Characterisation of Bacitracin Stress Response and Undecaprenyl pyrophosphate phosphatase in Enterococcus faecalis
Bacitracin is a mixture of closely related polypeptide antibiotics and is widely used as a topical antibiotic in humans and extensively for prophylaxis and therapy in food animals. Enterococcus faecalis is a microorganism that has an exceptional ability to survive under a variety of stressful conditions. Bacitracin stress response of E. faecalis has not been studied at a global level (such as a proteomic analysis). Low-level bacitracin resistance of E. faecalis has been reported, but the molecular mechanism of resistance remains unclear. Undecaprenyl pyrophosphate phosphatases (UppPs) are enzymes important in the functioning of the essential lipid carrier undecaprenyl phosphate (UP) that is required for the cell wall synthesis. UppP dephosphorylate undecaprenyl pyrophosphate (UPP) to UP. UppPs are reported to contribute to bacitracin resistance in some bacterial genera. Two types of UppPs, the BacA-type and PAP2-type have been described. The UppP proteins in the genus Enterococcus have not been studied. The first main aims of this thesis were to characterise the BacA-type uppP in E. faecalis and to determine the role of BacA-type uppP (uppP) in low-level bacitracin resistance in E. faecalis. The uppP gene was identified in the genomes of laboratory (JH2-2) and clinical (V583) strains of E. faecalis. The BacA-type UppP protein from E. faecalis showed high sequence identity to the E. coli BacA-type UppP and was predicted to be a membrane protein with eight transmembrane helices. When uppPs in E. faecalis strains JH2-2 and V583 were inactivated, uppP mutants showed no significant changes in growth rate, colony morphology, biofilm formation and incorporation of N-acetyl-D-[1-3H] glucosamine into the peptidoglycan. The bacitracin MICs of JH2-2 uppP and V583 uppP mutants were found to be 3-4 and 4-6 µg/ml respectively. The MICs for the wild-type strains JH2-2 and V583 were 32 and 48 µg/ml respectively. When uppP was expressed in the wild-type backgrounds, the MIC for bacitracin increased to 128-≥256 µg/ml. Inactivation of uppP had no effect on the MICs for cefoxitin, teicoplanin, vancomycin, gentamicin, enrofloxacin, D-cycloserine, oxacillin and no effect on susceptibility to selected stress agents (glycine, lysozyme, NaCl, sodium dodecyl sulfate, low and high pH, oxidative and ethanol). Gene fusions (uppP-lacZ) and 5'-RACE were used to study uppP expression. The genetic organisation of uppP in E. faecalis strains JH2-2 and V583 (and 2 other strains analysed) suggested it was not likely to be co-transcribed with other genes and expression was driven from a σ70 promoter. The expression of uppP-lacZ was constitutive and was not affected by bacitracin or cell wall-active antimicrobials. This study demonstrated that low-level bacitracin resistance in E. faecalis was mediated by uppP; multiple copies of uppP lead to increased bacitracin resistance; uppP was not essential for growth; and uppP mutants were not compromised under a variety of stresses. Attempts to overproduce the UppP protein from E. faecalis for biochemical and functional studies proved unsuccessful. It is known that E. faecalis bcrAB provides high-level bacitracin resistance and that bcrD (in the same operon with bcrAB) is not required for high-level bacitracin resistance. E. faecalis BcrD protein showed high sequence similarity to E. faecalis BacA-type UppP. This study showed that bcrD does not contribute to low-level bacitracin resistance in E. faecalis and does not complement uppP mutant. The second main aim of this thesis was to investigate the bacitracin stress response of E. faecalis by means of a proteomic approach. Strains JH2-2 and V583 were treated with 64 µg/ml bacitracin for 1 hour and protein extracts displayed using 2-D electrophoresis. MALDI-TOF/TOF and MASCOT was used to identify differentially regulated proteins due to bacitracin stress and which were statistically significant. Within these proteins 72 % (38/53) from strain V583 and 65 % (26/40) from strain JH2-2 were up-regulated. Proteins known to be associated with response to other stresses were up-regulated, indicating that there was a general stress response due to bacitracin stress. A probable mechanism used in response to bacitracin stress is the incorporation of rhamnopolysaccharides into the cell wall. This possibly forms a barrier and reinforces the cell wall. This study suggests that in response to bacitracin stress strain V583 synthesises more UPP and detoxifies the cell from bacitracin by using EF1753 (a YvlB like protein) and a Bacillus subtilis BceAB like ABC transporter (EF2050-EF2049). This study has identified new candidate proteins and probable mechanisms used by E. faecalis under bacitracin stress.
Advisor: Cook, Gregory; Fineran, Peter; Kalamorz, Falk
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: Bacitracin Stress; Undecaprenyl pyrophosphate phosphatase
Research Type: Thesis