The foodborne bacterium Listeria monocytogenes is transmitted to humans from various environmental sources through consumption of contaminated plant and animal-based food. L. monocytogenes uses ATP-binding cassette (ABC)-type drug transporters to resist antimicrobial compounds produced by competitors co-residing in its environmental reservoirs. We have shown previously that the TimAB transporter confers resistance of L. monocytogenes to tartrolon B, a boron containing macrodiolide produced by myxo- and proteobacterial species. Tartrolon B acts as a potassium ionophore and is sensed by TimR, the transcriptional repressor of timABR operon. We here have isolated tartrolon B resistant suppressor mutations outside the timABR locus. These mutations inactivated the clpP2 gene, which encodes the main proteolytic component of house-keeping Clp proteases. Deletion of clpP2 impaired growth and virulence but caused tartrolon B hyper-resistance. This phenotype was timAB-dependent, but neither production nor degradation of TimAB was affected upon clpP2 inactivation. Combinatorial deletions of the genes encoding the three Clp ATPases showed that ClpCP2 and ClpXP2 proteasomes jointly promote tartrolon B hyper-resistance. Genetic follow-up experiments identified the ClpP2 substrate and transcription factor SpxA1 and its protease adaptor YjbH as further tartrolon B resistance determinants. SpxA1 activates transcription of the cydABCD operon encoding cytochrome oxidase and in accordance with this transposon mutants with impaired cytochrome oxidase function were depleted from a transposon mutant library during tartrolon B exposure. Our work demonstrates novel roles of Clp proteasomes, SpxA1 and cytochrome oxidase CydAB in the resistance against compounds dissipating transmembrane ion gradients and helps to better understand the genetic and chemical basis of the manifold ecological interactions of an important human pathogen in its natural ecologic niches.

The facultative pathogen Listeria monocytogenes uses a master regulator, PrfA, to tightly control the fitness-costly expression of its virulence factors. We found that PrfA activity is repressed via competitive occupancy of the binding site for the PrfA-activating cofactor, glutathione, by exogenous nutritional oligopeptides. The inhibitory peptides show different sequence and physicochemical properties, but how such a wide variety of oligopeptides can bind PrfA was unclear. Using crystal structure analysis of PrfA complexed with inhibitory tri- and tetrapeptides, we show here that the binding promiscuity is due to the ability of PrfA β5 in the glutathione-binding inter-domain tunnel to establish parallel or antiparallel β sheet-like interactions with the peptide backbone. Spacious tunnel pockets provide additional flexibility for unspecific peptide accommodation while providing selectivity for hydrophobic residues. Hydrophobic contributions from two adjacent peptide residues appear to be critical for efficient PrfA inhibitory binding. In contrast to glutathione, peptide binding prevents the conformational change required for the correct positioning of the DNA-binding helix-turn-helix motifs of PrfA, effectively inhibiting virulence gene expression.

Listeria monocytogenes are facultative intracellular bacterial pathogens that cause foodborne disease in humans. The bacteria can use the surface protein InlA to invade intestinal epithelial cells or transcytose across M cells in the gut, but it is not well understood how the bacteria traffic from the underlying lamina propria to the draining mesenteric lymph nodes (MLN). Previous studies indicated that L. monocytogenes associated with both monocytes and dendritic cells in the intestinal lamina propria. We show here that CCR2-/- mice had a significant reduction in Ly6Chi monocytes in the MLN but no change in bacterial burden following foodborne infection; thus, dissemination of L. monocytogenes associated with monocytes is not required for colonization of the MLN. To block CCR7-mediated trafficking of dendritic cells from the lamina propria, we treated mice with anti-VEGFR3 antibody (clone AFL4) prior to and during infection but did not see a change in dendritic numbers in the MLN as had been previously reported with other anti-VEGFR3-specific antibodies. However, increasing the number of circulating dendritic cells by treating mice with rFlt3L resulted in a significant increase in L. monocytogenes in the lymph nodes that drain the small intestine and the spleen. Whole-mount fluorescent microscopy of lymphatic vessels following ligated loop infection revealed both free-floating L. monocytogenes and cell-associated bacteria within lymphatic vessels. Together, these results suggest that L. monocytogenes can use multiple, redundant mechanisms to disseminate from the gut tissue to the MLN.

Consumption of the foodborne bacterial pathogen Listeria monocytogenes results in a wide spectrum of human disease from mild self-limiting gastroenteritis to life-threatening infections of the bloodstream, brain, and placenta. It is not well understood how the bacteria migrate from the intestines to the draining mesenteric lymph nodes, which are thought to serve as the last barrier to prevent systemic infections. Results presented here reveal multiple redundant mechanisms L. monocytogenes can use to disseminate from the ileum or colon to the mesenteric lymph nodes.

