Motility genes in Listeria monocytogenes are expressed under saprophytic conditions (30°C or less) but are repressed within the host at 37°C. Motility is costly on cellular resources due to the large molecular structures that need to be synthesized, and the proton motive force required for flagellar rotation. Here, we investigated the impact of the SigB-mediated general stress response on the regulation of motility in L. monocytogenes and sought to elucidate the regulatory steps involved. We show that an rsbX mutation that is unable to inactivate the stressosome, the sensory hub at the top of the SigB activation pathway, results in motility repression. Escape from this repressed state occurred through the acquisition of spontaneous mutations that decreased SigB activity. Flagellar expression was abolished in strains lacking RsbX, and a transcriptomic analysis revealed that the entire flagellar operon was strongly repressed, including the motility anti-repressor gene gmaR. These effects could be reversed by providing functional copies of rsbX or gmaR in trans. Abolishing expression of an antisense RNA lying opposite the large flagella operon or deleting the mogR transcriptional repressor restored the ability to produce flagellin in the ΔrsbX background. Stressosome mutations that negatively affect SigB activity resulted in a derepressed motility phenotype, whereas those that increase SigB activity resulted in a decreased motility phenotype similar to the ΔrsbX strain. Our data indicate that stress sensing via the stressosome negatively impacts motility and shows that the general stress response is prioritized when L. monocytogenes encounters osmotic and light stress conditions.IMPORTANCEMotility involves the synthesis and operation of the flagella, which come at a high energy cost for the bacterium and need to be carefully controlled. The human pathogen Listeria monocytogenes senses and responds to various stresses, in part through the alternative sigma factor σB (SigB), which controls the general stress response regulon. Since the SigB-regulon harbor hundreds of genes, the activity of SigB needs to be carefully controlled under non-stressed conditions to save energy. On the other hand, upon stress, the bacterium needs to invest a large amount of energy to synthesize a myriad of proteins to cope with the increased stress. In this study, we examined how motility is regulated under osmotic and visible light stress. Our data imply that increased SigB activity negatively impacts motility gene expression by a signal that is conveyed through the stressosome multiprotein complex.

Food waste due to perishable and unsafe food products is a major issue worldwide. For some high-quality perishable food products, such as fresh fish and cold-smoked salmon, traditional food preservation techniques are unsuitable as they can compromise sensory qualities such as flavor, texture, and freshness. These products often support the growth of the human pathogen Listeria monocytogenes, which can be present if thermal treatment is not applied. Thus, antilisterial bacteriocins, such as garvicin KS (GarKS), in combination with other technologies like high-pressure processing, are being investigated as hurdle strategies to increase the shelf life and food safety of fish products. In this study, we aimed to identify potential resistance development and genetic factors affecting the susceptibility of L. monocytogenes towards GarKS. We show that L. monocytogenes strains associated with fish products and fish processing plants are susceptible to GarKS with MIC values ranging from 20 to 275nM. By RNA sequencing, we showed that exposure to GarKS resulted in an upregulation of genes involved in the phage shock protein (psp) response. Furthermore, isolation of resistant mutants indicated a low frequency of resistance to GarKS (10⁻9 to 10⁻11). The GarKS-tolerant mutants isolated (2-fold increased MIC values) were shown to harbor disruption mutations in lmo2468, encoding a PspC-domain-containing protein. Overexpression of this gene increased susceptibility to GarKS two-fold and restored wild-type susceptibility in a disruption mutant. This study thus demonstrates that resistance development to GarKS is rare and identifies the phage shock protein response as a key player involved in susceptibility to GarKS.

The RNA chaperone Hfq is a novel regulator of catalase expression and hydrogen peroxide-induced oxidative stress response in Listeria monocytogenes EGD-e

Stressed bacteria can enter a dormant viable but non-culturable (VBNC) state. VBNC pathogens pose an increased health risk as they are undetectable by growth-based techniques and can wake up back into a virulent state. Although widespread in bacteria, the mechanisms governing this phenotypic switch remain elusive. Here, we investigate the VBNC state transition in the human pathogen Listeria monocytogenes. We show that bacteria starved in mineral water become VBNC by converting into osmotically stable cell wall-deficient coccoid forms, a phenomenon that occurs in other Listeria species. We reveal the bacterial stress response regulator SigB and the autolysin NamA as major actors of VBNC state transition. We lastly show that VBNC Listeria revert to a walled and virulent state after passage in chicken embryos. Our study provides more detail on the VBNC state transition mechanisms, revealing wall-free bacteria naturally arising in aquatic environments as a potential survival strategy in hypoosmotic and oligotrophic conditions.

