The nitric oxide synthase gene negatively regulates biofilm formation in Staphylococcus epidermidis

Abstract

Staphylococcus epidermidis (S. epidermidis) is a clinically important conditioned pathogen that can cause a troublesome chronic implant-related infection once a biofilm is formed. The nitric oxide synthase (NOS) gene, which is responsible for endogenous nitric oxide synthesis, has already been found in the genome of S. epidermidis; however, the specific mechanisms associated with the effects of NOS on S. epidermidis pathogenicity are still unknown. The purpose of the current study was to investigate whether the NOS gene has an impact on biofilm formation in S. epidermidis. Bioinformatics analysis of the NOS gene was performed, and homologous recombination was subsequently employed to delete this gene. The effects of the NOS gene on biofilm formation of S. epidermidis and its underlying mechanisms were analyzed by bacterial growth assays, biofilm semiquantitative determination, Triton X-100-induced autolysis assays, and bacterial biofilm dispersal assays. Additionally, the transcription levels of fbe, aap, icaA, icaR and sigB, which are related to biofilm formation, were further investigated by qRT-PCR following NOS deletion. Phylogenetic analysis revealed that the NOS gene was conserved between bacterial species originating from different genera. The NOS deletion strain of S. epidermidis 1457 and its counterpart were successfully constructed. Disruption of the NOS gene resulted in significantly enhanced biofilm formation, slightly retarded bacterial growth, a markedly decreased autolysis rate, and drastically weakened bacterial biofilm dispersal. Our data showed that the fbe, aap and icaA genes were significantly upregulated, while the icaR and sigB genes were significantly downregulated, compared with the wild strain. Therefore, these data strongly suggested that the NOS gene can negatively regulate biofilm formation in S. epidermidis by affecting biofilm aggregation and dispersal.

Keywords: S. epidermidis; biofilm; chronic infection; nitric oxide; nitric oxide synthase gene.

Figure 1

Nitric oxide donor (SNP) inhibits NOS gene expression and biofilm formation in S. epidermidis. (A) SNPs of 0, 0.25, 0.5 and 1 mM exhibited no significant difference in the growth of S. epidermidis (p > 0.1). (B) The effect of SNP on the NOS gene was analyzed through fluorescent quantitative PCR (2^-△△Ct method). (C, D) The effect of SNP on the biofilm formation of S. epidermidis was analyzed by crystal violet staining. Experimental data are presented as the mean ± SD obtained from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Figure 2

Phylogenetic tree of the NOS gene in S. epidermidis 1457. The phylogenetic tree of NOS of S. epidermidis 1457 showed that it had the closest homology with NOS of S. epidermidis RP62A and S. epidermidis ATCC 12228.

Figure 3

Determination of the bacterial growth curves and observation of bacterial colony morphology. (A) The bacterial growth ability was significantly attenuated following NOS deletion. (B) NOS deletion apparently affected the size of bacterial colonies. SE1457: the wild-type strain; SE1457-ΔNOS: the deletion strain; SE1457-ΔNOS: cNOS: the complementation strain. These curves represent three independent experiments. **p < 0.01.

Figure 4

Determination of bacterial biofilm formation. Experimental data are presented as the mean ± SD obtained from three independent experiments. SE1457, SE1457-ΔNOS and SE1457-ΔNOS:cNOS are the wild strain, deletion strain and complement strain, respectively. *p < 0.05, **p < 0.01, ***p < 0.001. (A) Semi-quantitative determination of the biofilm formation of S. epidermidis at different culture time points. (B) Qualitative determination of the biofilm formation of S. epidermidis by crystal violet staining.

Figure 5

Determination of the bacterial autolysis induced by 0.01% Triton X-100 and bacterial biofilm dispersal capacity. (A) Experimental data are presented as the mean ± SD obtained from three independent experiments. SE1457, SE1457-ΔNOS and SE1457-ΔNOS:cNOS are the wild strain, deletion strain and complement strain, respectively. (B) Experimental data are presented as the mean ± SD obtained from three independent experiments. SE1457, SE1457-ΔNOS and SE1457-ΔNOS:cNOS are the wild strain, deletion strain and complement strain, respectively. *p < 0.05, **p < 0.01.

Figure 6

(A) Effects of NOS deletion on the transcriptional levels of fbe. (B) Effects of NOS deletion on the transcriptional levels of aap. (C) Effects of NOS deletion on the transcriptional levels of icaR. (D) Effects of NOS deletion on the transcriptional levels of SigB. (E) Effects of NOS deletion on the transcriptional levels of icaA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.