flexible arm of HU is involved in the bending and compaction activities of HU.
Asunto(s)
Ácidos/toxicidad , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/fisiología , Estrés Fisiológico , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Perfilación de la Expresión Génica , Genes Bacterianos , Concentración de Iones de Hidrógeno , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Unión ProteicaRESUMEN
BACKGROUND: Helicobacter pylori is a motile microaerophilic bacterium that colonizes the human stomach. H. pylori infection triggers gastric diseases, such as gastritis, peptic ulcer and gastric cancer. Stomach represents a barrier for microorganism colonization, particularly because of its high hydrochloric acid concentration. The main mechanism developed by H. pylori to maintain intracellular pH homeostasis in this environment is the urease activity. However, urease negative strains can be also isolated from clinical samples, suggesting that H. pylori presents other components involved in acid resistance. OBJECTIVE: Here, we present some evidence that the arginine decarboxylase gene (speA) in H. pylori could be involved in an acid adaptation mechanism similar to the one in Enterobacteriaceae, which is dependent on the presence of arginine. METHODS: Indeed, speA mRNA and protein expression are acutely induced by acid stress. RESULTS: Moreover, we showed that H. pylori uses arginine in an acid response mechanism required for its growth in acid conditions. CONCLUSION: Altogether, these results provide novel information regarding the H. pylori physiology and acid response mechanism.
Asunto(s)
Ácidos/toxicidad , Carboxiliasas/metabolismo , Tolerancia a Medicamentos , Helicobacter pylori/enzimología , Helicobacter pylori/fisiología , Carboxiliasas/genética , Perfilación de la Expresión Génica , Helicobacter pylori/genética , Homeostasis , Humanos , Concentración de Iones de HidrógenoRESUMEN
Helicobacter pylori is the etiologic agent of a series of gastric pathologies that may culminate in the development of gastric adenocarcinoma. An initial step in this process is the loss of glandular structures in the gastric mucosa, presumably as the consequence of increased apoptosis and reduced cellular regeneration, which may be attributed to the combination of several bacterial and host factors and to an unfavorable proinflammatory environment. In a previous study, we showed that survivin, a member of the inhibitor of apoptosis protein family, is expressed in the normal human gastric mucosa and that its levels decrease in the mucosa of infected patients and in gastric cells exposed in culture to the bacteria, coincident with increased cell death in the latter case. We investigated the bacterial factors responsible for loss of survivin in gastric cells exposed to H. pylori. The results of this study indicated that the loss of survivin due to H. pylori infection involves proteasome-mediated degradation of the protein. Studies with isogenic mutants deficient in either CagA, VacA, lipopolysaccharide, or gamma-glutamyl transpeptidase (GGT) implicated the latter in H. pylori-induced loss of survivin and cell viability. Moreover, experiments with the GGT inhibitor 6-diazo-5-oxo-l-norleucine and purified recombinant GGT protein indicated that secreted bacterial GGT activity was required and sufficient to induce these effects.