Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant

Abstract

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

FIG 1

Numbers of S. aureus genes with altered expression in cultures with serum or pulmonary surfactant. S. aureus Newman was aerobically cultured in TSB medium containing 10% calf serum or 0.3 mg/ml Surfacten, and total RNAs extracted from bacteria at an OD₆₀₀ of 1 were subjected to RNA-seq analysis using a next-generation sequencer. Circles written with dashed and solid lines indicate the results for samples treated with serum and surfactant, respectively. Numbers in blue circles show downregulated genes, and those in red circles indicate upregulated genes for each treatment.

FIG 2

Pulmonary surfactant-dependent induction of S. aureus essC, psiA, and hlgB genes. (A) S. aureus Newman was cultured in TSB medium containing either 10% serum or 0.3 mg/ml Surfacten to an OD₆₀₀ of 1. Amounts of essC, psiA, and hlgB mRNAs were determined by quantitative RT-PCR. Each value was normalized to 16S rRNA, and the ratio of the surfactant-treated to the nontreated group was calculated. Data represent means ± standard deviations (SD) for 4 experiments. For each gene, statistical differences compared with the nontreated group were analyzed by one-way analysis of variance (ANOVA) with Dunnett's multiple-comparison test (*, P < 0. 01). (B) Dose dependency of pulmonary surfactant for the induction of S. aureus gene expression. RNAs were collected from bacteria at an OD₆₀₀ of 1 and subjected to quantitative RT-PCR analysis. The expression level of each gene was normalized to that of 16S rRNA, and the induction ratio relative to the nontreated group is indicated. (C) Induction of gene expression by pulmonary surfactant in CA-MRSA. MW2 (USA400) and FRP3757 (USA300) were cultured in the presence of 0.3 mg/ml Surfacten to an OD₆₀₀ of 1, and quantitative RT-PCR analysis of each gene was performed. Data represent means ± SD for 3 or 4 experiments. Statistical differences for each gene compared with nontreated groups were analyzed by Student's t test (*, P < 0.05).

FIG 3

Induction of essC, psiA, and hlgB genes in S. aureus by pulmonary surfactant lipid components. (A) Effects of lipid fractions of cow lung homogenates on gene expression of S. aureus. Cow lung homogenates were fractionated by the Bligh-Dyer method to an organic phase (Organic-fr.) and a water phase (Water-fr.). S. aureus Newman was cultured in TSB medium supplied with either the organic or water phase to an OD₆₀₀ of 1, and the expression levels of the essC, psiA, and hlgB genes relative to those of the nontreated group were determined by quantitative RT-PCR. Data represent means ± SD for 4 experiments. Statistical differences were analyzed by one-way ANOVA with Tukey's multiple-comparison test (*, P < 0.001). (B) Effects of lipids contained in pulmonary surfactant on gene expression of S. aureus. S. aureus Newman was cultured in TSB medium supplied with 0.1 (left) or 1 (right) mg/ml of either DPPC, POPC, POPG, or palmitate to an OD₆₀₀ of 1. mRNA levels of the essC, psiA, and hlgB genes were measured by quantitative RT-PCR. The expression level of each gene was normalized with that of 16S rRNA, and the induction ratio relative to the nontreated group is indicated. Data represent means ± SD for 3 or 4 experiments. Statistical differences were analyzed by one-way ANOVA with Tukey's multiple-comparison test (*, P < 0.05). (C) Effects of oleate and detergents on gene expression of S. aureus. S. aureus Newman was cultured in TSB medium containing either 0.1 mg/ml oleate, 0.0003% SDS, 0.006% Triton X-100, or 0.006% Tween 20 to an OD₆₀₀ of 1. Expression levels of the three genes relative to those in the nontreated group were determined by quantitative RT-PCR as described above. Data represent means ± SD for 7 experiments (*, P < 0.05; #, P < 0.005).

FIG 4

Palmitate and pulmonary surfactant upregulate essC and hlgB promoter activities. Promoter sequences of either the essC (A) or hlgB (B) gene were inserted into a reporter plasmid upstream of the luciferase gene. S. aureus Newman harboring each plasmid was cultured in TSB medium supplied with 0.1 mg/ml palmitate or 0.3 mg/ml Surfacten to an OD₆₀₀ of 0.7. The luciferase activities were measured with a luminometer, and the luminescence units were normalized to the protein amounts. Data represent means and SD for 3 experiments. Statistical differences compared with the nontreated group were analyzed by one-way ANOVA with Dunnett's multiple-comparison test (*, P < 0.05). RLU, relative light units.

