Mannose Phosphotransferase Research Articles

Metformin reduced the alkaline resistance of Enterococcus faecalis against calcium hydroxide via Man-PTS EII: in vitro and in vivo studies.

The mannose phosphotransferase system (Man-PTS) plays crucial roles in the adaptive metabolic activity of Enterococcus faecalis (E. faecalis) in adverse environments. The aim of this study was to evaluate the role of Man-PTS in the alkaline resistance of E. faecalis against calcium hydroxide (CH) and the effect of metformin (Met) on the alkaline resistance of E. faecalis to CH. The regulatory role of Man-PTS EII in the alkaline resistance of E. faecalis was firstly investigated using a wild-type highly alkaline-resistant E. faecalis XS 003, standard ATCC 29212 and Man-PTS EIID gene deficient (△mptD) and overexpressing (+mptD) strains of E. faecalis. RNA sequencing of Met-treated E. faecalis was performed to further validate the effect of Met on Man-PTS. The effect of Met on CH resistance of E. faecalis was verified by evaluating the survival, membrane potential and permeability, intracellular pH and ATP, and the expression of Man-PTS EII and membrane transporter-related genes of E. faecalis. The effect of Met on the ability of CH to remove E. faecalis biofilm on the dentin surface was also tested. The in vivo therapeutic effect of Met plus CH (CHM) was further investigated in a rat apical periodontitis model induced by E. faecalis XS 003. Man-PTS EII significantly promoted the survival ability of E. faecalis in CH and enhanced its resistance to CH. The inhibition of Man-PTS EII by Met resulted in reduced alkaline resistance of E. faecalis in the presence of CH, while also enhancing the antimicrobial properties of CH against E. faecalis biofilm on dentin. Additionally, Met plus CH showed the synergistically promoted intra-canal E. faecalis infection control and healing of periapical lesion in rats. Met could significantly reduce the alkaline resistance of E. faecalis against CH through the modulation of Man-PTS EII, and improved the antibacterial effect of CH against E. faecalis infection both in vitro and in vivo. Met could significantly enhance the ability of CH to control E. faecalis infection through reducing the alkaline resistance of E. faecalis.

Subclass IId bacteriocins targeting mannose phosphotransferase system-Structural diversity and implications for receptor interaction and antimicrobial activity.

The bacterial mannose phosphotransferase system (Man-PTS) mediates uptake of selected monosaccharides. Simultaneously, it is a receptor for diverse bacteriocins such as subclass IIa pediocin-like bacteriocins and some subclass IId ones (garvicins ABCQ, lactococcins ABZ, BacSJ, ubericin K, and angicin). So far, no attempt has been made to categorize this ever-expanding group of bacteriocins. Here, we identified Man-PTS as a receptor for a number of previously uncharacterized bacteriocins, and demonstrated that they all belong to a large family of Man-PTS-binding nonpediocin-like peptides, providing new insights into their structure and function. Based on amino acid sequence similarities between members of this family, we propose their classification into five groups. This classification conveniently distinguishes bacteriocins with specific structures and properties regarding their spectrum of antimicrobial activity and pattern of interaction with Man-PTS. With respect to the latter, we indicate individual amino acid residues or regions of Man-PTS and the bacteriocin responsible for their interaction. In Man-PTS, these residues localize to the exterior of the transport complex, specifically the extracellular loop of the so-called Vmotif domain-containing regions γ and/or γ+, and to the interior of the transport complex, specifically the interface between the Core and Vmotif domains. Finally, we propose that while the bacteriocins from separate groups display specific binding patterns to Man-PTS, the general mechanism of their interaction with the receptor is universal despite significant differences in their predicted structures, i.e. after initial docking on the bacterial cell through an interaction with the Man-PTS regions γ and/or γ+, they pull away its Core and Vmotif from one another to form a pore across the membrane.

Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality.

