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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ItemFarewell Stan Stanley Falkow: 1934-2018.(Wiley, 2018-07)
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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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ItemInterleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells(NATL ACAD SCIENCES, 2018-10-02)Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection.
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ItemNew Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam(AMER SOC MICROBIOLOGY, 2018-09-04)Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S Typhimurium (serovar I:4,[5],12:i:- sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.IMPORTANCESalmonella Typhimurium is a major diarrheal pathogen and associated with invasive nontyphoid Salmonella (iNTS) disease in vulnerable populations. We present the first characterization of iNTS organisms in Southeast Asia and describe a different evolutionary trajectory from that of organisms causing iNTS in sub-Saharan Africa. In Vietnam, the globally distributed monophasic variant of Salmonella Typhimurium, the serovar I:4,[5],12:i:- ST34 clone, has reacquired a phase 2 flagellum and gained a multidrug-resistant plasmid to become associated with iNTS disease in HIV-infected patients. We document distinct communities of S Typhimurium and I:4,[5],12:i:- in animals and humans in Vietnam, despite the greater mixing of these host populations here. These data highlight the importance of whole-genome sequencing surveillance in a One Health context in understanding the evolution and spread of resistant bacterial infections.
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ItemDNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development.(BMJ, 2019-01)OBJECTIVE: Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited. DESIGN: We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology. RESULTS: We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models. CONCLUSIONS: Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.
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ItemInduction of Cell Cycle and NK Cell Responses by Live-Attenuated Oral Vaccines against Typhoid Fever(FRONTIERS MEDIA SA, 2017-10-12)The mechanisms by which oral, live-attenuated vaccines protect against typhoid fever are poorly understood. Here, we analyze transcriptional responses after vaccination with Ty21a or vaccine candidate, M01ZH09. Alterations in response profiles were related to vaccine-induced immune responses and subsequent outcome after wild-type Salmonella Typhi challenge. Despite broad genetic similarity, we detected differences in transcriptional responses to each vaccine. Seven days after M01ZH09 vaccination, marked cell cycle activation was identified and associated with humoral immunogenicity. By contrast, vaccination with Ty21a was associated with NK cell activity and validated in peripheral blood mononuclear cell stimulation assays confirming superior induction of an NK cell response. Moreover, transcriptional signatures of amino acid metabolism in Ty21a recipients were associated with protection against infection, including increased incubation time and decreased severity. Our data provide detailed insight into molecular immune responses to typhoid vaccines, which could aid the rational design of improved oral, live-attenuated vaccines against enteric pathogens.
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ItemGenome Sequence of Porcine Escherichia coli Strain IMT8073, an Atypical Enteropathogenic E. coli Strain Isolated from a Piglet with Diarrhea.(American Society for Microbiology, 2013-08-01)Escherichia coli is a highly diverse bacterial species, with atypical enteropathogenic E. coli (aEPEC) causing intestinal disease in both human and animal hosts. Here, we report the first complete genome sequence of an aEPEC strain of sequence type ST794 and serotype Ont:H7, isolated from a diseased piglet.
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ItemCorrection: Signatures of Adaptation in Human Invasive Salmonella Typhimurium ST313 Populations from Sub-Saharan Africa.(Public Library of Science (PLoS), 2015-06)
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ItemMultiple Introductions of Salmonella enterica Serovar Typhi H58 with Reduced Fluoroquinolone Susceptibility into Chile(CENTERS DISEASE CONTROL & PREVENTION, 2020-11)Salmonella enterica serovar Typhi H58, an antimicrobial-resistant lineage, is globally disseminated but has not been reported in Latin America. Genomic analysis revealed 3 independent introductions of Salmonella Typhi H58 with reduced fluoroquinolone susceptibility into Chile. Our findings highlight the utility of enhanced genomic surveillance for typhoid fever in this region.