FIG 5

Involvement of SigB in S. aureus gene induction by palmitate and pulmonary surfactant. (A) Requirement of the sigB gene in palmitate- and surfactant-dependent gene induction. S. aureus Newman as the parent strain (wild type [WT]) or a disruption mutant of the sigB gene (ΔsigB) was cultured in TSB medium containing 0.1 mg/ml palmitate or 0.3 mg/ml Surfacten to an OD₆₀₀ of 1. Expression levels of the three genes relative to those of the nontreated group were determined by quantitative RT-PCR as described in the text. Data represent means ± SD for 3 experiments. Statistical differences between the WT and ΔsigB strains were analyzed by Student's t test (*, P < 0.05). (B) Complementation of phenotypes of the S. aureus sigB disruption mutant. Either a control plasmid (pHY300E) or a complementation plasmid containing an open reading frame of the sigB gene (pHYsigB) was introduced into the ΔsigB mutant, resulting in ΔsigB/pHY or ΔsigB/pHYsigB, respectively. Cells were cultured in the presence of 0.1 mg/ml palmitate or 0.3 mg/ml Surfacten to an OD₆₀₀ of 1, and quantitative RT-PCR analysis of the three genes was performed as described in the text. Data represent means ± SD for 3 experiments. Statistical differences between the ΔsigB/pHY and ΔsigB/pHYsigB strains were analyzed by Student's t test (*, P < 0.05). (C) Induction of sigB gene expression in S. aureus by palmitate and pulmonary surfactant. S. aureus Newman was cultured in TSB medium supplied with either 0.1 mg/ml palmitate or 0.3 mg/ml Surfacten to an OD₆₀₀ of 1. The expression level of the sigB gene relative to that for the nontreated group was determined by quantitative RT-PCR. Data represent means ± SD for 3 experiments. Statistical differences compared to the nontreated group were analyzed by Student's t test (*, P < 0.05). (D) Induction of SigB-regulated genes by palmitate and pulmonary surfactant. S. aureus Newman was cultured in TSB medium supplied with either 0.1 mg/ml palmitate or 0.3 mg/ml Surfacten to an OD₆₀₀ of 1. Expression levels of the asp23, clfA, csb7, and NWMN_2089 genes relative to those for the nontreated group were determined by quantitative RT-PCR. Data represent means ± SD for 3 experiments. For each gene, statistical differences compared with the nontreated group were analyzed by one-way ANOVA with Dunnett's multiple-comparison test (#, P < 0.01; *, P < 0.001).

FIG 6

Involvement of essC, psiA, and hlgB genes in S. aureus virulence in a mouse pneumonia model. (A) Survival of S. aureus mutants in mouse lungs. Sublethal doses of either S. aureus Newman (WT) or a mutant strain (ΔessC, ΔpsiA, or ΔhlgB strain) were administered into the lungs of anesthetized mice through the nasal cavities. After 2 days, the lungs were dissected and numbers of viable bacteria in the homogenates were determined. Data represent means ± SD for 5 mice. Statistical differences compared with the WT were analyzed by one-way ANOVA with Dunnett's multiple-comparison test (*, P < 0.05). (B) Survival of S. aureus mutants and complementation strains in mouse lungs. Mice were infected with either S. aureus mutants harboring empty vectors (ΔessC/pHY, ΔpsiA/pHY, and ΔhlgB/pHY strains) or mutants harboring complementation vectors (ΔessC/pHYessC, ΔpsiA/pHYpsiA, and ΔhlgB/pHYhlgB strains). After 2 days, the numbers of viable bacteria in lung homogenates were determined. Data represent means ± SD for 3 to 6 mice. Statistical differences of the mutants compared with each complemented strain were analyzed by Student's t test (*, P < 0.001). (C) Host-killing ability in a mouse pneumonia model by mutant and complementation strains of surfactant-inducible genes. Bacterial cell suspensions of S. aureus Newman harboring an empty vector (×; WT/pHY), disruption mutants of each surfactant-inducible gene containing empty vectors (open symbols; ΔessC/pHY, ΔpsiA/pHY, and ΔhlgB/pHY strains), or mutants complemented with each gene (closed symbols; ΔessC/pHYessC, ΔpsiA/pHYpsiA, and ΔhlgB/pHYhlgB strains) were administered into the lungs of anesthetized mice through the nasal cavities (n = 7). Statistical differences between the wild type and each mutant or between each mutant and its complementation strain were determined to be significant (P < 0.05) by the log rank test.

FIG 7

Proposed mechanism of S. aureus virulence gene induction by free fatty acids in pulmonary surfactant.