The naturally attached phyllosphere microbiota play a crucial role in plant-derived fermentation, but the structure and function of phyllosphere endophytes remain largely unidentified. Here, we reveal the diversity, specificity, and functionality of phyllosphere endophytes in alfalfa (Medicago sativa L.) through combining typical microbial culture, high-throughput sequencing, and genomic comparative analysis. In comparison to phyllosphere bacteria (PB), the fermentation of alfalfa solely with endophytes (EN) enhances the fermentation characteristics, primarily due to the dominance of specific lactic acid bacteria (LAB) such as Lactiplantibacillus, Weissella, and Pediococcus. The inoculant with selected endophytic LAB strains also enhances the fermentation quality compared to epiphytic LAB treatment. Especially, one key endophytic LAB named Pediococcus pentosaceus EN5 shows enrichment of genes related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes and higher utilization of carbohydrates. Representing phyllosphere, endophytic LAB shows great potential of promoting ensiling and provides a novel direction for developing microbial inoculant.

The Klebsiella mannose phosphotransferase system promotes proliferation and the production of extracellular polymeric substances from mannose, facilitating adaptation to the host environment

ObjectivesKlebsiella spp., an opportunistic infectious organism, is commensal in the nasal and oral cavities of humans. Recently, it has been reported that oral Klebsiella spp. ectopically colonize the intestinal tract and induce the accumulation of intestinal Th1 cells. For oral bacteria to colonize the intestinal tract, they need to compete for nutrients with indigenous intestinal bacteria. Therefore, we focused on mannose, a mucus-derived sugar, and the mannose phosphotransferase system (PTS) (ManXYZ), a mechanism for mannose uptake, in terms of their effects on intestinal colonization and immune responses to Klebsiella spp. MethodsWe generated a Klebsiella manXYZ-deficient strain and investigated whether the utilization of intestinal mucus-derived sugars is associated with the growth. Furthermore, we examine the virulence of this organism in the mouse intestinal tract, especially the ability to colonize the host using competition assay. ResultsWe found that Klebsiella ManXYZ is a PTS that specifically takes up mannose and glucosamine. Through ManXYZ, mannose was used for bacterial growth and the upregulated production of extracellular polymeric substances. In vivo competition assays showed that mannose metabolism promoted intestinal colonization. However, ManXYZ was not involved in Th1 and Th17 induction in the intestinal tract. ConclusionThe fundamental roles of ManXYZ were to ensure that mannose, which is present in the host, is made available for bacterial growth, to promote the production of extracellular polymeric substances, thus facilitating bacterial adaptation to the host environment.

Lactococcus lactis mutants resistant to lactococcin A and garvicin Q reveal missense mutations in the sugar transport domain of the mannose phosphotransferase system.

Many bacteriocins target the sugar transporter mannose phosphotransferase system (man-PTS) to exert their antibacterial activity. The elucidation in recent years of the structure of man-PTS has facilitated our understanding of how bacteriocins might interact with the receptor and which domains of the transporter are involved in bacteriocin resistance. Here, we show that missense mutations in the sugar-binding domain of the man-PTS not only impede the uptake of sugars but also prevent the antibacterial activity of the bacteriocins lactococcin A and garvicin Q.

Structural Basis of the Mechanisms of Action and Immunity of Lactococcin A, a Class IId Bacteriocin.

