Fatty acids can inhibit Staphylococcus aureus SaeS activity at the membrane independent of alterations in respiration

Abstract

In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.

Keywords: S. aureus; MRSA; SaeRS; two-component system; virulence factor.

Figure 1.. Impact of different fatty acids on Sae activity.

P_coa-lacZ activity of exponential phase (six hours) S. aureus strain AH1263 grown in TSB supplemented with the indicated fatty acids (μM) represented as Modified Miller Units (M.M.U.) Fatty acids are as follows: 12:0 lauric acid, 14:0 myristic acid, 14:1 trans- myristelaidic acid, 16:1 cis- palmitoleic acid, 18:1 cis- oleic acid, 18:2 cis cis- linoleic acid, 16:0- palmitic acid, 18:0- stearic acid, 18:1 trans- elaidic acid, and 18:2 trans trans- linoelaidic acid. Data are representative of multiple experiments and are the mean (n=3) with SEM. *p<0.05, **p<0.01 compared to TSB alone by One-way ANOVA.

Figure 2.. Impact of fatty acids on S. aureus respiratory activity.

A) Respiratory analysis using CTC for wild-type S. aureus grown to exponential phase (six hours) in TSB supplemented with 3, 31, or 314μM oleic acid (C18:1 cis) or elaidic acid (18:1 trans). Data is the mean (n=3) with SEM. B) Ratio of NAD+ to NADH was determined for wild-type S. aureus grown in the presence of 31 or 314 μM oleic acid (C18:1 cis). Data is the mean (n=4) with SEM. C) CTC analysis of wildtype grown in either 30 μM linoleic acid (C18:2 cis) or 62 μM linoelaidic acid (C18:2 trans). Data is the mean (n=9) with SEM. Data is representative of multiple experiments. **p<0.01 compared to TSB alone by One-way ANOVA.

Figure 3.. SaeP and SaeQ do not impact SaeRS sensitivity to fatty acid treatment.

A) P_P3-lacZ activity of S. aureus strain AH1263. B) P_coa-lacZ activity of S. aureus AH1263, ΔsaeP, ΔsaeQ, and saeR::Tn. C) P_coa-lacZ activity of S. aureus AH1263, AH1263 with non-native P1 promoter of SaeRS, and AH1263 non-native P1 promoter ΔsaePQ. All cultures were grown in TSB with or without 31μM oleic acid (OA) to exponential phase (six hours). Data is represented as Modified Miller Units (M.M.U.) and is the mean (n=3) with SEM. All data are representative of multiple experiments. *p<0.01 compared to TSB alone by student’s t-test.

Figure 4.. Constitutive SaeS is not suppressed by oleic acid..

P_hla-lacZ (A) and P_coa-lacZ (B) activity (M.M.U) of wild-type AH1263, AH1263 SaeS^L18P, wild-type Newman, and Newman SaeS^P18L grown to late exponential phase (six hours) in TSB (solid bars) or TSB supplemented with 31 μM oleic acid (empty bars). Data are representative of multiple experiments and is the mean (n=3) with SEM. *p<0.01 compared to TSB alone by student’s t-test.

Figure 5.. Replacement or removal of native SaeS transmembrane domains desensitizes SaeRS to oleic acid.

Strains were grown to late exponential phase (six hours) in TSB (filled bars) or TSB supplemented with 31μM oleic acid (empty bars). A) Graphical representation of the different SaeS constructs used in this study. Blue indicates SaeS domains and green indicates GraS domains. B) Newman Δsae strain expressing chimeric the USA300 SaeS, chimeric GraS/SaeS, or SaeS^cyto. Activity was measured via P_coa-gfp and is represented as relative fluorescent units normalized to OD600 and is the mean (n=6) with SEM. C) AH1263 wildtype (WT) or ΔsaeRS strain expressing chimeric GraS/SaeS or SaeS^cyto. Activity was measured via P_coa-lacZ. Data are represented as Modified Miller Units (M.M.U.). Data are representative of multiple experiments and is the mean (n=3) with SEM. For both panels, *p<0.01 compared to TSB alone by student’s t-test.