Listeria brain abscess: a therapeutically challenging rare presentation of listeriosis.

We report a very rare case of Listeria multiple brain abscesses manifested as delirium, which represented diagnostic and therapeutic challenges overcome only by the close cooperation between Infectious Diseases and Neuroradiology, without which a satisfactory outcome would not be achieved.An elderly man presented with confusion and drowsiness with a background of type-II diabetes mellitus. Although computed tomography of the brain only showed frontal lobe oedema, contrast magnetic resonance (MR) imaging showed numerous irregular rim-enhancing lesions containing central diffusion restriction, suggesting multiple pyogenic cerebral abscesses of unclear aetiology. Thereafter, Listeria monocytogenes was isolated from blood cultures, suggesting this as the causative organism. Deemed unsuitable for neurosurgical drainage, the patient received medical management with a protracted course of antibiotics. This case was extremely challenging, due to 1) the impossibility of source control, 2) the small number of effective antibiotics available to treat this condition, and 3) the inevitable antibiotic side-effects, derived from long-term exposure. A successful outcome was only possible thanks to strict close multidisciplinary follow up, requiring frequent MR imaging and a judicious antibiotic choice, including monitoring of their side-effects. Due to the rarity of this condition, there is lack of guidance on its management, hence the importance of multidisciplinary involvement with very close imaging and antibiotic monitoring.

Outbreak of Listeria monocytogenes in hospital linked to a fava bean product, Finland, 2015 to 2019.

Listeria monocytogenes (Lm) is a bacterium widely distributed in the environment. Listeriosis is a severe disease associated with high hospitalisation and mortality rates. In April 2019, listeriosis was diagnosed in two hospital patients in Finland. We conducted a descriptive study to identify the source of the infection and defined a case as a person with a laboratory-confirmed Lm serogroup IIa sequence type (ST) 37. Six cases with Lm ST 37 were notified to the Finnish Infectious Diseases Registry between 2015 and 2019. Patient interviews and hospital menus were used to target traceback investigation of the implicated foods. In 2021 and 2022, similar Lm ST 37 was detected from samples of a ready-to-eat plant-based food product including fava beans. Inspections by the manufacturer and the local food control authority indicated that the food products were contaminated with Lm after pasteurisation. Our investigation highlights the importance that companies producing plant-based food are subject to similar controls as those producing food of animal origin. Hospital menus can be a useful source of information that is not dependent on patient recall.

Designing a Multiepitope Vaccine against the Foodborne Pathogenic Bacteria Listeria monocytogenes Using Subtractive Immunoinformatics Approaches.

BACKGROUND: Listeria monocytogenes, a Gram-positive bacterium, is a prominent foodborne pathogen that causes listeriosis and poses substantial health hazards worldwide. The continuing risk of listeriosis outbreaks underlies the importance of designing an effective prevention strategy and developing a robust immune response by reverse vaccinology approaches. This study aimed to provide a critical approach for developing a potent multiepitope vaccine against this foodborne disease. METHODS: A chimeric peptide construct containing 5 B-cell epitopes, 16 major histocompatibility complex I (MHC-I) epitopes, and 18 MHC-II epitopes were used to create a subunit vaccination against L. monocytogenes. The vaccine safety was evaluated by several online methods, and molecular docking was performed using ClusPro to determine the binding affinity. Immune simulation was performed using the C-ImmSimm server to demonstrate the immune response. RESULTS: The results validated the antigenicity, non-allergenicity, and nontoxicity of the chimeric peptide construct, confirming its suitability as a subunit vaccine. Molecular docking showed a good score of 1276.5 and molecular dynamics simulations confirmed the construct's efficacy, demonstrating its promise as a good candidate for listeriosis prophylaxis. The population coverage was as high as 91.04% with a good immune response, indicating good antigen presentation with dendritic cells and production of memory cells. CONCLUSIONS: The findings of this study highlight the potential of the designed chimeric peptide construct as an effective subunit vaccine against Listeria, paving the way for future advances in preventive methods and vaccine design.

The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes. The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 102 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.

Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Temporal and spatial dynamics of Listeria monocytogenes central nervous system infection in mice.

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.

Listeria monocytogenes: the silent assassin.

Listeriosis is a foodborne infection in humans caused by Listeria monocytogenes. Consumption of contaminated food can lead to severe infection in vulnerable patients, that can be fatal. Clinical manifestations include sepsis and meningitis, and in pregnancy-associated infection, miscarriage and stillbirth. Diagnosis is confirmed by culture and identification of the pathogen from blood, cerebrospinal fluid, vaginal swab, placenta or amniotic fluid. Treatment regimens recommend amoxicillin, ampicillin or an aminoglycoside. Virulence factors mediate bacterial adhesion and invasion of gut epithelial cells. Other factors mediate biofilm formation and tolerance to low temperatures and high salt concentrations facilitating persistence and survival in the environment.

Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing Escherichia coli and Listeria monocytogenes by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

Eosinophils promote CD8+ T cell memory generation to potentiate anti-bacterial immunity.

Memory CD8+ T cell generation is crucial for pathogen elimination and effective vaccination against infection. The cellular and molecular circuitry that underlies the generation of memory CD8+ T cells remains elusive. Eosinophils can modulate inflammatory allergic responses and interact with lymphocytes to regulate their functions in immune defense. Here we report that eosinophils are required for the generation of memory CD8+ T cells by inhibiting CD8+ T cell apoptosis. Eosinophil-deficient mice display significantly impaired memory CD8+ T cell response and weakened resistance against Listeria monocytogenes (L.m.) infection. Mechanistically, eosinophils secrete interleukin-4 (IL-4) to inhibit JNK/Caspase-3 dependent apoptosis of CD8+ T cells upon L.m. infection in vitro. Furthermore, active eosinophils are recruited into the spleen and secrete more IL-4 to suppress CD8+ T cell apoptosis during early stage of L.m. infection in vivo. Adoptive transfer of wild-type (WT) eosinophils but not IL-4-deficient eosinophils into eosinophil-deficient mice could rescue the impaired CD8+ T cell memory responses. Together, our findings suggest that eosinophil-derived IL-4 promotes the generation of CD8+ T cell memory and enhances immune defense against L.m. infection. Our study reveals a new adjuvant role of eosinophils in memory T cell generation and provides clues for enhancing the vaccine potency via targeting eosinophils and related cytokines.

Gut Microbiota Protects Listeria monocytogenes-Infected Mice by Reducing the Inflammatory Cytokines Storm and Cell Apoptosis.

Gut microbiota (GM) has been proven to resist pathogenic infection through nutritional competition, colonization resistance and promotion of the host immune response. However, in clinical practice, GM is mainly used in intestinal diseases, such as Clostridium difficile infection, and there are few reports on its application in the treatment of pathogenic bacterial infections. In this study, GM from healthy mice was transplanted into mice infected with Listeria monocytogenes using fecal microbiota transplantation (FMT) and the effects were observed. We found that GM from healthy mice could reduce the mortality of infected mice and decrease the counts of L. monocytogenes in their liver and spleen. In addition, FMT inhibited the expression of inflammatory factors in the liver and spleen of infected mice. In vitro cell experiments revealed that GM can reduce the count of L. monocytogenes invading Caco-2 cells and inhibit the L. monocytogenes-caused apoptosis. These results indicate that GM can be used to protect mice infected with L. monocytogenes by eliminating the amount of L. monocytogenes in the host and inhibiting the overexpression of inflammatory factors. Hence, this method can potentially replace antibiotics in the treatment of L. monocytogenes infection.

Genomic characterization of Listeria monocytogenes and Listeria innocua isolated from milk and dairy samples in Ethiopia.

Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0-10 SNP differences, except for the ST 1619, which displayed a greater diversity.

Clinical and genomic features of Listeria monocytogenes-associated mesenteric lymphadenitis in a cat.

