Contribution of the enterococcal surface protein Esp to pathogenesis of Enterococcus faecium endocarditis

The enterococcal surface protein Esp, specifically linked to nosocomial Enterococcus faecium, is involved in biofilm formation. To assess the role of Esp in endocarditis, a biofilm-associated infection, an Esp-expressing E. faecium strain (E1162) or its Esp-deficient mutant (E1162Δesp) were inoculated through a catheter into the left ventricle of rats. After 24 h, less E1162Δesp than E1162 were recovered from heart valve vegetations. In addition, anti-Esp antibodies were detected in Esp-positive E. faecium bacteremia and endocarditis patient sera. In conclusion, Esp contributes to colonization of E. faecium at the heart valves. Furthermore, systemic infection elicits an Esp-specific antibody response in humans.     

Calibration plot for proteomics: A graphical tool to visually check the assumptions underlying FDR control in quantitative experiments

In MS‐based quantitative proteomics, the FDR control (i.e. the limitation of the number of proteins that are wrongly claimed as differentially abundant between several conditions) is a major postanalysis step. It is classically achieved thanks to a specific statistical procedure that computes the adjusted p‐values of the putative differentially abundant proteins. Unfortunately, such adjustment is conservative only if the p‐values are well‐calibrated; the false discovery control being spuriously underestimated otherwise. However, well‐calibration is a property that can be violated in some practical cases. To overcome this limitation, we propose a graphical method to straightforwardly and visually assess the p‐value well‐calibration, as well as the R codes to embed it in any pipeline. All MS data have been deposited in the ProteomeXchange with identifier PXD002370 ( http://proteomecentral.proteomexchange.org/dataset/PXD002370 ).     

Five genes encoding surface-exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium clonal complex 17 isolates

Most Enterococcus faecium isolates associated with hospital outbreaks and invasive infections belong to a distinct genetic subpopulation called clonal complex 17 (CC17). It has been postulated that the genetic evolution of CC17 involves the acquisition of various genes involved in antibiotic resistance, metabolic pathways, and virulence. To gain insight into additional genes that may have favored the rapid emergence of this nosocomial pathogen, we aimed to identify surface-exposed LPXTG cell wall-anchored proteins (CWAPs) specifically enriched in CC17 E. faecium. Using PCR and Southern and dot blot hybridizations, 131 E. faecium isolates (40 CC17 and 91 non-CC17) were screened for the presence of 22 putative CWAP genes identified from the E. faecium TX0016 genome. Five genes encoding LPXTG surface proteins were specifically enriched in E. faecium CC17 isolates. These five LPXTG surface protein genes were found in 28 to 40 (70 to 100%) of CC17 and in only 7 to 24 (8 to 26%) of non-CC17 isolates (P < 0.05). Three of these CWAP genes clustered together on the E. faecium TX0016 genome, which may comprise a novel enterococcal pathogenicity island covering E. faecium contig 609. Expression at the mRNA level was demonstrated, and immunotransmission electron microscopy revealed an association of the five LPXTG surface proteins with the cell wall. Minimal spanning tree analysis based on the presence and absence of 22 CWAP genes revealed grouping of all 40 CC17 strains together with 18 hospital-derived but evolutionary unrelated non-CC17 isolates in a distinct CWAP-enriched cluster, suggesting horizontal transfer of CWAP genes and a role of these CWAPs in hospital adaptation.     

A peptide resource for the analysis of Staphylococcus aureus in host–pathogen interaction studies