Bacteria adapt the biosynthesis of their envelopes to specific growth conditions and prevailing stress factors. Peptidoglycan (PG) is the major component of the cell wall in Gram-positive bacteria, where PASTA kinases play a central role in PG biosynthesis regulation. Despite their importance for growth, cell division and antibiotic resistance, the mechanisms of PASTA kinase activation are not fully understood. ReoM, a recently discovered cytosolic phosphoprotein, is one of the main substrates of the PASTA kinase PrkA in the Gram-positive human pathogen Listeria monocytogenes. Depending on its phosphorylation, ReoM controls proteolytic stability of MurA, the first enzyme in the PG biosynthesis pathway. The late cell division protein GpsB has been implicated in PASTA kinase signalling. Consistently, we show that L. monocytogenes prkA and gpsB mutants phenocopied each other. Analysis of invivo ReoM phosphorylation confirmed GpsB as an activator of PrkA leading to the description of structural features in GpsB that are important for kinase activation. We further show that ReoM phosphorylation is growth phase-dependent and that this kinetic is reliant on the protein phosphatase PrpC. ReoM phosphorylation was inhibited in mutants with defects in MurA degradation, leading to the discovery that MurA overexpression prevented ReoM phosphorylation. Overexpressed MurA must be able to bind its substrates and interact with ReoM to exert this effect, but the extracellular PASTA domains of PrkA or MurJ flippases were not required. Our results indicate that intracellular signals control ReoM phosphorylation and extend current models describing the mechanisms of PASTA kinase activation.

Bacteria have evolved numerous regulatory pathways to survive in changing environments. The SOS response is an inducible DNA damage repair system that plays an indispensable role in bacterial adaptation and pathogenesis. Here we report a discovery of the previously uncharacterized protein Lmo0946 as an SOS response interfering factor (Sif) in the human pathogen Listeria monocytogenes. Functional genetic studies demonstrated that sif is indispensable for normal growth of L. monocytogenes in stress-free as well as multi-stress conditions, and sif contributes to susceptibility to β-lactam antibiotics, biofilm formation and virulence. Absence of Sif promoted the SOS response and elevated expression of mobilome genes accompanied by mobilization of the A118 prophage and ICELm-1 mobile genetic elements (MGEs). These changes were found to be associated with decreased expression of general stress response genes from the σB regulon as well as virulence genes, including the PrfA regulon. Together, this study uncovers an unexpected role of a previously uncharacterized factor, Sif, as an inhibitor of the SOS response in L. monocytogenes.

Biofilms provide an environment that protects microorganisms from external stresses such as nutrient deprivation, antibiotic treatments, and immune defences, thereby creating favorable conditions for bacterial survival and pathogenesis. Here we show that the RNA-binding protein and ribonuclease polynucleotide phosphorylase (PNPase) is a positive regulator of biofilm formation in the human pathogen Listeria monocytogenes, a major responsible for food contamination in food-processing environments. The PNPase mutant strain produces less biofilm biomass and exhibits an altered biofilm morphology that is more susceptible to antibiotic treatment. Through biochemical assays and microscopical analysis, we demonstrate that PNPase is a previously unrecognized regulator of the composition of the biofilm extracellular matrix, greatly affecting the levels of proteins, extracellular DNA, and sugars. Noteworthy, we have adapted the use of the fluorescent complex ruthenium red-phenanthroline for the detection of polysaccharides in Listeria biofilms. Transcriptomic analysis of wild-type and PNPase mutant biofilms reveals that PNPase impacts many regulatory pathways associated with biofilm formation, particularly by affecting the expression of genes involved in the metabolism of carbohydrates (e.g., lmo0096 and lmo0783, encoding PTS components), of amino acids (e.g., lmo1984 and lmo2006, encoding biosynthetic enzymes) and in the Agr quorum sensing-like system (lmo0048-49). Moreover, we show that PNPase affects mRNA levels of the master regulator of virulence PrfA and PrfA-regulated genes, and these results could help to explain the reduced bacterial internalization in human cells of the ΔpnpA mutant. Overall, this work demonstrates that PNPase is an important post-transcriptional regulator for virulence and adaptation to the biofilm lifestyle of Gram-positive bacteria and highlights the expanding role of ribonucleases as critical players in pathogenicity.

