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 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 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 Identification of the agr Locus of Listeria monocytogenes: Role in Bacterial Virulence

2003 ◽  
Vol 71 (8) ◽  
pp. 4463-4471 ◽  
Author(s):  
Nicolas Autret ◽  
Catherine Raynaud ◽  
Iharilalao Dubail ◽  
Patrick Berche ◽  
Alain Charbit
Keyword(s):  
Listeria Monocytogenes ◽  
Response Regulator ◽  
Lethal Dose ◽  
Fold Increase ◽  
Bacterial Virulence ◽  
Intravenous Route ◽  
Two Component System ◽  
Food Borne ◽  
Severe Infections

ABSTRACT Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced protein secretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L. monocytogenes.

scholarly journals In VitroProperties of a Listeria monocytogenes Bacteriophage-Resistant Mutant Predict Its Efficacy as a Live Oral Vaccine Strain

2011 ◽  
Vol 79 (12) ◽  
pp. 5001-5009 ◽  
Author(s):  
Patricia A. Spears ◽  
M. Mitsu Suyemoto ◽  
Terri S. Hamrick ◽  
Rebecca L. Wolf ◽  
Edward A. Havell ◽  
...  
Keyword(s):  
Growth Rate ◽  
Listeria Monocytogenes ◽  
Time Course ◽  
Oral Vaccine ◽  
Fold Increase ◽  
Resistant Mutant ◽  
Oral Immunization ◽  
Intracellular Growth ◽  
Content Type ◽  
Live Oral Vaccine

ABSTRACTAListeria monocytogenesglcVmutation precludes the binding of certain listerial phages and produces a profound attenuation characterized by the absence of detectable mutants in the livers and spleens of orally inoculated mice.Invitro, we found that the mutant formed plaques on mouse enterocyte monolayers as efficiently as the parent but the plaques formed were smaller. Intracellular growth rate determinations and examination of infected enterocytes by light and fluorescence microscopy established that the mutant was impaired not in intracellular growth rate but in cell-to-cell spreading. Because this property is shared by other immunogenic mutants (e.g.,actAmutants), ourglcVmutant was tested for vaccine efficacy. Oral immunization with the mutant and subsequent oral challenge (22 days postvaccination) with the parent revealed a ca. 10,000-fold increase in protection afforded by the mutant compared to sham-vaccinated controls. TheglcVmutant did not stimulate innate immunity under the dose and route employed for vaccination, and an infectivity index time course experiment revealed pronounced mutant persistence in Peyer's patches. The immunogenicity of theglcVmutant compared to an isogenicactAmutant reference strain was next tested in an experiment with a challenge given 52 days postvaccination. Both mutant strains showed scant vital organ infectivity and high levels of protection similar to those seen using theglcVmutant in the 22-day postvaccination challenge. Our results indicate that oral administration of a profoundly attenuated listerial mutant can safely elicit solid protective immunity.

scholarly journals Population structure-guided profiling of antibiotic resistance patterns in clinical Listeria monocytogenes isolates from Germany identifies pbpB3 alleles associated with low levels of cephalosporin resistance

2020 ◽  
Author(s):  
Martin A. Fischer ◽  
Sabrina Wamp ◽  
Angelika Fruth ◽  
Franz Allerberger ◽  
Antje Flieger ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Population Structure ◽  
Listeria Monocytogenes ◽  
Allelic Variation ◽  
Binding Protein ◽  
Population Based ◽  
The European Union ◽  
Penicillin Binding Protein ◽  
Minimal Inhibitory Concentrations ◽  
Cephalosporin Resistance

AbstractCase numbers of listeriosis have been increasing in Germany and the European Union during the last decade. In addition reports on the occurrence of antibiotic resistance in Listeria monocytogenes in clinical and environmental isolates are accumulating. The susceptibility towards 14 antibiotics was tested in a selection of clinical L. monocytogenes isolates to get a more precise picture of the development and manifestation of antibiotic resistance in the L. monocytogenes population. Based on the population structure determined by core genome multi locus sequence typing (cgMLST) 544 out of 1,220 sequenced strains collected in Germany between 2009 and 2019 were selected to cover the phylogenetic diversity observed in the clinical L. monocytogenes population. All isolates tested were susceptible towards ampicillin, penicillin and co-trimoxazole - the most relevant antibiotics in the treatment of listeriosis. Resistance to daptomycin and ciprofloxacin was observed in 493 (91%) and in 71 (13%) of 544 isolates, respectively. While all tested strains showed resistance towards ceftriaxone, the minimal inhibitory concentrations (MIC) observed varied widely between 4 mg/L up to >128 mg/L. An allelic variation of the penicillin binding protein gene pbpB3 could be identified as the cause of this difference in ceftriaxone resistance levels. This study is the first population structure-guided analysis of antimicrobial resistance in recent clinical isolates and confirms the importance of penicillin binding protein B3 (PBP B3) for the high level of intrinsic cephalosporin resistance of L. monocytogenes on a population-wide scale.

