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Intracellular Growth (intracellular + growth)
Selected AbstractsInvasiveness and Intracellular Growth of Multidrug-Resistant Salmonella and Other Pathogens in Caco-2 CellsJOURNAL OF FOOD SCIENCE, Issue 2 2007S.-H. Kim ABSTRACT:, The increase of multidrug-resistant pathogens of human and animal origins is a major public health concern. For a better understanding of the health consequences of multidrug-resistant bacteria transmitted from animal products to humans, the host interaction of zoonotic Salmonella isolates along with other pathogenic and commensal bacteria was evaluated using a human intestinal Caco-2 cell system. Multidrug-resistant S. Agona, S. Heidelberg, and S. Typhimurium possessed plasmid-mediated class 1 integrons. The S. Typhimurium DT104 isolate from ground beef showed the well-known genotypic and phenotypic resistance characteristics of the species, and contained the chromosomally located class 1 integron. Among the multidrug-resistant Salmonella isolates, the S. Heidelberg 219 had the highest invasion number at 1.0 × 104 CFU/mL, followed by the S. Typhimurium DT104 isolate at 7.7 × 103 CFU/mL. Listeria monocytogenes was the best performer among the tested species in invading the Caco-2 cell. Multidrug-resistant opportunistic pathogens Klebsiella pneumoniae and Pseudomonas aeruginosa were also able to invade the cells. The invasion of S. Heidelberg 219, S. Typhimurium DT104, L. monocytogenes, K. pneumoniae, and P. aeruginosa into the Caco-2 cells was not affected even in the presence of commensal E. coli. During the intracellular growth of S. Heidelberg 219, S. Typhimurium DT104, and L. monocytogenes, the bacterial counts increased 2 log cycles in 9 h in the Caco-2 cells. Therefore, these strains could rapidly proliferate after their invasion into the cells. [source] Ultrastructural and Cytochemical Studies on Cellulose, Xylan and Pectin Degradation in Wheat Spikes Infected by Fusarium culmorumJOURNAL OF PHYTOPATHOLOGY, Issue 5 2000Z. Kang The cell wall components cellulose, xylan and pectin in different tissues of noninoculated healthy and Fusarium culmorum (W. G. Smith) Sacc-infected wheat spikes were localized by means of enzyme-gold and immuno-gold labelling techniques. The cell walls in the ovary, lemma and rachis of the healthy wheat spike showed labellings in different patterns and densities with cellulase-gold and xylanase-gold probes, as well as with the antipectin monoclonal antibody JIM7. The inter- and intracellular growth of the pathogen in the ovary, lemma and rachis of the infected wheat spike, not only caused pronounced alterations of cell walls and middle lamella matrices, but also led to marked modifications of cell wall components. The enzyme-gold and immuno-gold labellings in the infected host tissues revealed that the labelling densities for cellulose, xylan and pectin were significantly reduced in the cell walls of infected ovary, lemma and rachis as compared with corresponding healthy host tissues. The host cell walls in contact with or close to hyphae of the pathogen showed more marked morphological changes and much greater reduction of the labelling density than those in distance from the hyphae. These results provide evidence that F. culmorum may produce cell-wall-degrading enzymes such as cellulases, xylanases and pectinases during infection and colonization of wheat spikes tissues. Furthermore, at the early stage of infection (e.g. 3 days after inoculation), the degradation of pectin was greater than that of cellulose and xylan in the cell walls of the same infected host tissues, indirectly suggesting that the pectinases may be secreted earlier or exert higher activities than cellulases and xylanases. Zusammenfassung Die Zellwandkomponenten Zellulose, Xylan und Pektin in verschiedenen Geweben von nicht inokulierten gesunden und Fusarium culmorum (W. G. Smith) Sacc. infizierten Weizenähren wurden mit Hilfe von Enzym-Gold- und Immun-Gold-Markierungstechniken nachgewiesen. Die Zellwände des Fruchtknotens der gesunden Ähre wiesen unterschiedliche Markierungsmuster und -dichten mit Zellulase-Gold- und Xylanase-Gold-Proben sowie mit dem monoklonalen Anti-Pektin Antikörper JIM7 auf. Das inter-und intrazelluläre Wachstum des Pathogens im Fruchtknoten, in der Deckspelze und Spindel der infizierten Ähre verursachte nicht nur ausgeprägte Veränderungen der Zellwände und der Matrix der Mittellamelle sondern führte auch zu deutlichen Modifikationen der Zellwandkomponenten. Die Enzym-Gold- und die Immun-Gold-Markierungen in den infizierten Wirtsgeweben ergaben deutlich verminderte Markierungsdichten für Zellulose, Xylan und Pektin in den Zellwänden des infizierten Fruchtknotens, der Deckspelze und Spindel im Vergleich zum entsprechenden gesunden Wirtsgewebe. Wirtszellwände, die sich in Kontakt mit den Hyphen oder in der Nähe der Hyphen des Pathogens befanden, zeigten deutlichere morphologische Veränderungen und stärkere Reduktionen der Markierungsdichten als die, die entfernt von Hyphen waren. Diese Ergebnisse weisen darauf hin, da,F. culmorum