Antifungal Activity of the Enterococcus faecalis Peptide EntV Requires Protease Cleavage and Disulfide Bond Formation

Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge. ABSTRACT Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other’s virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans. Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans. IMPORTANCE Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.

[1]  J. Beckwith,et al.  Disulfide bond formation in prokaryotes , 2018, Nature Microbiology.

[2]  M. Lorenz,et al.  Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans , 2017, Proceedings of the National Academy of Sciences.

[3]  H. Ton-That,et al.  Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria , 2015, Journal of bacteriology.

[4]  Alexander D. Johnson,et al.  Candida albicans Biofilms and Human Disease. , 2015, Annual review of microbiology.

[5]  T. Zendo,et al.  Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin , 2015, Applied and Environmental Microbiology.

[6]  D. Brede,et al.  The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16 , 2015, Journal of bacteriology.

[7]  A. Mitchell,et al.  Fungal biofilms, drug resistance, and recurrent infection. , 2014, Cold Spring Harbor perspectives in medicine.

[8]  S. Solomon,et al.  Antibiotic resistance threats in the United States: stepping back from the brink. , 2014, American family physician.

[9]  L. Hancock,et al.  Enterococcal Biofilm Structure and Role in Colonization and Disease , 2014 .

[10]  F. Ausubel,et al.  Pathogenesis and Models of Enterococcal Infection , 2014 .

[11]  D. Angus,et al.  Epidemiology of severe sepsis , 2013, Virulence.

[12]  Michael S. Gilmore,et al.  Enterococci: From Commensals to Leading Causes of Drug Resistant Infection , 2014 .

[13]  M. Gilmore,et al.  Enterococcal Disease, Epidemiology, and Implications for Treatment -- Enterococci: From Commensals to Leading Causes of Drug Resistant Infection , 2014 .

[14]  M. Lorenz,et al.  Candida albicans and Enterococcus faecalis in the gut , 2013, Gut microbes.

[15]  Nicholas J. Jacobs,et al.  Control of Candida albicans Metabolism and Biofilm Formation by Pseudomonas aeruginosa Phenazines , 2013, mBio.

[16]  M. Lorenz,et al.  Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans , 2012, Infection and Immunity.

[17]  J. V. van Dijl,et al.  Requirement of Signal Peptidase ComC and Thiol-Disulfide Oxidoreductase DsbA for Optimal Cell Surface Display of Pseudopilin ComGC in Staphylococcus aureus , 2012, Applied and Environmental Microbiology.

[18]  B. Peters,et al.  Polymicrobial Interactions: Impact on Pathogenesis and Human Disease , 2012, Clinical Microbiology Reviews.

[19]  V. Young,et al.  Interplay between the Gastric Bacterial Microbiota and Candida albicans during Postantibiotic Recolonization and Gastritis , 2011, Infection and Immunity.

[20]  J. Sillanpää,et al.  The Fsr Quorum-Sensing System of Enterococcus faecalisModulates Surface Display of the Collagen-Binding MSCRAMM Ace through Regulation of gelE , 2011, Journal of bacteriology.

[21]  B. Peters,et al.  Microbial interactions and differential protein expression in Staphylococcus aureus –Candida albicans dual-species biofilms , 2010, FEMS immunology and medical microbiology.

[22]  Deborah A. Hogan,et al.  Medically important bacterial–fungal interactions , 2010, Nature Reviews Microbiology.

[23]  G. von Heijne,et al.  Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria , 2009, The Journal of Biological Chemistry.

[24]  L. C. Dutton,et al.  Streptococcus gordonii Modulates Candida albicans Biofilm Formation through Intergeneric Communication , 2009, Infection and Immunity.

[25]  L. Hancock,et al.  A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis , 2009, Molecular microbiology.

[26]  J. Beckwith,et al.  Bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation , 2008, Proceedings of the National Academy of Sciences.

[27]  L. Hancock,et al.  Regulation of Autolysis-Dependent Extracellular DNA Release by Enterococcus faecalis Extracellular Proteases Influences Biofilm Development , 2008, Journal of bacteriology.

[28]  D. Stagliano,et al.  Polymicrobial Bloodstream Infection in Pediatric Patients: Risk Factors, Microbiology, and Antimicrobial Management , 2008, The Pediatric infectious disease journal.

[29]  P. Lipke,et al.  Polymicrobial bloodstream infections involving Candida species: analysis of patients and review of the literature. , 2007, Diagnostic microbiology and infectious disease.

[30]  L. Hancock,et al.  Full Activation of Enterococcus faecalis Gelatinase by a C-Terminal Proteolytic Cleavage , 2007, Journal of bacteriology.

