Role of ethanolamine utilization and bacterial microcompartment formation in Listeria monocytogenes intracellular infection.

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.

Role of Ethanolamine Utilization and Bacterial Microcompartment Formation in Listeria monocytogenes Intracellular Infection.

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.

Membrane-localized expression, production and assembly of Vibrio parahaemolyticus T3SS2 provides evidence for transertion.

It has been proposed that bacterial membrane proteins may be synthesized and inserted into the membrane by a process known as transertion, which involves membrane association of their encoding genes, followed by coupled transcription, translation and membrane insertion. Here, we provide evidence supporting that the pathogen Vibrio parahaemolyticus uses transertion to assemble its type III secretion system (T3SS2), to inject virulence factors into host cells. We propose a two-step transertion process where the membrane-bound co-component receptor (VtrA/VtrC) is first activated by bile acids, leading to membrane association and expression of its target gene, vtrB, located in the T3SS2 pathogenicity island. VtrB, the transmembrane transcriptional activator of T3SS2, then induces the localized expression and membrane assembly of the T3SS2 structural components and its effectors. We hypothesize that the proposed transertion process may be used by other enteric bacteria for efficient assembly of membrane-bound molecular complexes in response to extracellular signals.

Enzymatic Specificity of Conserved Rho GTPase Deamidases Promotes Invasion of Vibrio parahaemolyticus at the Expense of Infection.

Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens. IMPORTANCE Many species of intracellular bacterial pathogens target host small Rho GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Escherichia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor 1 (CNF1) drives UPEC's internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed that the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. Moreover, they pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape and, more broadly, the regulation of successful pathogenesis.

Cardiac Microlesions Form During Severe Bacteremic Enterococcus faecalis Infection.

Enterococcus  faecalis is a significant cause of hospital-acquired bacteremia. Herein, the discovery is reported that cardiac microlesions form during severe bacteremic E. faecalis infection in mice. The cardiac microlesions were identical in appearance to those formed by Streptococcus pneumoniae during invasive pneumococcal disease. However, E. faecalis does not encode the virulence determinants implicated in pneumococcal microlesion formation. Rather, disulfide bond forming protein A (DsbA) was found to be required for E. faecalis virulence in a Caenorhabditis elegans model and was necessary for efficient cardiac microlesion formation. Furthermore, E. faecalis promoted cardiomyocyte apoptotic and necroptotic cell death at sites of microlesion formation. Additionally, loss of DsbA caused an increase in proinflammatory cytokines, unlike the wild-type strain, which suppressed the immune response. In conclusion, we establish that E. faecalis is capable of forming cardiac microlesions and identify features of both the bacterium and the host response that are mechanistically involved.

Ethanolamine Utilization and Bacterial Microcompartment Formation Are Subject to Carbon Catabolite Repression.

Ethanolamine (EA) is a compound prevalent in the gastrointestinal (GI) tract that can be used as a carbon, nitrogen, and/or energy source. Enterococcus faecalis, a GI commensal and opportunistic pathogen, contains approximately 20 ethanolamine utilization (eut) genes encoding the necessary regulatory, enzymatic, and structural proteins for this process. Here, using a chemically defined medium, two regulatory factors that affect EA utilization were examined. First, the functional consequences of loss of the small RNA (sRNA) EutX on the efficacy of EA utilization were investigated. One effect observed, as loss of this negative regulator causes an increase in eut gene expression, was a concomitant increase in the number of catabolic bacterial microcompartments (BMCs) formed. However, despite this increase, the growth of the strain was repressed, suggesting that the overall efficacy of EA utilization was negatively affected. Second, utilizing a deletion mutant and a complement, carbon catabolite control protein A (CcpA) was shown to be responsible for the repression of EA utilization in the presence of glucose. A predicted cre site in one of the three EA-inducible promoters, PeutS, was identified as the target of CcpA. However, CcpA was shown to affect the activation of all the promoters indirectly through the two-component system EutV and EutW, whose genes are under the control of the PeutS promoter. Moreover, a bioinformatics analysis of bacteria predicted to contain CcpA and cre sites revealed that a preponderance of BMC-containing operons are likely regulated by carbon catabolite repression (CCR).IMPORTANCE Ethanolamine (EA) is a compound commonly found in the gastrointestinal (GI) tract that can affect the behavior of human pathogens that can sense and utilize it, such as Enterococcus faecalis and Salmonella Therefore, it is important to understand how the genes that govern EA utilization are regulated. In this work, we investigated two regulatory factors that control this process. One factor, a small RNA (sRNA), is shown to be important for generating the right levels of gene expression for maximum efficiency. The second factor, a transcriptional repressor, is important for preventing expression when other preferred sources of energy are available. Furthermore, a global bioinformatics analysis revealed that this second mechanism of transcriptional regulation likely operates on similar genes in related bacteria.

Heme peroxidase HPX-2 protects Caenorhabditis elegans from pathogens.

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx.

Loss of Ethanolamine Utilization in Enterococcus faecalis Increases Gastrointestinal Tract Colonization.

