Enterococci are found in soil, sewage, food, water, and are commensal to the gastrointestinal tracts of mammals, insects, and birds. Enterococci often become nosocomial pathogens that cause a wide variety of diseases including urinary tract infections, endocarditis, and septicemia. These infections are often difficult to treat with antibiotics because most of the nosocomial strains are multi-drug resistant. Enterococcal plasmids function as reservoirs for resistance genes because they are extremely stable, allow for specific and efficient transfer, and can acquire resistance determinants from the chromosome and other plasmids. Additionally, enterococcal plasmids transfer across species boundaries transferring resistance genes like vancomycin to species like Staphylococcus aureus. There are two types of enterococcal plasmids, pheromone-responsive and broad host range. Pheromone-responsive plasmids are extremely stable, have a limited host range, and are primarily found in Enterococcus faecalis. Broad host range plasmids of E. faecalis and Enterococcus faecium are less stable than pheromone-responsive plasmids, but have an expanded host range into other Gram-positive species. E. faecalis has at least 25 known pheromone-responsive conjugative plasmids. One of the most extensively studied pheromone-responsive conjugative plasmids, pCF10. Conjugation of pCF10 from donor to recipient cell is induced by pheromone cCF10. cCF10 is contained within n the lipoprotein signal sequence encoded by the E. faecalis chromosomal gene ccfA. The lipoprotein signal sequence is processed by a series of proteolytic cleavage events to produce mature cCF10. Maturation of pheromone cCF10 produces three peptides: pre-cCF10 (CcfA1-22), cCF10 (CcfA13-19), and CcfA1-12. Cells containing pCF10 continue to produce cell membrane associated precursor pheromone of cCF10 (pre-cCF10), as well as, secreted and cell wall-associated cCF10. The presence of cCF10 does not self-induce conjugation by the donor cell because of two inhibitory molecules, PrgY and iCF10. Transmembrane protein PrgY is encoded by pCF10 and reduces cell wall associated cCF10, iCF10 is a pCF10 encoded inhibitory peptide (AITLIFI) that binds to PrgX, preventing cCF10 binding. While cCF10 controls pCF10 conjugation, pre-cCF10 controls host range of pCF10 by interacting with pCF10 replication initiation protein PrgW. cCF10 can initiate conjugation and mobilize the transfer of plasmids into other species, including Lactococcus lactis, but pCF10 cannot be maintained within the cell. However, if L. lactis is engineered to produce pre-cCF10, pCF10 can be maintained. The pre-cCF10 involvement in the establishment of pCF10 into other species might be related to the observation that it binds to the pCF10 replication initiation protein PrgW. By in vitro affinity chromatography experiments, interaction of cCF10 and pre-cCF10 with PrgW induced changes in PrgW mobility in gel electrophoresis that caused by formation of doublets and formation of aggregates which were thought to be mediated by disulfide bonds. Initial evidence of regulation of PrgW conformation by disulfide bonds was seen in Western blots of E. faecalis whole cell lysates where PrgW migration is sensitive to reduction. Sequence alignment comparisons between PrgW and a group of 54 of 59 known RepA_N superfamily proteins in E. faecalis revealed three highly conserved cysteines; these RepA_N proteins had a limited host range to E. faecalis. To study the importance of theses cysteines in pCF10 maintenance and host range limitation, prgW single, double, and triple cysteine to alanine (C to A) substitutions were generated. The cysteine mutant prgW was cloned into a plasmid functioning as either a contained the prgW alone (pORI10), or containing prgW with genes necessary for efficient pCF10 maintenance (pMSP6050). While all cysteine mutant plasmids of pORI10 and pMSP6050 were still capable of replicating in E. faecalis, the plasmid stability and copy number decreased, providing evidence that the cysteines were important to PrgW function. Additionally, Western blot analysis revealed PrgW C to A substitutions decreased PrgW aggregation. Mutations of PrgW cysteines reduced pMSP6050 stability and aggregation, but increased host range to L. lactis. Both L. lactis engineered to produce pre-cCF10 and the mutation of the conserved cysteines of PrgW extended host range of pMSP6050 into L. lactis. These data taken together with the observations that pre-cCF10 induced PrgW aggregation suggested that pre-cCF10 regulated the activity of the PrgW replication initiation protein through disulfide bonds. While the conserved cysteines of RepA_N proteins are found only in E. faecalis, phylogenetic analysis revealed that RepA_N homologs lacking the three cysteines are also found in E. faecium or S. aureus, suggesting that the host range of multiple plasmids might be affected by cysteine bond formation. Phylogenetic analysis also showed that the RepA_N proteins of enterococci and staphylococci appear to have evolved to determine host range based on the presence of two of the three conserved cysteines. Modular evolution of E. faecalis plasmids, like pCF10, that contained RepA_N proteins with three conserved cysteines, might have determined the fate of the plasmid as a limited host range, stable reservoir for antibiotic resistance. / Microbiology and Immunology

