scholarly journals A Multiorgan Trafficking Circuit Provides Purifying Selection of Listeria monocytogenes Virulence Genes

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Alexander Louie ◽  
Ting Zhang ◽  
Simone Becattini ◽  
Matthew K. Waldor ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Population ◽  
Virulence Genes ◽  
Purifying Selection ◽  
Host Population ◽  
Wild Type ◽  
Content Type ◽  
Systemic Dissemination ◽  
Systemic Spread ◽  
Selection Of

ABSTRACT Listeria monocytogenes can cause a life-threatening illness when the foodborne pathogen spreads beyond the intestinal tract to distant organs. Many aspects of the intestinal phase of L. monocytogenes pathogenesis remain unknown. Here, we present a foodborne infection model using C57BL/6 mice that have been pretreated with streptomycin. In this model, as few as 100 L. monocytogenes CFU were required to cause self-limiting enterocolitis, and systemic dissemination followed previously reported routes. Using this model, we report that listeriolysin O (LLO) and actin assembly-inducing protein (ActA), two critical virulence determinants, were necessary for intestinal pathology and systemic spread but were dispensable for intestinal growth. Sequence tag-based analysis of microbial populations (STAMP) was used to investigate the within-host population dynamics of wild-type and LLO-deficient strains. The wild-type bacterial population experienced severe bottlenecks over the course of infection, and by 5 days, the intestinal population was highly enriched for bacteria originating from the gallbladder. In contrast, LLO-deficient strains did not efficiently disseminate and gain access to the gallbladder, and the intestinal population remained diverse. These findings suggest that systemic spread and establishment of a bacterial reservoir in the gallbladder imparts an intraspecies advantage in intestinal occupancy. Since intestinal L. monocytogenes is ultimately released into the environment, within-host population bottlenecks may provide purifying selection of virulence genes. IMPORTANCE Listeria monocytogenes maintains capabilities for free-living growth in the environment and for intracellular replication in a wide range of hosts, including livestock and humans. Here, we characterized an enterocolitis model of foodborne L. monocytogenes infection. This work highlights a multiorgan trafficking circuit and reveals a fitness advantage for bacteria that successfully complete this cycle. Because virulence factors play critical roles in systemic dissemination and multiple bottlenecks occur as the bacterial population colonizes different tissue sites, this multiorgan trafficking circuit likely provides purifying selection of virulence genes. This study also serves as a foundation for future work using the L. monocytogenes-induced enterocolitis model to investigate the biology of L. monocytogenes in the intestinal environment.

scholarly journals Avoidance of Autophagy Mediated by PlcA or ActA Is Required for Listeria monocytogenes Growth in Macrophages

2015 ◽  
Vol 83 (5) ◽  
pp. 2175-2184 ◽  
Author(s):  
Gabriel Mitchell ◽  
Liang Ge ◽  
Qiongying Huang ◽  
Chen Chen ◽  
Sara Kianian ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Wild Type Strain ◽  
Fold Increase ◽  
Host Cells ◽  
Listeriolysin O ◽  
Wild Type ◽  
Conjugation System ◽  
Content Type ◽  
A Cell ◽  
Gain Access

Listeria monocytogenesis a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ± 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ± 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ± 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy byL. monocytogenesprimarily involves PlcA and ActA and that either one of these factors must be present forL. monocytogenesgrowth in macrophages.

scholarly journals Nisin Resistance of Listeria monocytogenes Is Increased by Exposure to Salt Stress and Is Mediated via LiaR

2013 ◽  
Vol 79 (18) ◽  
pp. 5682-5688 ◽  
Author(s):  
Teresa M. Bergholz ◽  
Silin Tang ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Salt Stress ◽  
Listeria Monocytogenes ◽  
Safety Concern ◽  
Response Regulator ◽  
Brain Heart Infusion ◽  
Control Measures ◽  
Protective Effects ◽  
Wild Type ◽  
Content Type ◽  
Type Strains