Lactococcin A (LcnA), a class IId bacteriocin, induces membrane leakage and cell death by specifically binding to the membrane receptor-mannose phosphotransferase system (man-PTS), as is the case for pediocin-like (class IIa) bacteriocins. The cognate immunity protein of bacteriocins, which protects the producer cell from its own bacteriocin, recognizes and binds to the bacteriocin-man-PTS complex, consequently blocking membrane leakage. We previously deciphered the mode of action and immunity of class IIa bacteriocins. Here, we determined the structure of the ternary complex of LcnA, LciA (i.e., the immunity protein), and its receptor, i.e., the man-PTS of Lactococcus lactis (ll-man-PTS). An external loop on the membrane-located component IIC of ll-man-PTS was found to prevent specific binding of the N-terminal region of LcnA to the site recognized by pediocin-like bacteriocins. Thus, the N-terminal β-sheet region of LcnA recognized an adjacent site on the extracellular side of ll-man-PTS, with the LcnA C-terminal hydrophobic helix penetrating into the membrane. The cytoplasmic cleft formed within the man-PTS Core and Vmotif domains induced by embedded LcnA from the periplasmic side is adopted by the appropriate angle between helices H3 and H4 of the N terminus of LciA. The flexible C terminus of LciA then blocks membrane leakage. To summarize, our findings reveal the molecular mechanisms of action and immunity of LcnA and LciA, laying a foundation for further design of class IId bacteriocins. IMPORTANCE Class IId (lactococcin-like) bacteriocins and class IIa (pediocin-like) bacteriocins share a few similarities: (i) both induce membrane leakage and cell death by specifically binding the mannose phosphotransferase system (man-PTS) on their target cells, and (ii) cognate immunity proteins recognize and bind to the bacteriocin-man-PTS complex to block membrane leakage. However, class IId bacteriocins lack the "pediocin box" motif, which is typical of class IIa bacteriocins, and basically target only lactococcal cells; in contrast, class IIa bacteriocins target diverse bacterial cells, but not lactococcal cells. We previously solved the structure of class IIa bacteriocin-receptor-immunity ternary complex from Lactobacillus sakei. Here, we determined the structure of the ternary complex of class IId bacteriocin LcnA, its cognate immunity protein LciA, and its receptor, the man-PTS of Lactococcus lactis. By comparing the interactions between man-PTS and class IIa and class IId bacteriocins, this study affords some clues to better understand the specificity of bacteriocins targeting the mannose phosphotransferase system.

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment

BackgroundIFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown.MethodsBMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics.ResultsIFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), β-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG).ConclusionsThese results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Rcs phosphorelay affects the sensitivity of Escherichia coli to plantaricin BM-1 by regulating biofilm formation.

Introduction: Plantaricin BM-1 is a class IIa bacteriocin produced by Lactobacillus plantarum BM-1 that exerts significant antibacterial activity against many foodborne bacteria. Studies have shown that class IIa bacteriocins inhibit Gram-positive bacteria via the mannose phosphotransferase system; however, their mechanism of action against Gram-negative bacteria remains unknown. In this study, we explored the mechanism through which the Rcs phosphorelay affects the sensitivity of Escherichia coli K12 cells to plantaricin BM-1. Methods and Results: The minimum inhibitory concentrations of plantaricin BM-1 against E. coli K12, E. coli JW5917 (rcsC mutant), E. coli JW2204 (rcsD mutant), and E. coli JW2205 (rcsB mutant) were 1.25, 0.59, 1.31, and 1.22 mg/ml, respectively. Growth curves showed that E. coli JW5917 sensitivity to plantaricin BM-1 increased to the same level as that of E. coli K12 after complementation. Meanwhile, scanning electron microscopy and transmission electron microscopy revealed that, under the action of plantaricin BM-1, the appearance of E. coli JW5917 cells did not significantly differ from that of E. coli K12 cells; however, cell contents were significantly reduced and plasmolysis and shrinkage were observed at both ends. Crystal violet staining and laser scanning confocal microscopy showed that biofilm formation was significantly reduced after rcsC mutation, while proteomic analysis identified 382 upregulated and 260 downregulated proteins in E. coli JW5917. In particular, rcsC mutation was found to affect the expression of proteins related to biofilm formation, with growth curve assays showing that the deletion of these proteins increased E. coli sensitivity to plantaricin BM-1. Discussion: Consequently, we speculated that the Rcs phosphorelay may regulate the sensitivity of E. coli to plantaricin BM-1 by affecting biofilm formation. This finding of class IIa bacteriocin against Gram-negative bacteria mechanism provides new insights.

The bacteriocin Angicin interferes with bacterial membrane integrity through interaction with the mannose phosphotransferase system.

In a natural environment, bacteria are members of multispecies communities. To compete with rival species, bacteria produce antimicrobial peptides (AMPs), called bacteriocins. Bacteriocins are small, cationic, ribosomally synthesized peptides, which normally inhibit closely related species of the producing organism. Bacteriocin production is best studied in lactic bacteria (LAB). Streptococcus anginosus, belonging to LAB, produces the potent bacteriocin Angicin, which shows inhibitory activity against other streptococci, Listeria monocytogenes and vancomycin resistant Enterococcus faecium (VRE). Furthermore, Angicin shows a high resistance toward pH changes and heat, rendering it an interesting candidate for food preservation or clinical applications. The inhibitory activity of Angicin depends on the presence of a mannose phosphotransferase system (Man-PTS) in target cells, since L. monocytogenes harboring a deletion in an extracellular loop of this system is no longer sensitive to Angicin. Furthermore, we demonstrated by liposome leakage and pHluorin assays that Angicin destroys membrane integrity but shows only low cytotoxicity against human cell lines. In conclusion, we show that Angicin has a detrimental effect on the membrane of target organisms by using the Man-PTS as a receptor.

