Novel Functions for the Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori.
Infect Immun. 2012 Jan 30;
Authors: Pohl MA, Kienesberger S, Blaser MJ
Abstract
Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase, encoded by β-(1,3)galT is essential for production of type 1 (Le(a) and Le(b)) antigens. The upstream gene jhp0562, present in many, but not all, H. pylori strains is homologous to β-(1,3)galT, but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galT expression in all wild type and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Le(b) production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways, and that jhp0562 provides substrate for intragenomic recombination to generate diverse Le synthesis enzymes.
PMID: 22290141 [PubMed - as supplied by publisher]
Intravascular hemolysis is a hallmark event in the immunopathology of malaria that results in increased systemic concentrations of free hemoglobin (Hb). The oxidation of Hb by free radicals causes the release of heme, which amplifies inflammation. To circumvent the detrimental effects of free heme, hosts have developed several homeostatic mechanisms, including the enzyme haptoglobin (Hp), which scavenges cell-free Hb, the monocyte receptor CD163, which binds to Hb-Hp complexes, and heme oxygenase-1 (HO-1), which degrades intracellular free heme. We tested the association between these three main components of the host response to hemolysis and susceptibility to malaria in a Brazilian population. The genetic profiles of the HMOX1 and Hp genes and the plasma levels of the serum inflammatory marker sCD163 were studied in 264 subjects, including 78 individuals with symptomatic malaria, 106 individuals with asymptomatic malaria and 80 uninfected individuals. We found that long (GT)n repeats in the microsatellite polymorphism region of the HMOX1 gene, the Hp2 allele and the Hp2.2 genotype were associated with symptomatic malaria. Moreover, increased plasma concentrations of heme, Hp, HO-1 and sCD163 were associated with susceptibility to malaria. The validation of these results could support the development of targeted therapies and aid in reducing the severity of malaria.
PMID: 22290142 [PubMed - as supplied by publisher]
Microevolution in fimH gene of mucosa-associated Escherichia coli strains isolated from Inflammatory Bowel Disease pediatric patients.
Infect Immun. 2012 Jan 30;
Authors: Iebba V, Conte MP, Lepanto MS, Di Nardo G, Santangelo F, Aloi M, Totino V, Proietti Checchi M, Longhi C, Cucchiara S, Schippa S
Abstract
Several studies reported increased numbers of mucosa-associated Escherichia coli strains in patients with inflammatory bowel disease (IBD), encompassing Crohn’s disease (CD) and ulcerative colitis (UC). Majority of E. coli strains possess type 1 fimbriae, whose tip fibrillum protein, FimH, naturally undergoes through amino acid replacements, an important process in the adaptation of commensal E. coli strains to environmental changes, like observed in IBD and urinary tract infections. In this study we analyzed mutational patterns in fimH gene of 52 mucosa-associated E. coli strains isolated from IBD and non-IBD pediatric patients, in order to investigate microevolution in this genetic trait. FimH positive strains were also phylogenetically typed, and tested for their adhesive ability on Caco-2 cell. Specific FimH allele for each grouping feature were found. Mutations G66S and V27A were related to CD, whilst A242V, V163A and T74I mutations were attributed to UC. Otherwise G66S, N70S and S78N mutations were specifically attributed to B2/D phylogroups. N70S and A119V mutations were related to adhesive E. coli strains. Phylogroup B2, adhesive and IBD E. coli strains showed an higher site substitution rate (SSR) in fimH gene, together with an higher number of mutations. Naïve mucosal inflammation degree was related to specific FimH alleles. Moreover, we could suggest V27A mutation as pathoadaptive for the CD intestinal habitat, whilst both N70S and S78N mutations as related to the more aggressive E. coli B2 phylogroup. In conclusion, we found some FimH variants that seems to be more involved than others to IBD pathogenesis evolution.
PMID: 22290143 [PubMed - as supplied by publisher]
PDIMs and PGLs are both required for virulence of Mycobacterium marinum.
Infect Immun. 2012 Jan 30;
Authors: Yu J, Tran V, Li M, Huang X, Niu C, Wang D, Zhu J, Wang J, Gao Q, Liu J
Abstract
Phthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence of Mycobacterium tuberculosis (M. tb). This has been complicated by the natural deficiency of PGLs in many clinical strains of M. tb and the frequent loss of PDIMs in laboratory M. tb strains. In this study, we isolated seven mutants of Mycobacterium marinum deficient in PDIMs and/or PGLs, in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed that M. marinum strains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help to understand the role of PDIMs and PGLs in M. tb virulence.
