Survey of Genomic Diversity among Enterococcus faecalis Strains by Microarray-Based Comparative Genomic Hybridization

We have compared nine Enterococcus faecalis strains with E. faecalis V583 by comparative genomic hybridization using microarrays (CGH). The strains used in this study (the “test” strains) originated from various environments. CGH is a powerful and promising tool for obtaining novel information on genome diversity in bacteria. By CGH, one obtains clues about which genes are present or divergent in the strains, compared to a reference strain (here, V583). The information obtained by CGH is important from both ecological and systematic points of view. CGH of E. faecalis showed considerable diversity in gene content: Compared to V583, the percentage of divergent genes in the test strains varied from 15% to 23%, and 154 genes were divergent in all strains. The main variation was found in regions corresponding to exogenously acquired or mobile DNA in V583. Antibiotic resistance genes, virulence factors, and integrated plasmid genes dominated among the divergent genes. The strains examined showed various contents of genes corresponding to the pTEF1, pTEF2, and pTEF3 genes in V583. The extensive transport and metabolic capabilities of V583 appeared similar in the test strains; CGH indicated that the ability to transport and metabolize various carbohydrates was similar in the test strains (verified by API 50 CH assays). The contents of genes related to stress tolerance appeared similar in V583 and the nine test strains, supporting the view of E. faecalis as an organism able to resist harsh conditions.     

Enterococcus faecalis Prophage Dynamics and Contributions to Pathogenic Traits

Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among isolates.     

Impact of actin on adhesion and translocation of Enterococcus faecalis

This study focuses on the impact of actin on adhesion and translocation of Enterococcus (E.) faecalis OG1RF, E. faecalis Symbioflor^®, and E. faecalis V583. Insight into the role of actin aggregation in the mediation of bacterial adhesion and translocation was provided by a two-chamber translocation assay, which employed Ptk6 cells. Determination of translocation rates, cytochalasin D treatment, and laser scanning confocal microscopic observation revealed actin as a predominant brace for enterococci to pass through the epithelial cell layer. As the three enterococci had moderate adhesion ability to actin, actin-binding proteins were isolated and characterized by LC–MS/MS. The isolated proteins were identified as pyruvate formate lyase, enolase, glyceraldehyde-3-phosphate dehydrogenase, and GroEL. All these proteins belong to two major groups of moonlighting proteins, i.e., proteins, which display additional functions other than their described major biochemical catalysis. Both groups of moonlight proteins were determined to be associated with epithelial cell binding.     

Mapping the Pathways to Staphylococcal Pathogenesis by Comparative Secretomics

The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative “secretomics” approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the “gadgets” that S. aureus needs to conquer these well-protected niches.     

Novel insights into the vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F

The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3 Å resolution structure of the NTD_N/C domain reveals a unique loop-helix stretch (₄₀₉ILKDTEPAKELLYGIEKM₄₂₆) not present in homologue domains of other prokaryotic AhpFs. Deletion of the unique ₄₁₅PAKELLY₄₂₁-helix or of ₄₁₅PAKELL₄₂₀ affects protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described to be essential for E. faecalis AhpF's optimal NADH-oxidative activity.     

Screening of In Vivo Activated Genes in Enterococcus faecalis during Insect and Mouse Infections and Growth in Urine

Enterococcus faecalis is part of the commensal microbiota of humans and its main habitat is the gastrointestinal tract. Although harmless in healthy individuals, E. faecalis has emerged as a major cause of nosocomial infections. In order to better understand the transformation of a harmless commensal into a life-threatening pathogen, we developed a Recombination-based In Vivo Expression Technology for E. faecalis. Two R-IVET systems with different levels of sensitivity have been constructed in a E. faecalis V583 derivative strain and tested in the insect model Galleria mellonella, during growth in urine, in a mouse bacteremia and in a mouse peritonitis model. Our combined results led to the identification of 81 in vivo activated genes. Among them, the ef_3196/7 operon was shown to be strongly induced in the insect host model. Deletion of this operonic structure demonstrated that this two-component system was essential to the E. faecalis pathogenic potential in Galleria. Gene ef_0377, induced in insect and mammalian models, has also been further analyzed and it has been demonstrated that this ankyrin-encoding gene was also involved in E. faecalis virulence. Thus these R-IVET screenings led to the identification of new E. faecalis factors implied in in vivo persistence and pathogenic potential of this opportunistic pathogen.     