Targeted Transcriptomic Screen of Pneumococcal Genes Expressed during Murine and Human Infection.

The advent of pneumococcal conjugate vaccines led to the near disappearance of most of the included serotypes in high-income settings but also the rise of nonvaccine-type colonization and disease. Alternative strategies, using genetically conserved proteins as antigens, have been evaluated preclinically and clinically for years, so far unsuccessfully. One possible explanation for the failure of these efforts is that the choice of antigens may not have been sufficiently guided by an understanding of the gene expression pattern in the context of infection. Here, we present a targeted transcriptomic analysis of 160 pneumococcal genes encoding bacterial surface-exposed proteins in mouse models, human colonization, and human meningitis. We present the overlap of these different transcriptomic profiles. We identify two bacterial genes that are highly expressed in the context of mouse and human infection: SP_0282, an IID component of a mannose phosphotransferase system (PTS), and SP_1739, encoding RNase Y. We show that these two proteins can confer protection against pneumococcal nasopharyngeal colonization and intraperitoneal challenge in a murine model and generate opsonophagocytic antibodies. This study emphasizes and confirms the importance of studies of pneumococcal gene expression of bacterial surface proteins during human infection and colonization and may pave the way for the selection of a protein-based vaccine candidate.

Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46–47 and orf48–49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Structural Basis of Pore Formation in the Mannose Phosphotransferase System by Pediocin PA-1.

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal β-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

Ubericin K, a New Pore-Forming Bacteriocin Targeting mannose-PTS.

ABSTRACTBovine mastitis infection in dairy cattle is a significant economic burden for the dairy industry globally. To reduce the use of antibiotics in treatment of clinical mastitis, new alternative treatment options are needed. Antimicrobial peptides from bacteria, also known as bacteriocins, are potential alternatives for combating mastitis pathogens. In search of novel bacteriocins against mastitis pathogens, we screened samples of Norwegian bovine raw milk and found a Streptococcus uberis strain with potent antimicrobial activity toward Enterococcus, Streptococcus, Listeria, and Lactococcus. Whole-genome sequencing of the strain revealed a multibacteriocin gene cluster encoding one class IIb bacteriocin, two class IId bacteriocins, in addition to a three-component regulatory system and a dedicated ABC transporter. Isolation and purification of the antimicrobial activity from culture supernatants resulted in the detection of a 6.3-kDa mass peak by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry, a mass corresponding to the predicted size of one of the class IId bacteriocins. The identification of this bacteriocin, called ubericin K, was further confirmed by in vitro protein synthesis, which showed the same inhibitory spectrum as the purified antimicrobial compound. Ubericin K shows highest sequence similarity to the class IId bacteriocins bovicin 255, lactococcin A, and garvieacin Q. We found that ubericin K uses the sugar transporter mannose phosphotransferase (PTS) as a target receptor. Further, by using the pHlourin sensor system to detect intracellular pH changes due to leakage across the membrane, ubericin K was shown to be a pore former, killing target cells by membrane disruption.IMPORTANCE Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance. Compounds other than antibiotics also exist that kill bacteria causing infections in cows; these compounds, known as bacteriocins, are natural products produced by other bacteria in the environment. In this work, we discover a new bacteriocin that we call ubericin K, which kills several species of bacteria known to cause infections in dairy cows. We also use in vitro synthesis as a novel method for rapidly characterizing bacteriocins directly from genomic data, which could be useful for other researchers. We believe that ubericin K and the methods described in this work will aid in the transition away from antibiotics in the dairy industry.

Mining and Statistical Modeling of Natural and Variant Class IIa Bacteriocins Elucidate Activity and Selectivity Profiles across Species.

