Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis.

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 µg/mL) and vancomycin (MIC = 2 µg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

Substance P enhances lactic acid and tyramine production in Enterococcus faecalis V583 and promotes its cytotoxic effect on intestinal Caco-2/TC7 cells.

BACKGROUND: Enterococcus faecalis, generally considered as a saprophytic bowel commensal, has recently emerged as an important nosocomial pathogen causing severe urinary tract infections, surgical wound infections, bacteremia, and bacterial endocarditis. This bacterium is capable of forming biofilms on various surfaces and its high level of antibiotic resistance contributes to its pathogenicity. The aim of this study was to evaluate the effect on E. faecalis, of Substance P (SP), an antimicrobial peptide that is produced in the gut and skin. RESULTS: We found that SP did not have antibacterial activity against E. faecalis V583 (MIC >1000 µg/ml). Conversely, SP stimulated aggregation, hydrophobicity, lactic acid and tyramine production in this bacterium. The cytotoxicity and bacterial translocation were also accelerated when E. faecalis V583 were pretreated with SP before infection of intestinal Caco-2/TC7 cells. CONCLUSION: SP can modulate the physiology of E. faecalis. Extensive studies are now needed to screen within the human microbiota which bacteria are responsive to host molecules, and to identify their sensors.

Interaction with enzyme IIBMpo (EIIBMpo) and phosphorylation by phosphorylated EIIBMpo exert antagonistic effects on the transcriptional activator ManR of Listeria monocytogenes.

UNLABELLED: Listeriae take up glucose and mannose predominantly through a mannose class phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS(Man)), whose three components are encoded by the manLMN genes. The expression of these genes is controlled by ManR, a LevR-type transcription activator containing two PTS regulation domains (PRDs) and two PTS-like domains (enzyme IIA(Man) [EIIA(Man)]- and EIIB(Gat)-like). We demonstrate here that in Listeria monocytogenes, ManR is activated via the phosphorylation of His585 in the EIIA(Man)-like domain by the general PTS components enzyme I and HPr. We also show that ManR is regulated by the PTS(Mpo) and that EIIB(Mpo) plays a dual role in ManR regulation. First, yeast two-hybrid experiments revealed that unphosphorylated EIIB(Mpo) interacts with the two C-terminal domains of ManR (EIIB(Gat)-like and PRD2) and that this interaction is required for ManR activity. Second, in the absence of glucose/mannose, phosphorylated EIIB(Mpo) (P∼EIIB(Mpo)) inhibits ManR activity by phosphorylating His871 in PRD2. The presence of glucose/mannose causes the dephosphorylation of P∼EIIB(Mpo) and P∼PRD2 of ManR, which together lead to the induction of the manLMN operon. Complementation of a ΔmanR mutant with various manR alleles confirmed the antagonistic effects of PTS-catalyzed phosphorylation at the two different histidine residues of ManR. Deletion of manR prevented not only the expression of the manLMN operon but also glucose-mediated repression of virulence gene expression; however, repression by other carbohydrates was unaffected. Interestingly, the expression of manLMN in Listeria innocua was reported to require not only ManR but also the Crp-like transcription activator Lin0142. Unlike Lin0142, the L. monocytogenes homologue, Lmo0095, is not required for manLMN expression; its absence rather stimulates man expression. IMPORTANCE: Listeria monocytogenes is a human pathogen causing the foodborne disease listeriosis. The expression of most virulence genes is controlled by the transcription activator PrfA. Its activity is strongly repressed by carbohydrates, including glucose, which is transported into L. monocytogenes mainly via a mannose/glucose-specific phosphotransferase system (PTS(Man)). Expression of the man operon is regulated by the transcription activator ManR, the activity of which is controlled by a second, low-efficiency PTS of the mannose family, which functions as glucose sensor. Here we demonstrate that the EIIB(Mpo) component plays a dual role in ManR regulation: it inactivates ManR by phosphorylating its His871 residue and stimulates ManR by interacting with its two C-terminal domains.

The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.

Transcription regulators controlled by interaction with enzyme IIB components of the phosphoenolpyruvate: sugar phosphotransferase system.

Numerous bacteria possess transcription activators and antiterminators composed of regulatory domains phosphorylated by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). These domains, called PTS regulation domains (PRDs), usually contain two conserved histidines as potential phosphorylation sites. While antiterminators possess two PRDs with four phosphorylation sites, transcription activators contain two PRDs plus two regulatory domains resembling PTS components (EIIA and EIIB). The activity of these transcription regulators is controlled by up to five phosphorylations catalyzed by PTS proteins. Phosphorylation by the general PTS components EI and HPr is usually essential for the activity of PRD-containing transcription regulators, whereas phosphorylation by the sugar-specific components EIIA or EIIB lowers their activity. For a specific regulator, for example the Bacillus subtilis mtl operon activator MtlR, the functional phosphorylation sites can be different in other bacteria and consequently the detailed mode of regulation varies. Some of these transcription regulators are also controlled by an interaction with a sugar-specific EIIB PTS component. The EIIBs are frequently fused to the membrane-spanning EIIC and EIIB-mediated membrane sequestration is sometimes crucial for the control of a transcription regulator. This is also true for the Escherichia coli repressor Mlc, which does not contain a PRD but nevertheless interacts with the EIIB domain of the glucose-specific PTS. In addition, some PRD-containing transcription activators interact with a distinct EIIB protein located in the cytoplasm. The phosphorylation state of the EIIB components, which changes in response to the presence or absence of the corresponding carbon source, affects their interaction with transcription regulators. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Novel listerial glycerol dehydrogenase- and phosphoenolpyruvate-dependent dihydroxyacetone kinase system connected to the pentose phosphate pathway.

Several bacteria use glycerol dehydrogenase to transform glycerol into dihydroxyacetone (Dha). Dha is subsequently converted into Dha phosphate (Dha-P) by an ATP- or phosphoenolpyruvate (PEP)-dependent Dha kinase. Listeria innocua possesses two potential PEP-dependent Dha kinases. One is encoded by 3 of the 11 genes forming the glycerol (gol) operon. This operon also contains golD (lin0362), which codes for a new type of Dha-forming NAD(+)-dependent glycerol dehydrogenase. The subsequent metabolism of Dha requires its phosphorylation via the PEP:sugar phosphotransferase system components enzyme I, HPr, and EIIA(Dha)-2 (Lin0369). P∼EIIA(Dha)-2 transfers its phosphoryl group to DhaL-2, which phosphorylates Dha bound to DhaK-2. The resulting Dha-P is probably metabolized mainly via the pentose phosphate pathway, because two genes of the gol operon encode proteins resembling transketolases and transaldolases. In addition, purified Lin0363 and Lin0364 exhibit ribose-5-P isomerase (RipB) and triosephosphate isomerase activities, respectively. The latter enzyme converts part of the Dha-P into glyceraldehyde-3-P, which, together with Dha-P, is metabolized via gluconeogenesis to form fructose-6-P. Together with another glyceraldehyde-3-P molecule, the transketolase transforms fructose-6-P into intermediates of the pentose phosphate pathway. The gol operon is preceded by golR, transcribed in the opposite orientation and encoding a DeoR-type repressor. Its inactivation causes the constitutive but glucose-repressible expression of the entire gol operon, including the last gene, encoding a pediocin immunity-like (PedB-like) protein. Its elevated level of synthesis in the golR mutant causes slightly increased immunity against pediocin PA-1 compared to the wild-type strain or a pedB-like deletion mutant.