Peracetic acid (PAA), a strong oxidizing agent, has been widely used as a disinfectant in food processing settings as it does not produce harmful chlorinated by-products. In the present study, the transcriptional response of Listeria monocytogenes to a sub-lethal concentration of PAA (2.5 ppm) was assessed using RNA-sequencing (RNA-seq). Our analysis revealed 12 differentially expressed protein-coding genes, of which nine were upregulated (ohrR, ohrA, rpsN, lmo0637, lmo1973, fur, lmo2492, zurM, and lmo1007), and three were down-regulated (argG, lmo0604 and lmo2156) in PAA-treated samples compared to the control samples. A non-coding small RNA gene (rli32) was also found to be down-regulated. In detail, the organic peroxide toxicity protection (OhrA-OhrR) system, the metal homeostasis genes fur and zurM, the SbrE-regulated lmo0636-lmo0637 operon and a carbohydrate phosphotransferase system (PTS) operon component were induced under exposure of L. monocytogenes to PAA. Hence, this study identified key elements involved in the primary response of L. monocytogenes to oxidative stress caused by PAA, including the expression of the peroxide detoxification system and fine-tuning the levels of redox-active metals in the cell. The investigation of the molecular mechanism of PAA response in L. monocytogenes is of utmost importance for the food industry, as residual PAA can lead to stress tolerance in pathogens.

RNAs are often studied in nonnative sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods-a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added postprobing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5' and 3' ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer-only construct but introduced nonnative interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for all nucleotides in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is a straightforward alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to nonnative sequence elements.

Microbial pathogenesis is mediated by the expression of virulence genes. However, as microbes with identical virulence gene content can differ in their pathogenic potential, other virulence determinants must be involved. Here, by combining comparative genomics and transcriptomics of a large collection of isolates of the model pathogen Listeria monocytogenes, time-lapse microscopy, in vitro evolution and in vivo experiments, we show that the individual stress responsiveness of L. monocytogenes isolates determines their respective levels of virulence in vivo and reflects their degree of host adaptation. The transcriptional signature that accounts for the heterogeneity in the virulence of L. monocytogenes species is mediated by the stress response regulator SigB and driven by differential stress responsiveness. The tuning of SigB pathway responsiveness is polygenic and influenced by multiple, individually rare gene variations. This study reveals an overarching determinant of microbial virulence, challenging the paradigm of accessory virulence gene content as the major determinant of intraspecies virulence heterogeneity.

Biofilm is a dynamic structure from which individual bacteria and micro-aggregates are released to subsequently colonize new niches by either detachment or dispersal. Screening of a transposon mutant library identified genes associated with the alteration of Klebsiella pneumoniae biofilm including fabR, which encodes a transcriptional regulator involved in membrane lipid homeostasis. An isogenic ∆fabR mutant formed more biofilm than the wild-type (WT) strain and its trans-complemented strain. The thick and round aggregates observed with ∆fabR were resistant to extensive washes, unlike those of the WT strain. Confocal microscopy and BioFlux microfluidic observations showed that fabR deletion was associated with biofilm robustness and impaired erosion over time. The genes fabB and yqfA associated with fatty acid metabolism were significantly overexpressed in the ∆fabR strain, in both planktonic and biofilm conditions. Two monounsaturated fatty acids, palmitoleic acid (C16:1) and oleic acid (C18:1), were found in higher proportion in biofilm cells than in planktonic forms, whereas heptadecenoic acid (C17:1) and octadecanoic acid, 11-methoxy (C18:0-OCH3) were found in higher proportion in the planktonic lifestyle. The fabR mutation induced variations in the fatty acid composition, with no clear differences in the amounts of saturated fatty acids (SFA) and unsaturated fatty acids for the planktonic lifestyle but lower SFA in the biofilm form. Atomic force microscopy showed that deletion of fabR is associated with decreased K. pneumoniae cell rigidity in the biofilm lifestyle, as well as a softer, more elastic biofilm with increased cell cohesion compared to the wild-type strain.IMPORTANCEKlebsiella pneumoniae is an opportunistic pathogen responsible for a wide range of nosocomial infections. The success of this pathogen is due to its high resistance to antibiotics and its ability to form biofilms. The molecular mechanisms involved in biofilm formation have been largely described but the dispersal process that releases individual and aggregate cells from mature biofilm is less well documented while it is associated with the colonization of new environments and thus new threats. Using a multidisciplinary approach, we show that modifications of bacterial membrane fatty acid composition lead to variations in the biofilm robustness, and subsequent bacterial detachment and biofilm erosion over time. These results enhance our understanding of the genetic requirements for biofilm formation in K. pneumoniae that affect the time course of biofilm development and the embrittlement step preceding its dispersal that will make it possible to control K. pneumoniae infections.

