Monocytogenes Mutants Research Articles

C-di-AMP accumulation disrupts glutathione metabolism in Listeria monocytogenes.

C-di-AMP homeostasis is critical for bacterial stress response, cell wall integrity, and virulence. Except for osmotic stress response, the molecular mechanisms underlying other processes are not well defined. A Listeria monocytogenes mutant lacking both c-di-AMP phosphodiesterases, denoted as the ΔPDE mutant, is significantly attenuated in the mouse model of systemic infection. We utilized the ΔPDE mutant to define the molecular functions of c-di-AMP. RNAseq revealed that the ΔPDE mutant is significantly impaired for the expression of virulence genes regulated by the master transcription factor PrfA, which is activated by reduced glutathione (GSH) during infection. Subsequent quantitative gene expression analyses revealed that the ΔPDE strain is defective for PrfA-regulated gene expression both at the basal level and upon activation by GSH. We further found the ΔPDE strain to be significantly depleted for cytoplasmic GSH and impaired for GSH uptake. The ΔPDE strain was also deficient in GSH under conditions that activate GSH synthesis by the synthase GshF and upon constitutive expression of gshF, suggesting that c-di-AMP accumulation inhibits GSH synthesis activity or promotes GSH catabolism. A constitutively active PrfA* variant restored virulence gene expression in ΔPDE in broth cultures supplemented with GSH but did not rescue virulence defect in vivo. Therefore, virulence attenuation at high c-di-AMP is likely associated with defects outside of the PrfA regulon. For instance, the ΔPDE strain was sensitive to oxidative stress, a phenotype exacerbated in the absence of GshF. Our data reveal GSH metabolism as another pathway that is regulated by c-di-AMP.IMPORTANCEC-di-AMP regulates both bacterial pathogenesis and interactions with the host. Although c-di-AMP is essential in many bacteria, its accumulation also attenuates the virulence of many bacterial pathogens. Therefore, disrupting c-di-AMP homeostasis is a promising antibacterial treatment strategy and has inspired several studies that screened for chemical inhibitors of c-di-AMP phosphodiesterases. However, the molecular functions of c-di-AMP are still not fully defined, and the underlying mechanisms for attenuated virulence at high c-di-AMP levels are unclear. Our analyses in Listeria monocytogenes indicate that virulence-related defects are likely outside of the virulence gene regulon. We found c-di-AMP accumulation to impair L. monocytogenes virulence gene expression and disrupt GSH metabolism. Further studies are necessary to establish the relative contributions of these regulations to virulence and host adaptation.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

TLR2 and endosomal TLR-mediated secretion of IL-10 and immune suppression in response to phagosome-confined Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.

The Man-PTS subunit ⅡC is responsible for the sensitivity of Listeria monocytogenes to durancin GL.

Target cell recognition is an important issue in the realization of bacteriocin's activity. In this report, we provide genetic and biochemical evidence of durancin GL, a new bacteriocin produced by Enterococcus durans 41D, and use ⅡC subunit in the mannose phosphotransferase system (Man‐PTS) of Listeria monocytogenes as target/receptor. First, the L. monocytogenes mutants with Man‐PTS IIC or IID deletion were constructed with the vector pHoss1. Then, the utilization of glucose and mannose and the sensitivity to durancin GL of the mutant strains were investigated. Afterward, the interactions between durancin GL and the subunits of IIC or IID in Man‐PTS of L. monocytogenes were characterized by yeast two‐hybrid system. The results showed that the L. monocytogenes mutants with either IIC or IID deletion were not only resistant to durancin GL, but also their absorption and utilization of glucose and mannose were not disturbed by the presence of durancin GL. Finally, in situ detection of the interaction between durancin GL and Man‐PTS subunits of IIC or IID by yeast two‐hybrid system revealed that there was a strong interaction between durancin GL and Man‐PTS subunit IIC. However, the interaction between durancin GL and Man‐PTS subunit IID was not present or weak. Based on the experimental evidence above, the Man‐PTS subunit IIC is responsible for the sensitivity of L. monocytogenes to bacteriocin durancin GL.