Class IIa bacteriocin antimicrobial peptides (AMPs) are a compelling alternative to current antimicrobials because of potential specific activity toward antibiotic-resistant bacteria, including vancomycin-resistant enterococci. Engineering of these molecules would be enhanced by a better understanding of AMP sequence-activity relationships to improve efficacy in vivo and limit effects of off-target activity. Toward this goal, we experimentally evaluated 210 natural and variant class IIa bacteriocins for antimicrobial activity against six strains of enterococci. Inhibitory activity was ridge regressed to AMP sequence to predict performance, achieving an area under the curve of 0.70 and demonstrating the potential of statistical models for identifying and designing AMPs. Active AMPs were individually produced and evaluated against eight enterococcus strains and four Listeria strains to elucidate trends in susceptibility. It was determined that the mannose phosphotransferase system (manPTS) sequence is informative of susceptibility to class IIa bacteriocins, yet other factors, such as membrane composition, also contribute strongly to susceptibility. A broadly potent bacteriocin variant (lactocin DT1) from a Lactobacillus ruminis genome was identified as the only variant with inhibitory activity toward all tested strains, while a novel enterocin variant (DT2) from an Enterococcus faecium genome demonstrated specificity toward Listeria strains. Eight AMPs were evaluated for proteolytic stability to trypsin, chymotrypsin, and pepsin, and three C-terminal disulfide-containing variants, including divercin V41, were identified as compelling for future in vivo studies, given their high potency and proteolytic stability.IMPORTANCE Class IIa bacteriocin antimicrobial peptides (AMPs), an alternative to traditional small-molecule antibiotics, are capable of selective activity toward various Gram-positive bacteria, limiting negative side effects associated with broad-spectrum activity. This selective activity is achieved through targeting of the mannose phosphotransferase system (manPTS) of a subset of Gram-positive bacteria, although factors affecting this mechanism are not entirely understood. Peptides identified from genomic data, as well as variants of previously characterized AMPs, can offer insight into how peptide sequence affects activity and selectivity. The experimental methods presented here identify promising potent and selective bacteriocins for further evaluation, highlight the potential of simple computational modeling for prediction of AMP performance, and demonstrate that factors beyond manPTS sequence affect bacterial susceptibility to class IIa bacteriocins.

Crystal Structure of Mannose Specific IIA Subunit of Phosphotransferase System from Streptococcus pneumoniae.

Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae.

The Man-PTS subunit ⅡC is responsible for the sensitivity of Listeria monocytogenes to durancin GL.

Target cell recognition is an important issue in the realization of bacteriocin's activity. In this report, we provide genetic and biochemical evidence of durancin GL, a new bacteriocin produced by Enterococcus durans 41D, and use ⅡC subunit in the mannose phosphotransferase system (Man‐PTS) of Listeria monocytogenes as target/receptor. First, the L. monocytogenes mutants with Man‐PTS IIC or IID deletion were constructed with the vector pHoss1. Then, the utilization of glucose and mannose and the sensitivity to durancin GL of the mutant strains were investigated. Afterward, the interactions between durancin GL and the subunits of IIC or IID in Man‐PTS of L. monocytogenes were characterized by yeast two‐hybrid system. The results showed that the L. monocytogenes mutants with either IIC or IID deletion were not only resistant to durancin GL, but also their absorption and utilization of glucose and mannose were not disturbed by the presence of durancin GL. Finally, in situ detection of the interaction between durancin GL and Man‐PTS subunits of IIC or IID by yeast two‐hybrid system revealed that there was a strong interaction between durancin GL and Man‐PTS subunit IIC. However, the interaction between durancin GL and Man‐PTS subunit IID was not present or weak. Based on the experimental evidence above, the Man‐PTS subunit IIC is responsible for the sensitivity of L. monocytogenes to bacteriocin durancin GL.

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor

BackgroundThe scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria. MethodsL. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches. ResultsThe mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain. ConclusionsOur results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins. General significanceThis is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.

