Streptococcus mutans SpxA2 relays the signal of cell envelope stress from LiaR to effectors that maintain cell wall and membrane homeostasis

Abstract

Streptococcus mutans is a major etiologic agent of dental caries, which is the most common chronic infectious disease worldwide. S. mutans is particularly adept at causing caries due to its exceptional capacity to form biofilms and its ability to survive acidic conditions that arrest acid production and growth in many more benign members of the oral microbiota. Two mechanisms utilized by S. mutans to tolerate acid are: modulation of the membrane fatty acid content and utilization of the F₁ F₀ -ATPase to pump protons out of the cytosol. In this study, the role of the spxA2 transcriptional regulator in these two pathways, and overall cell envelope homeostasis, was examined. Loss of spxA2 resulted in an increase in the proportion of saturated fatty acids in the S. mutans membrane and altered transcription of several genes involved in the production of these membrane fatty acids, including fabT and fabM. Furthermore, activity of the F₁ F₀ -ATPase was increased in the ∆spxA2 strain. Transcription of spxA2 was elevated in the presence of a variety of membrane stressors, and highly dependent on the liaR component of the LiaFSR system, which is known to sense cell envelope stress in many Gram-positive bacteria. Finally, deletion of ∆spxA2 led to altered susceptibility of S. mutans to membrane stressors. Overall, the results of this study indicate that spxA2 serves a crucial role in transmitting the signal of cell wall/membrane damage from the LiaFSR sensor to downstream effectors in the SpxA2 regulon which restore and maintain membrane and cell wall homeostasis.

Keywords: Streptococcus mutans; Spx; dental caries; environmental regulation; fatty acids.

Figure 1:. Membrane fatty acid composition is altered in ΔspxA2.

(A) GC-FAME analysis was used to determine membrane fatty acid composition from cultures of S. mutans UA159 and ΔspxA2 grown to steady-state pH values of 7 and 5 as described in Materials and Methods. Percentage of membrane composition is denoted in each sector where saturated fatty acids are shown in blue and unsaturated fatty acids in orange. All values are statistically significant in pairwise comparisons, except for ^ns, as determined by Student’s t-test, p < 0.05. Below each pie graph is the UFA:SFA ratio, and values are statistically significant in pairwise comparisons, the fold-change between pH 7 and pH 5 is also indicated. (B) Fatty acid chain lengths are displayed for cultures of S. mutans UA159 and ΔspxA2 grown to steady-state pH values of 7 and 5. Percentage of membrane composition representing fatty acids composed of indicated carbon chain-lengths is denoted in each sector. Only chain lengths representing at least 1% of the total are shown. Statistical significance is designated for major chain length groups, between pairs of like symbols, determined by Student’s t-test, p < 0.05. Below each pie graph is the ratio of C14 + C16 carbon chains to C18 + C20 carbon chains. The fold-change in this ratio between pH 7 and pH 5 is also indicated. Ratios were statistically significant in pairwise comparisons.

Figure 2:. spxA2 affects transcription of fabT and fabM.

(A) qRT-PCR enumerating fabT transcripts in RNA extracted from the indicated strains during steady-state growth at pH 7 or pH 5. (B) CAT assay quantifying transcription of fabT in the indicated strain from batch cultures buffered to either pH 7 or pH 5. (C) CAT assay quantifying transcription of fabM in the indicated strain from batch cultures buffered to either pH 7 or pH 5. All data is derived from 3 independent cultures assayed in triplicate. Asterisks indicate expression levels which are statistically significant between indicated conditions based on a Tukey’s Multiple Comparisons Test following a two-way ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****P < 0.0001; n = 3)

Figure 3:. ATPase activity is increased in the ΔspxA2 strain.

Graph illustrating ATPase activity, measured as described in Materials and Methods, in the indicated strains over time. Data is derived from 3 independent cultures assayed in triplicate. The ATPase activities of UA159 and ΔspxA2 were significantly different based on a paired Student’s t-test (p = 0.0162; n = 3)

Figure 4:. spxA2 transcription is dependent on LiaR, and responds to membrane stressors.

(A) CAT assay quantifying transcription of spxA2 in the indicated strain from batch cultures buffered to either pH 7 or pH 5. (B) CAT assay quantifying transcription of spxA2 in the indicated strain from batch cultures containing the indicated membrane stressor, as described in Materials and Methods. Data is derived from 3 independent cultures assayed in triplicate. Asterisks indicate expression levels which are statistically significant between indicated conditions based on a Tukey’s Multiple Comparisons Test following a two-way ANOVA (**, P < 0.01; ****P < 0.0001; n = 3).

Figure 5:. The ΔspxA2 strain is resistant to osmotic stress (NaCl) and the fatty acid biosynthesis inhibitor, cerulenin.

Growth of the indicated strain with or without 2.5% NaCl (A) or 10 μg/mL cerulenin (B). n = 10 for each condition.