SreA is one of seven candidate S-adenosyl methionine (SAM) class I riboswitches identified in Listeria monocytogenes, a saprophyte and opportunistic foodborne pathogen. SreA precedes genes encoding a methionine ATP-binding cassette (ABC) transporter, which imports methionine and is presumed to regulate transcription of its downstream genes in a SAM-dependent manner. The proposed role of SreA in controlling the transcription of genes encoding an ABC transporter complex may have important implications for how the bacteria senses and responds to the availability of the metabolite SAM in the diverse environments in which L. monocytogenes persists. Here we validate SreA as a functional SAM-I riboswitch through ligand binding studies, structure characterization, and transcription termination assays. We determined that SreA has both a structure and SAM binding properties similar to those of other well-characterized SAM-I riboswitches. Despite the apparent structural similarities to previously described SAM-I riboswitches, SreA induces transcription termination in response to comparatively lower (nanomolar) ligand concentrations. Furthermore, SreA is a leaky riboswitch that permits some transcription of the downstream gene even in the presence of millimolar SAM, suggesting that L. monocytogenes may "dampen" the expression of genes for methionine import but likely does not turn them "OFF".

Microbes were the only form of life on Earth for most of its history, and they still account for the vast majority of life's diversity. They convert rocks to soil, produce much of the oxygen we breathe, remediate our sewage, and sustain agriculture. Microbes are vital to planetary health as they maintain biogeochemical cycles that produce and consume major greenhouse gases and support large food webs. Modern microbiologists analyze nucleic acids, proteins, and metabolites; leverage sophisticated genetic tools, software, and bioinformatic algorithms; and process and integrate complex and heterogeneous datasets so that microbial systems may be harnessed to address contemporary challenges in health, the environment, and basic science. Here, we consider an inevitably incomplete list of emergent themes in our discipline and highlight those that we recognize as the archetypes of its modern era that aim to address the most pressing problems of the 21st century.

Mycobacterium tuberculosis (MTB) is a pathogen that is known for its ability to persist in harsh environments and cause chronic infections. Understanding the regulatory networks of MTB is crucial for developing effective treatments. Small regulatory RNAs (sRNAs) play important roles in gene expression regulation in all kingdoms of life, and their classification based solely on genomic location can be imprecise due to the computational-based prediction of protein-coding genes in bacteria, which often neglects segments of mRNA such as 5'UTRs, 3'UTRs, and intercistronic regions of operons. To address this issue, our study simultaneously discovered genomic features such as TSSs, UTRs, and operons together with sRNAs in the M. tuberculosis H37Rv strain (ATCC 27294) across multiple stress conditions. Our analysis identified 1,376 sRNA candidates and 8,173 TSSs in MTB, providing valuable insights into its complex regulatory landscape. TSS mapping enabled us to classify these sRNAs into more specific categories, including promoter-associated sRNAs, 5'UTR-derived sRNAs, 3'UTR-derived sRNAs, true intergenic sRNAs, and antisense sRNAs. Three of these sRNA candidates were experimentally validated using 3'-RACE-PCR: predictedRNA_0240, predictedRNA_0325, and predictedRNA_0578. Future characterization and validation are necessary to fully elucidate the functions and roles of these sRNAs in MTB. Our study is the first to simultaneously unravel TSSs and sRNAs in MTB and demonstrate that the identification of other genomic features, such as TSSs, UTRs, and operons, allows for more accurate and specific classification of sRNAs.

Listeria monocytogenes is a ubiquitous and psychrotrophic foodborne pathogen commonly found in raw materials, ready-to-eat products, and food environments. We previously demonstrated that L. monocytogenes can grow faster at low temperature when unsaturated fatty acids (UFA) are present in its environment. This could question the maintenance of food safety for refrigerated foods, especially those reformulated with a higher ratio of UFA versus saturated fatty acids (SFA) to fit with nutritional recommendations. In this study, we used transcriptomics to understand the impact of UFA on the behavior of L. monocytogenes at low temperature. We first demonstrated that fabK, a key gene in SFA synthesis, is up-regulated in the presence of UFA but not SFA at low temperature. L. monocytogenes can thus regulate the synthesis of SFA in its membrane according to the type of FA available in its environment. Interestingly, we also observed up-regulation of genes involved in chemotaxis and flagellar assembly (especially cheY and flaA) in the presence of UFA but not SFA at low temperature. TEM observations confirmed that L. monocytogenes acquired a remarkable phenotype with numerous and long-looped flagella only in the presence of UFA at 5°C but not at 37°C. As flagella are well known to be involved in biofilm formation, this new finding raises questions about the structure and persistence of biofilms settled in refrigerated environments using unsaturated lipid-rich products.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Dissemination of food-borne L. monocytogenes in the host relies on internalin-mediated invasion, but the underlying invasion strategies remain elusive. Here we use live-cell microscopy to follow single cell interactions between individual human cells and L. monocytogenes and elucidate mechanisms associated with internalin B (InlB)-mediated invasion. We demonstrate that whilst a replicative invasion of nonphagocytic cells is a rare event even at high multiplicities of invasion, L. monocytogenes overcomes this by utilising a strategy relaying on PrfA-mediated ActA-based aggregation. We show that L. monocytogenes forms aggregates in extracellular host cell environment, which promote approximately 5-fold more host cell adhesions than the non-aggregating actA-ΔC mutant (which lacks the C-terminus coding region), with the adhering bacteria inducing 3-fold more intracellular invasions. Aggregation is associated with robust MET tyrosine kinase receptor clustering in the host cells, a hallmark of InlB-mediated invasion, something not observed with the actA-ΔC mutant. Finally, we show via RNA-seq analyses that aggregation involves a global adaptive response to host cell environment (including iron depletion), resulting in metabolic changes in L. monocytogenes and upregulation of the PrfA virulence regulon. Overall, our analyses provide new mechanistic insights into internalin-mediated host-pathogen interactions of L. monocytogenes.