Characterization of extracellular proteins in members of the Paracoccidioides complex

Paracoccidioides is a thermodimorphic fungus that causes Paracoccidioidomycosis (PCM) – an endemic systemic mycosis in Latin America. The genus comprises several phylogenetic species which present some genetic and serological differences. The diversity presented among isolates of the same genus has been explored in several microorganisms. There have also been attempts to clarify differences that might be related to virulence existing in isolates that cause the same disease. In this work, we analyzed the secretome of two isolates in the Paracoccidioides genus, isolates Pb01 and PbEpm83, and performed infection assays in macrophages to evaluate the influence of the secretomes of those isolates upon an in vitro model of infection. The use of a label-free proteomics approach (LC-MS^E) allowed us to identify 92 proteins that are secreted by those strains. Of those proteins, 35 were differentially secreted in Pb01, and 36 in PbEpm83. According to the functional annotation, most of the identified proteins are related to adhesion and virulence processes. These results provide evidence that different members of the Paracoccidioides complex can quantitatively secrete different proteins, which may influence the characteristics of virulence, as well as host-related processes.     

Modulation of the Bacillus anthracis Secretome by the Immune Inhibitor A1 Protease

The Bacillus anthracis secretome includes protective antigen, lethal factor, and edema factor, which are the components of anthrax toxin, and other proteins with known or potential roles in anthrax disease. Immune inhibitor A1 (InhA1) is a secreted metalloprotease that is unique to pathogenic members of the Bacillus genus and has been associated with cleavage of host proteins during infection. Here, we report the effect of InhA1 on the B. anthracis secretome. Differential in-gel electrophoresis of proteins present in culture supernatants from a parent strain and an isogenic inhA1-null mutant revealed multiple differences. Of the 1,340 protein spots observed, approximately one-third were less abundant and one-third were more abundant in the inhA1 secretome than in the parent strain secretome. Proteases were strongly represented among those proteins exhibiting a 9-fold or greater change. InhA1 purified from a B. anthracis culture supernatant directly cleaved each of the anthrax toxin proteins as well as an additional secreted protease, Npr599. The conserved zinc binding motif HEXXH of InhA1 (HEYGH) was critical for its proteolytic activity. Our data reveal that InhA1 directly and indirectly modulates the form and/or abundance of over half of all the secreted proteins of B. anthracis. The proteolytic activity of InhA1 on established secreted virulence factors, additional proteases, and other secreted proteins suggests that this major protease plays an important role in virulence not only by cleaving mammalian substrates but also by modulating the B. anthracis secretome itself.     

Role of Glycolipids in the Pathogenesis of Enterococcus faecalis Urinary Tract Infection

Background

After uropathogenic Escherichia coli (UPEC), Enterococcus faecalis is the second most common pathogen causing urinary tract infections. Monoglucosyl-diacylglycerol (MGlcDAG) and diglucosyl-diacylglycerol (DGlcDAG) are the main glycolipids of the E. faecalis cell membrane. Examination of two mutants in genes bgsB and bgsA (both glycosyltransferases) showed that these genes are involved in cell membrane glycolipid biosynthesis, and that their inactivation leads to loss of glycolipids DGlcDAG (bgsA) or both MGlcDAG and DGlcDAG (bgsB). Here we investigate the function of bgsB and bgsA regarding their role in the pathogenesis in a mouse model of urinary tract infection and in bacterial adhesion to T24 bladder epithelial cells.

Results

In a mouse model of urinary tract infection, we showed that E. faecalis 12030ΔbgsB and E. faecalis 12030ΔbgsA mutants, colonize uroepithelial surfaces more efficiently than wild-type bacteria. We also demonstrated that these mutants showed a more than three-fold increased binding to human bladder carcinoma cells line T24 compared to the wild-type strain. Bacterial binding could be specifically inhibited by purified glycolipids. Lipoteichoic acid (LTA), wall-teichoic acid (WTA), and glycosaminoglycans (GAGs) were not significantly involved in binding of E. faecalis to the bladder epithelial cell line.

Conclusions

Our data show that the deletion of bgsB and bgsA and the absence of the major glycolipid diglucosyl-diacylglycerol increases colonization and binding to uroepithelial cells. We hypothesize that secreted diglucosyl-diacylglycerol blocks host binding sites, thereby preventing bacterial adhesion. Further experiments will be needed to clarify the exact mechanism underlying the adhesion through glycolipids and their cognate receptors.     