BACKGROUND: Listeriosis is a severe foodborne infection caused by Listeria monocytogenes, an important foodborne pathogen of animals and humans. Listeriosis is a rare disease in cats. OBJECTIVE: To describe the clinical, diagnostic imaging, histological, and microbiological features of L. monocytogenes-associated mesenteric lymphadenitis in a cat. ANIMALS: Listeria monocytogenes-associated mesenteric lymphadenitis was confirmed in a cat by histology and microbiology. RESULTS: Two distinct isolates of L. monocytogenes were cultured from the affected mesenteric lymph node and whole genome sequencing was performed. CONCLUSION AND CLINICAL IMPORTANCE: This report should alert veterinary clinicians and microbiologists to the syndrome, which may have implications for health and food safety in animals and humans.

Impact of vitamin B12 on rhamnose metabolism, stress defense and in-vitro virulence of Listeria monocytogenes.

Listeria monocytogenes is a facultative anaerobe which can cause a severe food-borne infection known as listeriosis. L. monocytogenes is capable of utilizing various nutrient sources including rhamnose, a naturally occurring deoxy sugar abundant in foods. L. monocytogenes can degrade rhamnose into lactate, acetate and 1,2-propanediol. Our previous study showed that addition of vitamin B12 stimulated anaerobic growth of L. monocytogenes on rhamnose due to the activation of bacterial microcompartments for 1,2-propanediol utilization (pdu BMC) with concomitant production of propionate and propanol. Notably, anaerobic 1,2-propanediol metabolism has been linked to virulence of enteric pathogens including Salmonella spp. and L. monocytogenes. In this study we investigated the impact of B12 and BMC activation on i) aerobic and anerobic growth of L. monocytogenes on rhamnose and ii) the level of virulence. We observed B12-induced pdu BMC activation and growth stimulation only in anaerobically grown cells. Comparative Caco-2 virulence assays showed that these pdu BMC-induced cells have significantly higher translocation efficiency compared to non-induced cells (anaerobic growth without B12; aerobic growth with or without B12), while adhesion and invasion capacity is similar for all cells. Comparative proteome analysis showed specific and overlapping responses linked to metabolic shifts, activation of stress defense proteins and virulence factors, with RNA polymerase sigma factor SigL, teichoic acid export ATP-binding protein TagH, DNA repair and protection proteins, RadA and DPS, and glutathione synthase GshAB, previously linked to activation of virulence response in L. monocytogenes, uniquely upregulated in anaerobically rhamnose grown pdu-induced cells. Our results shed light on possible effects of B12 on L. monocytogenes competitive fitness and virulence activation when utilizing rhamnose in anaerobic conditions encountered during transmission and the human intestine.

PoreGlow: A split green fluorescent protein-based system for rapid detection of Listeria monocytogenes.

Listeria monocytogenes, a severe foodborne pathogen causing severe diseases underscores the necessity for the development of a detection system with high specificity, sensitivity and utility. Herein, the PoreGlow system, based on split green fluorescent protein (GFP), was developed and assessed for the fast and accurate detection of L. monocytogenes. Split GFP-encapsulated liposomes were optimized for targeted analysis. The system utilizes listeriolysin O (LLO), a toxin produced by L. monocytogenes that enlarges the pores split GFP-encapsulated liposomes, to detect L. monocytogenes by measuring the fluorescent signal generated when the encapsulated GFP is released and reacted with the externally added fragment of the split GFP. The system exhibited a limit of detection of 0.17 µg/ml for LLO toxin and 10 CFU/mL for L. monocytogenes with high sensitivity and specificity and no cross-reactivity with other bacteria. The PoreGlow system is practical, rapid, and does not require sample pre-treatment, making it a promising tool for the early detection of L. monocytogenes in food products, which is crucial for preventing outbreaks and protecting public health.

Synergistic effect of kanamycin and amikacin with setomimycin on biofilm formation inhibition of Listeria monocytogenes.