Listeria monocytogenes (Lm) is a highly pathogenic bacterium that can cause listeriosis, a relatively rare food-borne infectious disease that affects farm, domestic, wild animals and humans as well. The infected livestock is the frequent sources of Lm. Vaccination is one of the methods of controlling listeriosis in target farm animals to prevent Lm-associated food contamination. Here we report the complete sequence of the Lm strain AUF attenuated from a fully-virulent Lm strain by ultraviolet irradiation, successfully used since the 1960s as a live whole-cell veterinary vaccine. The de novo assembled genome consists of a circular chromosome of 2,942,932 bp length, including more than 2,800 CDSs, 17 pseudogenes, 5 antibiotic resistance genes, and 56/92 virulence genes. Two wild Lm strains, the EGD and the 10403S that is also used in cancer Immunotherapy, were the closest homologs for the Lm strain AUF. Although all three strains belonged to different sequence types (ST), namely ST12, ST85, and ST1538, they were placed in the same genetic lineage II, CC7.

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium (S. Typhimurium). The EA utilization (eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant (ΔeutV) was equally deficient in intracellular replication compared to an EA metabolism mutant (ΔeutB), and this was consistent across Caco-2, RAW 264.7, and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that ΔeutB and ΔeutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.IMPORTANCEListeria monocytogenes (LMO) is a bacterial pathogen that can cause severe disease in immunocompromised individuals when consumed in contaminated food. It can replicate inside of mammalian cells, escaping detection by the immune system. Therefore, understanding the features of this human pathogen that contribute to its infectiousness and intracellular lifestyle is important. In this work we demonstrate that genes encoding both regulators and enzymes of EA metabolism are important for optimal growth inside mammalian cells. Moreover, the formation of specialized compartments to enable EA metabolism were visualized by tagging with a fluorescent protein and found to form when LMO infects some mammalian cell types, but not others. Interestingly, the formation of the compartments was associated with features consistent with an early stage of the intracellular infection. By characterizing bacterial metabolic pathways that contribute to survival in host environments, we hope to positively impact knowledge and facilitate new treatment strategies.

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host-cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium ( S. Typhimurium). The EA utilization ( eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant ( ΔeutV) was equally deficient in intracellular replication compared to an EA metabolism mutant ( ΔeutB ), and this was consistent across Caco-2, RAW 264.7 and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro, and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that ΔeutB and ΔeutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.

Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome. We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin-antitoxin systems, and control of the phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

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.

Glabridin is a prenylated isoflavan which can be extracted from liquorice roots and has shown antimicrobial activity against foodborne pathogens and spoilage microorganisms. However, its application may be hindered due to limited information about its mode of action. In this study, we aimed to investigate the mode of action of glabridin using a combined phenotypic and proteomic approach on Listeria monocytogenes. Fluorescence and transmission electron microscopy of cells exposed to glabridin showed membrane permeabilization upon treatment with lethal concentrations of glabridin. Comparative proteomics analysis of control cells and cells exposed to sub-lethal concentrations of glabridin showed upregulation of proteins related to the two-component systems LiaSR and VirRS, confirming cell envelope damage during glabridin treatment. Additional upregulation of SigmaB regulon members signified activation of the general stress response in L. monocytogenes during this treatment. In line with the observed upregulation of cell envelope and general stress response proteins, sub-lethal treatment of glabridin induced (cross)protection against lethal heat and low pH stress and against antimicrobials such as nisin and glabridin itself. Overall, this study sheds light on the mode of action of glabridin and activation of the main stress responses to this antimicrobial isoflavan and highlights possible implications of its use as a naturally derived antimicrobial compound.