zellwandabbauende Enzyme wie Zellulasen, Xylanasen und Pektinasen während der Infektion und Besiedlung der Gewebe von Weizenähren ausscheidet. Au,ierdem war im frühen Infektionsstadium (z. B. 3 Tage nach Inokulation) der Abbau von Pektin stärker als der von Zellulose und Xylan in den Zellwänden infizierter Wirtsgewebe. Dies deutet darauf hin, da, Pektinasen früher ausgeschieden werden oder stärkere Aktivität als Zellulasen und Xylanasen besitzen. [source] A host-vector system for molecular study of the intracellular growth of Mycobacterium tuberculosis in phagocytic cellsMICROBIOLOGY AND IMMUNOLOGY, Issue 10 2009Mari Nomoto ABSTRACT The mechanisms by which Mycobacterium tuberculosis survives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host-vector system is indispensable. In this study it has been shown that, in contrast with M. smegmatis strain mc2155 which has been widely used for molecular analysis, M. smegmatis strain J15cs is able to survive even at day 6 post-infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from an Escherichia coli - mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp-, hyg- and km-resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence on in vitro growth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria-Bertani broth and at day 5 post-infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes of M. tuberculosis and intracellular growth was demonstrated. In this study, M. smegmatis strain J15cs and pYT923hyg were found to be capable of serving as an appropriate host-vector system for molecular study of the intracellular growth of M. tuberculosis in phagocytic cells; this system may be useful as a screening tool for M. tuberculosis genes. [source] The RNA chaperone Hfq is essential for the virulence of Salmonella typhimuriumMOLECULAR MICROBIOLOGY, Issue 1 2007Alexandra Sittka Summary The RNA chaperone, Hfq, plays a diverse role in bacterial physiology beyond its original role as a host factor required for replication of Q, RNA bacteriophage. In this study, we show that Hfq is involved in the expression and secretion of virulence factors in the facultative intracellular pathogen, Salmonella typhimurium. A Salmonella hfq deletion strain is highly attenuated in mice after both oral and intraperitoneal infection, and shows a severe defect in invasion of epithelial cells and a growth defect in both epithelial cells and macrophages in vitro. Surprisingly, we find that these phenotypes are largely independent of the previously reported requirement of Hfq for expression of the stationary phase sigma factor, RpoS. Our results implicate Hfq as a key regulator of multiple aspects of virulence including regulation of motility and outer membrane protein (OmpD) expression in addition to invasion and intracellular growth. These pleiotropic effects are suggested to involve a network of regulatory small non-coding RNAs, placing Hfq at the centre of post-transcriptional regulation of virulence gene expression in Salmonella. In addition, the hfq mutation appears to cause a chronic activation of the RpoE-mediated envelope stress response which is likely due to a misregulation of membrane protein expression. [source] A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretionMOLECULAR MICROBIOLOGY, Issue 6 2004Lian-Yong Gao Summary Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871,Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host. [source] CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenesMOLECULAR MICROBIOLOGY, Issue 4 2000Shamila Nair Stress proteins play an important role in virulence, yet little is known about the regulation of stress response in pathogens. In the facultative intracellular pathogen Listeria monocytogenes, the Clp ATPases, including ClpC, ClpP and ClpE, are required for stress survival and intracellular growth. The first gene of the clpC operon of L. monocytogenes encodes a homologue of the Bacillus subtilis CtsR repressor of stress response genes. An L. monocytogenes ctsR -deleted mutant displayed enhanced survival under stress conditions (growth in the presence of 2% NaCl or at 42°C), but its level of virulence in the mouse was not affected. The virulence of a wild-type strain constitutively expressing CtsR is significantly attenuated, presumably because of repression of the stress response. Regulation of the L. monocytogenes clpC, clpP and clpE genes was investigated using transcriptional fusions in B. subtilis as a host. The L. monocytogenes ctsR gene was placed under the control of an inducible promoter, and regulation by CtsR and heat shock was demonstrated in vivo in B. subtilis. The purified CtsR protein of L. monocytogenes binds specifically to the clpC, clpP and clpE regulatory regions, and the extent of the CtsR binding sites was defined by DNase I footprinting. Our results demonstrate that this human pathogen possesses a CtsR regulon controlling class III heat shock genes, strikingly similar to that of the saprophyte B. subtilis. This is the first description of a stress response regulatory gene in a pathogen. [source] Novel infection strategies of