Staphylococcus aureus Overcomes Anaerobe-Derived Short-Chain Fatty Acid Stress via FadX and the CodY Regulon

Abstract

Chronic rhinosinusitis (CRS) is characterized by immune dysfunction, mucus hypersecretion, and persistent infection of the paranasal sinuses. While Staphylococcus aureus is a primary CRS pathogen, recent sequence-based surveys have found increased relative abundances of anaerobic bacteria, suggesting that S. aureus may experience altered metabolic landscapes in CRS relative to healthy airways. To test this possibility, we characterized the growth kinetics and transcriptome of S. aureus in supernatants of the abundant CRS anaerobe Fusobacterium nucleatum. While growth was initially delayed, S. aureus ultimately grew to similar levels as in the control medium. The transcriptome was significantly affected by F. nucleatum metabolites, with the agr quorum sensing system notably repressed. Conversely, expression of fadX, encoding a putative propionate coenzyme A (CoA)-transferase, was significantly increased, leading to our hypothesis that short-chain fatty acids (SCFAs) produced by F. nucleatum could mediate S. aureus growth behavior and gene expression. Supplementation with propionate and butyrate, but not acetate, recapitulated delayed growth phenotypes observed in F. nucleatum supernatants. A fadX mutant was found to be more sensitive than wild type to propionate, suggesting a role for FadX in the S. aureus SCFA stress response. Interestingly, spontaneous resistance to butyrate, but not propionate, was observed frequently. Whole-genome sequencing and targeted mutagenesis identified codY mutants as resistant to butyrate inhibition. Together, these data show that S. aureus physiology is dependent on its cocolonizing microbiota and metabolites they exchange and indicate that propionate and butyrate may act on different targets in S. aureus to suppress its growth. IMPORTANCE Staphylococcus aureus is an important CRS pathogen, and yet it is found in the upper airways of 30% to 50% of people without complications. The presence of strict and facultative anaerobic bacteria in CRS sinuses has recently spurred research into bacterial interactions and how they influence S. aureus physiology and pathogenesis. We show here that propionate and butyrate produced by one such CRS anaerobe, namely, Fusobacterium nucleatum, alter the growth and gene expression of S. aureus. We show that fadX is important for S. aureus to resist propionate stress and that the CodY regulon mediates growth in inhibitory concentrations of butyrate. This work highlights the possible complexity of S. aureus-anaerobe interactions and implicates membrane stress as a possible mechanism influencing S. aureus behavior in CRS sinuses.

Keywords: Fusobacterium; Staphylococcus aureus; anaerobes; microbiome; short-chain fatty acids; sinusitis.

FIG 1

S. aureus growth is impaired in F. nucleatum supernatants. (A) Relative abundances of Fusobacterium and Staphylococcus in sinus mucus from patients with chronic rhinosinusitis are inversely correlated (6). (B) Representative growth curve of S. aureus USA300 in Brucella broth (BB; control); BB supplemented with 5 mM acetate, 5 mM propionate, and 15 mM butyrate; and cell-free supernatants from F. nucleatum (Fn CFS). (C) Production of SCFAs by F. nucleatum after 48 h (Fn CFS) and their levels after S. aureus (USA300) growth in Fn CFS. SCFAs were below the limit of detection in sterile BB. All data shown in B and C are the mean ± standard deviation of three biological replicates.

FIG 2

F. nucleatum metabolites significantly impact the S. aureus transcriptome. (A) Heatmap depicting log₁₀-transformed S. aureus gene expression in control media (BB) and F. nucleatum supernatant (CFS) as detected by NanoString. Genes were clustered with unsupervised hierarchical clustering. (B) MA plot representation of S. aureus gene expression in F. nucleatum CFS relative to the control medium. Genes were considered significant if they had a log₂-fold change of ≥1 and a Benjamini-Hochberg-adjusted P value of <0.05.

FIG 3

Propionate and butyrate repress the S. aureus agr system but fail to induce biofilm. (A) S. aureus carrying pAH1 (P_agr-mCherry) was grown for 24 h in LB supplemented with 100 mM sodium acetate, propionate, or butyrate (n = 3 biological replicates with n = 3 cultures per replicate). Fluorescence was measured and normalized to culture density for each replicate and then normalized to the LB controls. Significance was determined by. (B, C) Expression of cidA and lrgA from S. aureus in LB supplemented with SCFAs (n = 3). Copy number was determined via standard curve and normalized to the gmk housekeeping gene. (D) Crystal violet assay quantifying biofilm formation in LB, LB supplemented with glucose (positive control for increased biofilm formation), or LB supplemented with 100 mM each SCFA. All data are the mean ± standard deviation of three biological replicates. Significance was determined by Kruskal-Wallis one-way ANOVA with Dunn’s multiple-comparison test for A and ordinary one-way ANOVA with Holm-Sidak’s multiple comparison’s test for B to D. **, P < 0.01; ****, P < 0.0001.

FIG 4

The fad operon is induced by propionate and butyrate. (A and B) Reverse transcription-quantitative PCR was used to detect fad operon expression in control (BB) or F. nucleatum CFS (A) or in LB supplemented with 100 mM sodium propionate (B). Cultures were grown to an OD₆₀₀ of approximately 0.2 to 0.3 prior to RNA extraction. Data shown are mean ± standard deviation of three biological replicates. Significance was determined by two-way ANOVA with Siadak’s multiple-comparison test. **, P < 0.01; ****, P < 0.0001.

FIG 5

The ΔfadX mutant is more susceptible to growth inhibition by propionate than the wild type. Combined growth curves (n = 4) of wild-type USA300 or the ΔfadX mutant in 100 mM of the sodium salts of acetate (A), propionate (B), or butyrate (C). Data shown are the mean ± standard deviation of three biological replicates.

FIG 6

Spontaneous S. aureus codY mutants are not inhibited by butyrate. Combined growth curves of the ΔfadX mutant and butyrate-resistant derivatives in 100 mM sodium acetate (A), sodium propionate (B), or sodium butyrate (C). (D) Alignment of CodY protein sequences from diverse Gram-positive bacteria and S. aureus, including butyrate-resistant mutants (but^R1 and but^R2); but^R1 encodes a premature stop codon at position 66 (blue), while but^R2-4 mutants have a 20-amino acid deletion from positions 171 to 190 (red). The but^R3 and but^R4 mutants were omitted from the alignment as their codY mutations are identical to but^R2. All data shown in A to C are the mean ± standard deviation of three biological replicates.

FIG 7

codY mutation is sufficient to escape butyrate growth inhibition, although not likely through Fad activity. (A) Growth of wild-type S. aureus and a codY::tn mutant in 100 mM sodium butyrate. (B) Expression of the fad operon from the same strains as in A, grown in LB to an OD₆₀₀ of approximately 0.2 to 0.3. All data shown are the mean ± standard deviation of three biological replicates.