The tight and coordinated regulation of virulence gene expression is crucial to ensure the survival and persistence of bacterial pathogens in different contexts within their hosts. Considering this, bacteria do not express virulence factors homogenously in time and space, either due to their associated fitness cost or to their detrimental effect at specific infection stages. To efficiently infect and persist into their hosts, bacteria have thus to monitor environmental cues or chemical cell-to-cell signaling mechanisms that allow their transition from the external environment to the host, and therefore adjust gene expression levels, intrinsic biological activities, and appropriate behaviors. Listeria monocytogenes (Lm), a major Gram-positive facultative intracellular pathogen, stands out for its adaptability and capacity to thrive in a wide range of environments. Because of that, Lm presents itself as a significant concern in food safety and public health, that can lead to potentially life-threatening infections in humans. A deeper understanding of the intricate bacterial virulence mechanisms and the signals that control them provide valuable insights into the dynamic interplay between Lm and the host. Therefore, this review addresses the role of some crucial signals behind Lm pathogenic virulence mechanisms and explores how the ability to assimilate and interpret these signals is fundamental for pathogenesis, identifying potential targets for innovative antimicrobial strategies.

The adaption and tolerance to various environmental stresses are the fundamental factors for the widespread existence of Listeria monocytogenes. Anti-oxidative stress is the critical mechanism for the survival and pathogenesis of L. monocytogenes. The thioredoxin (Trx) and glutaredoxin (Grx) systems are known to contribute to the anti-oxidative stress of L. monocytogenes, but whether the Dsb system has similar roles remains unknown. This study demonstrated that the DsbA family protein Lmo1059 of L. monocytogenes participates in bacterial oxidative stress tolerance, with Cys36 as the key amino acid of its catalytic activity and anti-oxidative stress ability. It is worth noting that Lmo1059 was involved in the invading and cell-to-cell spread of L. monocytogenes. This study lays a foundation for further understanding the specific mechanisms of oxidative cysteine repair and antioxidant stress regulation of L. monocytogenes, which contributes to an in-depth understanding of the environmental adaptation mechanisms for foodborne bacterial pathogens.

A detailed understanding of how host fitness changes in response to variations in microbe density (an ecological measure of disease tolerance) is an important aim of infection biology. Here, we applied dose-response curves to study Aedes aegypti survival upon exposure to different microbes. We challenged female mosquitoes with Listeria monocytogenes, a model bacterial pathogen, Dengue 4 virus and Zika virus, two medically relevant arboviruses, to understand the distribution of mosquito survival following microbe exposure. By correlating microbe loads and host health, we found that a blood meal promotes disease tolerance in our systemic bacterial infection model and that mosquitoes orally infected with bacteria had an enhanced defensive capacity than insects infected through injection. We also showed that Aedes aegypti displays a higher survival profile following arbovirus infection when compared to bacterial infections. Here, we applied a framework for investigating microbe-induced mosquito mortality and details how the lifespan of Aedes aegypti varies with different inoculum sizes of bacteria and arboviruses.

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. However, available strategies to follow gene expression in bacteria within their natural ecosystem that can be typically rich and complex are scarce. In this work, we designed a plasmid-based tool ensuring both the identification of a strain of interest in complex environments and the monitoring of gene expression through the combination of two distinct fluorescent proteins as reporter genes. The tool was validated in Escherichia coli to monitor the expression of eut genes involved in the catabolism of ethanolamine. We demonstrated that the constructed reporter strain gradually responds with a bimodal output to increasing ethanolamine concentrations during in vitro cultures. The reporter strain was next inoculated to mice, and flow cytometry was used to detect the reporter strain among the dense microbiota of intestinal samples and to analyze specifically the expression of eut genes. This novel dual-fluorescent reporter system would be helpful to evaluate transcriptional processes in bacteria within complex environments. KEY POINTS: • A reporter tool was developed to monitor bacterial gene expression in complex environments. • Ethanolamine utilization (eut) genes are expressed by commensal E. coli in the mouse gut. • Expression of eut genes follows a bimodal distribution.

Listeria monocytogenes is a globally distributed bacterium that exhibits genetic diversity and trait heterogeneity. The alternative sigma factor SigB serves as a crucial transcriptional regulator essential for responding to environmental stress conditions and facilitating host infection.

We employed a comprehensive genetic analysis of sigB in a dataset comprising 46,921 L. monocytogenes genomes. The functional attributes of SigB were evaluated by phenotypic experiments.

Our study revealed the presence of two predominant SigB factors (SigBT1 and SigBT2) in L. monocytogenes, with a robust correlation between SigBT1 and lineages I and III, as well as SigBT2 and lineage II. Furthermore, SigBT1 exhibits superior performance in promoting cellular invasion, cytotoxicity and enhancing biofilm formation and cold tolerance abilities under minimally defined media conditions compared to SigBT2.

