(680h) Balancing Antibiotic Resistant Subpopulation in Enterococcus Faecalis Via a Dual Signal Mating-Sensing and Self-Sensing System

Shu, C. C., University of Minnesota
Chatterjee, A., University of Minnesota
Dunny, G. M., University of Minnesota
Ramkrishna, D., Purdue University
Hu, W. S., University of Minnesota, Twin Cities

Enterococcus faecalis is a pathogen causing frequent hospital acquired infection.  It transmits the plasmid encoding antibiotic resistance from donors to plasmid-free recipient cells through conjugation. The process of conjugation and plasmid transfer is regulated by two peptides:  iCF10 and cCF10.  We uncovered the dual functions of these two peptides recently through a combined experimental discovery and modeling expedition. Intracellularly these two peptides regulate the genetic circuit for conjugative plasmid transfer, while intercellularly they serve as signals of donor and recipient cell concentrations. iCF10 produced by the donor suppresses conjugation also enables the donor to sense its own concentration; cCF10 produced by the recipient allows the donor to sense recipient concentration and induces conjugation. By sensing the self-signal iCF10 and mate signal cCF10 the donor titrates its response according to the abundance level of the recipient: a vigorous response when recipient cells are abundant and a more restrained reaction when recipients are in short supplies. Such calibrated response minimizes futile replication of the plasmid and wasteful synthesis of conjugation machinery.

We formulated a mathematical model that spans from molecular regulation of the genetic circuit to the balancing and dynamics of the donor-recipient populations. At the molecular level the model incorporates mechanistic genetic regulation of the initiation of conjugation by iCF10 and cCF10; at the population level, the model considers the dynamics of signal peptides and donor-recipient populations. Various parameters of the model have been previously estimated using reporter genes. The mechanism was cross examined using mutants which were impaired in cCF10 and/or iCF10 production. To further shed light on this dual signaling system the dynamics of transcriptome and proteome were evaluated by RNAseq and iTRAQ proteomics respectively. The genome wide survey enabled us to fine tune the parameter values so that the model truly reflect experimentally observed intracellular variables.

The model was used to prescribe the dynamic behavior of a mixed culture of donor and recipient cells at different abundance levels. Intriguingly the model predicts an incompatibility of a complete conversion of recipient cells to plasmid bearing donors. This was attributed from the  suppression of conjugation exerted by the self-sensing signal. We hypothesize that the self-sensing signal plays a donor/recipient balancing role to ensure that the population is endowed with antibiotic resistance while maintaining a subpopulation that is free from the metabolic burden of harboring a particular plasmid. Such a dual signaling system may be rather common in nature. Understanding the balancing act of the signaling system may shed light on new ways of controlling the spread of antibiotic resistance.