Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner.

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

A sensor histidine kinase from a plant-endosymbiont bacterium restores the virulence of a mammalian intracellular pathogen.

Alphaproteobacteria include organisms living in close association with plants or animals. This interaction relies partly on orthologous two-component regulatory systems (TCS), with sensor and regulator proteins modulating the expression of conserved genes related to symbiosis/virulence. We assessed the ability of the exoS+Sm gene, encoding a sensor protein from the plant endosymbiont Sinorhizobium meliloti to substitute its orthologous bvrS in the related animal/human pathogen Brucella abortus. ExoS phosphorylated the B. abortus regulator BvrR in vitro and in cultured bacteria, showing conserved biological function. Production of ExoS in a B. abortus bvrS mutant reestablished replication in host cells and the capacity to infect mice. Bacterial outer membrane properties, the production of the type IV secretion system VirB, and its transcriptional regulators VjbR and BvrR were restored as compared to parental B. abortus. These results indicate that conserved traits of orthologous TCS from bacteria living in and sensing different environments are sufficient to achieve phenotypic plasticity and support bacterial survival. The knowledge of bacterial genetic networks regulating host interactions allows for an understanding of the subtle differences between symbiosis and parasitism. Rewiring these networks could provide new alternatives to control and prevent bacterial infection.

Brucella abortus Senses the Intracellular Environment through the BvrR/BvrS Two-Component System, Which Allows B. abortus To Adapt to Its Replicative Niche.

Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of Alphaproteobacteria.

Phenotypes controlled by the Brucella abortus two component system BvrR/BvrS are differentially impacted by BvrR phosphorylation.

Brucella abortus is a zoonotic pathogen whose virulence depends on its ability to survive intracellularly at the endoplasmic reticulum derived compartment. The two-component system BvrR/BvrS (BvrRS) is essential for intracellular survival due to the transcriptional control of the type IV secretion system VirB and its transcriptional regulator VjbR. It is a master regulator of several traits including membrane homeostasis by controlling gene expression of membrane components, such as Omp25. BvrR phosphorylation is related to DNA binding at target regions, thereby repressing or activating gene transcription. To understand the role of BvrR phosphorylation we generated dominant positive and negative versions of this response regulator, mimicking phosphorylated and non-phosphorylated BvrR states and, in addition to the wild-type version, these variants were introduced in a BvrR negative background. We then characterized BvrRS-controlled phenotypes and assessed the expression of proteins regulated by the system. We found two regulatory patterns exerted by BvrR. The first pattern was represented by resistance to polymyxin and expression of Omp25 (membrane conformation) which were restored to normal levels by the dominant positive and the wild-type version, but not the dominant negative BvrR. The second pattern was represented by intracellular survival and expression of VjbR and VirB (virulence) which were, again, complemented by the wild-type and the dominant positive variants of BvrR but were also significantly restored by complementation with the dominant negative BvrR. These results indicate a differential transcriptional response of the genes controlled to the phosphorylation status of BvrR and suggest that unphosphorylated BvrR binds and impacts the expression of a subset of genes. We confirmed this hypothesis by showing that the dominant negative BvrR did not interact with the omp25 promoter whereas it could interact with vjbR promoter. Furthermore, a global transcriptional analysis revealed that a subset of genes responds to the presence of the dominant negative BvrR. Thus, BvrR possesses diverse strategies to exert transcriptional control on the genes it regulates and, consequently, impacting on the phenotypes controlled by this response regulator.

Wolf in Sheep's Clothing: Clostridioides difficile Biofilm as a Reservoir for Recurrent Infections.

The microbiota inhabiting the intestinal tract provide several critical functions to its host. Microorganisms found at the mucosal layer form organized three-dimensional structures which are considered to be biofilms. Their development and functions are influenced by host factors, host-microbe interactions, and microbe-microbe interactions. These structures can dictate the health of their host by strengthening the natural defenses of the gut epithelium or cause disease by exacerbating underlying conditions. Biofilm communities can also block the establishment of pathogens and prevent infectious diseases. Although these biofilms are important for colonization resistance, new data provide evidence that gut biofilms can act as a reservoir for pathogens such as Clostridioides difficile. In this review, we will look at the biofilms of the intestinal tract, their contribution to health and disease, and the factors influencing their formation. We will then focus on the factors contributing to biofilm formation in C. difficile, how these biofilms are formed, and their properties. In the last section, we will look at how the gut microbiota and the gut biofilm influence C. difficile biofilm formation, persistence, and transmission.

Virulence potential of Listeria monocytogenes strains recovered from pigs in Spain.

BACKGROUND: Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis, an infectious disease in animals and people, with pigs acting as asymptomatic reservoirs. In August 2019 an outbreak associated with the consumption of pork meat caused 222 human cases of listeriosis in Spain. Determining the diversity as well as the virulence potential of strains from pigs is important to public health. METHODS: The behaviour of 23 L monocytogenes strains recovered from pig tonsils, meat and skin was compared by studying (1) internalin A, internalin B, listeriolysin O, actin assembly-inducing protein and PrfA expression levels, and (2) their invasion and intracellular growth in eukaryotic cells. RESULTS: Marked differences were found in the expression of the selected virulence factors and the invasion and intracellular replication phenotypes of L monocytogenes strains. Strains obtained from meat samples and belonging to serotype 1/2a did not have internalin A anchored to the peptidoglycan. Some strains expressed higher levels of the studied virulence factors and invaded and replicated intracellularly more efficiently than an epidemic L monocytogenes reference strain (F2365). CONCLUSION: This study demonstrates the presence of virulent L monocytogenes strains with virulent potential in pigs, with valuable implications in veterinary medicine and food safety.

Listeriolysin S: A bacteriocin from epidemic Listeria monocytogenes strains that targets the gut microbiota.

Listeria monocytogenes is a Gram-positive food-borne pathogen that in humans may traverse the intestinal, placental and blood/brain barriers, causing gastroenteritis, abortions and meningitis. Crossing of these barriers is dependent on the bacterial ability to enter host cells, and several L. monocytogenes surface and secreted virulence factors are known to facilitate entry and the intracellular lifecycle. The study of L. monocytogenes strains associated to human listeriosis epidemics has revealed the presence of novel virulence factors. One such factor is Listeriolysin S, a thiazole/oxazole modified microcin that displays bactericidal activity and modifies the host microbiota during infection. Our recent results therefore highlight the interaction of L. monocytogenes with gut microbes as a crucial step in epidemic listeriosis. In this article, we will discuss novel implications for this family of toxins in the pathogenesis of diverse medically relevant microorganisms.

Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo.

Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections.IMPORTANCE The most severe human listeriosis outbreaks are caused by L. monocytogenes strains harboring listeriolysin S (LLS), previously described as a cytotoxin that plays a critical role in host inner tissue infection. Cytotoxic activities have been proposed as a general mode of action for streptolysin S (SLS)-like toxins, including clostridiolysin S and LLS. We now challenge this dogma by demonstrating that LLS does not contribute to virulence in vivo once the intestinal barrier has been crossed. Importantly, we show that intravenous L. monocytogenes inoculation leads to bacterial translocation to the gastrointestinal system, where LLS is specifically expressed, targeting the host gut microbiota. Our study highlights the heterogeneous modes of action of SLS-like toxins, and we demonstrate for the first time a further level of complexity for SLS-like biosynthetic clusters as we reveal that the putative posttranslational modification enzyme LlsB is actually required for inner organ colonization, independently of the LLS activity.