Rational Design of a Robust G-Quadruplex Aptamer as an Inhibitor to Alleviate Listeria monocytogenes Infection.

Listeria monocytogenes (LM) is one of the most invasive foodborne pathogens that cause listeriosis, making it imperative to explore novel inhibiting strategies for alleviating its infection. The adhesion and invasion of LM within host cells are partly orchestrated by an invasin protein internalin A (InlA), which facilitates bacterial passage by interacting with the host cell E-cadherin (E-Cad). Hence, in this work, we proposed an aptamer blocking strategy by binding to the region on InlA that directly mediated E-Cad receptor engagement, thereby alleviating LM infection. An aptamer GA8 with a robust G-quadruplex (G4) structural feature was designed through truncation and base mutation from the original aptamer A8. The molecular docking and dynamics analysis showed that the InlA/aptamer GA8 binding interface was highly overlapping with the natural InlA/E-Cad binding interface, which confirmed that GA8 can tightly and stably bind InlA and block more distinct epitopes on InlA that involved the interaction with E-Cad. On the cellular level, it was confirmed that GA8 effectively blocked LM adhesion with an inhibition rate of 78%. Overall, the robust G4 aptamer-mediated design provides a new direction for the development of inhibitors against other wide-ranging and emerging pathogens.

Mechanistic insights into the alternative ribosome recycling by HflXr.

During stress conditions such as heat shock and antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, a homolog of the conserved housekeeping GTPase HflX that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use time-resolved cryo-electron microscopy to elucidate the mechanism of ribosome recycling by Listeria monocytogenes HflXr. Within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported here is universal in the prokaryotic kingdom.

Rombocin, a Short Stable Natural Nisin Variant, Displays Selective Antimicrobial Activity against Listeria monocytogenes and Employs a Dual Mode of Action to Kill Target Bacterial Strains.

Nisin, with its unique mode of action and potent antimicrobial activity, serves as a remarkable inspiration for the design of novel antibiotics. However, peptides possess inherent weaknesses, particularly their susceptibility to proteolytic degradation, such as by trypsin, which limits their broader applications. This led us to speculate that natural variants of nisin produced by underexplored bacterial species can potentially overcome these limitations. We carried out genome mining of two Romboutsia sedimentorum strains, RC001 and RC002, leading to the discovery of rombocin A, which is a 25 amino acid residue short nisin variant that is predicted to have only four macrocycles compared to the known 31-35 amino acids long nisin variants with five macrocycles. Using the nisin-controlled expression system, we heterologously expressed fully modified and functional rombocin A in Lactococcus lactis and demonstrated its selective antimicrobial activity against Listeria monocytogenes. Rombocin A uses a dual mode of action involving lipid II binding activity and dissipation of the membrane potential to kill target bacteria. Stability tests confirmed its high stability at different pH values, temperatures, and in particular, against enzymatic degradation. With its gene-encoded characteristic, rombocin A is amenable to bioengineering to generate novel derivatives. Further mutation studies led to the identification of rombocin K, a mutant with enhanced bioactivity against L. monocytogenes. Our findings suggest that rombocin A and its bioengineered variant, rombocin K, are promising candidates for development as food preservatives or antibiotics against L. monocytogenes.

Two-component system LiaSR negatively regulated the acid resistance and pathogenicity of Listeria monocytogenes 10403S.

The glutamate decarboxylase (GAD) system is one of the acid-resistant systems of Listeria monocytogenes (L. monocytogenes), while the regulatory mechanism of GadT2/GadD2, which plays the major role in the GAD system for acid resistance, is not clear. The two-component system (TCS) is a signal transduction system that is also involved in regulating acid resistance in bacteria. By screening the TCSs of L. monocytogenes 10403S, we found that knocking out the TCS LisSR (encoded by lmo1021/lmo1022) led to a significant increase in the transcription and expression of the gadT2/gadD2 cluster. Subsequently, we constructed a complemental strain CΔliaSR. and a complemental strain with LiaS His157 to Ala, which was designated as CΔliaSRH157A. Survival assay, transcriptional and expression analysis and pathogenicity assay revealed that liaSR deletion significantly enhanced the acid resistance and pathogenicity of 10403S and significantly increased the gadT2/gadD2 transcription and expression. Mutating LiaS His157 to Ala significantly enhanced the acid resistance and pathogenicity of CΔliaSR and significantly increased the gadT2/gadD2 transcription and expression. The results suggest that the two-component system LiaSR mediates the acid resistance and pathogenicity in 10403S by inhibiting the gadT2/gadD2 cluster, and the key activation site of LiaS is His157. This study provides novel knowledge on the regulation of GAD system and the control of this foodborne pathogen.

