scholarly journals VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts

2015 ◽  
Vol 81 (13) ◽  
pp. 4553-4562 ◽  
Author(s):  
Jihun Kang ◽  
Martin Wiedmann ◽  
Kathryn J. Boor ◽  
Teresa M. Bergholz
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Resistance Mechanism ◽  
Cross Protection ◽  
System Response ◽  
Antimicrobial Compounds ◽  
Content Type ◽  
Lauric Arginate ◽  
Increased Sensitivity

ABSTRACTFormulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such asListeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed byL. monocytogenesto counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, ε-polylysine, and chitosan). AvirRdeletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or ε-polylysine (P< 0.05). The VirR-regulated operon,dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene,mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a β-glucuronidase (GUS) reporter fusion with thedltpromoter indicated that nisin does not specifically induce VirR-dependent upregulation ofdltABCD. Lastly, prior exposure ofL. monocytogenesparent strain H7858 and the ΔvirRmutant to 2% potassium lactate enhanced subsequent resistance against nisin and ε-polylysine (P< 0.05). These data demonstrate that VirRS-mediated regulation ofdltABCDis the major resistance mechanism used byL. monocytogenesagainst cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods.

scholarly journals c-di-AMP Accumulation Impairs Muropeptide Synthesis in Listeria monocytogenes

2020 ◽  
Vol 202 (24) ◽  
Author(s):  
Steven M. Massa ◽  
Amar Deep Sharma ◽  
Cheta Siletti ◽  
Zepeng Tu ◽  
Jared J. Godfrey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Cell Envelope ◽  
Second Messenger ◽  
Fold Increase ◽  
Bacterial Cells ◽  
Content Type ◽  
Bacterial Physiology ◽  
Peptidoglycan Synthesis ◽  
Increased Sensitivity

ABSTRACT Cyclic di-AMP (c-di-AMP) is an essential and ubiquitous second messenger among bacteria. c-di-AMP regulates many cellular pathways through direct binding to several molecular targets in bacterial cells. c-di-AMP depletion is well known to destabilize the bacterial cell wall, resulting in increased bacteriolysis and enhanced susceptibility to cell wall targeting antibiotics. Using the human pathogen Listeria monocytogenes as a model, we found that c-di-AMP accumulation also impaired cell envelope integrity. An L. monocytogenes mutant deleted for c-di-AMP phosphodiesterases (pdeA pgpH mutant) exhibited a 4-fold increase in c-di-AMP levels and several cell wall defects. For instance, the pdeA pgpH mutant was defective for the synthesis of peptidoglycan muropeptides and was susceptible to cell wall-targeting antimicrobials. Among different muropeptide precursors, we found that the pdeA pgpH strain was particularly impaired in the synthesis of d-Ala–d-Ala, which is required to complete the pentapeptide stem associated with UDP–N-acetylmuramic acid (MurNAc). This was consistent with an increased sensitivity to d-cycloserine, which inhibits the d-alanine branch of peptidoglycan synthesis. Finally, upon examining d-Ala:d-Ala ligase (Ddl), which catalyzes the conversion of d-Ala to d-Ala–d-Ala, we found that its activity was activated by K+. Based on previous reports that c-di-AMP inhibits K+ uptake, we propose that c-di-AMP accumulation impairs peptidoglycan synthesis, partially through the deprivation of cytoplasmic K+ levels, which are required for cell wall-synthetic enzymes. IMPORTANCE The bacterial second messenger c-di-AMP is produced by a large number of bacteria and conditionally essential to many species. Conversely, c-di-AMP accumulation is also toxic to bacterial physiology and pathogenesis, but its mechanisms are largely undefined. We found that in Listeria monocytogenes, elevated c-di-AMP levels diminished muropeptide synthesis and increased susceptibility to cell wall-targeting antimicrobials. Cell wall defects might be an important mechanism for attenuated virulence in bacteria with high c-di-AMP levels.

scholarly journals Assessing the Contributions of the LiaS Histidine Kinase to the Innate Resistance of Listeria monocytogenes to Nisin, Cephalosporins, and Disinfectants

2012 ◽  
Vol 78 (8) ◽  
pp. 2923-2929 ◽  
Author(s):  
Barry Collins ◽  
Caitriona M. Guinane ◽  
Paul D. Cotter ◽  
Colin Hill ◽  
R. Paul Ross
Keyword(s):  
Listeria Monocytogenes ◽  
Histidine Kinase ◽  
Fold Increase ◽  
Penicillin Binding Protein ◽  
Two Component System ◽  
Food Preservative ◽  
Content Type ◽  
Cephalosporin Resistance ◽  
Increased Sensitivity ◽  
Cephalosporin Antibiotics

