Research Article: The Cell Wall-Targeting Antibiotic Stimulon of Enterococcus faecalis

Date Published: June 3, 2013

Publisher: Public Library of Science

Author(s): Jacqueline Abranches, Pamella Tijerina, Alejandro Avilés-Reyes, Anthony O. Gaca, Jessica K. Kajfasz, José A. Lemos, Willem van Schaik.


Enterococcus faecalis is an opportunistic nosocomial pathogen that is highly resistant to a variety of environmental insults, including an intrinsic tolerance to antimicrobials that target the cell wall (CW). With the goal of determining the CW-stress stimulon of E. faecalis, the global transcriptional profile of E. faecalis OG1RF exposed to ampicillin, bacitracin, cephalotin or vancomycin was obtained via microarrays. Exposure to the β-lactams ampicillin and cephalotin resulted in the fewest transcriptional changes with 50 and 192 genes differentially expressed 60 min after treatment, respectively. On the other hand, treatment with bacitracin or vancomycin for 60 min affected the expression of, respectively, 377 and 297 genes. Despite the differences in the total number of genes affected, all antibiotics induced a very similar gene expression pattern with an overrepresentation of genes encoding hypothetical proteins, followed by genes encoding proteins associated with cell envelope metabolism as well as transport and binding proteins. In particular, all drug treatments, most notably bacitracin and vancomycin, resulted in an apparent metabolic downshift based on the repression of genes involved in translation, energy metabolism, transport and binding. Only 19 genes were up-regulated by all conditions at both the 30 and 60 min time points. Among those 19 genes, 4 genes encoding hypothetical proteins (EF0026, EF0797, EF1533 and EF3245) were inactivated and the respective mutant strains characterized in relation to antibiotic tolerance and virulence in the Galleria mellonella model. The phenotypes obtained for two of these mutants, ΔEF1533 and ΔEF3245, support further characterization of these genes as potential candidates for the development of novel preventive or therapeutic approaches.

Partial Text

Enterococci are normal inhabitants of the gastrointestinal (GI) tract of humans and animals. While typically harmless to healthy individuals, two enterococcal species, Enterococcus faecalis and E. faecium, are the leading organisms involved in hospital-acquired infections such as catheter-associated urinary tract infections, endocarditis, and surgical and burn wound infections [1], [2], [3]. Notably, the risk of death for patients infected with multidrug resistant strains, such as vancomycin-resistant enterococci (VRE), is considerably higher than for those infected with antibiotic susceptible strains [4]. In addition, multidrug resistant enterococci pose an additional health care threat as these strains may function as reservoirs for the dissemination of antibiotic resistance determinants to other opportunistic pathogens [5].

In this study, we used microarrays to obtain a snapshot of the transcriptional responses of E. faecalis to four different antibiotics that target CW biosynthesis, namely ampicillin, bacitracin, cephalotin and vancomycin. Treatment with all four drugs, most notably bacitracin and vancomycin, resulted in an apparent metabolic downshift based on the repression of a number of genes involved in translation, energy metabolism, transport and binding. This may be an indication that cells are saving or redirecting energy resources to maintain CW integrity and survive the physiological stress imposed by the antibiotics. In fact, genes involved in CW metabolism were highly affected, and overwhelmingly up-regulated during antibiotic treatment. For example, several genes coding for penicillin binding proteins (PBPs), responsible for CW cross-linking, and genes from the dlt operon, responsible for D-alanylation of lipoteichoic acids (LTA), were highly induced by bacitracin, cephalotin and vancomycin. Notably, esterification of LTA with D-alanine has been directly linked to vancomycin tolerance in both S. aureus and E. faecium[29], [30].