C-di-AMP assists osmoadaptation by regulating the Listeria monocytogenes potassium transporters KimA and KtrCD

Many bacteria and some archaea produce the second messenger cyclic diadenosine monophosphate (c-di-AMP). c-di-AMP controls the uptake of osmolytes in Firmicutes, including the human pathogen Listeria monocytogenes, making it essential for growth. c-di-AMP is known to directly regulate several potassium channels involved in osmolyte transport in species such as Bacillus subtilis and Streptococcus pneumoniae, but whether this same mechanism is involved in L. monocytogenes, or even whether similar ion channels were present, was not known. Here, we have identified and characterized the putative L. monocytogenes' potassium transporters KimA, KtrCD, and KdpABC. We demonstrate that Escherichia coli expressing KimA and KtrCD, but not KdpABC, transport potassium into the cell, and both KimA and KtrCD are inhibited by c-di-AMP in vivo For KimA, c-di-AMP-dependent regulation requires the C-terminal domain. In vitro assays demonstrated that the dinucleotide binds to the cytoplasmic regulatory subunit KtrC and to the KdpD sensor kinase of the KdpDE two-component system, which in Staphylococcus aureus regulates the corresponding KdpABC transporter. Finally, we also show that S. aureus contains a homolog of KimA, which mediates potassium transport. Thus, the c-di-AMP-dependent control of systems involved in potassium homeostasis seems to be conserved in phylogenetically related bacteria. Surprisingly, the growth of an L. monocytogenes mutant lacking the c-di-AMP-synthesizing enzyme cdaA is only weakly inhibited by potassium. Thus, the physiological impact of the c-di-AMP-dependent control of potassium uptake seems to be less pronounced in L. monocytogenes than in other Firmicutes.

Control of Bacterial Virulence through the Peptide Signature of the Habitat

SummaryTo optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent-treated (PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione (GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Opp-imported oligopeptides promote PrfA induction intracellularly and PrfA repression outside the host.

Characterization of novel actin‐associated proteins at enteropathogenic Escherichia coli and Listeria monocytogenes actin‐rich structures

Enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes generate actin‐rich structures that are used for their colonization of host cells. EPEC remains extracellular, attaches to the host plasma membrane and secretes a variety of effector proteins that are used to control the host cells. The most dramatic morphological phenotype generated during these infections is the formation of actin‐rich protrusions called pedestals that are needed for E. coli‐based disease. L. monocytogenes enters its host cell and once in the cytosol it generates a bacterial protein (called ActA) that recruits actin polymerizing proteins to 1 pole of the microbe forming a comet tail that is used for movement within and amongst the host cells Previously, our lab conducted a mass spectrometry analysis of concentrated EPEC pedestals and over 90 novel proteins were identified. From this list, we selected a subset for confirmatory analysis and to determine whether their presence required the pre‐formation of the actin‐rich structures. These proteins included a calponin protein (CNN), dihydropyrimidinase‐like protein (CRMP4), nucleoside kinase (NK), mitogen‐activated protein pathway kinase (MK), and a ubiquitin‐conjugating enzyme (Ube2N). Because these novel proteins were observed in EPEC pedestals, we hypothesized that these five protein candidates are important for EPEC pedestals and L. monocytogenes comet tails. To study this, we first confirmed that these proteins were enriched in EPEC pedestals by immunolocalizing the proteins in EPEC‐infected cells. CNN, and NK were found throughout the full length of the pedestal while CRMP4, MK, and Ube2N were concentrated at the apical tip of EPEC pedestals. In L. monocytogenes‐infected cells, CNN and NK were found in actin clouds, comet tails, and listeriopods. CRMP4 immunolocalized only at invasion sites and listeriopods while MK was only present at listeriopods. To ensure that recruitment of these proteins were independent of bacterial attachment on the host cell and required formation of the actin‐rich structures, we infected cells with either a tir‐deficient EPEC mutant or an actA‐deficient L. monocytogenes mutant as both mutants cannot generate actin‐structures. None of the five proteins were recruited to the mutants suggesting that their presences are dependent on the formation of the actin‐rich structures. From these results, we have identified novel proteins that are important for the various stages of EPEC and L. monocytogenes infections. By identifying these proteins, we have begun to elucidate the complex web of proteins involved in the formation and maintenance of actin structures during these infections.Support or Funding InformationThis study was funded through NSERC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of a Lipoteichoic Acid Glycosyltransferase Enzyme Reveals that GW-Domain-Containing Proteins Can Be Retained in the Cell Wall of Listeria monocytogenes in the Absence of Lipoteichoic Acid or Its Modifications.