ABSTRACTTheListeria monocytogenesLiaSR two-component system (2CS) encoded bylmo1021andlmo1022plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (ΔliaS) resulted in a 4-fold increase in nisin resistance. In contrast, the ΔliaSstrain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance inL. monocytogeneshas previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator ofliaSand a penicillin binding protein encoded bylmo2229. We noted thatlmo2229transcription is increased in the ΔliaSmutant and in a ΔliaSΔlisKdouble mutant and that disruption oflmo2229in the ΔliaSΔlisKmutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance ofL. monocytogenescould facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.

scholarly journals Appropriate Regulation of the σE-Dependent Envelope Stress Response Is Necessary To Maintain Cell Envelope Integrity and Stationary-Phase Survival in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Hervé Nicoloff ◽  
Saumya Gopalkrishnan ◽  
Sarah E. Ades
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Outer Membrane ◽  
Stress Responses ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Point Mutations ◽  
Content Type ◽  
Envelope Stress ◽  
Outer Membrane Porins

ABSTRACT The alternative sigma factor σE is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σE increases during entry into stationary phase, suggesting an important role for σE when nutrients are limiting. Elevated σE activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σE-directed cell lysis and the role of σE in stationary phase, we investigated the effects of elevated σE activity in cultures grown for 10 days. We demonstrate that high σE activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σE activity, due primarily to point mutations in the region of σE that binds the −35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σE activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg2+ and buffering the growth medium or by deleting the σE-dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σE activity. Our results demonstrate that appropriate regulation of σE activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σE response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σE is tightly regulated to match the production of σE regulon members to the needs of the cell. In E. coli, loss of σE results in lethality. Here we demonstrate that excessive σE activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent.

scholarly journals Listeria monocytogenes Shows Temperature-Dependent and -Independent Responses to Salt Stress, Including Responses That Induce Cross-Protection against Other Stresses

2012 ◽  
Vol 78 (8) ◽  
pp. 2602-2612 ◽  
Author(s):  
Teresa M. Bergholz ◽  
Barbara Bowen ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Salt Stress ◽  
Listeria Monocytogenes ◽  
Osmotic Stress ◽  
Parent Strain ◽  
Cross Protection ◽  
Temperature Dependent ◽  
Short Term ◽  
Transcript Levels ◽  
Content Type ◽  
Short Term Adaptation

ABSTRACTThe food-borne pathogenListeria monocytogenesexperiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. During transmission,L. monocytogenesis likely to experience osmotic stress at different temperatures and may adapt to osmotic stress in a temperature-dependent manner. To understand the impact of temperature on the responses this pathogen uses to adapt to osmotic stress, we assessed genome-wide changes in theL. monocytogenesH7858 transcriptome during short-term and long-term adaptation to salt stress at 7°C and 37°C. At both temperatures, the short-term response to salt stress included increased transcript levels ofsigBand SigB-regulated genes, as well asmrpABCDEFG, encoding a sodium/proton antiporter. This antiporter was found to play a role in adaptation to salt stress at both temperatures; ΔmrpABCDEFGhad a significantly longer lag phase than the parent strain in BHI plus 6% NaCl at 7°C and 37°C. The short-term adaptation to salt stress at 7°C included increased transcript levels of two genes encoding carboxypeptidases that modify peptidoglycan. These carboxypeptidases play a role in the short-term adaptation to salt stress only at 7°C, where the deletion mutants had significantly different lag phases than the parent strain. Changes in the transcriptome at both temperatures suggested that exposure to salt stress could provide cross-protection to other stresses, including peroxide stress. Short-term exposure to salt stress significantly increased H2O2resistance at both temperatures. These results provide information for the development of knowledge-based intervention methods against this pathogen, as well as provide insight into potential mechanisms of cross-protection.

scholarly journals Transcriptomic Response of Listeria monocytogenes during the Transition to the Long-Term-Survival Phase

2011 ◽  
Vol 77 (17) ◽  
pp. 5966-5972 ◽  
Author(s):  
Jia Wen ◽  
Xiangyu Deng ◽  
Zengxin Li ◽  
Edward G. Dudley ◽  
Ramaswamy C. Anantheswaran ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Cell Envelope ◽  
Growth Stages ◽  
Transcriptomic Response ◽  
Term Survival ◽  
Long Term Survival ◽  
Content Type ◽  
Serotype 4B ◽  
Underlying Mechanisms

