No Holes Barred: Invasion of the Intestinal Mucosa by Mycobacterium avium subsp. paratuberculosis

ABSTRACTThe infection biology ofMycobacterium aviumsubsp.paratuberculosishas recently crystallized, with added details surrounding intestinal invasion. The involvement of pathogen-derived effector proteins such as the major membrane protein, oxidoreductase, and fibronectin attachment proteins have been uncovered. Mutations constructed in this pathogen have also shed light on genes needed for invasion. The host cell types that are susceptible to invasion have been defined, along with their transcriptional response. Recent details have given a new appreciation for the dynamic interplay between the host and bacterium that occurs at the outset of infection. An initial look at the global expression pathways of the host has shown a circumvention of the cell communication pathway byM. aviumsubsp.paratuberculosis, which loosens the integrity of the tight junctions. We now know thatM. aviumsubsp.paratuberculosisactivates the epithelial layer and also actively recruits macrophages to the site of infection. These notable findings are summarized along with added mechanistic details of the early infection model. We conclude by proposing critical next steps to further elucidate the process ofM. aviumsubsp.paratuberculosisinvasion.

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Modulating Pathogenesis with Mobile-CRISPRi

Journal of Bacteriology ◽

10.1128/jb.00304-19 ◽

2019 ◽

Vol 201 (22) ◽

Cited By ~ 7

Author(s):

Jiuxin Qu ◽

Neha K. Prasad ◽

Michelle A. Yu ◽

Shuyan Chen ◽

Amy Lyden ◽

...

Keyword(s):

Gene Expression ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Effector Proteins ◽

Infection Model ◽

Secretion Systems ◽

Bacterial Genomes ◽

Gene Encoding ◽

Content Type ◽

Animal Infection

ABSTRACT Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models. IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

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Substrate Interaction with the EssC Coupling Protein of the Type VIIb Secretion System

Journal of Bacteriology ◽

10.1128/jb.00646-19 ◽

2020 ◽

Vol 202 (7) ◽

Cited By ~ 1

Author(s):

Nicole Mietrach ◽

Diana Damián-Aparicio ◽

Benjamin Mielich-Süss ◽

Daniel Lopez ◽

Sebastian Geibel

Keyword(s):

Critical Role ◽

Secretion System ◽

Effector Proteins ◽

Resistant Bacteria ◽

Infection Model ◽

Atpase Domain ◽

Bacterial Persistence ◽

Substrate Transport ◽

Content Type ◽

Coupling Protein

ABSTRACT Staphylococcus aureus employs the type VIIb secretion system (T7SSb) to secrete effector proteins that either have antibacterial activities or promote bacterial persistence in mouse infection models. Here, we present the crystal structure of the ATPase domain D3 of the EssC coupling protein from S. aureus USA300_FPR3757, an integral component of the T7SSb complex, resolved at a 1.7-Å resolution. EssC-D3 shares structural homology with FtsK/SpoIII-like ATPase domains of T7SSa and T7SSb and exhibits a conserved pocket on the surface with differential amino acid composition. In T7SSa, substrate EsxB interacts with the D3 domain through this pocket. Here, we identify amino acids in this pocket that are essential for effector protein secretion in the T7SSb. Our results reveal that the adjacent ATPase domain D2 is a substrate binding site on EssC and that substrates bound to D2 require domain D3 for further transport. Point mutations in the Walker B motif of domain D3 have diametric effects on secretion activity, either abolishing or boosting it, pointing to a critical role of domain D3 in the substrate transport. Finally, we identify ATPase domain D3 as a virulence determinant of S. aureus USA300_FPR3757 using an invertebrate in vivo infection model. IMPORTANCE The emergence of antibiotic-resistant bacteria poses a rising problem in antibiotic treatment (S. Boyle-Vavra and R. S. Daum, Lab Invest 87:3–9, 2007, https://doi.org/10.1038/labinvest.3700501). We have used the multidrug-resistant S. aureus USA300_FPR3757 as a model organism to study the T7SSb. Effector proteins of this system have been associated with abscess formation and bacterial persistence in mouse models (M. L. Burts, A. C. DeDent, and D. M. Missiakas, Mol Microbiol 69:736–746, 2008, https://doi.org/10.1111/j.1365-2958.2008.06324.x; M. L. Burts, W. A. Williams, K. DeBord, and D. M. Missiakas, Proc Natl Acad Sci U S A 102:1169–1174, 2005, https://doi.org/10.1073/pnas.0405620102). We determined the structure of the essential ATPase domain D3 of the T7SSb at atomic resolution and validated a surface-exposed pocket as a potential drug target to block secretion. Furthermore, our study provides new mechanistic insights into the T7SSb substrate transport.

