Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

This is the published version. Copyright 2007 American Society for MicrobiologyConjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently

Acceleration of Enterococcus faecalis Biofilm Formation by Aggregation Substance Expression in an Ex Vivo Model of Cardiac Valve Colonization

Infectious endocarditis involves formation of a microbial biofilm in vivo. Enterococcus faecalis Aggregation Substance (Asc10) protein enhances the severity of experimental endocarditis, where it has been implicated in formation of large vegetations and in microbial persistence during infection. In the current study, we developed an ex vivo porcine heart valve adherence model to study the initial interactions between Asc10+ and Asc10− E. faecalis and valve tissue, and to examine formation of E. faecalis biofilms on a relevant tissue surface. Scanning electron microscopy of the infected valve tissue provided evidence for biofilm formation, including growing masses of bacterial cells and the increasing presence of exopolymeric matrix over time; accumulation of adherent biofilm populations on the cardiac valve surfaces during the first 2–4 h of incubation was over 10-fold higher than was observed on abiotic membranes incubated in the same culture medium. Asc10 expression accelerated biofilm formation via aggregation between E. faecalis cells; the results also suggested that in vivo adherence to host tissue and biofilm development by E. faecalis can proceed by Asc10-dependent or Asc10-independent pathways. Mutations in either of two Asc10 subdomains previously implicated in endocarditis virulence reduced levels of adherent bacterial populations in the ex vivo system. Interference with the molecular interactions involved in adherence and initiation of biofilm development in vivo with specific inhibitory compounds could lead to more effective treatment of infectious endocarditis

Buoyant densities of DNA from various strains of Streptococcus mutans

The buoyant densities of eight strains of Streptococcus mutans were determined by analytical ultracentrifugation in caesium chloride. The results were as follows (in g/cm3): K-1-R(1.7044); SL-1(1.7040); E-49(1.7010); BHT(1.7009); FA-1(1.7000); Ingbritt(1.6967); NCTC-10449(1.6964); GS-5(1.6962). These data add to the mounting evidence for genetic heterogeneity in Strept. mutans.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34097/1/0000379.pd

Enterococcal sex pheromone precursors are part of signal sequences for surface lipoproteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73811/1/j.1365-2958.2000.01687.x.pd

Enterococcal sex pheromone precursors are part of signal sequences for surface lipoproteins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73811/1/j.1365-2958.2000.01687.x.pd

AhrC and Eep are biofilm infection-associated virulence factors in enterococcus faecalis

Enterococcus faecalis is part of the human intestinal microbiome and is a prominent cause of health care-associated infections. The pathogenesis of many E. faecalis infections, including endocarditis and catheter-associated urinary tract infection (CAUTI), is related to the ability of clinical isolates to form biofilms. To identify chromosomal genetic determinants responsible for E. faecalis biofilm-mediated infection, we used a rabbit model of endocarditis to test strains with transposon insertions or in-frame deletions in biofilm-associated loci: ahrC, argR, atlA, opuBC, pyrC, recN, and sepF. Only the ahrC mutant was significantly attenuated in endocarditis. We demonstrate that the transcriptional regulator AhrC and the protease Eep, which we showed previously to be an endocarditis virulence factor, are also required for full virulence in murine CAUTI. Therefore, AhrC and Eep can be classified as enterococcal biofilm-associated virulence factors. Loss of ahrC caused defects in early attachment and accumulation of biofilm biomass. Characterization of ahrC transcription revealed that the temporal expression of this locus observed in wild-type cells promotes initiation of early biofilm formation and the establishment of endocarditis. This is the first report of AhrC serving as a virulence factor in any bacterial species

Genomic Dissection of an Icelandic Epidemic of Respiratory Disease in Horses and Associated Zoonotic Cases.

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country's native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans.IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent

Bistability versus Bimodal Distributions in Gene Regulatory Processes from Population Balance

In recent times, stochastic treatments of gene regulatory processes have appeared in the literature in which a cell exposed to a signaling molecule in its environment triggers the synthesis of a specific protein through a network of intracellular reactions. The stochastic nature of this process leads to a distribution of protein levels in a population of cells as determined by a Fokker-Planck equation. Often instability occurs as a consequence of two (stable) steady state protein levels, one at the low end representing the “off” state, and the other at the high end representing the “on” state for a given concentration of the signaling molecule within a suitable range. A consequence of such bistability has been the appearance of bimodal distributions indicating two different populations, one in the “off” state and the other in the “on” state. The bimodal distribution can come about from stochastic analysis of a single cell. However, the concerted action of the population altering the extracellular concentration in the environment of individual cells and hence their behavior can only be accomplished by an appropriate population balance model which accounts for the reciprocal effects of interaction between the population and its environment. In this study, we show how to formulate a population balance model in which stochastic gene expression in individual cells is incorporated. Interestingly, the simulation of the model shows that bistability is neither sufficient nor necessary for bimodal distributions in a population. The original notion of linking bistability with bimodal distribution from single cell stochastic model is therefore only a special consequence of a population balance model

Enterococcus faecalis Endocarditis Severity in Rabbits Is Reduced by IgG Fabs Interfering with Aggregation Substance

Background: Enterococcus faecalis is a significant cause of infective endocarditis, an infection of the heart endothelium leading to vegetation formation (microbes, fibrin, platelets, and host cells attached to underlying endothelial tissue). Our previous research determined that enterococcal aggregation substance (AS) is an important virulence factor in causation of endocarditis, although endocarditis may occur in the absence of AS production. Production of AS by E. faecalis causes the organism to form aggregates through AS binding to enterococcal binding substance. In this study, we assessed the ability of IgGs and IgG Fabs against AS to provide protection against AS + E. faecalis endocarditis. Methodology/Principal Findings: When challenged with AS + E. faecalis, 10 rabbits actively immunized against AS + E. faecalis developed more significant vegetations than 9 animals immunized against AS 2 E. faecalis, and 9/10 succumbed compared to 2/9 (p,0.005), suggesting enhanced aggregation by IgG contributes significantly to disease. IgG antibodies against AS also enhanced enterococcal aggregation as tested in vitro. In contrast, Fab fragments of IgG from rabbits immunized against purified AS, when passively administered to rabbits (6/group) immediately before challenge with AS + E. faecalis, reduced total vegetation (endocarditis lesion) microbial counts (7.9610 6 versus 2.0610 5, p = 0.02) and size (40 mg versus 10, p = 0.05). In vitro, the Fabs prevented enterococcal aggregation. Conclusions/Significance: The data confirm the role of AS in infective endocarditis formation and suggest that use of Fab

Gene Transfer In Streptococcus Faecalis: Conjugation, Mating Aggregates, And Sex Pheromones.

PhDMicrobiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/178497/2/7907064.pd