Structure of the mannose phosphotransferase system (man-PTS) complexed with microcin E492, a pore-forming bacteriocin.
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ABSTRACT:
SUBMITTER: Huang K
PROVIDER: S-EPMC8021565 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bovine 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.
| S-EPMC8515946 | biostudies-literature
Project description:Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this phenomenon as endogenous MceA bactericidal activity to differentiate it from the action of extracellularly secreted MccE492. The toxicity of endogenous MceA is enhanced by an efficient targeting to the inner membrane. However, a periplasmic intermediate state is not required for MceA toxicity. Indeed, endogenous MceA remains fully active when it is fused to thioredoxin-1, a fast-folding protein that promotes retention of the C terminus of MceA in the cytoplasm. The C-terminal domain of MccE492 is required only for delivery from the extracellular environment to the periplasm, and it is not required for inner membrane damage. A common component is absolutely essential for the bactericidal activity of both endogenous MceA and extracellular MccE492. Indeed, toxicity is strictly dependent on the presence of ManYZ, an inner membrane protein complex involved in mannose uptake. Based on these findings, we propose a new model for cell entry, inner membrane insertion, and toxic activity of MccE492.
| S-EPMC1636244 | biostudies-literature
Project description:Lactic acid bacteria produce diverse antimicrobial peptides called bacteriocins. Most bacteriocins target sensitive bacteria by binding to specific receptors. Although a plethora of bacteriocins have been identified, for only a few of them the receptors they recognize are known. Here, we identified permease IIC and surface protein IID, two membrane subunits of the mannose-specific quaternary phosphotransferase system (Man-PTS), as a receptor for BacSJ, a subclass IId bacteriocin produced by Lactobacillus paracasei subsp. paracasei BGSJ2-8. BacSJ shares 45% identity with another Man-PTS binding bacteriocin, garvicin Q (GarQ). Similarly to GarQ, BacSJ has a relatively broad activity spectrum acting against several Gram-positive bacteria, such as Lactococcus lactis and Listeria monocytogenes, harboring fairly similar Man-PTSs, but not against Lactococcus garvieae. To identify specific Man-PTS amino acids responsible for the L.lactis sensitivity to BacSJ, and thus likely involved in the interaction with this bacteriocin, we generated eight independent BacSJ resistant L.lactis mutants harboring five distinct missense mutations in the ptnC or ptnD genes encoding the IIC and IID subunits. Concurrently with the resistance to BacSJ, the mutants efficiently utilized mannose as a carbon source, which indicated functionality of their mutated Man-PTS. The amino acid substitutions in the mutants localized to the intracellular region of the IIC permease or to the extracellular parts of IID. This localization coincides with regions targeted by GarQ and some other Man-PTS-binding garvicins, pointing to similarities between all these bacteriocins in the mechanism of their interaction with Man-PTS. During the attack by these bacteriocins, subunits IID and IIC are assumed to function sequentially as a docking and an entry module allowing the toxic peptide to bind the cell and then open the pore. However, since not all of the BacSJ-resistant mutants exhibited cross-resistance to GarQ, we propose that BacSJ interacts with Man-PTS in a manner slightly different from that of GarQ.
| S-EPMC7660280 | biostudies-literature
Project description:Identification of the major PEP-phosphotransferase systems (PTS) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth. Transcriptional profile of prfA, the dependent genes and the PTS genes after growth in MM (minimal medium (Premaratne et al. 2001)) (supplemented with 10 mM Glucose). Listeria monocytogenes EGD-e possesses 86 pts genes encoding 29 complete and several incomplete PEP-dependent phosphotransferase systems (PTS) for the transport of carbohydrates and sugar alcohols. By a systematic deletion analysis we identified the major PTS involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of either of these carbohydrates. Under these conditions two of the four PTSMan and at least one of the five PTSGlc function as glucose transporter with different affinities. Cellobiose is transported (with different efficiencies) mainly by two of the six PTSLac and by a PTSGlc that also transports glucose. One of the PTSMan and both PTSLac are regulated by LevR-homologous PRD containing transcriptional activators, detected with PRD deletion mutants. The growth rates of mutants that are lacking these PTS are drastically reduced compared to the wild-type strain upon growth in a defined medium with low concentrations of glucose and cellobiose, respectively. In contrast, replication of these PTS mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain. Keywords: pts and prd deletion mutants
2009-12-01 | GSE18363 | GEO
Project description:The class IIa bacteriocin, pediocin PA-1, has clear potential as food preservative and in the medical field to be used against Gram negative pathogen species as Enterococcus faecalis and Listeria monocytogenes. Resistance towards class IIa bacteriocins appear in laboratory and characterization of these phenotypes is important for their application. To gain insight into bacteriocin resistance we studied mutants of E. faecalis V583 resistant to pediocin PA-1 by use of transcriptomic analyses.Mutants of E. faecalis V583 resistant to pediocin PA-1 were isolated, and their gene expression profiles were analyzed and compared to the wild type using whole-genome microarray. Significantly altered transcription was detected from about 200 genes; most of them encoding proteins involved in energy metabolism and transport. Glycolytic genes were down-regulated in the mutants, but most of the genes showing differential expression were up-regulated. The data indicate that the mutants were relieved from glucose repression and putative catabolic responsive elements (cre) could be identified in the upstream regions of 70% of the differentially expressed genes. Bacteriocin resistance was caused by reduced expression of the mpt operon encoding the mannose-specific phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), and the same transcriptional changes were seen in a mptD-inactivated mutant. This mutant also had decreased transcription of the whole mpt operon, showing that the PTS is involved in its own transcriptional regulation.Our data confirm the important role of mannose PTS in class IIa bacteriocin sensitivity and we demonstrate its importance involving global carbon catabolite control.