ABSTRACTGrowth ofListeria monocytogeneson refrigerated, ready-to-eat food is a significant food safety concern. Natural antimicrobials, such as nisin, can be used to control this pathogen on food, but little is known about how other food-related stresses may impact how the pathogen responds to these compounds. Prior work demonstrated that exposure ofL. monocytogenesto salt stress at 7°C led to increased expression of genes involved in nisin resistance, including the response regulatorliaR. We hypothesized that exposure to salt stress would increase subsequent resistance to nisin and that LiaR would contribute to increased nisin resistance. Isogenic deletion mutations inliaRwere constructed in 7 strains ofL. monocytogenes, and strains were exposed to 6% NaCl in brain heart infusion broth and then tested for resistance to nisin (2 mg/ml Nisaplin) at 7°C. For the wild-type strains, exposure to salt significantly increased subsequent nisin resistance (P< 0.0001) over innate levels of resistance. Compared to the salt-induced nisin resistance of wild-type strains, ΔliaRstrains were significantly more sensitive to nisin (P< 0.001), indicating that induction of LiaFSR led to cross-protection ofL. monocytogenesagainst subsequent inactivation by nisin. Transcript levels of LiaR-regulated genes were induced by salt stress, and lmo1746 andtelAwere found to contribute to LiaR-mediated salt-induced nisin resistance. These data suggest that environmental stresses similar to those on foods can influence the resistance ofL. monocytogenesto antimicrobials such as nisin, and potential cross-protective effects should be considered when selecting and applying control measures for this pathogen on ready-to-eat foods.

scholarly journals Sigma B Contributes to Listeria monocytogenes Gastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model

2006 ◽  
Vol 74 (2) ◽  
pp. 876-886 ◽  
Author(s):  
M. R. Garner ◽  
B. L. Njaa ◽  
M. Wiedmann ◽  
K. J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Guinea Pig ◽  
Guinea Pigs ◽  
Type Strain ◽  
Wild Type Strain ◽  
Critical Role ◽  
Host Cells ◽  
Gastrointestinal Infection ◽  
Wild Type ◽  
Systemic Spread

ABSTRACT Contributions of the alternative sigma factor σB to Listeria monocytogenes infection were investigated using strains bearing null mutations in sigB, prfA, or inlA or in selected inlA or prfA promoter regions. The ΔP4 inlA strain, which has a deletion in the σB-dependent P4 inlA promoter, and the ΔsigB strain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σB dependent P2 prfA promoter region did not differ from that of the wild type. The virulence of the ΔsigB and ΔP4 inlA strains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigB strain showing greater attenuation, while the virulence capacity of the ΔP2 prfA strain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigB mutation does not result from loss of σB-dependent prfA transcription. Our results show that σB-dependent activation of inlA is important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition to inlA appear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigB strain was not attenuated in intravenously infected guinea pigs. We conclude that (i) L. monocytogenes σB plays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects on inlA transcription but not on prfA transcription, and (iii) σB plays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.

scholarly journals The bvr Locus of Listeria monocytogenes Mediates Virulence Gene Repression by β-Glucosides

1999 ◽  
Vol 181 (16) ◽  
pp. 5024-5032 ◽  
Author(s):  
Klaus Brehm ◽  
María-Teresa Ripio ◽  
Jürgen Kreft ◽  
José-Antonio Vázquez-Boland
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Genes ◽  
Natural Habitat ◽  
Sensory System ◽  
Virulence Gene ◽  
Gene Repression ◽  
Signal Molecules ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Virulence Gene Expression

ABSTRACT The β-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence genes hly andplcA in Listeria monocytogenes NCTC 7973. This led to the hypothesis that β-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes if L. monocytogenes is living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressed hly. This suggested that the downregulation of virulence genes by β-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C geneplcB as a reporter. Utilization of any fermentable sugar caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973, plcB was only repressed by β-glucosides. Thus, the regulation of L. monocytogenes virulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to β-glucosides. We have identified in L. monocytogenes a 4-kb operon, bvrABC, encoding an antiterminator of the BglG family (bvrA), a β-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from other Listeria spp. Transcription ofbvrB was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part ofbvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that the bvr locus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of virulence genes.

