Biofilm Formation in Streptococcus agalactiae Is Inhibited by a Small Regulatory RNA Regulated by the Two-Component System CiaRH

Abstract

Regulatory small RNAs (sRNAs) are involved in the adaptation of bacteria to their environment. CiaR-dependent sRNAs (csRNAs) are controlled by the regulatory two-component system (TCS) CiaRH, which is widely conserved in streptococci. Except for Streptococcus pneumoniae and Streptococcus sanguinis, the targets of these csRNAs have not yet been investigated. Streptococcus agalactiae, the leading cause of neonatal infections, has four conserved csRNA genes, namely, srn015, srn024, srn070, and srn085. Here, we demonstrate the importance of the direct repeat TTTAAG-N5-TTTAAG in the regulation of these csRNAs by CiaRH. A 24-nucleotide Srn024-sap RNA base-pairing region is predicted in silico. The sap gene encodes a LPXTG-cell wall-anchored pullulanase. This protein cleaves α-glucan polysaccharides such as pullulan and glycogen present in the environment to release glucose and is involved in adhesion to human cervical epithelial cells. Inactivation of S. agalactiae pullulanase (SAP) leads to no bacterial growth in a medium with only pullulan as a carbon source and reduced biofilm formation, while deletion of ciaRH and srn024 genes significantly increases bacterial growth and biofilm formation. Using a new translational fusion vector, we demonstrated that Srn024 is involved in the posttranscriptional regulation of sap expression. Complementary base pair exchanges in S. agalactiae suggest that Srn024 interacts directly with sap mRNA and that disruption of this RNA pairing is sufficient to yield the biofilm phenotype of Srn024 deletion. These results suggest the involvement of Srn024 in the adaptation of S. agalactiae to environmental changes and biofilm formation, likely through the regulation of the sap gene. IMPORTANCE Although Streptococcus agalactiae is a commensal bacterium of the human digestive and genitourinary tracts, it is also an opportunistic pathogen for humans and other animals. As the main cause of neonatal infections, it is responsible for pneumonia, bacteremia, and meningitis. However, its adaptation to these different ecological niches is not fully understood. Bacterial regulatory networks are involved in this adaptation, and the regulatory TCSs (e.g., CiaRH), as well as the regulatory sRNAs, are part of it. This study is the first step to understand the role of csRNAs in the adaptation of S. agalactiae. This bacterium does not currently exhibit extensive antibiotic resistance. However, it is crucial to find alternatives before multidrug resistance emerges. Therefore, we propose that drugs targeting regulatory RNAs with Srn024-like activities would affect pathogens by reducing their abilities to form biofilm and to adapt to host niches.

Keywords: CiaRH; SAP pullulanase; Streptococcus agalactiae; adaptation; biofilm; csRNAs; regulation; sRNAs.

FIG 1

Activation of the four csRNA promoters by the TCS CiaRH. (A) Sequence logo of S. agalactiae promoters for csRNA predicted genes. The CiaR-related repeats are underlined by arrows. The transcriptional start site and the −10 region are underlined. WebLogo produced the logo. (B and C) The promoter activities of srn015 and srn024 (B) and srn070 and srn085 (C) were measured in S. agalactiae NEM316, ΔciaRH, and ΔciaRH::ciaRH^{in situ} strains. Results are presented as means ± standard deviations of three independent experiments. The significance was determined by ANOVAs and Student t tests. **, P < 0.01; ****, P < 0.0001.

FIG 2

Involvement of the CiaR binding site in the regulation of Srn024. (A) Sequences of native (P_srn024) and mutated (P_srn024* and P_srn024**) promoters of the srn024 gene. The performed substitutions are shown in bold and indicated by stars. In the promoter sequences, the CiaR-related repeat is underlined by arrows and boxed. The transcriptional start site is shown in bold. The putative −35 sequence is shown in red, and the −10 region is boxed. (B) The activity of native and mutated promoters of srn024 was measured in S. agalactiae strain NEM316. Results are presented as means ± standard deviations of three independent experiments. The significance was determined by ANOVAs and Student t tests. ***, P < 0.001.

FIG 3

Predicted interaction between Srn024 and sap mRNA. (A) Putative secondary structure of the csRNA Srn024, as predicted by RNAfold (43). (B) Putative interaction between csRNA Srn024 and the sap mRNA target as predicted by IntaRNA. The first codon of sap mRNA is boxed.

FIG 4

Posttranscriptional regulation of sap by Srn024. (A) Sequences of native sap mRNA, sapΔ24 (24-nt deletion is boxed), and sapS (7-nt substitutions are shown in bold and underlined). (B) Translation of SAP was measured in S. agalactiae NEM316, ΔSrn024, and ΔSrn024::Srn024^{in situ} strains. Results are presented as means ± standard deviations of three independent experiments. The significance was determined by ANOVAs and Student t tests. **, P < 0.01.

FIG 5

Posttranscriptional regulation of sap by Srn024. (A) Putative interaction sequence between sap mRNA and Srn024. Performed substitutions are shown in bold and underlined. (B) Translation of SAP was measured in S. agalactiae NEM316 and Srn024S strains. Results are presented as means ± standard deviations of three independent experiments. The significance was determined by ANOVAs and Student t tests. **, P < 0.01.

FIG 6

SAP protein is essential for S. agalactiae growth in the presence of pullulan as a unique carbon source, while CiaRH and Srn024 repress its growth in the same medium. Growth of NEM316, sap*, and sap*::sap^{in situ} strains (A), growth of NEM316, Δsrn024, and Δsrn024::Δsrn024^{in situ} strains (B), and growth of NEM316, ΔciaRH, and ΔciaRH::ΔciaRH^{in situ} strains (C) in CDM with 1% pullulan was measured. Results are presented as means ± standard deviations of three independent experiments. Significance was determined by ANOVAs and Student t tests using the WT strain NEM316 as a reference.

FIG 7

SAP increases biofilm formation, while CiaRH and Srn024 inhibit it. Biofilm formation by NEM316, sap*, and sap*::sap^{in situ} strains (A), NEM316, Δsrn024, Δsrn024::Srn024^{in situ}, ΔciaRH, and ΔciaRH::ciaRH^{in situ} strains (B), and NEM316, Δsrn024, and srn024S strains (C) in TH broth supplemented with 1% glucose was measured. Results are presented as means ± standard deviations of at least three independent experiments. The significance was determined by ANOVAs and Student t tests. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.