Streptococcus suis Encodes Multiple Allelic Variants of a Phase-Variable Type III DNA Methyltransferase, ModS, That Control Distinct Phasevarions

Abstract

Streptococcus suis is a significant cause of bacterial meningitis in humans, particularly in Southeast Asia, and is a leading cause of respiratory and invasive disease in pigs. Phase-variable DNA methyltransferases, associated with restriction-modification (R-M) systems, are a source of epigenetic gene regulation, controlling the expression of multiple genes. These systems are known as phasevarions (phase-variable regulons) and have been characterized in many host-adapted bacterial pathogens. We recently described the presence of a Type III DNA methyltransferase in S. suis, ModS, which contains a simple sequence repeat (SSR) tract within the open reading frame of the modS gene and which differed in length between individual strains. We also observed that multiple allelic variants of the modS gene were present in a population of S. suis isolates. Here, we demonstrate that a biphasic ON-OFF switching of expression occurs in the two most common ModS alleles, ModS1 and ModS2, and that switching is dependent on SSR tract length. Furthermore, we show using single-molecule real-time (SMRT) sequencing that ModS1 and ModS2 are active methyltransferases in S. suis ON-OFF switching of each ModS allele results in the regulation of distinct phasevarions, with the ModS2 phasevarion impacting growth patterns and antibiotic resistance. This is the first demonstration of a phase-variable Type III DNA methyltransferase in a Gram-positive organism that controls a phasevarion. Characterizing the phenotypic effects of phasevarions in S. suis is key to understanding pathogenesis and the development of future vaccines.IMPORTANCEStreptococcus suis is a causative agent of meningitis, polyarthritis, and polyserositis in swine, and it is a major cause of zoonotic meningitis in humans. Here, we investigate epigenetic gene regulation in S. suis by multiple phasevarions controlled by the phase-variable Type III DNA methyltransferase ModS. This is the first characterized example of a Type III R-M system regulating a phasevarion in a Gram-positive organism. We demonstrate that biphasic ON-OFF switching of ModS expression results in differences in bacterial growth and antibiotic resistance. Understanding the effects of ModS phase variation is required to determine the stably expressed antigenic repertoire of S. suis, which will direct and inform the development of antimicrobial treatments and vaccines against this important pathogen.

Keywords: R-M systems; Streptococcus suis; methyltransferase; phase variation; phasevarion; veterinary pathogen.

FIG 1

Expression of ModS alleles. (A) The modS gene contains a variable-length GAGCA_(n) simple sequence repeat (SSR) tract (gray box) near the start of the gene and a variable central target recognition domain (TRD), represented by the hatched box. The 5′ and 3′ regions of the modS gene are highly (>95% nucleotide identity) conserved (white). PCR over the SSR tract was determined by 6-carboxyfluorescein (FAM)-labeled PCR using the primers SsuT3-F-FAM and SsuT3-R and analyzed using fragment length analysis. (B) Alignment of the TRD regions of ModS1 and ModS2 showing <25% amino acid identity. An asterisk (*) represents identical amino acid residues; dots (.) represents similar amino acid residues (basic, acidic, and neutral); the TRD region is underlined. Alignments carried out in ClustalW. (C) Fragment length analysis traces of the enriched modS1 and modS2 populations of strains LSS89 and SS1056, respectively, containing three consecutive GAGCA_(n) SSR tract lengths. (D) Western blot analysis using ModS antiserum demonstrates that the ModS protein is only present in S. suis populations enriched for 21 repeats in ModS1 (LSS89) and 18 repeats in ModS2 (SS1056), demonstrating phase-variable expression of this protein.

FIG 2

Volcano plot demonstrating changes to protein expression as a result of ModS. SWATH-MS proteomics demonstrated a coverage of 450 of 1,964 identified proteins (∼23%) in the LSS89 ModS1 ON-OFF strain pair, and 411 of 1,905 (∼22%) identified proteins in the SS1056 ModS2 ON-OFF strain pair. The x axis indicates relative fold difference in protein abundance in ON compared to OFF; the y axis indicates statistical significance.

FIG 3

Growth curves of S. suis populations enriched for modS1 and modS2. ON-OFF strain pairs for ModS1 (strain LSS89) and ModS2 (SS1056) were grown in rich medium (THB-Y broth) for 18 h with shaking. Statistically significant differences (P < 0.05) in absorbance at each time point are indicated by asterisks, assessed using Student’s t test.