Probiotic bacteria reduce the intestinal colonization of pathogens. Yet, their use in preventing fatal infection caused by foodborne Listeria monocytogenes (Lm), is inconsistent. Here, we bioengineered Lactobacillusprobiotics (BLP) to express the Listeria adhesion protein (LAP) from a non-pathogenic Listeria (L. innocua) and a pathogenic Listeria (Lm) on the surface of Lactobacillus casei. The BLP strains colonize the intestine, reduce Lm mucosal colonization and systemic dissemination, and protect mice from lethal infection. The BLP competitively excludes Lm by occupying the surface presented LAP receptor, heat shock protein 60 and ameliorates the Lm-induced intestinal barrier dysfunction by blocking the nuclear factor-κB and myosin light chain kinase-mediated redistribution of the major epithelial junctional proteins. Additionally, the BLP increases intestinal immunomodulatory functions by recruiting FOXP3⁺T cells, CD11c⁺ dendritic cells and natural killer cells. Engineering a probiotic strain with an adhesion protein from a non-pathogenic bacterium provides a new paradigm to exclude pathogens and amplify their inherent health benefits.

The emergence of bacterial resistance to therapeutic antibiotics limits options for treatment of common microbial diseases. Subinhibitory antibiotics dosing, often aid in the emergence of resistance, but its impact on pathogen’s physiology and pathogenesis is not well understood. Here we investigated the effect of tunicamycin, a cell wall teichoic acid (WTA) biosynthesis inhibiting antibiotic at the subinhibitory dosage on Staphylococcus aureus and Listeria monocytogenes physiology, antibiotic cross-resistance, biofilm-formation, and virulence. Minimum inhibitory concentration (MIC) of tunicamycin to S. aureus and L. monocytogenes was 20–40 μg/ml and 2.5–5 μg/ml, respectively, and the subinhibitory concentration was 2.5–5 μg/ml and 0.31–0.62 μg/ml, respectively. Tunicamycin pre-exposure reduced cellular WTA levels by 18–20% and affected bacterial cell wall ultrastructure, cell membrane permeability, morphology, laser-induced colony scatter signature, and bacterial ability to form biofilms. It also induced a moderate level of cross-resistance to tetracycline, ampicillin, erythromycin, and meropenem for S. aureus, and ampicillin, erythromycin, vancomycin, and meropenem for L. monocytogenes. Pre-treatment of bacterial cells with subinhibitory concentrations of tunicamycin also significantly reduced bacterial adhesion to and invasion into an enterocyte-like Caco-2 cell line, which is supported by reduced expression of key virulence factors, Internalin B (InlB) and Listeria adhesion protein (LAP) in L. monocytogenes, and a S. aureus surface protein A (SasA) in S. aureus. Tunicamycin-treated bacteria or the bacterial WTA preparation suppressed NF-κB and inflammatory cytokine production (TNFα, and IL-6) from murine macrophage cell line (RAW 264.7) indicating the reduced WTA level possibly attenuates an inflammatory response. These results suggest that at the subinhibitory dosage, tunicamycin-mediated inhibition of WTA biosynthesis interferes with cell wall structure, pathogens infectivity and inflammatory response, and ability to form biofilms but promotes the development of antibiotic cross-resistance.

Environmental cues promote microbial biofilm formation and physiological and genetic heterogeneity. In food production facilities, biofilms produced by pathogens are a major source for food contamination; however, the pathogenesis of biofilm-isolated sessile cells is not well understood. We investigated the pathogenesis of sessile Listeria monocytogenes (Lm) using cell culture and mouse models. Lm sessile cells express reduced levels of the lap, inlA, hly, prfA, and sigB and show reduced adhesion, invasion, translocation, and cytotoxicity in the cell culture model than the planktonic cells. Oral challenge of C57BL/6 mice with food, clinical, or murinized-InlA (InlA^m) strains reveals that at 12 and 24 h post-infection (hpi), Lm burdens are lower in tissues of mice infected with sessile cells than those infected with planktonic cells. However, these differences are negligible at 48 hpi. Besides, the expressions of inlA and lap mRNA in sessile Lm from intestinal content are about 6.0- and 280-fold higher than the sessle inoculum, respectively, suggesting sessile Lm can still upregulate virulence genes shortly after ingestion (12 h). Similarly, exposure to simulated gastric fluid (SGF, pH 3) and intestinal fluid (SIF, pH 7) for 13 h shows equal reduction in sessile and planktonic cell counts, but induces LAP and InlA expression and pathogenic phenotypes. Our data show that the virulence of biofilm-isolated Lm is temporarily attenuated and can be upregulated in mice during the early stage (12–24 hpi) but fully restored at a later stage (48 hpi) of infection. Our study further demonstrates that in vitro cell culture assay is unreliable; therefore, an animal model is essential for studying the pathogenesis of biofilm-isolated bacteria.

Within mammalian cells, diverse endocytic mechanisms, including phagocytosis, pinocytosis, and receptor-mediated endocytosis, serve as gateways exploited by many bacterial pathogens and toxins. Among these, caveolae-mediated endocytosis is characterized by lipid-rich caveolae and dimeric caveolin proteins. Caveolae are specialized microdomains on cell surfaces that impact cell signaling. Caveolin proteins facilitate the creation of caveolae and have three members in vertebrates: caveolin-1, caveolin-2, and caveolin-3. Many bacterial pathogens hijack caveolin machinery to invade host cells. For example, the Gram-positive facultative model intracellular bacterial pathogen Listeria monocytogenes exploits caveolin-mediated endocytosis for efficient cellular entry, translocation across the intestinal barrier, and cell–cell spread. Caveolin facilitates the internalization of group A streptococci by promoting the formation of invaginations in the plasma membrane and avoiding fusion with lysosomes, thereby aiding intracellular survival. Caveolin plays a crucial role in internalizing and modulation of host immune responses by Gram-negative bacterial pathogens, such as Escherichia coli K1, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium. Here, we summarize how bacterial pathogens manipulate the host’s caveolin system to facilitate bacterial entry and movement within and between host cells, to support intracellular survival, to evade immune responses, and to trigger inflammation. This knowledge enhances the intervention of new therapeutic targets against caveolin in microbial invasion and immune evasion processes.