ABSTRACTListeria monocytogenes is a foodborne Gram-positive bacterial pathogen, and many of its virulence factors are either secreted proteins or proteins covalently or noncovalently attached to the cell wall. Previous work has indicated that noncovalently attached proteins with GW (glycine-tryptophan) domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerol phosphate polymer, which is modified in L. monocytogenes with galactose and d-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA, for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or d-alanine modifications or the complete LTA polymer, we show that GW domain proteins are retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW domain proteins. Further experiments revealed peptidoglycan as the binding receptor as a purified GW domain fusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identify the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of noncovalently attached cell wall proteins. IMPORTANCE Over the past 20 years, a large number of bacterial genome sequences have become available. Computational approaches are used for the genome annotation and identification of genes and encoded proteins. However, the function of many proteins is still unknown and often cannot be predicted bioinformatically. Here, we show that the previously uncharacterized Listeria monocytogenes gene lmo0933 likely codes for a glycosyltransferase required for the decoration of the cell wall polymer lipoteichoic acid (LTA) with galactose residues. Using L. monocytogenes mutants lacking LTA modifications or the complete polymer, we show that specific cell wall proteins, often associated with virulence, are retained within the cell wall, indicating that additional cell wall polymers are involved in their retention.

PrfA regulation offsets the cost of Listeria virulence outside the host.

SummaryVirulence traits are essential for pathogen fitness, but whether they affect microbial performance in the environment, where they are not needed, remains experimentally unconfirmed. We investigated this question with the facultative pathogen L isteria monocytogenes and its PrfA virulence regulon. PrfA‐regulated genes are activated intracellularly (PrfA ‘ON’) but shut down outside the host (PrfA ‘OFF’). Using a mutant PrfA regulator locked ON (PrfA*) and thus causing PrfA‐controlled genes to be constitutively activated, we show that virulence gene expression significantly impairs the listerial growth rate (μ) and maximum growth (A) in rich medium. Deletion analysis of the PrfA regulon and complementation of a L. monocytogenes mutant lacking all PrfA‐regulated genes with PrfA* indicated that the growth reduction was specifically due to the unneeded virulence determinants and not to pleiotropic regulatory effects of PrfA ON. No PrfA*‐associated fitness disadvantage was observed in infected eukaryotic cells, where PrfA‐regulated virulence gene expression is critical for survival. Microcosm experiments demonstrated that the constitutively virulent state strongly impaired L . monocytogenes performance in soil, the natural habitat of these bacteria. Our findings provide empirical proof that virulence carries a significant cost to the pathogen. They also experimentally substantiate the assumed, although not proven, key role of virulence gene regulation systems in suppressing the cost of bacterial virulence outside the host.

Intracellular Listeria monocytogenes comprises a minimal but vital fraction of the intestinal burden following foodborne infection.

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlA(m)-expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens of L. monocytogenes were recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication of L. monocytogenes was essential during the intestinal phase of infection. We found that lipoate protein ligase-deficient L. monocytogenes (ΔlplA1) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of the L. monocytogenes burden in the gastrointestinal tract is extracellular, but the small proportion of intracellular L. monocytogenes is essential for dissemination to the MLN and systemic organs.

An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence.

The nucleotide cyclic di-3',5'- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP-producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5'-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network.