Secretome Analysis of the Pine Wood Nematode Bursaphelenchus xylophilus Reveals the Tangled Roots of Parasitism and Its Potential for Molecular Mimicry

Since it was first introduced into Asia from North America in the early 20^th century, the pine wood nematode Bursaphelenchus xylophilus has caused the devastating forest disease called pine wilt. The emerging pathogen spread to parts of Europe and has since been found as the causal agent of pine wilt disease in Portugal and Spain. In 2011, the entire genome sequence of B. xylophilus was determined, and it allowed us to perform a more detailed analysis of B. xylophilus parasitism. Here, we identified 1,515 proteins secreted by B. xylophilus using a highly sensitive proteomics method combined with the available genomic sequence. The catalogue of secreted proteins contained proteins involved in nutrient uptake, migration, and evasion from host defenses. A comparative functional analysis of the secretome profiles among parasitic nematodes revealed a marked expansion of secreted peptidases and peptidase inhibitors in B. xylophilus via gene duplication and horizontal gene transfer from fungi and bacteria. Furthermore, we showed that B. xylophilus secreted the potential host mimicry proteins that closely resemble the host pine’s proteins. These proteins could have been acquired by host–parasite co-evolution and might mimic the host defense systems in susceptible pine trees during infection. This study contributes to an understanding of their unique parasitism and its tangled roots, and provides new perspectives on the evolution of plant parasitism among nematodes.     

Targeted Proteomics of the Secretory Pathway Reveals the Secretome of Mouse Embryonic Fibroblasts and Human Embryonic Stem Cells

Proteins endogenously secreted by human embryonic stem cells (hESCs) and those present in hESC culture medium are critical regulators of hESC self-renewal and differentiation. Current MS-based approaches for identifying secreted proteins rely predominantly on MS analysis of cell culture supernatants. Here we show that targeted proteomics of secretory pathway organelles is a powerful alternate approach for interrogating the cellular secretome. We have developed procedures to obtain subcellular fractions from mouse embryonic fibroblasts (MEFs) and hESCs that are enriched in secretory pathway organelles while ensuring retention of the secretory cargo. MS analysis of these fractions from hESCs cultured in MEF conditioned medium (MEF-CM) or MEFs exposed to hESC medium revealed 99 and 129 proteins putatively secreted by hESCs and MEFs, respectively. Of these, 53 and 62 proteins have been previously identified in cell culture supernatants of MEFs and hESCs, respectively, thus establishing the validity of our approach. Furthermore, 76 and 37 putatively secreted proteins identified in this study in MEFs and hESCs, respectively, have not been reported in previous MS analyses.The identification of low abundance secreted proteins via MS analysis of cell culture supernatants typically necessitates the use of altered culture conditions such as serum-free medium. However, an altered medium formulation might directly influence the cellular secretome. Indeed, we observed significant differences between the abundances of several secreted proteins in subcellular fractions isolated from hESCs cultured in MEF-CM and those exposed to unconditioned hESC medium for 24 h. In contrast, targeted proteomics of secretory pathway organelles does not require the use of customized media. We expect that our approach will be particularly valuable in two contexts highly relevant to hESC biology: obtaining a temporal snapshot of proteins secreted in response to a differentiation trigger, and identifying proteins secreted by cells that are isolated from a heterogeneous population.Human embryonic stem cells (hESCs)¹ are pluripotent cells isolated from the inner cell mass of a pre-implantation blastocyst stage embryo (1). They have potential applications in regenerative medicine, are an attractive source of human cells for drug evaluation, and are useful models for understanding human development. The self-renewal or differentiation of hESCs is controlled by endogenous proteins secreted by hESCs and by exogenous factors present in cell culture medium (2, 3). For instance, hESCs are routinely cultured on feeder layers of mouse embryonic fibroblasts (MEFs) or on Matrigel-coated plates in mouse embryonic fibroblast–conditioned medium (MEF-CM). In these cases, cytokines secreted by MEFs and present in MEF-CM, together with cytokines and extracellular matrix (ECM) proteins secreted by hESCs, form a localized microenvironment that regulates hESC fate.The comprehensive identification of proteins secreted by MEFs and hESCs—their cellular secretome—can help unravel the molecular mechanisms that regulate hESC fate. Yet the use of MS-based approaches for secretome analysis remains challenging. In general, secretome studies of various cell types have relied on MS analysis of cell culture supernatants (reviewed in Ref. 4). However, such an approach typically results in the identification of small numbers of extracellular proteins. This was indeed the case with MS analysis of conditioned medium (CM) from MEFs or other feeder cells that support the maintenance of undifferentiated hESCs (5–8). A low abundance of secreted proteins of interest and a high concentration of serum proteins in cell culture media significantly impede MS analysis. To overcome these limitations, Bendall et al. implemented an iterative-exclusion MS (IE-MS) strategy, in conjunction with the use of medium without serum or serum replacer, for the identification of proteins secreted by MEFs and hESCs (2). Using this approach, large numbers of previously unreported proteins secreted by MEFs and hESCs could be identified, showing that IE-MS is a powerful strategy for the identification of low abundance proteins. However, the use of medium without serum or serum replacer for secretomic analysis can be problematic. Specifically, the use of a “blank” or serum-free medium might alter cellular physiology and, consequently, the profile of secreted proteins. Indeed, we observe that hESCs are highly prone to apoptosis under such growth conditions. Moreover, an analysis of the cell culture supernatant is not specifically targeted toward endogenously secreted ECM proteins, which are also an important component of the cellular microenvironment. ECM proteins form a matrix that associates with the cell and might not be present in the cell culture supernatant. Moreover, many growth factors are known to be sequestered by ECM proteins and might not be released into the culture medium (9). Here we present a rigorous evaluation of an alternate strategy to interrogate the entire cellular secretome, including cytokines and ECM proteins. Notably, our approach does not require the use of customized media lacking serum and serum replacers, and it is compatible with cell culture systems utilizing media of unknown or poorly defined composition, such as CM from MEFs.To identify the secretome of MEFs and hESCs, we carried out an MS analysis of their subcellular fractions that were enriched in secretory pathway organelles. The secretory pathway comprises the endoplasmic reticulum (ER), the Golgi apparatus, and the associated transport vesicles. Detailed MS analysis of these organelles identifies the secretory cargo (i.e. proteins destined to be secreted) in addition to the secretory pathway proteome (10). Indeed, we have previously identified several secreted proteins in hESCs as a result of contamination by the ER and Golgi (11) in our subcellular fractions. In light of these reports, we hypothesized that targeted proteomic analysis of the secretory pathway is a viable approach for comprehensive characterization of the cellular secretome. Accordingly, we developed protocols to isolate subcellular fractions enriched in the ER and Golgi compartments from MEFs and hESCs, and we subsequently carried out MS analysis on these samples. Several proteins secreted by MEFs and hESCs could be identified in this manner. Strikingly, the numbers of proteins identified were comparable to those obtained with the highly efficient IE-MS approach. Furthermore, we also show that short-term changes in medium composition affect the profile and quantitative levels of several proteins that transit through the secretory pathway, including secreted and membrane proteins. Taken together, our results validate the use of targeted secretory pathway proteomics as a powerful alternate approach to interrogate the cellular secretome.     