Staphylococcus aureus is an opportunistic human pathogen, which can cause life‐threatening disease. Proteome analyses of the bacterium can provide new insights into its pathophysiology and important facets of metabolic adaptation and, thus, aid the recognition of targets for intervention. However, the value of such proteome studies increases with their comprehensiveness. We present an MS–driven, proteome‐wide characterization of the strain S. aureus HG001. Combining 144 high precision proteomic data sets, we identified 19 109 peptides from 2088 distinct S. aureus HG001 proteins, which account for 72% of the predicted ORFs. Peptides were further characterized concerning pI, GRAVY, and detectability scores in order to understand the low peptide coverage of 8.7% (19 109 out of 220 245 theoretical peptides). The high quality peptide‐centric spectra have been organized into a comprehensive peptide fragmentation library (SpectraST) and used for identification of S. aureus‐typic peptides in highly complex host–pathogen interaction experiments, which significantly improved the number of identified S. aureus proteins compared to a MASCOT search. This effort now allows the elucidation of crucial pathophysiological questions in S. aureus‐specific host–pathogen interaction studies through comprehensive proteome analysis. The S. aureus‐specific spectra resource developed here also represents an important spectral repository for SRM or for data‐independent acquisition MS approaches. All MS data have been deposited in the ProteomeXchange with identifier PXD000702 ( http://proteomecentral.proteomexchange.org/dataset/PXD000702 ).     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

Heat-Resistance and Heat-Shock Response in the Nosocomial Pathogen <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50°C, and no growth at 52°C or 55°C. In agreement, a marked decrease of general protein synthesis was observed at 52°C, and very light synthesis was detected at 55°C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52°C and, after 2 h of incubation, viable cells were still observed at 70°C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment. Received: 29 March 2002 / Accepted: 5 July 2002     

Identification of Peptidoglycan-Associated Proteins as Vaccine Candidates for Enterococcal Infections

Infections by opportunistic bacteria have significant contributions to morbidity and mortality of hospitalized patients and also lead to high expenses in healthcare. In this setting, one of the major clinical problems is caused by Gram-positive bacteria such as enterococci and staphylococci. In this study we extract, purify, identify and characterize immunogenic surface-exposed proteins present in the vancomycin resistant enterococci (VRE) strain Enterococcus faecium E155 using three different extraction methods: trypsin shaving, biotinylation and elution at high pH. Proteomic profiling was carried out by gel-free and gel-nanoLC-MS/MS analyses. The total proteins found with each method were 390 by the trypsin shaving, 329 by the elution at high pH, and 45 using biotinylation. An exclusively extracytoplasmic localization was predicted in 39 (10%) by trypsin shaving, in 47 (15%) by elution at high pH, and 27 (63%) by biotinylation. Comparison between the three extraction methods by Venn diagram and subcellular localization predictors (CELLO v.2.5 and Gpos-mPLoc) allowed us to identify six proteins that are most likely surface-exposed: the SCP-like extracellular protein, a low affinity penicillin-binding protein 5 (PBP5), a basic membrane lipoprotein, a peptidoglycan-binding protein LysM (LysM), a D-alanyl-D-alanine carboxypeptidase (DdcP) and the peptidyl-prolyl cis-trans isomerase (PpiC). Due to their close relationship with the peptidoglycan, we chose PBP5, LysM, DdcP and PpiC to test their potential as vaccine candidates. These putative surface-exposed proteins were overexpressed in Escherichia coli and purified. Rabbit polyclonal antibodies raised against the purified proteins were able to induce specific opsonic antibodies that mediated killing of the homologous strain E. faecium E155 as well as clinical strains E. faecium E1162, Enterococcus faecalis 12030, type 2 and type 5. Passive immunization with rabbit antibodies raised against these proteins reduced significantly the colony counts of E. faecium E155 in mice, indicating the effectiveness of these surface-related proteins as promising vaccine candidates to target different enterococcal pathogens.     

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram‐negative bacteria and have a relevant role in bacteria–host interactions. Using 1D SDS–PAGE and highly sensitive LC–MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain‐linked genes and 57 were common to pathogen‐derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic‐derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 ( http://proteomecentral.proteomexchange.org/dataset/PXD000367 ).     

Proteomic identification of Listeria monocytogenes surface‐associated proteins

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano‐LC and online‐MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy‐four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface‐exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.     

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.     