The Listeria monocytogenes LPXTG surface protein Lmo1413 is an invasin with capacity to bind mucin

Many Gram-positive bacterial pathogens use surface proteins covalently anchored to the peptidoglycan to cause disease. Bacteria of the genus Listeria have the largest number of surface proteins of this family. Every Listeria genome sequenced to date contains more than forty genes encoding surface proteins bearing anchoring-domains with an LPXTG motif that is recognized for covalent linkage to the peptidoglycan. About one-third of these proteins are present exclusively in pathogenic Listeria species, with some of them acting as adhesins or invasins that promote bacterial entry into eukaryotic cells. Here, we investigated two LPXTG surface proteins of the pathogen L. monocytogenes, Lmo1413 and Lmo2085, of unknown function and absent in non-pathogenic Listeria species. Lack of these two proteins does not affect bacterial adhesion or invasion of host cells using in vitro infection models. However, expression of Lmo1413 promotes entry of the non-invasive species L. innocua into non-phagocytic host cells, an effect not observed with Lmo2085. Moreover, overproduction of Lmo1413, but not Lmo2085, increases the invasion rate in non-phagocytic eukaryotic cells of an L. monocytogenes mutant deficient in the acting-binding protein ActA. Unexpectedly, production of full-length Lmo1413 and InlA exhibited opposite trends in a high percentage of L. monocytogenes isolates obtained from different sources. The idea of Lmo1413 playing a role as a new auxiliary invasin was also sustained by assays revealing that purified Lmo1413 binds to mucin via its MucBP domains. Taken together, these data indicate that Lmo1413, which we rename LmiA, for Listeria-mucin-binding invasin-A, may promote interaction of bacteria with adhesive host protective components and, in this manner, facilitate bacterial entry.

The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type.

Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

STING-Dependent Type I IFN Production Inhibits Cell-Mediated Immunity to Listeria monocytogenes

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-β and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.

Acanthamoeba feature a unique backpacking strategy to trap and feed on Listeria monocytogenes and other motile bacteria

Despite its prominent role as an intracellular human pathogen, Listeria monocytogenes normally features a saprophytic lifestyle, and shares many environmental habitats with predatory protozoa. Earlier studies claimed that Acanthamoeba may act as environmental reservoirs for L. monocytogenes, whereas others failed to confirm this hypothesis. Our findings support the latter and provide clear evidence that L. monocytogenes is unable to persist in Acanthamoeba castellanii and A. polyphaga. Instead, external Listeria cells are rapidly immobilized on the surface of Acanthamoeba trophozoites, forming large aggregates of densely packed bacteria that we termed backpacks. While the assembly of backpacks is dependent on bacterial motility, flagellation alone is not sufficient. Electron micrographs showed that the aggregates are held together by filaments of likely amoebal origin. Time-lapse microscopy revealed that shortly after the bacteria are collected, the amoeba can change direction of movement, phagocytose the backpack and continue to repeat the process. The phenomenon was also observed with avirulent L. monocytogenes mutants, non-pathogenic Listeria, and other motile bacteria, indicating that formation of backpacks is not specific for L. monocytogenes, and independent of bacterial pathogenicity or virulence. Hence, backpacking appears to represent a unique and highly effective strategy of Acanthamoeba to trap and feed on motile bacteria.

Construction of Listeria monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette (ABC) Transporters and Analysis of Their Growth under Stress Conditions

Listeria monocytogenes is a food-borne pathogen that is difficult to eliminate since it can survive under different stress conditions such as low pH and high salt. Understanding its survival under stress conditions is important in controlling this pathogen in food. ABC transporters have been shown to be induced in L. monocytogenes subjected to high pressure and nisin treatments; therefore, we hypothesized that genes encoding the ABC transporters may be involved in general stress responses. To study the function of these genes, deletion mutants of ABC transporter genes (LMOf2365_1875, LMOf2365_1877) were created in L. monocytogenes F2365, and these deletion mutants were tested under different stress conditions. Compared to the wild type, ΔLMOf2365_1875 and ΔLMOf2365_1877 showed slower growth under nisin (250 μg/ml) and acid (pH 5) treatments. Under salt treatment (5% NaCl in minimal medium), ΔLMOf2365_1877 showed slower growth whereas ΔLMOf2365_1875 had growth similar to the wild type. Moreover, ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type. Our results indicate that these deletion mutants may be more sensitive to multiple stress conditions compared to the wild type, suggesting that LMOf2365_1875 and LMOf2365_1877 may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions and their ability to form biofilms may help in the development of intervention strategies to control L. monocytogenes in food and in the environment.

