Summary Intracellular replication of Brucella requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that Brucella internalizes into macrophages by swimming on the cell surface with generalized membrane ruffling for several minutes, after which the bacteria are enclosed by macropinosomes. Lipid raft‐associated molecules such as glycosylphosphatidylinositol (GPI)‐anchored proteins, GM1 gangliosides and cholesterol were selectively incorporated into macropinosomes containing Brucella. In contrast, lysosomal glycoprotein LAMP‐1 and host cell transmembrane protein CD44 were excluded from the macropinosomes. Removing GPI‐anchored proteins from the macrophage surface and cholesterol sequestration markedly inhibited the VirB‐dependent macropinocytosis and intracellular replication. Our results suggest that the entry route of Brucella into the macrophage determines the intracellular fate of the bacteria that is modulated by lipid raft microdomains.
Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human -defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a ϳ2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-B (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-B activation and thus plays an important role in up-regulation of the innate immune response.Antimicrobial peptides play an important role in host defense at mucosal surfaces. The two major groups of vertebrate defensins, ␣-and -defensins, differ in the arrangements of their disulfide bonds. Six human ␣-defensins (HD-1 to HD-6) 1 and two -defensins (hBD-1 and hBD-2) have been reported to date. HD-1, HD-2, HD-3, and HD-4 are present in neutrophils, where they constitute 30 -50% of the total protein in azurophilic granules (1). HD-5 and HD-6 were identified in the Paneth cells of small intestinal crypts (2, 3) and in female reproductive tissue (4). hBD-1 was purified from plasma (5) and detected in a range of epithelial tissues (6). hBD-2 was purified from skin and shown to be expressed in the lung and uterus (7). hBD-2 was induced in mucosal tissues following bacterial infections (7-9).Salmonella species are Gram-negative organisms that cause gastroenteritis and enteric fever in humans. As pointed out by Bä umler et al. (10), an increase in numbers of human infections with Salmonella enteritidis began in the 1960s but was followed by an almost 50% decrease from 1970 to 1976. Another increase began in 1977, with signs of a decrease beginning in 1992. In contrast, the increasing frequency of S. enteritidis infection in poultry did not begin until 1989, and a decline started in 1996 (11). Reasons for the fluctuations are unknown, but the recent recognition of food-borne infection with S. enteritidis has renewed interest in how these organisms can invade, persist, and spread.To evaluate the role of defensins in S. enteritidis infection, we investigated the induction of ...
Many bacterial pathogens encode ADP-ribosyltransferase toxins. The authors identified an ADP-ribosyltransferase toxin homologue (ArtA, ArtB) in Salmonella enterica serovar Typhimurium (S. typhimurium) DT104. ArtA is most homologous to a putative pertussis-like toxin subunit present in Salmonella typhi (STY1890) and Salmonella paratyphi A (SPA1609), while ArtB shows homology to a hypothetical periplasmic protein of S. typhi (STY1364) and S. paratyphi A (SPA1188), and a putative pertussis-like toxin subunit in S. typhi (STY1891) and S. paratyphi A (SPA1610). The artA gene was detected from the phage particle fraction upon mitomycin C induction, and the flanking region of artAB contains a prophage-like sequence, suggesting that these putative toxin genes reside within a prophage. Southern blotting analysis revealed that artA is conserved in 12 confirmed DT104 strains and in four related strains which are not phage-typed but are classified into the same group as DT104 by both amplified-fragment length polymorphism and pulsed-field gel electrophoresis. Except for one strain, NCTC 73, all 13 S. typhimurium strains which were classified into different groups from that of DT104 lacked the artA locus. The results suggest that phage-mediated recombination has resulted in the acquisition of art genes in S. typhimurium DT104 strains.
Escherichia coli is a major causative agent of uncomplicated urinary tract infection (UTI) and these strains are designated as uropathogenic E. coli (UPEC). UTI occurs not only among humans, but is also observed in companion animals such as dogs and cats (7). Although the etiological bacterial agents of UTI are diverse, E. coli is the chief causative pathogen in dogs and cats suffering from UTI of bacterial origin. UTI caused by E. coli has been associated with urogenital disease such as cystitis, nephritis, metritis and prostatitis in dogs and cats (7). Seven virulence factors (VFs), including type 1 pilus (pil), pilus associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin I (afaI), hemolysin (hly), aerobactin (aer) and cytotoxic necrotizing factor 1 (cnfI) have been documented to play an important role in causing human UTI (2). Several serotypes such as O1, O2, O4, O6, O8, O16, O18, O22, O25 and O75 are preferentially associated with UPEC strains and each of the uropathogenic virulence factors is significantly associated with these serotypes (1,6,8). E. coli strains isolated from dogs and cats with UTI have high prevalence of hly and clustered in 5 serotypes including O2, O4, O6, O7 and O18 (7). These serotypes except for O7 are shared with human UPEC strains. Further, E. coli strains isolated from dogs and cats with UTI were similar to E. coli strains isolated from human UTI (4, 10, 11). These findings suggest that similar E. coli strains might be 797 Microbiol. Immunol., 47(10), [797][798][799][800][801][802] 2003 Editor-Communicated Paper Abstract: Uncomplicated urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a serious problem not only among humans but also in companion animals such as dogs and cats. The uropathogenic specific protein gene (usp) is preferentially distributed in UPEC isolates from dogs and cats compared with the distribution of usp in E. coli strains from feces of healthy dogs and cats and this pattern of distribution resembles that observed in human UPEC strains. The UPEC strains from companion animals share common O serotypes like O1, O2, O4, O6, O16, O18, O22, O25 and O75 as those reported for human UPEC. The size variation of the pathogenicity island that includes usp in UPEC from dogs and cats was almost similar to those seen in human UPEC. We propose that dogs and cats are the alternative reservoirs for UPEC strains that are associated with human UTI. Distribution of the usp Gene in Uropathogenic
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