ABSTRACTListeria monocytogenescan change its cellular morphology from bacilli to cocci during the transition to the long-term-survival (LTS) phase. The LTS cells demonstrated increased baro- and thermotolerance compared to their vegetative counterparts. So far, the underlying mechanisms that trigger this morphological and physiological transition remain largely unknown. In this study, we compared the transcriptomic profiles ofL. monocytogenesserotype 4b strain F2365 at different growth stages in tryptic soy broth with yeast extract (TSBYE) using a whole-genome DNA chip approach. We identified a total of 225 differentially expressed genes (≥4-fold;P< 0.05) during the transition to the LTS phase in TSBYE. Genes related to cell envelope structure, energy metabolism, and transport were most significantly upregulated in the LTS phase. The upregulation of compatible solute transporters may lead to the accumulation of cellular solutes, lowering intracellular water activity and thus increasing bacterial stress resistance during the transition to the LTS phase. The downregulation of genes associated with protein synthesis may indicate a status of metabolic dormancy of the LTS cells. The transcriptomic profiles of resuscitated LTS cells in fresh TSBYE resembled those of log-phase cells (r=0.94), as the LTS cells rapidly resume metabolic activities and transit back to log phase with decreased baro- and thermotolerance.

scholarly journals Short- and Long-Term Adaptation to Ethanol Stress and Its Cross-Protective Consequences in Lactobacillus plantarum

2011 ◽  
Vol 77 (15) ◽  
pp. 5247-5256 ◽  
Author(s):  
Hermien van Bokhorst-van de Veen ◽  
Tjakko Abee ◽  
Marcel Tempelaars ◽  
Peter A. Bron ◽  
Michiel Kleerebezem ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Stress Responses ◽  
Dna Microarrays ◽  
Cell Envelope ◽  
Transcriptome Profiling ◽  
Ethanol Exposure ◽  
Cross Protection ◽  
Ethanol Stress ◽  
Class Iii ◽  
Content Type

ABSTRACTThis paper describes the molecular responses ofLactobacillus plantarumWCFS1 toward ethanol exposure. Global transcriptome profiling using DNA microarrays demonstrated adaptation of the microorganism to the presence of 8% ethanol over short (10-min and 30-min) and long (24-h) time intervals. A total of 57 genes were differentially expressed at all time points. Expression levels of an additional 859 and 873 genes were modulated after 30 min and 24 h of exposure to the solvent, respectively. Ethanol exposure led to induced expression of genes involved in citrate metabolism and cell envelope architecture, as well as canonical stress response pathways controlled by the central stress regulators HrcA and CtsR. Correspondingly, cells grown for 24 h in medium containing 8% ethanol exhibited higher levels of citrate consumption and modified cell membrane fatty acid composition and showed invaginating septa compared with cells grown in liquid medium without ethanol. In addition, these physiological changes resulted in cross-protection against high temperatures but not against several other stresses tested. To evaluate the role of HrcA and CtsR in ethanol tolerance,ctsRandhrcAgene deletion mutants were constructed. The growth rate of theL. plantarumΔctsR::catstrain was impaired in de Man-Rogosa-Sharpe (MRS) medium containing 8% ethanol, whereas growth of theL. plantarumΔhrcA::catand ΔctsRΔhrcA::catmutants was indistinguishable from that of wild-type cells. Overall, these results suggest that the induction of CtsR class III stress responses provides cross-protection against heat stress.

scholarly journals Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
Benjamin I. Baarda ◽  
Sarah Emerson ◽  
Philip J. Proteau ◽  
Aleksandra E. Sikora
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Phenotype Microarray ◽  
Negative Effects ◽  
Content Type ◽  
Vaccine Candidates ◽  
Phenotype Microarrays

ABSTRACTThe function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such asNeisseria gonorrhoeae. A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates—NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344—in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamEmutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for anN. gonorrhoeaephenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such asN. gonorrhoeae.IMPORTANCEInnovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation intoNeisseria gonorrhoeaeto probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overallN. gonorrhoeaephysiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins ofN. gonorrhoeaediscovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.

scholarly journals Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis

2012 ◽  
Vol 56 (11) ◽  
pp. 5907-5915 ◽  
Author(s):  
Diana Wolf ◽  
Patricia Domínguez-Cuevas ◽  
Richard A. Daniel ◽  
Thorsten Mascher
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Membrane Integrity ◽  
Culture Conditions ◽  
Content Type ◽  
Damage Sensing ◽  
Controlled Switching ◽  
Gene Assay ◽  
Envelope Stress

ABSTRACTL-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacteriumBacillus subtiliswas constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response ofB. subtilisto the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show thatB. subtilisL-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established alacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

scholarly journals The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Renate Frantz ◽  
Lisa Teubner ◽  
Tilman Schultze ◽  
Luigi La Pietra ◽  
Christin Müller ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Bacterial Growth ◽  
Cell Envelope ◽  
Intracellular Pathogen ◽  
Type I ◽  
Type I Ifn ◽  
Intracellular Growth ◽  
Potent Inducer ◽  
Content Type ◽  
Bacterial Physiology

ABSTRACT Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response. IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.

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