PMID: 22290144 [PubMed - as supplied by publisher]
Toxoplasma gondii infection inhibits Th17-mediated spontaneous development of arthritis in IL-1 receptor antagonist-deficient mice.
Infect Immun. 2012 Jan 30;
Authors: Washino T, Moroda M, Iwakura Y, Aosai F
Abstract
IL-1 receptor antagonist (IL-1Ra)-deficient BALB/c mice develop spontaneous arthritis resembling human rheumatoid arthritis. We herein report that infection with Toxoplasma gondii, an intracellular protozoan, is capable of ameliorating the spontaneous development of arthritis in IL-1Ra-deficient mice. The onset of arthritis development was delayed and the severity score of arthritis was significantly suppressed in T. gondii-infected mice. Expression of IL-12p40 mRNA from CD11c(+) cells of mesenteric lymph nodes (mLN) and spleen markedly increased at 1 week after peroral infection. While CD11c(+) cells also produced IL-10, IL-1β and IL-6, CD4(+) T cells from T. gondii-infected mice expressed significantly high levels of T-bet and IFN- γ mRNA at both mLN and spleen. Levels of GATA-3/IL-4 mRNA or RORγt/IL-17 mRNA decreased in the infected mice, indicating Th1 polarization and the reduction of Th2 and Th17 polarization. The severity of arthritis was related to Th1 polarization accompanied with Th17 reduction, demonstrating the protective role of T. gondii-derived Th1 response against Th17-mediated arthritis in IL-1Ra-deficient mice.
PMID: 22290145 [PubMed - as supplied by publisher]
A serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibody requires FcγRIII and macrophages to mediate protection against pneumococcal pneumonia in mice.
Infect Immun. 2012 Jan 30;
Authors: Weber S, Tian H, van Rooijen N, Pirofski LA
Abstract
Antibodies to pneumococcal capsular polysaccharide (PPS) are required for PPS-based vaccine-mediated protection against Streptococcus pneumoniae. Previous work established that 1E2, a mouse IgG1 to PPS3 that does not induce ST3 killing by phagocytes in vitro protects mice from death after intranasal infection with serotype 3 S. pneumoniae (ST3), but its efficacy was abrogated in Fc(common)γR-deficient mice. In this study, we determined whether 1E2 efficacy against pulmonary ST3 infection requires FcγRIII. 1E2 did not protect FcγRIII-deficient (FcγRIII(-/-)) mice. Studies of the mechanism of 1E2-mediated effects showed that it resulted in a marked reduction in lung inflammation in ST3-infected wild-type (Wt, C57Bl/6) mice that was abrogated in FcγRIII(-/-) mice. 1E2 had no effect on early bacterial clearance in the lungs of ST3-infected Wt, FcγRIIB(-/-), or FcγRIII(-/-) mice, but it reduced bacteremia and serum MIP-2, IL-6, and TNF-α in Wt and FcγRIIB(-/-) mice, strains in which it is protective. As previous work showed that neutrophils were dispensable for 1E2 efficacy, we investigated whether macrophages are required for 1E2 efficacy against intranasal infection with ST3 and found that its efficacy was abrogated in Wt mice depleted of macrophages intranasally. In vitro studies revealed that1E2 promoted ST3 internalization by naïve alveolar macrophages but did not induce early intracellular killing. Macrophages from 1E2-treated ST3-infected mice studied ex vivo exhibited more apoptosis than those from FcγRIII(-/-) mice. These findings suggest that 1E2 mediates protection against ST3 in mice by affecting the inflammatory response, perhaps in part via macrophage apoptosis, rather than by inducing early bacterial clearance.
