Microbiology & Immunology - Research Publications
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ItemThe flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi-like protein tail fibre(WILEY, 2019-12)The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.
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ItemAn investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis(WILEY, 2018-09)Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.
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ItemPhylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan(CENTERS DISEASE CONTROL & PREVENTION, 2017-11)Klebsiella pneumoniae shows increasing emergence of multidrug-resistant lineages, including strains resistant to all available antimicrobial drugs. We conducted whole-genome sequencing of 178 highly drug-resistant isolates from a tertiary hospital in Lahore, Pakistan. Phylogenetic analyses to place these isolates into global context demonstrate the expansion of multiple independent lineages, including K. quasipneumoniae.
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ItemThe Hd, Hj, and Hz66 flagella variants of Salmonella enterica serovar Typhi modify host responses and cellular interactions(NATURE PORTFOLIO, 2015-01-22)Salmonella Typhi, the causative agent of typhoid fever, is a monophyletic, human-restricted bacterium that exhibits limited phenotypic variation. S. Typhi from Indonesia are a notable exception, with circulating strains expressing diverse flagella antigens including Hj, Hd and Hz66. Hypothesizing that S. Typhi flagella plays a key role during infection, we constructed an S. Typhi fliC mutant and otherwise isogenic S. Typhi strains expressing the Hj, Hd, Hz66 flagella antigens. Phenotyping revealed differences in flagellum structure, strain motility and immunogenicity, but not in the ability of flagellated isolates to induce TLR5 activity. Invasion assays using epithelial and macrophage cell lines revealed differences in the ability of these S. Typhi derivatives to invade cells or induce cellular restructuring in the form of ruffles. Notably, the Hj variant induced substantial ruffles that were not fully dependent on the GTPases that contribute to this process. These data highlight important differences in the phenotypic properties of S. Typhi flagella variation and how they impact on the pathogenesis of S. Typhi.
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ItemFusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants(PUBLIC LIBRARY SCIENCE, 2018-08)Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids.
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ItemResistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1(NATURE PORTFOLIO, 2019-02-20)Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance.
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ItemCentral role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium(AMER SOC MICROBIOLOGY, 2003-09)Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to C. rodentium. C57Bl/6 mice orally infected with C. rodentium resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1(-/-) mice orally infected with C. rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4(+) T cells, but not CD8(+) T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4(+) T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C. rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells ( micro MT mice), were highly susceptible to C. rodentium infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected micro MT mice. Adoptive transfer of immune B cells, but not naïve B cells, provided highly variable immunity to recipient micro MT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a C. rodentium infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.