Listeria monocytogenes Mutants with Altered Growth Phenotypes at Refrigeration Temperature and High Salt Concentrations

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

Listeria monocytogenes cell wall constituents exert a charge effect on electroporation threshold

Genetically engineered cells with mutations of relevance to electroporation, cell membrane permeabilization by electric pulses, can become a promising new tool for fundamental research on this important biotechnology. Listeria monocytogenes mutants lacking DltA or MprF and assayed for sensitivity to the cathelicidin like anti-microbial cationic peptide (mCRAMP), were developed to study the effect of cell wall charge on electroporation. Working in the irreversible electroporation regime (IRE), we found that application of a sequence of 50 pulses, each 50μs duration, 12.5kV/cm field, delivered at 2Hz led to 2.67±0.29 log reduction in wild-type L. monocytogenes, log 2.60±0.19 in the MprF-minus mutant, and log 1.33±0.13 in the DltA-minus mutant. The experimental observation that the DltA-minus mutant was highly susceptible to cationic mCRAMP and resistant to IRE suggests that the charge on the bacterial cell wall affects electroporation and shows that this approach may be promising for fundamental studies on electroporation.

A single substitution in 5′-untranslated region of <italic>plcB</italic> is involved in enhanced broad-range phospholipase C activity in <italic>Listeria monocytogenes</italic> strain H4

To examine whether the in vitro phospholipase activity in Listeria monocytogenes strain H4 was due to two nucleotide mutations (C to T at position -26 and A to G at position +1) in plcB or resulted from regulatory activation, two mutants H4-plcB-m1 (single mutation at position -26) and H4-plcB-m2 (substitution at both positions) were constructed by site-directed mutagenesis. It was found that the two mutants had significantly lower transcription of plcB than their parent strain H4 and did not show phospholipase activity on the egg yolk agar, implying that the apparent phospholipase activity of strain H4 could be related to single substitution at position -26 of plcB, most probably by its 5'-untranslated region (5'-UTR) regulation mechanism. Tn917-based transposon mutagenesis generated eight L. monocytogenes mutants lacking phospholipase activity among 560 mutant candidates. Seven mutants had transposon insertion into prfA (encoding positive regulatory factor A) open reading frame, whereas only one mutant (WF-L127) was inserted into the P1 promoter region of prfA (prfAP1). Transcription of major virulence genes was significantly lower in both types of mutants than in their parent strain H4. Disruption of prfAP1 in WF-L127 abolished its phospholipase C activity but did not change its hemolytic phenotype, indicating that plcB was more dependent on prfA regulation than hly. Taken together, this study presents some evidence for the regulation of plcB expression by its 5'-UTR mechanism.

Listeria monocytogenes Triggers AIM2-Mediated Pyroptosis upon Infrequent Bacteriolysis in the Macrophage Cytosol

A host defense strategy against pathogens is the induction of cell death, thereby eliminating the pathogen's intracellular niche. Pyroptosis, one such form of cell death, is dependent on inflammasome activation. In a genetic screen to identify Listeria monocytogenes mutants that induced altered levels of host cell death, we identified a mutation in lmo2473 that caused hyperstimulation of IL-1beta secretion and pyroptosis following bacteriolysis in the macrophage cytosol. In addition, strains engineered to lyse in the cytosol by expression of both bacteriophage holin and lysin or induced to lyse by treatment with ampicillin stimulated pyroptosis. Pyroptosis was independent of the Nlrp3 and Nlrc4 inflammasome receptors but dependent on the inflammasome adaptor ASC and the cytosolic DNA sensor AIM2. Importantly, wild-type L. monocytogenes were also found to lyse, albeit at low levels, and trigger AIM2-dependent pyroptosis. These data suggested that pyroptosis is triggered by bacterial DNA released during cytosolic lysis.