| S-EPMC2941500 | biostudies-literature
Project description:Microcin E492 (Mcc) is a pore-forming bacteriotoxin. Mcc activity is inhibited at the stationary phase by formation of amyloid-like aggregates in the culture. Here we report that, in a similar manner as prions, Mcc naturally exists as two conformers: a β-sheet-rich, protease-resistant, aggregated, inactive form (Mccia), and a soluble, protease-sensitive, active form (Mcca). The exogenous addition of culture medium containing Mccia or purified in vitro-generated Mccia into the culture induces the rapid and efficient conversion of Mcca into Mccia, which is maintained indefinitely after passaging, changing the bacterial phenotype. Mccia prion-like activity is conformation-dependent and could be reduced by immunodepleting Mccia. Interestingly, an internal region of Mcc shares sequence similarity with the central domain of the prion protein, which is key to the formation of mammalian prions. A synthetic peptide spanning this sequence forms amyloid-like fibrils in vitro and is capable of inducing the conversion of Mcca into Mccia in vivo, suggesting that this region corresponds to the prion domain of Mcc. Our findings suggest that Mcc is the first prokaryotic protein with prion properties which harnesses prion-like transmission to regulate protein function, suggesting that propagation of biological information using a prion-based conformational switch is an evolutionary conserved mechanism.
| S-EPMC5374632 | biostudies-literature
Project description: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.
| S-EPMC9486217 | biostudies-literature
Project description:Identification of the major PEP-phosphotransferase systems (PTS) for glucose, mannose and cellobiose of Listeria monocytogenes, and their significance for extra- and intracellular growth. Transcriptional profile of prfA, the dependent genes and the PTS genes after growth in MM (minimal medium (Premaratne et al. 2001)) (supplemented with 10 mM Glucose). Listeria monocytogenes EGD-e possesses 86 pts genes encoding 29 complete and several incomplete PEP-dependent phosphotransferase systems (PTS) for the transport of carbohydrates and sugar alcohols. By a systematic deletion analysis we identified the major PTS involved in glucose, mannose and cellobiose transport, when L. monocytogenes grows in a defined minimal medium in the presence of either of these carbohydrates. Under these conditions two of the four PTSMan and at least one of the five PTSGlc function as glucose transporter with different affinities. Cellobiose is transported (with different efficiencies) mainly by two of the six PTSLac and by a PTSGlc that also transports glucose. One of the PTSMan and both PTSLac are regulated by LevR-homologous PRD containing transcriptional activators, detected with PRD deletion mutants. The growth rates of mutants that are lacking these PTS are drastically reduced compared to the wild-type strain upon growth in a defined medium with low concentrations of glucose and cellobiose, respectively. In contrast, replication of these PTS mutants within epithelial cells or macrophages is as efficient as that of the wild-type strain. Keywords: pts and prd deletion mutants A total of three independently isolated RNA samples from each condition were used for the analysis. PTS Samples: GSM458191-GSM458208 PRD Samples: GSM458209-GSM458214
2010-05-19 | E-GEOD-18363 | biostudies-arrayexpress
Project description:Mannose phosphotransferase system (Man-PTS) is the main mannose permease in bacteria but it is also a known receptor for subclass IIa bacteriocins (pediocin-like group) as well as subclass IId lactococcin A (LcnA) and lactococcin B (LcnB) (LcnA-like group). Subclass IIa bacteriocins exhibit a strong activity against Listeria spp. but they are not against Lactococcus spp. In contrast, the LcnA-like bacteriocins act only against Lactococcus lactis strains. Garvicin Q (GarQ) is a subclass IId bacteriocin with minor similarity to LcnA-like bacteriocins and a relatively broad antimicrobial spectrum including, among others, Listeria and Lactococcus spp. To identify the GarQ receptor, we obtained GarQ-resistant mutants of Lactococcus garvieae IBB3403 and L. lactis IL1403 and sequenced their genomes that revealed mutations in genes encoding the membrane-bound Man-PTS IIC or IID subunits encoded by ptnCD in L. lactis and manCD in L. garvieae. This is the first time that a bacteriocin outside the pediocin- and LcnA-like groups is shown to target Man-PTS. The interaction between GarQ and Man-PTS may occur through a new binding pattern involving specific amino acids highly conserved among the GarQ-sensitive bacterial species located in the N-terminal part and extracellular loops of subunit IID and in transmembrane region of IIC.
| S-EPMC5566476 | biostudies-literature
Project description:The pore-forming microcin E492 was purified by solid-phase extraction and reversed-phase high-pressure liquid chromatography. Its molecular mass was 7,886 Da. The entire 84-amino-acid sequence was determined. There is no postranslational modification in the secreted microcin, and the sequence has homologies with the sequence of the microcin colicin V.
| S-EPMC126999 | biostudies-literature