Library Screen Identifies Enterococcus faecalis CcpA,the Catabolite Control Protein A,as an Effector of Ace,a Collagen Adhesion Protein Linked to Virulence

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis.     

Secretomes of medically important fungi reflect morphological and phylogenetic diversity

Secretome represents a main target for understanding the mechanisms of fungal adaptation. In the present study, we focus on the secretomes of fungi associated with infections in humans and other mammals in order to explore relationships between the diverse morphological and phylogenetic groups. Almost all the mammalian pathogenic fungi analyzed have secretome sizes smaller than 1000 proteins and, secreted proteins comprise between 5% and 10% of the total proteome. As expected, the correlation pattern between the secretome size and the total proteome was similar to that described in previous secretome studies of fungi. With regard to the morphological groups, minimum secretome sizes of less than 250 secreted proteins and low values for the fraction of secreted proteins are shown in mammalian pathogenic fungi with reduced proteomes such as microsporidia, atypical fungi and some species of yeasts and yeast-like fungi (Malassezia). On the other hand, filamentous fungi have significantly more secreted proteins and the highest numbers are present in species of filamentous fungi that also are plant or insect pathogens (Fusarium verticilloides, Fusarium oxysporum and Basidiobolus meristosporus). With respect to phylogeny, there are also variations in secretome size across fungal subphyla: Microsporidia, Taphrinomycotina, Ustilagomycotina and Saccharomycotina contain small secretomes; whereas larger secretomes are found in Agaricomycotina, Pezizomycotina, Mucoromycotina and Entomophthoromycotina. Finally, principal component analysis (PCA) was conducted on the complete secretomes. The PCA results revealed that, in general, secretomes of fungi belonging to the same morphological group or subphyla cluster together. In conclusion, our results point out that in medically important fungi there is a relationship between the secretome and the morphological group or phylogenetic classification.     

Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC‐homologue HgdD in protein secretion

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC‐homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst‐forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss‐of‐function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild‐type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin‐tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD‐mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD‐dependent pathway.