The human pathogen Listeria monocytogenes can cope with severe environmental challenges, for which the high molecular weight stressosome complex acts as the sensing hub in a complicated signal transduction pathway. Here, we show the dynamics and functional roles of the stressosome protein RsbR1 and its paralogue, the blue-light receptor RsbL, using photo-activated localization microscopy combined with single-particle tracking and single-molecule displacement mapping and supported by physiological studies. In live cells, RsbR1 is present in multiple states: in protomers with RsbS, large clusters of stressosome complexes, and in connection with the plasma membrane via Prli42. RsbL diffuses freely in the cytoplasm but forms clusters upon exposure to light. The clustering of RsbL is independent of the presence of Prli42. Our work provides a comprehensive view of the spatial organization and intracellular dynamics of the stressosome proteins in L. monocytogenes, which paves the way towards uncovering the stress-sensing mechanism of this signal transduction pathway.

Protein secretion plays a central role in modulating interactions of the human pathogen Listeria monocytogenes with its environment. Recently, secretion of RNA has emerged as an important strategy used by the pathogen to manipulate the host cell response to its advantage. In general, the Sec-dependent translocation pathway is a major route for protein secretion in L. monocytogenes, but mechanistic insights into the secretion of RNA by these pathways are lacking. Apart from the classical SecA1 secretion pathway, L. monocytogenes also encodes for a SecA paralogue (SecA2) which targets the export of a specific subset of proteins, some of which are involved in virulence. Here, we demonstrated that SecA2 co-sediments with translating ribosomes and provided evidence that it associates with a subset of secreted small non-coding RNAs (sRNAs) that induce high levels of IFN-β response in host cells. We found that enolase, which is translocated by a SecA2-dependent mechanism, binds to several sRNAs, suggesting a pathway by which sRNAs are targeted to the supernatant of L. monocytogenes.

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes

PurposeThe emerging biobased economy will require robust, adaptable, organisms for the production and processing of biomaterials as well as for bioremediation. Recently, the search for solvent tolerant organisms and solvent tolerant enzymes has intensified. Resilient organisms secreting solvent stable lipases are of particular interest for biotechnological applications.MethodsScreening of soil samples for lipase-producing organisms was carried out on Rhodamine B plates. The most productive lipase-producing organisms were further screened for their resistance to solvents commonly used in biotechnological applications.ResultsIn the course of screening, one of the isolated organisms that exhibited extracellular lipase activity, was identified as the human pathogen Listeria monocytogenes through 16S rRNA sequencing. Further exploration revealed that this organism was resistant to solvents ranging from log P − 0.81 to 4.0. Moreover, in the presence of these solvents, L. monocytogenes secreted an extracellular, solvent tolerant, lipase activity. This lipase retained approximately 80% activity when incubated in 30% (v/v) methanol for 24 h.ConclusionThese findings identify L. monocytogenes as a potentially useful organism for biotechnological applications. However, the fact that Listeria is a pathogen is problematic and it will require the use of non-pathogenic or attenuated Listeria strains for practical applications. Nonetheless, the ability to adapt to rapidly changing environmental conditions, to grow at low temperatures, to resist solvents and to secrete an extracellular solvent tolerant lipase are unique and highly useful characteristics. The potential application of L. monocytogenes in wastewater bioremediation and plastics degradation is discussed.

The human pathogen Listeria monocytogenes synthesizes and degrades c-di-AMP using the diadenylate cyclase CdaA and the phosphodiesterases PdeA and PgpH respectively. c-di-AMP is essential because it prevents the uncontrolled uptake of osmolytes. Here, we studied the phenotypes of cdaA, pdeA, pgpH and pdeA pgpH mutants with defects in c-di-AMP metabolism and characterized suppressor mutants restoring their growth defects. The characterization of the pdeA pgpH mutant revealed that the bacteria show growth defects in defined medium, a phenotype that is invariably suppressed by mutations in cdaA. The previously reported growth defect of the cdaA mutant in rich medium is suppressed by mutations that osmotically stabilize the c-di-AMP-free strain. We also found that the cdaA mutant has an increased sensitivity against isoleucine. The isoleucine-dependent growth inhibition of the cdaA mutant is suppressed by codY mutations that likely reduce the DNA-binding activity of encoded CodY variants. Moreover, the characterization of the cdaA suppressor mutants revealed that the Opp oligopeptide transport system is involved in the uptake of the antibiotic fosfomycin. In conclusion, the suppressor analysis corroborates a key function of c-di-AMP in controlling osmolyte homeostasis in L. monocytogenes.