scholarly journals Activation of Bvg-Repressed Genes in Bordetella pertussis by RisA Requires Cross Talk from Noncooperonic Histidine Kinase RisK

2017 ◽  
Vol 199 (22) ◽  
Author(s):  
Qing Chen ◽  
Victoria Ng ◽  
Jason M. Warfel ◽  
Tod J. Merkel ◽  
Scott Stibitz
Keyword(s):  
Bordetella Pertussis ◽  
Histidine Kinase ◽  
Genomic Sequence ◽  
Response Regulator ◽  
Two Component System ◽  
Kinase Gene ◽  
Content Type ◽  
Reading Frame ◽  
Two Component

ABSTRACT The two-component response regulator RisA, encoded by open reading frame BP3554 in the Bordetella pertussis Tohama I genomic sequence, is a known activator of vrg genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the bvgAS virulence regulon. Here we demonstrate that RisA is phosphorylated in vivo and that RisA phosphorylation is required for activation of vrg genes. An adjacent histidine kinase gene, risS, is truncated by frameshift mutation in B. pertussis but not in Bordetella bronchiseptica or Bordetella parapertussis. Neither deletion of risS′ or bvgAS nor phenotypic modulation with MgSO4 affected levels of phosphorylated RisA (RisA∼P) in B. pertussis. However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the vrg genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisAD60E mutant, indicating that an active conformation of RisA, but not phosphorylation per se, is crucial for vrg activation. Interestingly, expression of vrg genes is still modulated by MgSO4 in cells harboring the RisAD60E mutation, suggesting that the activated RisA senses additional signals to control vrg expression in response to environmental stimuli. IMPORTANCE In B. pertussis, the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally regulated vrg genes requires RisA and is also repressed by the Bvg-activated BvgR. RisA is an OmpR-like response regulator, but RisA phosphorylation was not expected because the gene for its presumed, cooperonic, histidine kinase is inactivated by mutation. In this study, we demonstrate phosphorylation of RisA in vivo by a noncooperonic histidine kinase. We also show that RisA phosphorylation is necessary but not sufficient for vrg activation but, importantly, is not affected by BvgAS status. Instead, we propose that vrg expression is controlled by BvgAS through its regulation of BvgR, a cyclic di-GMP (c-di-GMP) phosphodiesterase.

scholarly journals Suppressor Mutations Linking gpsB with the First Committed Step of Peptidoglycan Biosynthesis in Listeria monocytogenes

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Jeanine Rismondo ◽  
Jennifer K. Bender ◽  
Sven Halbedel
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Cell Wall Biosynthesis ◽  
Human Pathogen ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Cell Division Protein ◽  
Precursor Molecules

ABSTRACT The cell division protein GpsB is a regulator of the penicillin binding protein A1 (PBP A1) in the Gram-positive human pathogen Listeria monocytogenes. Penicillin binding proteins mediate the last two steps of peptidoglycan biosynthesis as they polymerize and cross-link peptidoglycan strands, the main components of the bacterial cell wall. It is not known what other processes are controlled by GpsB. L. monocytogenes gpsB mutants are unable to grow at 42°C, but we observed that spontaneous suppressors correcting this defect arise on agar plates with high frequency. We here describe a first set of gpsB suppressors that mapped to the clpC and murZ genes. While ClpC is the ATPase component of the Clp protease, MurZ is a paralogue of the listerial UDP–N-acetylglucosamine (UDP-GlcNAc) 1-carboxyvinyltransferase MurA. Both enzymes catalyze the enolpyruvyl transfer from phosphoenolpyruvate to UDP-GlcNAc, representing the first committed step of peptidoglycan biosynthesis. We confirmed that clean deletion of the clpC or murZ gene suppressed the ΔgpsB phenotype. It turned out that the absence of either gene leads to accumulation of MurA, and we show that artificial overexpression of MurA alone was sufficient for suppression. Inactivation of other UDP-GlcNAc-consuming pathways also suppressed the heat-sensitive growth of the ΔgpsB mutant, suggesting that an increased influx of precursor molecules into peptidoglycan biosynthesis can compensate for the lack of GpsB. Our results support a model according to which PBP A1 becomes misregulated and thus toxic in the absence of GpsB due to unproductive consumption of cell wall precursor molecules. IMPORTANCE The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

scholarly journals New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Inga Schweizer ◽  
Sebastian Blättner ◽  
Patrick Maurer ◽  
Katharina Peters ◽  
Daniela Vollmer ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Morphological Changes ◽  
Penicillin Resistance ◽  
Penicillin Binding Protein ◽  
Two Component System ◽  
Gene Encoding ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
High Level