The functional characteristics of SigBT1 suggest a potential association with the epidemiology of lineages I and III strains in both human hosts and the natural environment. Our findings highlight the important role of distinct SigB factors in influencing the biological traits of L. monocytogenes of different lineages, thus highlighting its distinct pathogenic and adaptive attributes.

Listeria monocytogenes is a foodborne bacterium that naturally occurs in the soil. Originating from there, it contaminates crops and infects farm animals and their consumption by humans may lead to listeriosis, a systemic life-threatening infectious disease. The adaptation of L. monocytogenes to such contrastive habitats is reflected by the presence of virulence genes for host infection and other genes for survival under environmental conditions. Among the latter are ABC transporters for excretion of antibiotics produced by environmental competitors; however, most of these transporters have not been characterized. Here, we generated a collection of promoter-lacZ fusions for genes encoding ABC-type drug transporters of L. monocytogenes and screened this reporter strain collection for induction using a library of natural compounds produced by various environmental microorganisms. We found that the timABR locus (lmo1964-lmo1962) was induced by the macrodiolide antibiotic tartrolon B, which is synthesized by the soil myxobacterium Sorangium cellulosum. Tartrolon B resistance of L. monocytogenes was dependent on timAB, encoding the ATPase and the permease component of a novel ABC transporter. Moreover, transplantation of timAB was sufficient to confer tartrolon B resistance to Bacillus subtilis. Expression of the timABR locus was found to be auto-repressed by the TimR repressor, whose repressing activity was lost in the presence of tartrolon B. We also demonstrate that tartrolon sensitivity was suppressed by high external potassium concentrations, suggesting that tartrolon acts as potassium ionophore. Our results help to map the ecological interactions of an important human pathogen with its co-residing species within their joint natural reservoir.

Listeria monocytogenes is a Gram positive foodborne pathogen that regularly causes outbreaks of systemic infectious diseases. The bacterium maintains a facultative intracellular lifestyle; it thrives under a variety of environmental conditions and is able to infect human host cells. L. monocytogenes is genetically tractable and therefore has become an attractive model system to study the mechanisms employed by facultative intracellular bacteria to invade eukaryotic cells and to replicate in their cytoplasm. Besides its importance for basic research, L. monocytogenes also serves as a paradigmatic pathogen in genomic epidemiology, where the relative stability of its genome facilitates successful outbreak detection and elucidation of transmission chains in genomic pathogen surveillance systems. In both terms, it is necessary to keep the annotation of the L. monocytogenes genome up to date. Therefore, we have created the database ListiWiki (http://listiwiki.uni-goettingen.de/) which stores comprehensive information on the widely used L. monocytogenes reference strain EDG-e. ListiWiki is designed to collect information on genes, proteins and RNAs and their relevant functional characteristics, but also further information such as mutant phenotypes, available biological material, and publications. In its present form, ListiWiki combines the most recent annotation of the EDG-e genome with published data on gene essentiality, gene expression and subcellular protein localization. ListiWiki also predicts protein-protein interactions networks based on protein homology to Bacillus subtilis proteins, for which detailed interaction maps have been compiled in the sibling database SubtiWiki. Furthermore, crystallographic information of proteins is made accessible through integration of Protein Structure Database codes and AlphaFold structure predictions. ListiWiki is an easy-to-use web interface that has been developed with a focus on an intuitive access to all information. Use of ListiWiki is free of charge and its content can be edited by all members of the scientific community after registration. In our labs, ListiWiki has already become an important and easy to use tool to quickly access genome annotation details that we can keep updated with advancing knowledge. It also might be useful to promote the comprehensive understanding of the physiology and virulence of an important human pathogen.

Genomic data-based machine learning tools are promising for real-time surveillance activities performing source attribution of foodborne bacteria such as Listeria monocytogenes. Given the heterogeneity of machine learning practices, our aim was to identify those influencing the source prediction performance of the usual holdout method combined with the repeated k-fold cross-validation method.

A large collection of 1 100 L. monocytogenes genomes with known sources was built according to several genomic metrics to ensure authenticity and completeness of genomic profiles. Based on these genomic profiles (i.e. 7-locus alleles, core alleles, accessory genes, core SNPs and pan kmers), we developed a versatile workflow assessing prediction performance of different combinations of training dataset splitting (i.e. 50, 60, 70, 80 and 90%), data preprocessing (i.e. with or without near-zero variance removal), and learning models (i.e. BLR, ERT, RF, SGB, SVM and XGB). The performance metrics included accuracy, Cohen's kappa, F1-score, area under the curves from receiver operating characteristic curve, precision recall curve or precision recall gain curve, and execution time.