Enterococcus faecalis is paradoxically a dangerous nosocomial pathogen and a normal constituent of the human gut microbiome, an environment rich in ethanolamine. E. faecalis carries the eut (ethanolamine utilization) genes, which enable the catabolism of ethanolamine (EA) as a valuable source of carbon and/or nitrogen. EA catabolism was previously shown to contribute to the colonization and growth of enteric pathogens, such as Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli (EHEC), in the gut environment. We tested the ability of eut mutants of E. faecalis to colonize the gut using a murine model of gastrointestinal (GI) tract competition and report the surprising observation that these mutants outcompete the wild-type strain.IMPORTANCE Some bacteria that are normal, harmless colonizers of the human body can cause disease in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote or negate these organisms' ability to colonize. Previously, ethanolamine, found in high concentrations in the GI tract, was shown to promote the colonization and growth of bacteria associated with food poisoning. Here, we report the surprising, opposite effect of ethanolamine utilization on the commensal colonizer E. faecalis, namely, that loss of this metabolic capacity made it a better colonizer.

Ethanolamine Utilization in Bacteria.

Ethanolamine (EA) is a valuable source of carbon and/or nitrogen for bacteria capable of its catabolism. Because it is derived from the membrane phospholipid phosphatidylethanolamine, it is particularly prevalent in the gastrointestinal tract, which is membrane rich due to turnover of the intestinal epithelium and the resident microbiota. Intriguingly, many gut pathogens carry the eut (ethanolamine utilization) genes. EA utilization has been studied for about 50 years, with most of the early work occurring in just a couple of species of Enterobacteriaceae Once the metabolic pathways and enzymes were characterized by biochemical approaches, genetic screens were used to map the various activities to the eut genes. With the rise of genomics, the diversity of bacteria containing the eut genes and surprising differences in eut gene content were recognized. Some species contain nearly 20 genes and encode many accessory proteins, while others contain only the core catabolic enzyme. Moreover, the eut genes are regulated by very different mechanisms, depending on the organism and the eut regulator encoded. In the last several years, exciting progress has been made in elucidating the complex regulatory mechanisms that govern eut gene expression. Furthermore, a new appreciation for how EA contributes to infection and colonization in the host is emerging. In addition to providing an overview of EA-related biology, this minireview will give special attention to these recent advances.

Genetic Dissection of DivIVA Functions in Listeria monocytogenes.

DivIVA is a membrane binding protein that clusters at curved membrane regions, such as the cell poles and the membrane invaginations occurring during cell division. DivIVA proteins recruit many other proteins to these subcellular sites through direct protein-protein interactions. DivIVA-dependent functions are typically associated with cell growth and division, even though species-specific differences in the spectrum of DivIVA functions and their causative interaction partners exist. DivIVA from the Gram-positive human pathogen Listeria monocytogenes has at least three different functions. In this bacterium, DivIVA is required for precise positioning of the septum at midcell, it contributes to the secretion of autolysins required for the breakdown of peptidoglycan at the septum after the completion of cell division, and it is essential for flagellar motility. While the DivIVA interaction partners for control of division site selection are well established, the proteins connecting DivIVA with autolysin secretion or swarming motility are completely unknown. We set out to identify divIVA alleles in which these three DivIVA functions could be separated, since the question of the degree to which the three functions of L. monocytogenes DivIVA are interlinked could not be answered before. Here, we identify such alleles, and our results show that division site selection, autolysin secretion, and swarming represent three discrete pathways that are independently influenced by DivIVA. These findings provide the required basis for the identification of DivIVA interaction partners controlling autolysin secretion and swarming in the future.IMPORTANCE DivIVA of the pathogenic bacterium Listeria monocytogenes is a central scaffold protein that influences at least three different cellular processes, namely, cell division, protein secretion, and bacterial motility. How DivIVA coordinates these rather unrelated processes is not known. We here identify variants of L. monocytogenes DivIVA, in which these functions are separated from each other. These results have important implications for the models explaining how DivIVA interacts with other proteins.

A function of DivIVA in Listeria monocytogenes division site selection.

The cell division protein DivIVA influences protein transport via the accessory SecA2 secretion route in Listeria monocytogenes. In contrast, DivIVA from the closely related bacterium Bacillus subtilis contributes to division site selection via the MinCDJ system. However, no classical min phenotype, i.e. filamentation and minicell production was observed with a listerial ΔdivIVA mutant. This has prompted the speculation that division site selection is DivIVA-independent in L. monocytogenes. We addressed this question with genetic, cytological and bacterial two-hybrid experiments and the data obtained correct this view. DivIVA not only binds to MinJ but also directly interacts with MinD. Experiments with fluorescently tagged proteins showed that localization of MinC and MinD was clearly DivIVA-dependent, whereas localization of MinJ was not. An impact of DivIVA on cell division was confirmed by careful comparisons of cell size distributions of divIVA and secA2 mutants. Gene deletion studies and epistasis experiments consistently reinforced these findings, and also revealed that MinJ must have a DivIVA-independent function. The frequency of minicell formation is low in L. monocytogenes min mutants. However, since listerial minicells might be useful as carriers for the introduction of therapeutic compounds into eukaryotic cells, we present a strategy how minicell frequency can be increased.

Protein-protein interaction domains of Bacillus subtilis DivIVA.

DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent protein-tagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.