Protein 2DE reference map of the anterior midgut of the blood‐sucking bug Rhodnius prolixus

Rhodnius prolixus is an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease, an illness that affects 20% of Latin America population. The obligatory course of the parasite in the vector digestive tract has made it an important target for investigation in order to control the parasite transmission and thus interrupt its biological cycle in the insect vector. Therefore, an insight into the vector midgut physiology is valuable for insect control as well as to provide potential novel targets for drugs and vaccines development and thus disease treatment. In this study, the first 2DE map of R. prolixus anterior midgut is described. Proteins were separated by 2DE and analyzed by nano‐LC MS/MS. The results yielded 489 proteins from 475 spots. These proteins were classified into 28 functional groups and their physiological roles in the insect midgut are discussed. All MS data have been deposited in the ProteomeXchange with identifiers PXD001488 and PXD001489 ( http://proteomecentral.proteomexchange.org/dataset/PXD001488 , http://proteomecentral.proteomexchange.org/dataset/PXD001489 ).     

Enterococcal surface protein Esp is not essential for cell adhesion and intestinal colonization of Enterococcus faecium in mice

Background  

Enterococcus faecium has globally emerged as a cause of hospital-acquired infections with high colonization rates in hospitalized patients. The enterococcal surface protein Esp, identified as a potential virulence factor, is specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous E. faecium strains. To investigate whether Esp facilitates bacterial adherence and intestinal colonization of E. faecium, we used human colorectal adenocarcinoma cells (Caco-2 cells) and an experimental colonization model in mice.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an <Emphasis Type="Italic">Enterococcus durans</Emphasis> to a Human <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.     

Interspecies diversity,safety and probiotic potential of bacteriocinogenic <Emphasis Type="Italic">Enterococcus faecium</Emphasis> isolated from dairy food and human faeces

In the present study 14 bacteriocinogenic strains of Enterococcus faecium isolated from dairy foods and faecal sample were evaluated for the presence of virulence determinants, production of biogenic amines and their susceptibility to various antibiotics. Genetic diversity among them was evaluated by RAPD-PCR method. Further, they were evaluated for their probiotic potential under in vitro trials. The efaAfm was the only virulence trait detected in all E. faecium and tyramine was the only biogenic amine produced by 9 tested strains. No strain was resistant to all antibiotics and for some strains, multiple resistances were observed. E. faecium FH 99 showed highest good ability to tolerate acid and bile, while good bile salt hydrolase activity and were able to assimilate cholesterol from growth media. These results suggest that the tested E. faecium are generally free from virulence traits and having good probiotic potential and may be exploit in dairy industry and probiotic preparations.     

Identification of inositol 1,4,5‐trisphosphate‐binding proteins by heparin‐agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa

Inositol 1,4,5‐trisphosohate (IP₃) and its receptors play a pivotal role in calcium signal transduction in mammals. However, no homologs of mammalian IP₃ receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP₃‐binding proteins by heparin‐agarose affinity purification. The IP₃‐binding activities of these protein fractions were determined by [³H] IP₃‐binding assay. SDS‐PAGE and MS analysis were then performed to characterize these proteins. We have identified 297 proteins from the eluates of heparin‐agarose column chromatography, which will provide insight into the IP₃ signaling pathways in plants. All MS data have been deposited in the ProteomeXchange with identifier PXD000763 ( http://proteomecentral.proteomexchange.org/dataset/PXD000763 ).     

Species distribution and antimicrobial resistance of enterococci isolated from surface and ocean water

Aims: The species identification and antimicrobial resistance profiles were determined for enterococci isolated from Southern California surface and ocean waters. Methods and Results: Species identification was determined for 1413 presumptive Enterococcus isolates from urban runoff, bay, ocean and sewage water samples. The most frequently isolated species were Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. All five of these species were isolated from ocean and bay water with a frequency ranging from 7% to 36%. Enterococcus casseliflavus was the most frequently isolated species in urban runoff making up 36–65% of isolates while E. faecium was the most frequently isolated species in sewage making up 53–78% of isolates. The similar distribution of species in urban runoff and receiving water suggests that urban runoff may be the source of Enterococcus. No vancomycin or high level gentamycin resistance was detected in E. faecalis and E. faecium isolates. Conclusions: Enterococcus faecalis, E. faecium, E. casseliflavus and E. mundtii are the most commonly isolated Enterococcus species from urban runoff and receiving waters in Southern California. Significance and Impact of the Study: Determination of the Enterococcus species isolated from receiving waters and potential pollution sources may assist in determining the sources of pollution.