ABSTRACT The Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0 ). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murM Hu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murM Hu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2x Hu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2x Hu17 and pbp1a of strain Hu17 (pbp1a Hu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.

scholarly journals MifS, a DctB family histidine kinase, is a specific regulator of α-ketoglutarate response in Pseudomonas aeruginosa PAO1

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 867-879 ◽  
Author(s):  
Zaara Sarwar ◽  
Michael X. Wang ◽  
Benjamin R. Lundgren ◽  
Christopher T. Nomura
Keyword(s):  
Signal Transduction ◽  
Pseudomonas Aeruginosa ◽  
Histidine Kinase ◽  
Type Species ◽  
Mutational Analysis ◽  
Homology Modelling ◽  
Fold Increase ◽  
Binding Pocket ◽  
Content Type ◽  
Link Type

The C5-dicarboxylate α-ketoglutarate (α-KG) is a preferred nutrient source for the opportunistic pathogen Pseudomonas aeruginosa . However, very little is known about how P. aeruginosa detects and responds to α-KG in the environment. Our laboratory has previously shown that the MifS/MifR two-component signal transduction system regulates α-KG assimilation in P. aeruginosa PAO1. In an effort to better understand how this bacterium detects α-KG, we characterized the MifS sensor histidine kinase. In this study we show that although MifS is a homologue of the C4-dicarboxylate sensor DctB, it specifically responds to the C5-dicarboxylate α-KG. MifS activity increased >10-fold in the presence of α-KG, while the related C5-dicarboxylate glutarate caused only a 2-fold increase in activity. All other dicarboxylates tested did not show any significant effect on MifS activity. Homology modelling of the MifS sensor domain revealed a substrate binding pocket for α-KG. Using protein modelling and mutational analysis, we identified nine residues that are important for α-KG response, including one residue that determines the substrate specificity of MifS. Further, we found that MifS has a novel cytoplasmic linker domain that is required for α-KG response and is probably involved in signal transduction from the sensor domain to the cytoplasmic transmitter domain. Until this study, DctB family histidine kinases were known to only respond to C4-dicarboxylates. Our work shows that MifS is a novel member of the DctB family histidine kinase that specifically responds to α-KG.

scholarly journals The cell envelope stress response mediated by the LiaFSR Lm three-component system of Listeria monocytogenes is controlled via the phosphatase activity of the bifunctional histidine kinase LiaS Lm

Microbiology ◽  
2011 ◽  
Vol 157 (2) ◽  
pp. 373-386 ◽  
Author(s):  
Frederike Fritsch ◽  
Norman Mauder ◽  
Tatjana Williams ◽  
Julia Weiser ◽  
Markus Oberle ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Phosphatase Activity ◽  
Histidine Kinase ◽  
Cell Envelope ◽  
Transcriptional Profiling ◽  
Two Component System ◽  
Component System ◽  
Lipid Ii ◽  
Envelope Stress ◽  
Sensor Protein

Most members of the phylum Firmicutes harbour a two-component system (TCS), LiaSR, which is involved in the response to cell envelope stress elicited most notably by inhibitors of the lipid II cycle. In all LiaSR systems studied in detail, LiaSR-mediated signal transduction has been shown to be negatively controlled by a membrane protein, LiaF, encoded upstream of liaSR. In this study we have analysed the LiaSR orthologue of Listeria monocytogenes (LiaSR Lm ). Whole-genome transcriptional profiling indicated that activation of LiaSR Lm results in a remodelling of the cell envelope via the massive upregulation of membrane-associated and extracytoplasmic proteins in the presence of inducing stimuli. As shown for other LiaSR TCSs, LiaSR Lm is activated by cell wall-active antibiotics. We demonstrate that the level of phosphorylated LiaR Lm , which is required for the induction of the LiaSR Lm regulon, is controlled by the interplay between the histidine kinase and phosphatase activities of the bifunctional sensor protein LiaS Lm . Our data suggest that the phosphatase activity of LiaS Lm is stimulated by LiaF Lm in the absence of cell envelope stress.

Sign in / Sign up

Export Citation Format

Share Document