Quantitative live imaging reveals a direct interaction between CD44v6 and MET in membrane domains upon activation with both MET ligands, HGF and internalin B.

Deregulation of the receptor tyrosine kinase MET/hepatocyte growth factor (HGF) pathway results in several pathological processes involved in tumor progression and metastasis. In a different context, MET can serve as an entry point for the bacterium Listeria monocytogenes, when activated by the internalin B (InlB) protein during infection of non-phagocytic cells. We have previously demonstrated that MET requires CD44v6 for its ligand-induced activation. However, the stoichiometry and the steps required for the formation of this complex, are still unknown. In this work, we studied the dynamics of the ligand-induced interaction of CD44v6 with MET at the plasma membrane. Using Förster resonance energy transfer-based fluorescence lifetime imaging microscopy in T-47D cells, we evidenced a direct interaction between MET and CD44v6 promoted by HGF and InlB in live cells. In the absence of MET, fluorescence correlation spectroscopy experiments further showed the dimerization of CD44v6 and the increase of its diffusion induced by HGF and InlB. In the presence of MET, stimulation of the cells by HGF or InlB significantly decreased the diffusion of CD44v6, in line with the formation of a ternary complex of MET with CD44v6 and HGF/InlB. Finally, similarly to HGF/InlB, disruption of liquid-ordered domains (Lo) by methyl-ß-cyclodextrin increased CD44v6 mobility suggesting that these factors induce the exit of CD44v6 from the Lo domains. Our data led us to propose a model for MET activation, where CD44v6 dimerizes and diffuses rapidly out of Lo domains to form an oligomeric MET/ligand/CD44v6 complex that is instrumental for MET activation.

Listeria-based vaccination against the pericyte antigen RGS5 elicits anti-vascular effects and colon cancer protection.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality despite efforts to improve standard interventions. As CRC patients can benefit from immunotherapeutic strategies that incite effector T cell action, cancer vaccines represent a safe and promising therapeutic approach to elicit protective and durable immune responses against components of the tumor microenvironment (TME). In this study, we investigate the pre-clinical potential of a Listeria monocytogenes (Lm)-based vaccine targeting the CRC-associated vasculature. CRC survival and progression are reliant on functioning blood vessels to effectively mediate various metabolic processes and oxygenate underlying tissues. We, therefore, advance the strategy of initiating immunity in syngeneic mouse models against the endogenous pericyte antigen RGS5, which is a critical mediator of pathological vascularization. Overall, Lm-based vaccination safely induced potent anti-tumor effects that consisted of recruiting functional Type-1-associated T cells into the TME and reducing tumor blood vessel content. This study underscores the promising clinical potential of targeting RGS5 against vascularized tumors like CRC.

Characterization of a DsbA family protein reveals its crucial role in oxidative stress tolerance of Listeria monocytogenes.

IMPORTANCE: 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.

Vγ9+Vδ2+ T cell control of Listeria monocytogenes growth in infected epithelial cells requires butyrophilin 3A genes.

Intracellular bacteria produce antigens, which serve as potent activators of γδ T cells. Phosphoantigens are presented via a complex of butyrophilins (BTN) to signal infection to human Vγ9+Vδ2+ T cells. Here, we established an in vitro system allowing for studies of Vγ9+Vδ2+ T cell activity in coculture with epithelial cells infected with the intracellular bacterial pathogen Listeria monocytogenes. We report that the Vγ9+Vδ2+ T cells efficiently control L. monocytogenes growth in such cultures. This effector function requires the expression of members of the BTN3A family on epithelial cells. Specifically, we observed a BTN3A1-independent BTN3A3 activity to present antigen to Vγ9+Vδ2+ T cells. Since BTN3A1 is the only BTN3A associated with phosphoantigen presentation, our study suggests that BTN3A3 may present different classes of antigens to mediate Vγ9+Vδ2+ T cell effector function against L. monocytogenes-infected epithelia.

Design of a novel affinity probe using the cell wall-binding domain of a Listeria monocytogenes autolysin for pathogen detection.