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Effect of Soil Slope on the Appearance of Mycobacterium avium subsp. paratuberculosis in Water Running off Grassland Soil after Application of Contaminated Slurry

Applied and Environmental Microbiology ◽

10.1128/aem.00610-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3544-3552 ◽

Cited By ~ 14

Author(s):

M. Salgado ◽

M. Alfaro ◽

F. Salazar ◽

E. Troncoso ◽

R. M. Mitchell ◽

...

Keyword(s):

Mycobacterium Avium ◽

Daily Rainfall ◽

Water Runoff ◽

Soil Slope ◽

Loamy Soil ◽

Monthly Basis ◽

Content Type ◽

Natural Rainfall ◽

Rainwater Runoff ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACTThe study assessed the effect of soil slope onMycobacterium aviumsubsp.paratuberculosistransport into rainwater runoff from agricultural soil after application ofM. aviumsubsp.paratuberculosis-contaminated slurry. Under field conditions, 24 plots of undisturbed loamy soil 1 by 2 m2were placed on platforms. Twelve plots were used for water runoff: 6 plots at a 3% slope and 6 plots at a 15% slope. Half of the plots of each slope were treated withM. aviumsubsp.paratuberculosis-contaminated slurry, and half were not treated. Using the same experimental design, 12 plots were established for soil sampling on a monthly basis using the same spiked slurry application and soil slopes. Runoff following natural rainfall was collected and analyzed forM. aviumsubsp.paratuberculosis, coliforms, and turbidity.M. aviumsubsp.paratuberculosiswas detected in runoff from all plots treated with contaminated slurry and one control plot. A higher slope (15%) increased the likelihood ofM. aviumsubsp.paratuberculosisdetection but did not affect the likelihood of finding coliforms. Daily rainfall increased the likelihood that runoff would have coliforms and the coliform concentration, but it decreased theM. aviumsubsp.paratuberculosisconcentration in the runoff. When there was no runoff, rain was associated with increasedM. aviumsubsp.paratuberculosisconcentrations. Coliform counts in runoff were related to runoff turbidity.M. aviumsubsp.paratuberculosispresence/absence, however, was related to turbidity. Study duration decreased bacterial detection and concentration. These findings demonstrate the high likelihood thatM. aviumsubsp.paratuberculosisin slurry spread on pastures will contaminate water runoff, particularly during seasons with high rainfall.M. aviumsubsp.paratuberculosiscontamination of water has potential consequences for both animal and human health.

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Complete Genome Sequence of Mycobacterium avium subsp. paratuberculosis Strain 42-13-1, Isolated in Japan

Microbiology Resource Announcements ◽

10.1128/mra.00084-21 ◽

2021 ◽

Vol 10 (15) ◽

Author(s):

Yuichi Ueno ◽

Yohsuke Ogawa ◽

Yuji Takamura ◽

Reiko Nagata ◽

Satoko Kawaji ◽

...

Keyword(s):

Genome Sequence ◽

Mycobacterium Avium ◽

Complete Genome Sequence ◽

Complete Genome ◽

Chronic Diarrhea ◽

Mycobacterium Avium Subsp ◽

Hybrid Assembly ◽

Short Read ◽

Content Type ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACT Here, we report the complete genome sequence of Mycobacterium avium subsp. paratuberculosis strain 42-13-1, isolated from cattle presenting with chronic diarrhea caused by Johne’s disease in Japan, which was assembled via long- and short-read hybrid assembly.

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Global Transcriptional Response to Organic Hydroperoxide and the Role of OhrR in the Control of Virulence Traits in Chromobacterium violaceum

Infection and Immunity ◽

10.1128/iai.00017-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 9

Author(s):

Maristela Previato-Mello ◽

Diogo de Abreu Meireles ◽

Luis Eduardo Soares Netto ◽

José Freire da Silva Neto

Keyword(s):

Cumene Hydroperoxide ◽

Transcriptional Response ◽

Transcriptional Regulator ◽

Opportunistic Pathogen ◽

Chromobacterium Violaceum ◽

Infection Model ◽

Diguanylate Cyclase ◽

Major Pathway ◽

Content Type ◽

Mouse Infection Model

ABSTRACT A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.

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Development and Evaluation of a Novel Multicopy-Element-Targeting Triplex PCR for Detection of Mycobacterium avium subsp. paratuberculosis in Feces

Applied and Environmental Microbiology ◽

10.1128/aem.01026-14 ◽

2014 ◽

Vol 80 (12) ◽

pp. 3757-3768 ◽

Cited By ~ 25

Author(s):

Iker A. Sevilla ◽

Joseba M. Garrido ◽

Elena Molina ◽

María V. Geijo ◽

Natalia Elguezabal ◽

...

