PhoU2 but Not PhoU1 as an Important Regulator of Biofilm Formation and Tolerance to Multiple Stresses by Participating in Various Fundamental Metabolic Processes in Staphylococcus epidermidis

Abstract

PhoU, a conserved protein that has been proposed to coordinate phosphate import, is a negative regulator of drug tolerance in most bacteria. In Staphylococcus epidermidis, the role of PhoU in biofilm formation and drug tolerance has not yet been investigated. Two PhoU homologs in the genome of S. epidermidis have been identified by the presence of the conserved motif E(D)XXXD of PhoU. We separately constructed ΔphoU1 and ΔphoU2 mutants of S. epidermidis strain 1457. The ΔphoU2 mutant displayed growth retardation, a weakened biofilm formation capacity, a higher sensitivity to H2O2, and reduced tolerance to multiple antibiotics. However, deletion of phoU1 had no effect on those. We compared the transcriptome profiles of the ΔphoU2 and ΔphoU1 mutants with that of the parent strain. In the ΔphoU2 mutant, expression of genes related to inorganic phosphate uptake was significantly upregulated (pst operon) and the levels of intracellular inorganic polyphosphate (polyP) were increased. In the ΔphoU2 mutant, expression of enzymes in the pentose phosphate pathway (PPP) was downregulated and less NADP (NADPH) was detected, consistent with the high sensitivity to H2O2 and the growth retardation of the ΔphoU2 mutant. The upregulated expression of ATP synthase was consistent with the high intracellular ATP content in the ΔphoU2 mutant, which may have been related to the lower drug tolerance of the ΔphoU2 mutant. This study demonstrates that PhoU2, but not PhoU1, in S. epidermidis regulates bacterial growth, biofilm formation, oxidative stress, and drug tolerance in association with alterations to inorganic phosphate metabolism, the pentose phosphate pathway, galactose metabolism, the tricarboxylic acid (TCA) or citric cycle, glycolysis and gluconeogenesis, and respiratory reactions.IMPORTANCE PhoU is widely conserved throughout the bacterial kingdom and plays an important role in response to stress and metabolic maintenance. In our study, two PhoU homologs were found in S. epidermidis. The function of phoU2, but not phoU1, in S. epidermidis is related to growth, drug tolerance, the oxidative stress response, polyP levels, and ATP accumulation. In addition, phoU2 regulates biofilm formation. Hence, phoU2 is a regulator of both drug tolerance and biofilm formation, which are two bacterial properties that present major challenges to the clinical treatment of infections. Analysis of differential gene expression revealed that phoU2 is involved in fundamental metabolic processes, such as the PPP pathway. These findings indicate that phoU2 is a crucial regulator in S. epidermidis.

ClpP participates in stress tolerance, biofilm formation, antimicrobial tolerance, and virulence of Enterococcus faecalis

“…To determine which antimicrobial concentrations ensured that only drug-tolerant bacterial cells survived, we performed time-killing assays for six antimicrobials. Based on previous research [28] and our preliminary results, the concentrations of six antimicrobials were set at 50× MIC. As shown in Fig.…”

“…E. faecalis ATCC 47077 (OG1RF; GenBank accession number CP002621.1) and ATCC 29212 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). E. faecalis strains were cultured in tryptic soya broth (TSB; Oxoid, Basingstoke, UK) as previously described [28]. TSBG (TSB medium added 0.25% glucose) for biofilm formation detection.…”

“…TSBG (TSB medium added 0.25% glucose) for biofilm formation detection. Electroporation was used for plasmid transformation, and B2 medium was used for bacteria recovery [28]. The antibiotics used in this study were purchased from Sigma Chemical Co. (St Louis, MO, USA) and used at concentrations of 20 mg/L for chloramphenicol and 750 or 25 mg/L for erythromycin.…”

“…Briefly, the upstream and downstream fragments of OG1RF_10505 (gene: clpP; product: ATP-dependent Clp protease proteolytic subunit), which is highly homologous (86.8%) to SA0723 (product as the ClpP protease) of S. aureus N315 strain [23], were amplified from OG1RF by PCR and separately cloned into the pJRS233 vector to generate pJRS233-ΔclpP. The recombinant plasmid pJRS233-ΔclpP was successively transferred and electroporated into wildtype OG1RF strain, then the pJRS233-ΔclpP clones were selected by variable temperature screening as previously described [28]. The complemented ΔclpP mutant strain was constructed using the E. coli -Streptococcus shuttle vector pIB166.…”

“…Overnight cultures of E. faecalis strains were diluted 1: 200 in fresh TSB medium and incubated at 37°C for 4 h until an OD 600 of 1.0 was reached. After 10-fold serial dilution, 5 μL of the aliquot was spotted onto a TSB agar plate containing 0.008% SDS and incubated at 37°C for 24 h. Bacterial colonies on the plates were photographed and counted [28]. Three independent experiments were performed, and representative results are shown.…”