IMPORTANCE: Human listeriosis is caused by consuming foods contaminated with the bacterial pathogen Listeria monocytogenes, leading to the development of a severe and life-threatening foodborne illness. Detection of L. monocytogenes present in food and food processing environments is crucial for preventing Listeria infection. The L. monocytogenes peptidoglycan hydrolase IspC anchors non-covalently to the bacterial surface through its C-terminal cell wall-binding domain (CWBD), CWBDIspC. This study explored the surface binding property of CWBDIspC to design, construct, characterize, and assess an affinity molecular probe for detecting L. monocytogenes. CWBDIspC recognized a cell wall ligand lipoteichoic acid that remains evenly displayed and mostly unoccupied on the bacterial surface for interaction with the exogenously added CWBDIspC. CWBDIspC, when fused to the enhanced green fluorescent protein reporter or covalently conjugated onto magnetic beads, exhibited the functionality as an antibody alternative for rapid detection and efficient separation of the pathogen.

Tartrolon sensing and detoxification by the Listeria monocytogenes timABR resistance operon.

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.

Cinnamaldehyde Targets the LytTR DNA-Binding Domain of the Response Regulator AgrA to Attenuate Biofilm Formation of Listeria monocytogenes.

The Agr quorum sensing (QS) system is known to contribute to biofilm formation in Listeria monocytogenes. Cinnamaldehyde, a natural food preservative, is considered an inhibitor of Agr-mediated QS in L. monocytogenes. However, the exact mechanism by which cinnamaldehyde acts on Agr remains unclear. In this study, we assessed the effects of cinnamaldehyde on the histidine kinase AgrC and the response regulator AgrA in the Agr system. AgrC kinase activity was not influenced by cinnamaldehyde, and binding between AgrC and cinnamaldehyde was not observed when microscale thermophoresis (MST) was performed, indicating that AgrC was not the target of cinnamaldehyde. AgrA is specifically bound to the agr promoter (P2) to activate the transcription of the Agr system. However, AgrA-P2 binding was prevented by cinnamaldehyde. The interaction between cinnamaldehyde and AgrA was further confirmed with MST. Two conserved amino acids, Asn-178 and Arg-179, located in the LytTR DNA-binding domain of AgrA, were identified as the key sites for cinnamaldehyde-AgrA binding by alanine mutagenesis and MST. Coincidentally, Asn-178 was also involved in the AgrA-P2 interaction. Taken together, these results suggest that cinnamaldehyde acts as a competitive inhibitor of AgrA in AgrA-P2 binding, which leads to suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. IMPORTANCE Listeria monocytogenes can form biofilms on various food contact surfaces, posing a serious threat to food safety. Biofilm formation of L. monocytogenes is positively regulated by the Agr quorum sensing system. Thus, an alternative strategy for controlling L. monocytogenes biofilms is interfering with the Agr system. Cinnamaldehyde is considered an inhibitor of the L. monocytogenes Agr system; however, its exact mechanism of action is still unclear. Here, we found that AgrA (response regulator), rather than AgrC (histidine kinase), was the target of cinnamaldehyde. The conserved Asn-178 in the LytTR DNA-binding domain of AgrA was involved in cinnamaldehyde-AgrA and AgrA-P2 binding. Therefore, the occupation of Asn-178 by cinnamaldehyde suppressed transcription of the Agr system and reduced biofilm formation in L. monocytogenes. Our findings could provide a better understanding of the mechanism by which cinnamaldehyde inhibits L. monocytogenes biofilm formation.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

[Knock-down of long intergenic noncoding RNA cyclooxygenase 2 (lincRNA-COX2) inhibits apoptosis and polarization into M1 in Listeria monocytogenes-infected macrophages].

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.

Experimental Evolution Reveals a Novel Ene Reductase That Detoxifies α,ß-Unsaturated Aldehydes in Listeria monocytogenes.