Keyword(s):

Mycobacterium Avium ◽

Control Program ◽

Detection Methods ◽

Bacterial Strains ◽

Fecal Samples ◽

Content Type ◽

Control Programs ◽

Worldwide Distribution ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Cattle Herds

ABSTRACTThe enteropathy called paratuberculosis (PTB), which mainly affects ruminants and has a worldwide distribution, is caused byMycobacterium aviumsubsp.paratuberculosis. This disease significantly reduces the cost-effectiveness of ruminant farms, and therefore, reliable and rapid detection methods are needed to control the spread of the bacterium in livestock and in the environment. The aim of this study was to identify a specific and sensitive combination of DNA extraction and amplification to detectM. aviumsubsp.paratuberculosisin feces. Negative bovine fecal samples were inoculated with increasing concentrations of two different bacterial strains (field and reference) to compare the performance of four extraction and five amplification protocols. The best results were obtained using the JohnePrep and MagMax extraction kits combined with an in-house triplex real-time PCR designed to detect IS900, ISMap02(an insertion sequence ofM. aviumsubsp.paratuberculosispresent in 6 copies per genome), and an internal amplification control DNA simultaneously. These combinations detected 10M. aviumsubsp.paratuberculosiscells/g of spiked feces. The triplex PCR detected 1 fg of genomic DNA extracted from the reference strain K10. The performance of the robotized version of the MagMax extraction kit combined with the IS900and ISMap02PCR was further evaluated using 615 archival fecal samples from the first sampling of nine Friesian cattle herds included in a PTB control program and followed up for at least 4 years. The analysis of the results obtained in this survey demonstrated that the diagnostic method was highly specific and sensitive for the detection ofM. aviumsubsp.paratuberculosisin fecal samples from cattle and a very valuable tool to be used in PTB control programs.

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Uptake and Persistence of Mycobacterium avium subsp. paratuberculosis in Human Monocytes

Infection and Immunity ◽

10.1128/iai.00534-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3768-3775 ◽

Cited By ~ 19

Author(s):

Dayle A. Keown ◽

David A. Collings ◽

Jacqueline I. Keenan

Keyword(s):

Mycobacterium Avium ◽

Human Cells ◽

Monocytic Cell ◽

Monocytic Cell Line ◽

Tissue Samples ◽

Content Type ◽

Human Monocytic Cell Line ◽

Human Monocytic Cell ◽

Mycobacterium Avium Subsp Paratuberculosis ◽

Lysosomal Protein

ABSTRACTMycobacterium aviumsubsp.paratuberculosisis a bacterium sometimes found in human blood and tissue samples that may have a role in the etiology of Crohn's disease in humans. To date, however, there have been few studies examining the interactions of these bacteria with human cells. Using the THP-1 human monocytic cell line, this study shows that the uptake and trafficking ofM. aviumsubsp.paratuberculosisin human cells are cholesterol dependent and that these bacteria localize to cholesterol-rich compartments that are slow to acidify.M. aviumsubsp.paratuberculosisbacteria containing phagosomes stain for the late endosomal marker Rab7, but recruitment of the Rab7-interacting lysosomal protein that regulates the fusion of bacterium-containing phagosomes with lysosomal compartments and facilitates subsequent bacterial clearance is significantly reduced. Disruption of phagosome acidification via this mechanism may contribute toM. aviumsubsp.paratuberculosispersistence in human cells, but there was no evidence that internalizedM. aviumsubsp.paratuberculosisalso affects the survival of bacteria taken up during a secondary phagocytic event.

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Survival of Mycobacterium avium subsp. paratuberculosis in Synthetic Human Gastric Juice and Acidified Porcine Bile

Applied and Environmental Microbiology ◽

10.1128/aem.03523-12 ◽

2012 ◽

Vol 79 (4) ◽

pp. 1418-1420

Author(s):

J. P. Dalton ◽

C. Hill

Keyword(s):

Quantitative Analysis ◽

Gastric Juice ◽

Mycobacterium Avium ◽

Reverse Transcription ◽

Propidium Monoazide ◽

Human Gastric Juice ◽

Content Type ◽

Reverse Transcription Pcr ◽

Bactericidal Activities ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACTThe bactericidal activities of synthetic gastric juice and acidified porcine bile onMycobacterium aviumsubsp.paratuberculosiswere assessed using propidium monoazide (PMA)-mediated quantitative reverse transcription-PCR, which allowed rapid relative quantitative analysis of viableM. aviumsubsp.paratuberculosiscells.

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