Involvement of Chromosomally Encoded Homologs of the RRNPP Protein Family in Enterococcus faecalis Biofilm Formation and Urinary Tract Infection Pathogenesis

Abstract

Enterococcus faecalis is an opportunistic pathogen capable of causing infections including endocarditis and urinary tract infections (UTI). One of the well characterized quorum sensing pathways in E. faecalis involves coordination of the conjugal transfer of pheromone-responsive plasmids by PrgX, a member of the RRNPP protein family. Members of this protein family in various Firmicutes have also been shown to contribute to numerous cellular processes including sporulation, competence, conjugation, nutrient sensing, biofilm formation and virulence. As PrgX is a plasmid-encoded RRNPP family member, we surveyed the genome of the multi-drug resistant strain V583 for additional RRNPP homologs using computational searches and refined those identified hits for predicted structural similarities to known RRNPP family members. This led us to investigate the contribution of the chromosomally encoded RRNPP homologs to biofilm processes and pathogenesis in a catheter associated urinary tract infection (CAUTI) model. In this study, we identified five such homologs and report that 3 of the 5 homologs, EF0073, EF1599 and EF1316, affect biofilm formation as well as outcomes in the CAUTI model. Importance Enterococcus faecalis causes healthcare-associated infections and displays resistance to a variety of broad spectrum antibiotics by acquisition of resistance traits as well as the ability to form biofilms. Even though a growing number of factors related to biofilm formation have been identified, mechanisms that contribute to biofilm formation are still largely unknown. The RRNPP protein family regulate a diverse set of biological reactions in low G-C Gram-positive bacteria (Firmicutes). Here we identify three predicted structural homologs of the RRNPP family, EF0073, EF1599 and EF1316, which affect biofilm formation and CAUTI pathogenesis.

Influence of the Alternative Sigma Factor RpoN on Global Gene Expression and Carbon Catabolism in Enterococcus faecalis V583

“…The biofilm conditions in the prior study were based on a static biofilm under nutrientrich conditions, and we were interested in knowing how RpoN and CcpA might contribute to biofilm formation under flow conditions under nutrient-poor conditions to more closely mimic the environment that is likely encountered at sites of infection. To assess the role of RpoN and CcpA in regulating biofilm formation, we quantified the level of biofilm formation under flow conditions of rpoN and ccpA mutant strains using a drip-flow bioreactor (DFBR) (28,29) in 0.1Â MM9YEGC medium (modified M9 medium supplemented with yeast extract, glucose, and casamino acids). In the absence of rpoN, there is a significant 6-fold decrease in biofilm formation relative to that of the parental V583 strain and rpoN complement, whereas in the absence of ccpA, there is a more drastic decrease in biofilm formation that is represented by an approximate 170fold decrease in biofilm formation compared to that of parental V583 or the ccpA-complemented strain (Fig.…”

“…Biofilm formation assessment using a drip-flow biofilm reactor. A drip-flow biofilm reactor (DFBR) was utilized as previously described (28,29) to assess V583, DrpoN, DrpoN::rpoN, DccpA, and DccpA::ccpA strains for their ability to form biofilms. Briefly, the channels of the growth chamber of the DFBR were seeded with diluted overnight cultures grown in THB (1:100) in either MM9YEGC (G = 15 mM glucose) or MM9YEFC (F = 15 mM fructose) medium and incubated at 37°C for 8 h to allow initial adherence.…”

Enterococcal biofilm—A nidus for antibiotic resistance transfer?

“…Nagasawa et al ( 2020 ) working with Streptococcus mutans biofilm showed that stress caused by sub‐MIC levels of antibiotics stimulated biofilm formation and this contributed to higher levels of horizontal gene transfer. This theme is reflected in the enterococci where a number of investigators have demonstrated a direct link between conjugation elements, biofilm formation and virulence (Bhatty et al, 2015 ; Parthasarathy et al, 2020 ; Schmitt et al, 2018 ). While the molecular mechanisms underlying HGT in biofilm are beginning to be elucidated there has been a shift in focus to define the impact of the process in vivo.…”

Large-Scale Identification of Known and Novel RRNPP Quorum-Sensing Systems by RRNPP_Detector Captures Novel Features of Bacterial, Plasmidic, and Viral Coevolution

“…Yet, the genetic diversity of RRNPP QSSs may not have been fully explored, as hinted, for instance, by the candidate receptors of E. faecalis reported to harbor local similarities with regions of Rap, PlcR, or Rgg ( Parthasarathy et al 2020 ). Hence, new communication codes as well as novel density-dependent evolutionary strategies likely await to be discovered.…”