Enterococcus faecalis is a Gram-positive opportunistic pathogen and causative agent of urinary tract and wound infections, bacteremia, and infectious endocarditis. Like many other Gram-positive pathogens, enterococci carry mobile DNA elements in their genomes that mediate the spread of antibiotic resistance and other virulence determinants, complicating efforts by the medical community to control associated infections. Mobile DNA elements code for type IV secretion systems (T4SS's);these are multicomponent translocation channels responsible for delivery of DNA substrates to bacterial target cells. In many important Gram-negative pathogens, T4SS's are employed not only for interbacterial DNA transfer but also for injection of effector proteins into eukaryotic target cell. Upon translocation, the effectors disrupt host cellular processes allowing for growth and proliferation of the invading pathogen. Recent studies suggest that T4SS's of Gram-negative and -positive bacteria display conserved mechanisms of action in their ability to recognize and translocate cognate substrates across their cell envelopes. Thus, it is surprising that no studies have yet examined the potential for Gram-positive pathogens to use T4SS's for delivery of effector proteins to eukaryotic host cells during infection. Here, we propose to test a hypothesis that T4SS's of Gram-positive bacteria, like their Gram-negative counterparts, contribute to the infection process through delivery of effector proteins into eukaryotic target cells or the milieu. Two initial findings strongly support our hypothesis: i) we have gained evidence for protein translocation by the pCF10-encoded T4SS from E. faecalis donor cells to Escherichia coli recipients, and ii) a functional T4SS encoded by an 89-kilobase pathogenicity island (89K PAI) was shown to be necessary for virulence of another Gram-positive pathogen, Streptococcus suis. We will combine the expertise of the Christie and Dunny groups to identify protein substrates, secretion signals, and host range for protein translocation by two E. faecalis T4SS's, one encoded by the pheromone-responsive plasmid pCF10 and the second by the chromosomal 153-kb pathogenicity island (PAI). We will integrate the goal of defining the substrate repertoire of these Gram-positive T4SS's with infection assays using a rabbit endocarditis infection model to define the contribution of T4SS-mediated protein translocation to E. faecalis pathogenesis. We anticipate that our investigations will establish a proof-of-principle for the contribution of T4SS-mediated protein translocation to virulence of a Gram-positive pathogen. Such an outcome will stimulate new research initiatives aimed at deciphering mechanistic and cellular consequences of intercellular protein translocation during infection by E. faecalis and other medically-important Gram-positive pathogens.

Public Health Relevance

Enterococci are Gram-positive, opportunistic pathogens and causative agents of urinary tract and wound infections, bacteremia, and >20 % of bacterial infectious endocarditis cases. Enterococci carry type IV secretion systems (T4SS's) in their genomes that are known to mediate the spread of antibiotic resistance and other virulence genes among bacterial populations. The goal of this project is to determine the extent to which Enterococcus faecalis employs T4SS's to deliver protein effectors to eukaryotic target cells to modulate host cellular processes during infection. Once we identify the substrate repertoire of T4SS's among E. faecalis and other Gram-positive pathogens, we can develop methods to disrupt effector functions within the eukaryotic host to mitigate disease progression.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IDM-A (80))
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Huntley, Clayton C
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University of Texas Health Science Center Houston
Schools of Medicine
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Schmitt, Andreas; Jiang, Kai; Camacho, Martha I et al. (2018) PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction. Mol Microbiol 109:291-305
Grohmann, Elisabeth; Christie, Peter J; Waksman, Gabriel et al. (2018) Type IV secretion in Gram-negative and Gram-positive bacteria. Mol Microbiol 107:455-471
Christie, Peter J; Gomez Valero, Laura; Buchrieser, Carmen (2017) Biological Diversity and Evolution of Type IV Secretion Systems. Curr Top Microbiol Immunol 413:1-30
Bhatty, Minny; Camacho, Martha I; Gonzalez-Rivera, Christian et al. (2017) PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis. Mol Microbiol 103:398-412
Christie, Peter J (2016) The Mosaic Type IV Secretion Systems. EcoSal Plus 7:
Gonzalez-Rivera, Christian; Bhatty, Minny; Christie, Peter J (2016) Mechanism and Function of Type IV Secretion During Infection of the Human Host. Microbiol Spectr 4:
Bhatty, Minny; Cruz, Melissa R; Frank, Kristi L et al. (2015) Enterococcus faecalis?pCF10-encoded surface proteins PrgA, PrgB (aggregation substance) and PrgC contribute to plasmid transfer, biofilm formation and virulence. Mol Microbiol 95:660-77
Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J et al. (2015) The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates. J Bacteriol 197:2335-49
Christie, Peter J; Whitaker, Neal; González-Rivera, Christian (2014) Mechanism and structure of the bacterial type IV secretion systems. Biochim Biophys Acta 1843:1578-91
Laverde Gomez, Jenny A; Bhatty, Minny; Christie, Peter J (2014) PrgK, a multidomain peptidoglycan hydrolase, is essential for conjugative transfer of the pheromone-responsive plasmid pCF10. J Bacteriol 196:527-39

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