Isolation and Whole‐Genome Sequencing of Environmental <i>Campylobacter</i>

Kelley, Brittni R.; Ellis, J. Christopher; Hyatt, Doreene R.; Jacobson, Dan; Johnson, Jeff S.

doi:10.1002/cpmc.64

Cited by 5 publications

(6 citation statements)

References 45 publications

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“…Campylobacter jejuni strains used in this study were isolated previously from human clinical samples by the Tennessee Department of Health using commercially available Campylobacter blood free selective media (CCDA) plates. After a 48 h incubation at 42 • C in a GasPak container with a Campy sachet, isolates were used to conduct a Gram stain, hippurate hydrolysis assay, catalase, indole, and oxidase test, in addition to MALDI-TOF confirmation (data not shown) as described previously (Kelley et al, 2018). Personal identifiable information was removed before isolates were shipped to researchers at the University of Tennessee in accordance with the IRB protocol: UTK IRB-17-03683-XP.…”

Section: Obtaining Regional Human Isolatesmentioning

confidence: 99%

“…Genomic DNA was obtained from growth of isolated colonies following the protocol described previously (Kelley et al, 2018). Briefly, growth was resuspended in sterile genomic lysis buffer (50 mM Tris Base -pH7.5, 50 mM EDTA, 1% SDS, 10 mM NaCl) before adding protein precipitation solution (Promega -A795A).…”

Section: Genomic Dna Preparation and Pcr-based Identificationmentioning

confidence: 99%

“…The initial analysis of the resulting sequence reads and reads obtained from the GenomeTrakr database was performed as previously described (Kelley et al, 2018). Briefly, read quality for each sample was analyzed and adapter sequences were trimmed using Atropos (Didion et al, 2017).…”

Section: Bioinformatic Analysis Of C Jejuni Genomesmentioning

confidence: 99%

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Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee

et al. 2020

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As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health in both the developed and developing worlds. Despite its prevalence as a human pathogen, the source of these infections remains poorly understood due to the mutation frequency of the organism and past limitations of whole genome analysis. Recent advances in both whole genome sequencing and computational methods have allowed for the high-resolution analysis of intraspecies diversity, leading multiple groups to postulate that these approaches may be used to identify the sources of Campylobacter jejuni infection. To address this hypothesis, our group conducted a regionally and temporally restricted sampling of agricultural and environmental Campylobacter sources and compared isolated C. jejuni genomes to those that caused human infections in the same region during the same time period. Through a network analysis comparing genomes from various sources, we found that human C. jejuni isolates clustered with those isolated from cattle and chickens, indicating these as potential sources of human infection in the region.

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Section: Obtaining Regional Human Isolatesmentioning

confidence: 99%

Section: Genomic Dna Preparation and Pcr-based Identificationmentioning

confidence: 99%

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Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee

et al. 2020

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“…Sequencing was conducted as previously described (62). Genome assembly was conducted with Geneious version 11.1.5.…”