Colletotrichum acutatum on ripe blueberry fruitPLANT PATHOLOGY, Issue 1 2008P. S. Wharton The infection and colonization process of Colletotrichum acutatum on ripe blueberry fruit from two cultivars with different susceptibility to anthracnose were examined using light and confocal laser scanning microscopy. Ripe fruit from susceptible cv. Jersey and resistant cv. Elliott were drop-inoculated with a conidial suspension of C. acutatum, and epidermal peels were evaluated at selected times after inoculation and incubation. Results from pre-penetration studies demonstrated that there were significant differences in the rate of formation of melanized appressoria between the two cultivars, with the rate of formation being faster in the susceptible one. In both cultivars, penetration by the pathogen occurred via appressoria 48 h post-inoculation (hpi). However, in the susceptible cv. Jersey, C. acutatum then adopted an intracellular hemibiotrophic-like infection strategy, whereas in the resistant cv. Elliott subcuticular intramural-like infection occurred. In cv. Jersey by 108 hpi, intracellular growth of the pathogen led to the formation of numerous acervuli, with orange conidial masses. By 120 hpi, the conidial masses had coalesced covering the entire inoculated area. In cv. Elliott, acervuli were not seen until 144 hpi and contained few conidia. These results demonstrate for the first time the ability of C. acutatum to adopt a different infection and colonization strategy depending on the susceptibility of the host tissue being colonized. [source] Iron acquisition within host cells and the pathogenicity of LeishmaniaCELLULAR MICROBIOLOGY, Issue 2 2008Chau Huynh Summary Iron is an essential cofactor for several enzymes and metabolic pathways, in both microbes and in their eukaryotic hosts. To avoid toxicity, iron acquisition is tightly regulated. This represents a particular challenge for pathogens that reside within the endocytic pathway of mammalian cells, because endosomes and lysosomes are gradually depleted in iron by host transporters. An important player in this process is Nramp1 (Slc11a1), a proton efflux pump that translocates Fe2+ and Mn2+ ions from macrophage lysosomes/phagolysosomes into the cytosol. Mutations in Nramp1 cause susceptibility to infection with the bacteria Salmonella and Mycobacteria and the protozoan Leishmania, indicating that an available pool of intraphagosomal iron is critical for the intracellular survival and replication of these pathogens. Salmonella and Mycobacteria are known to express iron transporter systems that effectively compete with host transporters for iron. Until recently, however, very little was known about the molecular strategy used by Leishmania for survival in the iron-poor environment of macrophage phagolysosomes. It is now clear that intracellular residence induces Leishmania amazonensis to express LIT1, a ZIP family membrane Fe2+ transporter that is required for intracellular growth and virulence. [source] New concepts in Salmonella virulence: the importance of reducing the intracellular growth rate in the hostCELLULAR MICROBIOLOGY, Issue 7 2005Alberto Tierrez Summary The literature refers to Salmonella enterica as an intracellular bacterial pathogen that proliferates within vacuoles of mammalian cells. However, recent in vivo studies have revealed that the vast majority of infected cells contain very few intracellular bacteria (three to four organisms). Salmonella intracellular growth is also limited in cultured dendritic cells and fibroblasts, two cell types abundant in tissues located underneath the intestinal epithelium. Recently, a Salmonella factor previously known for its role as a negative regulator of intracellular growth has been shown to tightly repress certain pathogen functions upon host colonization and to be critical for virulence. The connection between virulence and the negative control of intracellular growth is further sustained by the fact that some attenuated mutants overgrow in non-phagocytic cells located in the intestinal lamina propria. These findings are changing our classical view of Salmonella as a fast growing intracellular pathogen and suggest that this pathogen may trigger responses directed to reduce the growth rate within the infected cell. These responses could play a critical role in modulating the delicate balance between disease and persistence. [source] Growth of Brucella abortus in macrophages from resistant and susceptible mouse strainsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2000J. Sathiyaseelan C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice. While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance. The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B. abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants. While both BALB/c and C57Bl are Nramp -susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis. The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B. abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation. A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results. [source] | ||||||||||