PMID: 22290146 [PubMed - as supplied by publisher]
A new strategy to develop an effective vaccine is essential to control food-borne Salmonella Enteritidis (SE) infections. Bacterial ghosts (BG), which are non-living, Gram-negative bacterial cell envelopes, are generated by expulsion of the cytoplasmic contents from bacterial cells through controlled expression using the modified cI857/λ P(R/)gene E expression system. In the present study, the pJHL99 lysis plasmid carrying the mutated lambda pR37-cI857 repressor and PhiX174 lysis gene E was constructed and transformed in SE to produce a BG. Temperature induction of the lysis gene cassette at 42°C revealed a quantitative killing of SE. The SE ghost was characterized using scanning and transmission electron microscopy to visualize the trans-membrane tunnel structure and loss of cytoplasmic materials, respectively. The efficacy of the BG as a vaccine candidate was evaluated in a chicken model using 60 10-day-old chickens, which were divided into four groups (n = 15) as groups A, B, C and D. Group A was designated as non-immunized control group, whereas birds in groups B, C and D were immunized via intramuscular, subcutaneous, and oral routes, respectively. The chickens from all immunized groups showed significant increase in plasma IgG and intestinal secretory IgA levels. The lymphocyte proliferation response and CD3+CD4+ and CD3+CD8+ T-cell subpopulation, were also significantly increased in all immunized group. Data indicates that both humoral and cell mediated immune responses are robustly stimulated. By the examination of the protection efficacy measured by observations of gross lesions in the organs and bacterial recovery, the candidate can provide an efficient protection upon virulent challenge.
PMID: 22290147 [PubMed - as supplied by publisher]
Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of Type I interferons, including Interferon β (IFNβ), which, surprisingly, promotes L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFNβ. We have identified L. monocytogenes strain LO28 as able to hyper-induce IFNβ production in infected cells, ∼30-fold more than the common laboratory clone 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele in the transcriptional regulator BrtA, and correspondingly de-represses expression of MdrT. Surprisingly, while de-repression of MdrT resulted in hyper-stimulation of IFNβ, this results in significant attenuation in multiple mouse models of infection. While Type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo, by an unknown mechanism.
PMID: 22290148 [PubMed - as supplied by publisher]
Involvement of mannose receptor and the p38 MAPK signaling pathway of micro integral membrane protein after enteropathogenic Escherichia coli infection.
Infect Immun. 2012 Jan 30;
Authors: Liu Z, Ma Y, Moyer MP, Zhang P, Shi C, Qin H
Abstract
Micro integral membrane protein (MIMP) has been shown to adhere to mucin and antagonize the adhesion of enteropathogenic Escherichia coli (EPEC) to epithelial cells, however, the mechanism has not been fully elucidated. In this study, we further identified the receptor of MIMP on NCM460 cells, and investigated the mechanism (p38 MAPK pathway) following the interaction of MIMP and its corresponding receptor mannose receptor. We first identified the target receptor of MIMP on the surface of NCM460 cells using immunoprecipitation/mass spectrometry (IP/MS) technology. We also verified the mannose receptor and examined degradation and activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Results indicated that MIMP adhered to NCM460 cells by binding to the mannose receptor, and inhibited the phosphorylation of p38 MAPK stimulated after EPEC infection via inhibition of the toll-like receptor-5 pathway. These findings indicated that MIMP relieve the injury of NCM460 cells after enteropathogenic Escherichia coli infection through the mannose receptor and inhibition of the p38 MAPK signaling pathway, both of which may therefore be potential therapeutic targets for intestinal diseases, such as inflammatory bowel disease.
PMID: 22290149 [PubMed - as supplied by publisher]
Detection of established virulence genes and plasmids to differentiate Borrelia burgdorferi strains.
Infect Immun. 2012 Jan 30;
Authors: Chan K, Casjens S, Parveen N
Abstract
Borrelia burgdorferi sensu stricto is the major causative agent of Lyme disease in the United States while B. garinii and B. afzelii are more prevalent in the Europe. The highly complex genome of B. burgdorferi is comprised of a linear chromosome and a large number of variably sized linear and circular plasmids. Many plasmids of this spirochete are unstable during its culture in vitro. Given that many of the B. burgdorferi virulence factors identified to date are plasmid-encoded, spirochetal plasmid content determination is essential for genetic analysis of Lyme pathogenesis. Although Polymerase Chain Reaction (PCR)-based assays facilitate plasmid profiling of sequenced B. burgdorferi strains, the rapid genetic content determination strategy for non-sequenced strains has not been described yet. In this study, we combined pulse field gel electrophoresis and Southern hybridization for detection of genes encoding known virulence factors, ribosomal DNA spacer restriction fragment length polymorphism types (RST), ospC group determination, and sequencing of the variable dbpA and ospC genes. We show that two strains isolated from the same tick and both originally named N40, are in fact very distinct. Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin in one “N40″ strain. Thus, two distinct strains were isolated from the same tick that show different plasmid profile as determined by PFGE and PCR and vary in their ospC and dbpA sequence. However, both belong to RST3B group.
PMID: 22290150 [PubMed - as supplied by publisher]
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