The testing average accuracies from accessory genes and pan kmers were significantly higher than accuracies from core alleles or SNPs. While the accuracies from 70 and 80% of training dataset splitting were not significantly different, those from 80% were significantly higher than the other tested proportions. The near-zero variance removal did not allow to produce results for 7-locus alleles, did not impact significantly the accuracy for core alleles, accessory genes and pan kmers, and decreased significantly accuracy for core SNPs. The SVM and XGB models did not present significant differences in accuracy between each other and reached significantly higher accuracies than BLR, SGB, ERT and RF, in this order of magnitude. However, the SVM model required more computing power than the XGB model, especially for high amount of descriptors such like core SNPs and pan kmers.

In addition to recommendations about machine learning practices for L. monocytogenes source attribution based on genomic data, the present study also provides a freely available workflow to solve other balanced or unbalanced multiclass phenotypes from binary and categorical genomic profiles of other microorganisms without source code modifications.

My scientific career has resulted from key decisions and reorientations, sometimes taken rapidly but not always, guided by discussions or collaborations with amazing individuals from whom I learnt a lot scientifically and humanly. I had never anticipated that I would accomplish so much in what appeared as terra incognita when I started to interrogate the mechanisms underlying the virulence of the bacterium Listeria monocytogenes. All this has been possible thanks to a number of talented team members who ultimately became friends.

Bacteria adjust gene expression at the post-transcriptional level through an intricate network of small regulatory RNAs and RNA-binding proteins, including ribonucleases (RNases). RNases play an essential role in RNA metabolism, regulating RNA stability, decay, and activation. These enzymes exhibit species-specific effects on gene expression, bacterial physiology, and different strategies of target recognition. Recent advances in high-throughput RNA sequencing (RNA-seq) approaches have provided a better understanding of the roles and modes of action of bacterial RNases. Global studies aiming to identify direct targets of RNases have highlighted the diversity of RNase activity and RNA-based mechanisms of gene expression regulation. Here, we review recent RNA-seq approaches used to study bacterial RNases, with a focus on the methods for identifying direct RNase targets.

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed" stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

The number of multidrug-resistant bacteria is rapidly spreading worldwide. Among the various mechanisms determining resistance to antimicrobial agents, multidrug efflux pumps play a noteworthy role because they export extraneous and noxious substrates from the inside to the outside environment of the bacterial cell contributing to multidrug resistance (MDR) and, consequently, to the failure of anti-infective therapies. The expression of multidrug efflux pumps can be under the control of transcriptional regulators and two-component systems (TCS). TCS are a major mechanism by which microorganisms sense and reply to external and/or intramembrane stimuli by coordinating the expression of genes involved not only in pathogenic pathways but also in antibiotic resistance. In this review, we describe the influence of TCS on multidrug efflux pump expression and activity in some Gram-negative and Gram-positive bacteria. Taking into account the strict correlation between TCS and multidrug efflux pumps, the development of drugs targeting TCS, alone or together with already discovered efflux pump inhibitors, may represent a beneficial strategy to contribute to the fight against growing antibiotic resistance.

The mesenteric lymph nodes (MLN) function as a barrier to systemic spread for both commensal and pathogenic bacteria in the gut. Listeria monocytogenes, a facultative intracellular foodborne pathogen, readily overcomes this barrier and spreads into the bloodstream, causing life-threatening systemic infections. We show here that intracellular replication protected L. monocytogenes from clearance by monocytes and neutrophils and promoted colonization of the small intestine-draining MLN (sMLN) but was not required for dissemination to the colon-draining MLN (cMLN). Intestinal tissue had enough free lipoate to support LplA2-dependent extracellular growth of L. monocytogenes, but exogenous lipoate in the MLN was severely limited, and so the bacteria could replicate only inside cells, where they used LplA1 to scavenge lipoate from host peptides. When foodborne infection was manipulated to allow ΔlplA1 L. monocytogenes to colonize the MLN to the same extent as wild-type bacteria, the mutant was still never recovered in the spleen or liver of any animal. We found that intracellular replication in the MLN promoted actin-based motility and cell-to-cell spread of L. monocytogenes and that rapid efficient exit from the MLN was actA dependent. We conclude that intracellular replication of L. monocytogenes in intestinal tissues is not essential and serves primarily to amplify bacterial burdens above a critical threshold needed to efficiently colonize the cMLN. In contrast, intracellular replication in the MLN is absolutely required for further systemic spread and serves primarily to promote ActA-mediated cell-to-cell spread.