Enterococcus faecalis is a commensal bacterium, which upon acquisition of virulence factors on mobile genetic elements can cause sepsis, urinary tract infections and endocarditis. E. faecalis isolates can be multi-drug resistant and have been implicated in the dissemination of antibiotic resistance genes to other genera. Although the host range of pheromone inducible conjugative plasmids is restricted to Enterococci, they often carry transposons, which are capable of transposing into the chromosome of other genera. The plasmid pCF10 contains the antibiotic resistance gene tetM on a conjugative transposon Tn925. Tn925 is a Tn916-like plasmid and is capable of pCF10-independent conjugative transfer to multiple bacterial species at low levels. Biofilms are communities of bacteria growing within a matrix. In biofilms, bacteria are more difficult to kill because of their lower susceptibility to antibiotics. In hospital settings, biofilms can grow on medically implanted devices, catheters or even human tissue. In mixed species biofilms, antibiotic resistances are able to be transferred through horizontal gene transfer from E. faecalis to other bacterial species. In mixed species biofilms, it has been show that Tn925 can transpose into S. aureus at rates of 10-8 by Ella Massie Schuh. Using static mixed species biofilms, the transfer of tetM from E. faecalis to S. aureus was studied, hoping to better understand the underlying mechanisms. The goal of these studies was to determine if residence on pCF10 increased the transfer frequency of Tn925 in mixed species biofilms. Mixed species biofilms containing E. faecalis (pCF10) and S. aureus (pALC2073aPSM) were established and pCF10 conjugation was induced with pheromone cCF10. Transfer of Tn925 / Biomedical Sciences

Horizontal gene transfer transforms commensal E. faecalis into multidrug resistance (MDR) opportunistic pathogens causing diseases such as infective endocarditis (IE), septicemia, and urinary tract infections (UTI) (4,1). E. faecalis are among the top three leading causes of hospital-acquired infections and pheromone responsive plasmids (pCF10) are the most extensively characterized conjugative plasmids in E. faecalis infection (2,4). E. faecalis is a potential future public health concern because of the co-occurrence factors of antibiotic resistance and virulence traits (6)Plasmid-free commensal E. faecalis form homogenous biofilms that have a uniform distribution of the bacterial cell and a fluid-like movement (22). The introduction of the pheromone responsive plasmid pCF10 leads to the formation of heterologous rigid structures within the biofilm (22). In the current work, the timeline of biofilm tower formation was characterized. Tower formation was not observed in the commensal strain. The pCF10-containing bacteria formed a rigid base layer on day 1 and small aggregates on day 1. pCF10-containing biofilm forms heterologous towers on days two and three. Interestingly, mixed biofilms with both plasmid-containing and plasmid-free bacteria developed tower-like structures as early as day 1 and had larger resulting structures by day three. In the mixed population, we hypothesize that the induction of aggregation substance and cell clumping during plasmid transfer may further contribute to structure formation (5,10). Plasmid-free mCherry-labeled bacteria could be observed in the viscous biofilms between heterologous rigid structures; however, the rigid structures were predominantly composed of plasmid-containing cells. Occasionally, mCherry cells were observed in the rigid structures, we hypothesize that these cells represent transconjugants, where pCF10 was transferred by conjugation to mCherry-plasmid-free OG1RF. The formation of rigid structures can protect bacteria from antibiotics by reducing the penetration of the antibiotic but binding and sequestration of the antibiotic in the outer layers. Antibiotic resistance increased in the pCF10-containing biofilms as rigid structures were formed. We hypothesize that underflow, like that found in the gastrointestinal tract, the heterologous rigid structures may form protected microenvironments for sensitive regions of the biofilms. In future studies, fluorescently labeled antibiotics will be used to access the formation of protected microenvironments in biofilms underflow. Previous studies in the laboratory demonstrated that the presence of pCF10 protects E. faecalis from hydrogen peroxide oxidative stress. E. faecalis produces hydrogen peroxide. Higher levels of hydrogen peroxide can be detected in rigid structures. The presence of pCF10 is known to increase the size of heart vegetations during endocarditis and hydrogen peroxide is a known activator of platelet activation (12,19). In these studies, the presence of pCF10 increased platelet activation in pCF10 containing biofilms. Software toolboxes are currently being developed to quantitate visual observations. The role of hydrogen peroxide is supported in our preliminary experiment revealing catalase treatment reduced platelet activation. Studies are ongoing to mutate the aroc and menB gene of E. faecalis, which contribute to hydrogen peroxide production (35). We will compare platelet activation in knockout (double or single) E. faecalis and the wild-type strain. For future studies, several of the preliminary data need to be repeated to further the study. We will repeat quantitative hydrogen peroxide production in the catalase experiments. We will also finish knocking out the aroc and men B gene of E. faecalis responsible for hydrogen peroxide and then compare platelet activation to the control strain. / Microbiology and Immunology