The plant essential oil component trans-cinnamaldehyde (t-CIN) exhibits antibacterial activity against a broad range of foodborne pathogenic bacteria, including L. monocytogenes, but its mode of action is not fully understood. In this study, several independent mutants of L. monocytogenes with increased t-CIN tolerance were obtained via experimental evolution. Whole-genome sequencing (WGS) analysis revealed single-nucleotide-variation mutations in the yhfK gene, encoding an oxidoreductase of the short-chain dehydrogenases/reductases superfamily, in each mutant. The deletion of yhfK conferred increased sensitivity to t-CIN and several other α,ß-unsaturated aldehydes, including trans-2-hexenal, citral, and 4-hydroxy-2-nonenal. The t-CIN tolerance of the deletion mutant was restored via genetic complementation with yhfK. Based on a gas chromatography-mass spectrometry (GC-MS) analysis of the culture supernatants, it is proposed that YhfK is an ene reductase that converts t-CIN to 3-phenylpropanal by reducing the C=C double bond of the α,ß-unsaturated aldehyde moiety. YhfK homologs are widely distributed in Bacteria, and the deletion of the corresponding homolog in Bacillus subtilis also caused increased sensitivity to t-CIN and trans-2-hexenal, suggesting that this protein may have a conserved function to protect bacteria against toxic α,ß-unsaturated aldehydes in their environments. IMPORTANCE While bacterial resistance against clinically used antibiotics has been well studied, less is known about resistance against other antimicrobials, such as natural compounds that could replace traditional food preservatives. In this work, we report that the food pathogen Listeria monocytogenes can rapidly develop an elevated tolerance against t-cinnamaldehyde, a natural antimicrobial from cinnamon, by single base pair changes in the yhfK gene. The enzyme encoded by this gene is an oxidoreductase, but its substrates and precise role were hitherto unknown. We demonstrate that the enzyme reduces the double bond in t-cinnamaldehyde and thereby abolishes its antibacterial activity. Furthermore, the mutations linked to t-CIN tolerance increased bacterial sensitivity to a related compound, suggesting that they modify the substrate specificity of the enzyme. Since the family of oxidoreductases to which YhfK belongs is of great interest in the mediation of stereospecific reactions in biocatalysis, our work may also have unanticipated application potential in this field.

Distinct Energy-Coupling Factor Transporter Subunits Enable Flavin Acquisition and Extracytosolic Trafficking for Extracellular Electron Transfer in Listeria monocytogenes.

A variety of electron transfer mechanisms link bacterial cytosolic electron pools with functionally diverse redox activities in the cell envelope and extracellular space. In Listeria monocytogenes, the ApbE-like enzyme FmnB catalyzes extracytosolic protein flavinylation, covalently linking a flavin cofactor to proteins that transfer electrons to extracellular acceptors. L. monocytogenes uses an energy-coupling factor (ECF) transporter complex that contains distinct substrate-binding, transmembrane, ATPase A, and ATPase A' subunits (RibU, EcfT, EcfA, and EcfA') to import environmental flavins, but the basis of extracytosolic flavin trafficking for FmnB flavinylation remains poorly defined. In this study, we show that the EetB and FmnA proteins are related to ECF transporter substrate-binding and transmembrane subunits, respectively, and are essential for exporting flavins from the cytosol for flavinylation. Comparisons of the flavin import versus export capabilities of L. monocytogenes strains lacking different ECF transporter subunits demonstrate a strict directionality of substrate-binding subunit transport but partial functional redundancy of transmembrane and ATPase subunits. Based on these results, we propose that ECF transporter complexes with different subunit compositions execute directional flavin import/export through a broadly conserved mechanism. Finally, we present genomic context analyses that show that related ECF exporter genes are distributed across members of the phylum Firmicutes and frequently colocalize with genes encoding flavinylated extracytosolic proteins. These findings clarify the basis of ECF transporter export and extracytosolic flavin cofactor trafficking in Firmicutes. IMPORTANCE Bacteria import vitamins and other essential compounds from their surroundings but also traffic related compounds from the cytosol to the cell envelope where they serve various functions. Studying the foodborne pathogen Listeria monocytogenes, we find that the modular use of subunits from a prominent class of bacterial transporters enables the import of environmental vitamin B2 cofactors and the extracytosolic trafficking of a vitamin B2-derived cofactor that facilitates redox reactions in the cell envelope. These studies clarify the basis of bidirectional small-molecule transport across the cytoplasmic membrane and the assembly of redox-active proteins within the cell envelope and extracellular space.

Isoflavone glucoside genistin, an inhibitor targeting Sortase A and Listeriolysin O, attenuates the virulence of Listeria monocytogenes in vivo and in vitro.