Listeria monocytogenes is a facultatively intracellular pathogenic bacterium that can provoke invasive listeriosis, a severe foodborne infection in humans. Outside the host, this is capable to survive for long periods in soil, and water, as well as on plants, while, like many other microorganisms, this can also attach to abiotic surfaces, such as food contact ones, forming biofilms on them. It has been suggested that inside those sessile communities, L. monocytogenes cells not only display an increased stress tolerance but may also boost their pathogenicity. In this work, the expression of ten key stress response and/or virulence-related genes (i.e., groEL, hly, iap, inlA, inlB, lisK, mdrD, mdrL, prfA, and sigB) was studied in three different L. monocytogenes strains (AAL20066, AAL20107, and PL24), all isolated from foods and each belonging to a different listeriosis-associated serovar (1/2a, 1/2b, and 1/2c, respectively). For this, each strain was initially left to develop a mature biofilm on a model polystyrene surface (Petri dish) by incubating for 144 h (6 days) at 20 °C in tryptone soya broth (with medium renewal every 48 h). Following incubation, both biofilm and the surrounding free-swimming (planktonic) cells were recovered, and their gene expressions were comparatively evaluated through targeted reverse transcription-quantitative polymerase chain reactions (RT-qPCR). Results revealed a strain-dependent differential gene expression between the two cell types. Thus, for instance, in strain AAL20107 (ser. 1/2b) biofilm growth worryingly resulted in a significant overexpression of all the studied genes (P < 0.05), whereas in strain PL24 (ser. 1/2c), the expression of most genes (8/10) did not change upon biofilm growth, with only two of them (groEL and hly) being again significantly upregulated. Such transcriptomic strain variability in stress adaptation and/or virulence induction should be generally considered in the physiological studies of pathogenic biofilms and preferably upon designing and implementing novel and more efficient eradication methods.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that can cause severe invasive infections upon ingestion with contaminated food. Clinically, listerial disease, or listeriosis, most often presents as bacteremia, meningitis or meningoencephalitis, and pregnancy-associated infections manifesting as miscarriage or neonatal sepsis. Invasive listeriosis is life-threatening and a main cause of foodborne illness leading to hospital admissions in Western countries. Sources of contamination can be identified through international surveillance systems for foodborne bacteria and strains' genetic data sharing. Large-scale whole genome studies have increased our knowledge on the diversity and evolution of L. monocytogenes, while recent pathophysiological investigations have improved our mechanistic understanding of listeriosis. In this article, we present an overview of human listeriosis with particular focus on relevant features of the causative bacterium, epidemiology, risk groups, pathogenesis, clinical manifestations, and treatment and prevention.

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.

L. monocytogenes and L. ivanovii, the only two pathogens of Listeria, can survive in various environments, having different pathogenic characteristics. However, the genetic basis of their excellent adaptability and differences in pathogenicity has still not been completely elucidated.

We performed a comparative genomic analysis based on 275 L. monocytogenes, 10 L. ivanovii, and 22 non-pathogenic Listeria strains.

Core/pan-genome analysis revealed that 975 gene families were conserved in all the studied strains. Additionally, 204, 242, and 756 gene families existed uniquely in L. monocytogenes, L. ivanovii, and both, respectively. Functional annotation partially verified that these unique gene families were closely related to their adaptability and pathogenicity. Moreover, the protein-protein interaction (PPI) network analysis of these unique gene sets showed that plenty of carbohydrate transport systems and energy metabolism enzymes were clustered in the networks. Interestingly, ethanolamine-metabolic-process-related proteins were significantly enriched in the PPI network of the unique genes of the Listeria pathogens, which can be understood as a determining factor of their pathogenicity.

The utilization capacity of multiple carbon sources of Listeria pathogens, especially ethanolamine, is the key genetic basis for their ability to adapt to various environments and pathogenic lifestyles.

A long scientific journey has led to prominent technological advances in the RNA field, and several new types of molecules have been discovered, from non-coding RNAs (ncRNAs) to riboswitches, small interfering RNAs (siRNAs) and CRISPR systems. Such findings, together with the recognition of the advantages of RNA in terms of its functional performance, have attracted the attention of synthetic biologists to create potent RNA-based tools for biotechnological and medical applications. In this review, we have gathered the knowledge on the connection between RNA metabolism and pathogenesis in Gram-positive and Gram-negative bacteria. We further discuss how RNA techniques have contributed to the building of this knowledge and the development of new tools in synthetic biology for the diagnosis and treatment of diseases caused by pathogenic microorganisms. Infectious diseases are still a world-leading cause of death and morbidity, and RNA-based therapeutics have arisen as an alternative way to achieve success. There are still obstacles to overcome in its application, but much progress has been made in a fast and effective manner, paving the way for the solid establishment of RNA-based therapies in the future.

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety.

Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes pduCDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein's high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Salmonella Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of S. Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a pduC-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of Limosilactobacillus reuteri, but not a pduCDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 108 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.

Listeria monocytogenes is a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine against Listeria is available. Attenuated Listeria strains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screen for Listeria peptides presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detect 68 Listeria immunopeptides from 42 different bacterial proteins, including several known antigens. Peptides presented on different cell lines are often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations results in specific CD8+ T-cell responses and induces protection in vaccination challenge experiments in mice. Our results can serve as a starting point for the development of a clinical mRNA vaccine against Listeria and aid to improve attenuated Listeria vaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.