Section: Methodsmentioning

confidence: 99%

The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

Jackson

Hancock

LaPrade

et al. 2019

mBio

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Human cytomegalovirus (HCMV) is a betaherpesvirus that is a significant pathogen within newborn and immunocompromised populations. Morbidity associated with HCMV infection is the consequence of viral dissemination. HCMV has evolved to manipulate the host immune system to enhance viral dissemination and ensure long-term survival within the host. The immunomodulatory protein vCXCL-1, a viral chemokine functioning primarily through the CXCR2 chemokine receptor, is hypothesized to attract CXCR2+ neutrophils to infection sites, aiding viral dissemination. Neutrophils harbor HCMV in vivo; however, the interaction between vCXCL-1 and the neutrophil has not been evaluated in vivo. Using the mouse model and mouse cytomegalovirus (MCMV) infection, we show that murine neutrophils harbor and transfer infectious MCMV and that virus replication initiates within this cell type. Utilizing recombinant MCMVs expressing vCXCL-1 from the HCMV strain (Toledo), we demonstrated that vCXCL-1 significantly enhances MCMV dissemination kinetics. Through cellular depletion experiments, we observe that neutrophils impact dissemination but that overall dissemination is largely neutrophil independent. This work adds neutrophils to the list of innate cells (i.e., dendritic and macrophages/monocytes) that contribute to MCMV dissemination but refutes the hypothesis that neutrophils are the primary cell responding to vCXCL-1. IMPORTANCE An adequate in vivo analysis of HCMV’s viral chemokine vCXCL-1 has been lacking. Here we generate recombinant MCMVs expressing vCXCL-1 to study vCXCL-1 function in vivo using MCMV as a surrogate. We demonstrate that vCXCL-1 increases MCMV dissemination kinetics for both primary and secondary dissemination. Additionally, we provide evidence, that the murine neutrophil is largely a bystander in the mouse’s response to vCXCL-1. We confirm the hypothesis that vCXCL-1 is a HCMV virulence factor. Infection of severely immunocompromised mice with MCMVs expressing vCXCL-1 was lethal in more than 50% of infected animals, while all animals infected with parental virus survived during a 12-day period. This work provides needed insights into vCXCL-1 function in vivo.

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“…Genomic DNAs from a parental C. jejuni Δ flgX strain (DRH4803) and from corresponding suppressor mutant strains were prepared as previously described (65). Briefly, isolates were grown on Mueller-Hinton agar plates supplemented with 10 μg/ml trimethoprim at 37°C under microaerobic conditions.…”

Section: Methodsmentioning

confidence: 99%

A Chaperone for the Stator Units of a Bacterial Flagellum

Ribardo

Kelley

Johnson

et al. 2019

mBio

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The stator units of the flagellum supply power to the flagellar motor via ion transport across the cytoplasmic membrane and generate torque on the rotor for rotation. Flagellar motors across bacterial species have evolved adaptations that impact and enhance stator function to meet the demands of each species, including producing stator units using different fuel types or various stator units for different motility modalities. Campylobacter jejuni produces one of the most complex and powerful flagellar motors by positioning 17 stator units at a greater radial distance than in most other bacteria to increase power and torque for high velocity of motility. We report another evolutionary adaptation impacting flagellar stators by identifying FlgX as a chaperone for C. jejuni stator units to ensure sufficient power and torque for flagellar rotation and motility. We discovered that FlgX maintains MotA and MotB stator protein integrity likely through a direct interaction with MotA that prevents their degradation. Suppressor analysis suggested that the physiology of C. jejuni drives the requirement for FlgX to protect stator units from proteolysis by the FtsH protease complex. C. jejuni ΔflgX was strongly attenuated for colonization of the natural avian host, but colonization capacity was greatly restored by a single mutation in MotA. These findings suggest that the likely sole function of FlgX is to preserve stator unit integrity for the motility required for host interactions. Our findings demonstrate another evolved adaptation in motile bacteria to ensure the equipment of the flagellar motor with sufficient power to generate torque for motility. IMPORTANCE The bacterial flagellum is a reversible rotating motor powered by ion transport through stator units, which also exert torque on the rotor component to turn the flagellum for motility. Species-specific adaptations to flagellar motors impact stator function to meet the demands of each species to sufficiently power flagellar rotation. We identified another evolutionary adaptation by discovering that FlgX of Campylobacter jejuni preserves the integrity of stator units by functioning as a chaperone to protect stator proteins from degradation by the FtsH protease complex due to the physiology of the bacterium. FlgX is required to maintain a level of stator units sufficient to power the naturally high-torque flagellar motor of C. jejuni for motility in intestinal mucosal layers to colonize hosts. Our work continues to identify an increasing number of adaptations to flagellar motors across bacterial species that provide the mechanics necessary for producing an effective rotating nanomachine for motility.

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Isolation and Whole‐Genome Sequencing of Environmental Campylobacter

Cited by 5 publications

References 45 publications

Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee

Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee

The Human Cytomegalovirus Chemokine vCXCL-1 Modulates Normal Dissemination Kinetics of Murine Cytomegalovirus In Vivo

A Chaperone for the Stator Units of a Bacterial Flagellum

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