The extracellular loop of Man-PTS subunit IID is responsible for the sensitivity of Lactococcus garvieae to garvicins A, B and C

Mannose phosphotransferase system (Man-PTS) serves as a receptor for several bacteriocins in sensitive bacterial cells, namely subclass IIa bacteriocins (pediocin-like; pediocins) and subclass IId ones - lactococcin A (LcnA), lactococcin B (LcnB) and garvicin Q (GarQ). Here, to identify the receptor for three other narrow-spectrum subclass IId bacteriocins - garvicins A, B and C (GarA-C) Lactococcus garvieae mutants resistant to bacteriocins were generated and sequenced to look for mutations responsible for resistance. Spontaneous mutants had their whole genome sequenced while in mutants obtained by integration of pGhost9::ISS1 regions flanking the integration site were sequenced. For both types of mutants mutations were found in genes encoding Man-PTS components IIC and IID indicating that Man-PTS likely serves as the receptor for these bacteriocins as well. This was subsequently confirmed by deletion of the man-PTS operon in the bacteriocin-sensitive L. garvieae IBB3403, which resulted in resistant cells, and by heterologous expression of appropriate man-PTS genes in the resistant Lactococcus lactis strains, which resulted in sensitive cells. GarA, GarB, GarC and other Man-PTS-targeting bacteriocins differ in the amino acid sequence and activity spectrum, suggesting that they interact with the receptor through distinct binding patterns. Comparative analyses and genetic studies identified a previously unrecognized extracellular loop of Man-PTS subunit IID (γ+) implicated in the L. garvieae sensitivity to the bacteriocins studied here. Additionally, individual amino acids localized mostly in the sugar channel-forming transmembrane parts of subunit IIC or in the extracellular parts of IID likely involved in the interaction with each bacteriocin were specified. Finally, template-based 3D models of Man-PTS subunits IIC and IID were built to allow a deeper insight into the Man-PTS structure and functioning.

Increased Viability of Sugar Transport-Deficient Mutant of the Periodontal Pathogen, Aggregatibacter actinomycetemcomitans.

The periodontal pathogen, Aggregatibacter actinomycetemcomitans is extremely sensitive to even a mildly acidic pH resulting from metabolic acids secreted during growth, losing viability rapidly as the pH goes below 6.0. Cells grown at high glucose concentration grow fast but rapidly lose viability. However, if the cells are grown at low glucose concentration, the pH of the growth medium first decreases slowly for about 24h and then starts to increase. This increase of pH is indicative of cell death since the spontaneous rise of pH due to the presence of bicarbonate can no longer be opposed by secreted metabolic acids. By monitoring these pH changes on a petri dish, a method was developed to screen for sugar transport-deficient mutants from a library of transposon insertion mutants. Isolation of a mannose phosphotransferase mutant strain is described. The mutant cells were found to be more viable and for a longer period of time than wild-type cells both in high and low glucose concentrations due to slower metabolism and less acid secreted. This observation highlights the concern that spontaneous mutations in the sugar transport genes may be selected for in patients due to increased viability of the mutant cells especially in a biofilm.

Characterisation of the action mechanism of a Lactococcus-specific bacteriocin, lactococcin Z

Lactococcin Z is a novel Lactococcus-specific bacteriocin produced by Lactococcus lactis QU 7 that shares 55.6% identity with lactococcin A. To identify the receptor targeted by lactococcin Z, several lactococcin Z-resistant mutants were generated from the sensitive strain, L.lactis IL1403. The resistant mutants showed difficulties in utilising mannose and glucose as sole carbon sources, contrary to their pattern of growth in the presence of galactose as a sole carbon source. Mutations were found in the ptnC and ptnD genes of lactococcin Z-resistant mutants, which encode the mannose phosphotransferase system (Man-PTS) components, IIC and IID, respectively; therefore, IIC and IID are proposed as potential receptors employed by lactococcin Z and are the same receptors targeted by lactococcin A. Both lactococcins A and Z share a high percentage identity in their N-termini regions in contrast to their C-termini that show less similarity; this may explain the difference in their action mechanisms as well as the lack of cross-immunity between them. Although lactococcin Z showed bactericidal activity, it neither dissipated membrane potential nor formed pores on the membranes of sensitive cells, in sharp contrast to the pore-forming lactococcin A.