Listeria monocytogenes, a foodborne pathogen that causes listeriosis with high fatality rate, exhibits multidrug resistance (MDR) known to be progressively increasing. Alternative antibacterial strategies are in high demand for treating this well-known pathogen. Anti-biofilm and anti-virulence strategies are being explored as novel approaches to treat bacterial infections. In this study, one rare antibacterial named setomimycin was isolated from Streptomyces cyaneochromogenes, which showed potent antibacterial activity against L. monocytogenes. Next, the inhibition of biofilm formation and listeriolysin O (LLO) production against L. monocytogenes were investigated at sub-minimal inhibitory concentrations (sub-MICs) of setomimycin alone or combined with kanamycin and amikacin. Crystal violet staining confirmed that setomimycin combining with kanamycin or amikacin could dramatically reduce biofilm formation against L. monocytogenes at sub-MICs, which was further evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In the meantime, sub-MICs of setomimycin could significantly suppress the secretion of LLO. Furthermore, the transcription of genes associated with biofilms and main virulence factors, such as LLO, flagellum, and metalloprotease, were suppressed by setomimycin at sub-MICs. Hence, the study provided a deep insight into setomimycin as an alternative antibacterial agent against L. monocytogenes.

Multilab Validation Report for the Verification and Subtyping of Listeria monocytogenes Using qPCR.

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes invasive listeriosis, an illness with high mortality and hospitalization rates. Due to the severity of illness associated with Lm, rapid identification and characterization of isolates from foods and the food-processing environment are critical to properly identify and track the pathogen and quickly remove adulterated foods from the market. Prior methods can rely on time-consuming biochemical or sera-agglutination assays to perform these tasks. Development of a high-throughput method that would rapidly perform these tasks is critical to improve response to contamination events. Previously, a single laboratory validation of a qPCR-based method was presented that could rapidly verify Lm isolates and characterize them into six molecular serogroups. In the current study, a multi-laboratory validation (MLV) was performed to evaluate the reliability of the qPCR method for identification and serogrouping of Lm isolates. Sixteen collaborating laboratories independently analyzed a panel of 43 blinded isolates plus three control strains using the qPCR method. This panel was comprised of representatives for non-Listeria (n = 7), Listeria sp. (n = 8), and Lm (n = 28) strains. The Lm isolates contained representatives of the six serogroups: 2A, 2B, 2C, 4B, NT, and 4bV/IVb-v1, with five strains for each serogroup except 4bV/IVb-v1 (n = 3). The results generated by 16 laboratories showed high sensitivity, specificity, and accuracy, generally ≥97%, for both the genus-species and serogrouping qPCRs. Results from one laboratory lowered the sensitivity of the non-Listeria group to 93%. These results indicated the method was highly reliable. However, only the previously evaluated serogroups were tested within the MLV panel, though there is the potential for other serogroup results. Sequence Read Archive (SRA) files for Lm isolates were evaluated to determine the frequency of other potential serogroup profiles. This effort identified a low percentage of isolates with atypical qPCR serogroups (0.30%) that are consistent with Lm and were generally associated with lineage II and the natural environment. In summary, the results indicate that the proposed qPCR method is reliable and has a high degree of sensitivity, accuracy, and specificity, while also decreasing hands-on analysis time and increasing throughput of the analysis.

Structural basis for the unique molecular properties of broad-range phospholipase C from Listeria monocytogenes.

Listeriosis is one of the most serious foodborne diseases caused by the intracellular bacterium Listeria monocytogenes. Its two major virulence factors, broad-range phospholipase C (LmPC-PLC) and the pore-forming toxin listeriolysin O (LLO), enable the bacterium to spread in the host by destroying cell membranes. Here, we determine the crystal structure of LmPC-PLC and complement it with the functional analysis of this enzyme. This reveals that LmPC-PLC has evolved several structural features to regulate its activity, including the invariant position of the N-terminal tryptophan (W1), the structurally plastic active site, Zn2+-dependent activity, and the tendency to form oligomers with impaired enzymatic activity. We demonstrate that the enzymatic activity of LmPC-PLC can be specifically inhibited by its propeptide added in trans. Furthermore, we show that the phospholipase activity of LmPC-PLC facilitates the pore-forming activity of LLO and affects the morphology of LLO oligomerization on lipid membranes, revealing the multifaceted synergy of the two virulence factors.

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.