[31]  A. Tauch,et al.  ef1097 and ypkK encode enterococcin V583 and corynicin JK, members of a new family of antimicrobial proteins (bacteriocins) with modular structure from Gram-positive bacteria. , 2007, Microbiology.

[32]  J. Dubois,et al.  Thiol‐disulphide oxidoreductase modules in the low‐GC Gram‐positive bacteria , 2007, Molecular microbiology.

[33]  G. Dunny,et al.  Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. , 2007, Plasmid.

[34]  D. Garsin,et al.  Enterococcus faecalis Mutations Affecting Virulence in the Caenorhabditis elegans Model Host , 2007, Infection and Immunity.

[35]  F. Ausubel,et al.  Antifungal Chemical Compounds Identified Using a C. elegans Pathogenicity Assay , 2007, PLoS pathogens.

[36]  M. Bischoff,et al.  Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity , 2005, Archives of Microbiology.

[37]  A. Joachimiak,et al.  Structure of Conserved Protein of Unknown Function from Enterococcus faecalis V583 , 2005 .

[38]  F. Teng,et al.  Gelatinase Is Important for Translocation of Enterococcus faecalis across Polarized Human Enterocyte-Like T84 Cells , 2005, Infection and Immunity.

[39]  Y. Bahn,et al.  Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans. , 2003, Microbiology.

[40]  J. Lopez-Ribot,et al.  Engineered Control of Cell Morphology In Vivo Reveals Distinct Roles for Yeast and Filamentous Forms of Candida albicans during Infection , 2003, Eukaryotic Cell.

[41]  A. Schmidtchen,et al.  Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL‐37 , 2002, Molecular microbiology.

[42]  Frederick M. Ausubel,et al.  Virulence Effect of Enterococcus faecalis Protease Genes and the Quorum-Sensing Locus fsr in Caenorhabditis elegans and Mice , 2002, Infection and Immunity.

[43]  Gordon Ramage,et al.  The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans. , 2002, FEMS microbiology letters.

[44]  R. Kolter,et al.  Pseudomonas-Candida Interactions: An Ecological Role for Virulence Factors , 2002, Science.

[45]  S. Bron,et al.  Thiol-Disulfide Oxidoreductases Are Essential for the Production of the Lantibiotic Sublancin 168* , 2002, The Journal of Biological Chemistry.

[46]  M. Gilmore,et al.  The enterococci : pathogenesis, molecular biology, and antibiotic resistance , 2002 .

[47]  A. Shevchenko,et al.  Archived polyacrylamide gels as a resource for proteome characterization by mass spectrometry , 2001, Electrophoresis.

[48]  A. Schmidtchen,et al.  Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial α‐defensin , 2001, Molecular microbiology.

[49]  G. Weinstock,et al.  Effects of Enterococcus faecalis fsrGenes on Production of Gelatinase and a Serine Protease and Virulence , 2000, Infection and Immunity.

[50]  B. van den Burg,et al.  Two Allelic Forms of the Aureolysin Gene (aur) within Staphylococcus aureus , 2000, Infection and Immunity.

[51]  U. Munzel,et al.  Bacterial flora accompanying Candida yeasts in clinical specimens , 1999, Mycoses.

[52]  G. Weinstock,et al.  Generation and testing of mutants of Enterococcus faecalis in a mouse peritonitis model. , 1998, The Journal of infectious diseases.

[53]  G. Fink,et al.  Nonfilamentous C. albicans Mutants Are Avirulent , 1997, Cell.

[54]  B. Roques,et al.  Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin. , 1994, The Biochemical journal.

[55]  G. Weinstock,et al.  Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function , 1993, Journal of bacteriology.

[56]  D. Clewell,et al.  Purification and substrate specificity of a strongly hydrophobic extracellular metalloendopeptidase ("gelatinase") from Streptococcus faecalis (strain 0G1-10). , 1989, The Journal of biological chemistry.

[57]  J. Phair,et al.  Enterococcal bacteremia: analysis of 75 episodes. , 1989, Reviews of infectious diseases.

[58]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[59]  N. Yanaihara,et al.  Isolation and Characterization of Plasmids Carrying the dut Gene of Escherichia coli. , 1979 .

[60]  W. G. Maccallum,et al.  A CASE OF ACUTE ENDOCARDITIS CAUSED BY MICROCOCCUS ZYMOGENES (NOV. SPEC.), WITH A DESCRIPTION OF THE MICROORGANISM , 1899, The Journal of experimental medicine.