As a common intracellular facultative anaerobic Gram-positive bacterium, Listeria monocytogenes (L. monocytogenes) exhibits strong resistance to extreme environments, such as low temperature and a wide range of pH values, causing contamination in food production and processing. Sortase A (SrtA) and listeriolysin O (LLO), two crucial virulence factors of L. monocytogenes, are widely recognized as potential targets for the development of anti-L. monocytogenes infection drugs. In this study, we found that genistin simultaneously inhibits the peptidase activity of SrtA and the hemolytic activity of LLO without affecting the growth of L. monocytogenes, alleviating concerns about developing resistance. Furthermore, we demonstrated that genistin reduces L. monocytogenes biofilm formation and invasion of human colorectal cancer (Caco-2) cells. Subsequent mechanistic studies revealed that genistin inhibited LLO-mediated Caco-2 cell damage by blocking LLO oligomerization. Fluorescence quenching assay revealed the potential binding mode of SrtA and LLO to genistin. Genistin might bind to the active pocket of SrtA through residues Leu33, Asn29, and Met40, interacting with D1 domain of LLO involved in oligomerization and pore formation through residues Asn259. Studies in infection models revealed that genistin reduces mortality and pathological damage in mice infected with L. monocytogenes. These results indicate that genistin is a promising anti-virulence agent that could be considered an alternative candidate for the treatment of L. monocytogenes infection.

Marked inter-strain heterogeneity in the differential expression of some key stress response and virulence-related genes between planktonic and biofilm cells in Listeria monocytogenes.

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.

MicroRNAs induced by Listeria monocytogenes and their role in cells.

Listeria monocytogenes (Lm) causes abortions at high rates and threatens newborns' lives. Also, the elderly and immunocompromised individuals are particularly vulnerable neurologically. The bacterium exerts its pathogenesis intracellularly by manipulating cell organs. It manipulates nucleus elements, microRNAs (miRNAs), in order to increase survival and evade immunity. miRNAs are small non-coding RNAs that degrade gene expression post-transcriptionally. Any alteration to the expression of miRNAs affects various cascades in cells, especially immunity-related responses. Thus, utilizing miRNAs as a novel therapeutic agent not only restricts infection but enhances immunity reactions. This review provides an overview of miRNAs in listeriosis, their role in cells, and their prospects as therapy.

A Copper-Responsive Two-Component System Governs Lipoprotein Remodeling in Listeria monocytogenes.

Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.

The Extracellular Electron Transport Pathway Reduces Copper for Sensing by the CopRS Two-Component System under Anaerobic Conditions in Listeria monocytogenes.

The renowned antimicrobial activity of copper stems in part from its ability to undergo redox cycling between Cu1+/2+ oxidation states. Bacteria counter copper toxicity with a network of sensors that often include two-component signaling systems to direct transcriptional responses. As in typical two-component systems, ligand binding by the extracellular domain of the membrane bound copper sensor component leads to phosphorylation and activation of the cognate response regulator transcription factor. In Listeria monocytogenes, the plasmid-borne CopRS two-component system upregulates both copper resistance and lipoprotein remodeling genes upon copper challenge, but the oxidation state of copper bound by CopS is unknown. Herein, we show CopS utilizes a triad of key residues (His-His-Phe) that are predicted to be at the dimerization interface and that are analogous with the Escherichia coli CusS copper sensor to specifically bind Cu1+/Ag1+ and activate CopR transcription. We demonstrate Cu2+ only induces CopRS if first reduced by electron transport systems, as strains lacking menaquinone carriers were unable to respond to Cu2+. The flavin-dependent extracellular electron transport system (EET) was the main mechanism for metal reduction, capable of either generating inducing ligand (Cu2+ to Cu1+) or removing it by precipitation (Ag1+ to Ag0). We show that EET flux is directly proportional to the rate of Cu2+ reduction and that since EET activity is low under oxygenated conditions when a competing respiratory chain is operating, CopRS signaling in turn is activated only under anaerobic conditions. EET metal reduction thus sensitizes cells to copper while providing resistance to silver under anaerobic growth. IMPORTANCE Two-component extracellular copper sensing from the periplasm of Gram-negative bacteria has been well studied, but copper detection at the cell surface of the Gram-positive L. monocytogenes is less understood. Collectively, our results show that EET is most active under anaerobic conditions and reduces Cu2+ and Ag1+ to, respectively, generate or remove the monovalent ligands that directly bind to CopS and lead to the induction of lipoprotein remodeling genes. This reducing activity regulates CopRS signaling and links the upregulation of copper resistance genes with increasing EET flux. Our studies provide insight into how a two-component copper sensing system is integrated into a model monoderm Firmicute to take cues from the electron transport chain activity.