scholarly journals Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
C. D. Shaw ◽  
H. Imamura ◽  
T. Downing ◽  
G. Blackburn ◽  
G. D. Westrop ◽  
...  
Keyword(s):  
Leishmania Donovani ◽  
Wild Type ◽  
Genetic Changes ◽  
Content Type ◽  
Novel Treatments ◽  
Branched Chain Amino Acid ◽  
Amastigote Stage ◽  
Branched Chain ◽  
Intracellular Amastigote ◽  
Selection Of

ABSTRACT Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr. In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

scholarly journals Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use

2016 ◽  
Vol 55 (1) ◽  
pp. 183-198 ◽  
Author(s):  
Soumitesh Chakravorty ◽  
Sandy S. Roh ◽  
Jennifer Glass ◽  
Laura E. Smith ◽  
Ann Marie Simmons ◽  
...  
Keyword(s):  
Point Of Care ◽  
Stokes Shift ◽  
Drug Resistant ◽  
Wild Type ◽  
Resistance Mutations ◽  
Content Type ◽  
Automated Assay ◽  
Resistant Tuberculosis ◽  
Three Phase ◽  
Selection Of

ABSTRACTExtensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested ingyrA,gyrB,katG, andrrsgenes and the promoters ofinhAandeisgenes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU ofMycobacterium tuberculosisin 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

scholarly journals AprlMutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

2014 ◽  
Vol 197 (5) ◽  
pp. 932-942 ◽  
Author(s):  
Juliana Durack ◽  
Thomas P. Burke ◽  
Daniel A. Portnoy
Keyword(s):  
Listeria Monocytogenes ◽  
Atp Hydrolysis ◽  
Colony Morphology ◽  
Wild Type ◽  
Swarming Motility ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Culture Cells ◽  
Almost All

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogenListeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence inL. monocytogenes; however, the mechanism of export via this pathway is poorly understood.L. monocytogenessecA2mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility toL. monocytogenessecA2mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by thelmo2769operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation insecYwas identified that conferred smooth colony morphology tosecA2mutants, restored wild-type plaque formation, and increased virulence in mice. ThissecYmutation resembled aprlsuppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although theprlmutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1mutant was still less virulentin vivothan the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

σ B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3843-3855 ◽  
Author(s):  
David Sue ◽  
Daniel Fink ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Virulence Genes ◽  
Acid Stress ◽  
Exponential Phase ◽  
Wild Type ◽  
Transcript Accumulation ◽  
Gastrointestinal Infections ◽  
Stress Response Genes ◽  
Foodborne Infections

Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor σ B, encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0·3 M NaCl) or acid (pH 4·5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes ΔsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that σ B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that σ B contributes to the ability of L. monocytogenes to cause foodborne infections.

scholarly journals Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Christophe Bécavin ◽  
Christiane Bouchier ◽  
Pierre Lechat ◽  
Cristel Archambaud ◽  
Sophie Creno ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Biology ◽  
Virulence Genes ◽  
Constitutive Expression ◽  
Nucleotide Polymorphisms ◽  
Pathogenic Potential ◽  
Bacterial Gene ◽  
Content Type

ABSTRACTFor nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogenListeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strainin vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infectionin vivo, highlighting the fact thatin vitroinvasion assays are not sufficient for evaluating the pathogenic potential ofL. monocytogenesstrains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using differentL. monocytogenesstrains.IMPORTANCEOver the past 3 decades,Listeriahas become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studyingListeriause primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD’s PrfA, the main regulator ofListeriavirulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may useListeriaas a tool to study the pathophysiology of listeriosis and immune responses.

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