The use of foodborne infection to evaluate bacterial pathogenesis and host response.

Characterisation of a new cell wall teichoic acid produced by Listeria innocua ŽM39 and analysis of its biosynthesis genes

The requirement of a large input amount (500 ng) for Nanopore direct RNA‐seq presents a major challenge for low input transcriptomic analysis and early pathogen surveillance. The high RNA input requirement is attributed to significant sample loss associated with library preparation using solid‐phase reversible immobilization (SPRI) beads. A novel solid‐phase catalysis strategy for RNA library preparation to circumvent the need for SPRI bead purification to remove enzymes is reported here. This new approach leverages concurrent processing of non‐polyadenylated transcripts with immobilized poly(A) polymerase and T4 DNA ligase, followed by directly loading the prepared library onto a flow cell. Whole transcriptome sequencing, using a human pathogen Listeria monocytogenes as a model, demonstrates this new method displays little sample loss, takes much less time, and generates higher sequencing throughput correlated with reduced nanopore fouling compared to the current library preparation for 500 ng input. Consequently, this approach enables Nanopore low‐input direct RNA‐seq, improving pathogen detection and transcript identification in a microbial community standard with spike‐in transcript controls. Besides, as evident in the bioinformatic analysis, the new method provides accurate RNA consensus with high fidelity and identifies higher numbers of expressed genes for both high and low input RNA amounts.

Mitochondrial respiration restricts Listeria monocytogenes infection by slowing down host cell receptor recycling.

ABSTRACTThe survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multiprotein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins poststress. We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation levels under nonstress conditions and that it is required for appropriate SigB-mediated stress adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation and also an increased survival at low pH. Our results could suggest that absence of RsbX alters the multiprotein composition of the stressosome without dramatically affecting its phosphorylation status. Overall, the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under nonstressed conditions.IMPORTANCE Pathogenic bacteria need to sense and respond to stresses to survive harsh environments and also to turn off the response when no longer facing stress. Activity of the stress sigma factor SigB in the human pathogen Listeria monocytogenes is controlled by a hierarchic system having a large stress-sensing multiprotein complex known as the stressosome at the top. Following stress exposure, proteins in the stressosome become phosphorylated, leading to SigB activation. We have studied the role of a putative phosphatase, RsbX, which is hypothesized to dephosphorylate stressosome proteins. RsbX is critical not only to switch off the stress response poststress but also to keep the activity of SigB low at nonstressed conditions to prevent unnecessary gene expression and save energy.

Lysozyme is an important component of the innate immune system. It functions by hydrolyzing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, referred to here as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the MIC to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we showed that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant, indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB affects not only lysozyme resistance but also endogenous cell lysis, cell wall biosynthesis, cell division, and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is, by means of a yet-unknown mechanism, an important determinant for cell wall integrity in L. monocytogenes.

Queso fresco (QF) is a fresh Hispanic-style cheese that is commonly associated with the human foodborne pathogen Listeria monocytogenes and outbreaks of listeriosis in the United States. Endolysins, cell wall hydrolases derived from bacteriophages, are promising candidates for controlling bacterial pathogens in food systems. In this study, we characterized the lytic capabilities of 2 endolysins, PlyP40 and PlyPSA, under varying conditions (pH, temperature, salt concentration) and compared their activities with those of the previously described endolysin PlyP100. We showed that PlyP40 was effective, showing at least a 33% reduction in cellular debris, against a broader range of Listeria than PlyPSA, which showed little lytic activity toward Listeria strains not from serovar 4. Both endolysins were also capable of maintaining lytic activity to varying extents at refrigeration temperature. The effect of salt concentration and pH differed between PlyP40 and PlyPSA. Furthermore, we added the endolysins to QF and monitored their ability to control L. monocytogenes contamination over 28 d of cold storage. Both PlyP40 and PlyPSA were capable of lowering QF inoculum cell counts compared with the control; however, both were less effective than the previously characterized PlyP100. Further characterization of endolysins will continue to open opportunities to optimization and implementation in a variety of food matrices for controlling pathogen contamination.

Mitochondrial Respiration Restricts <i>Listeria</i> Monocytogenes Infection by Slowing Down Host Cell Receptor Recycling