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. 2014 Jun;82(6):2337–2344. doi: 10.1128/IAI.01411-13

DrsG from Streptococcus dysgalactiae subsp. equisimilis Inhibits the Antimicrobial Peptide LL-37

Danielle Smyth a,*, Ainslie Cameron a, Mark R Davies b,c, Celia McNeilly a, Louise Hafner d, Kadaba S Sriprakash a, David J McMillan a,e,✉
Editor: A Camilli
PMCID: PMC4019180  PMID: 24664506

Abstract

SIC and DRS are related proteins present in only 4 of the >200 Streptococcus pyogenes emm types. These proteins inhibit complement-mediated lysis and/or the activity of certain antimicrobial peptides (AMPs). A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp. equisimilis. Here we show that geographically dispersed isolates representing 14 of 50 emm types examined possess variants of drsG. However, not all isolates within the drsG-positive emm types possess the gene. Sequence comparisons also revealed a high degree of conservation in different S. dysgalactiae subsp. equisimilis emm types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatants. Unlike SIC, but similar to DRS, DrsG does not inhibit complement-mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelicidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. Conservation of prolines in the C-terminal region also suggests that these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP-inhibitory protein in S. dysgalactiae subsp. equisimilis and suggests that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of the complement-inhibitory activity of SIC may reflect its continuing evolution.

INTRODUCTION

Antimicrobial peptides (AMPs) are major components of the innate immune system that typically exert their antimicrobial activity via pore formation in the bacterial membrane (1). Some AMPs are also reported to have chemotactic properties, recruiting immune cells such as monocytes, neutrophils, and T cells to the site of bacterial colonization (2), or to directly stimulate chemokine or cytokine secretion (3). Active evasion or inhibition of AMP activity is therefore a strategy for the survival and multiplication of several pathogens that colonize the respiratory tract and skin (4).

Streptococcus pyogenes (group A streptococcus [GAS]) and S. dysgalactiae subspecies equisimilis (human group C and G streptococci) are related Gram-positive bacteria that colonize the skin and throat. While S. pyogenes is generally considered to be more virulent, the range of disease associated with S. dysgalactiae subsp. equisimilis is similar to that for GAS (5, 6). There is also evidence of increasing pathogenesis within the S. dysgalactiae subsp. equisimilis population (7–9). Both organisms must contend with the innate immune system at the colonization sites. Some strains of S. pyogenes express related secretory proteins, called SIC and DRS. SIC is found in only two S. pyogenes emm types, emm1 and emm57, and was first reported as a ligand for the complement proteins C6 and C7, inhibiting the formation of the membrane attack complex, thereby blocking complement-mediated lysis (10, 11). SIC was subsequently shown to inhibit the antimicrobial activity of numerous AMPs and proteins, including LL-37, lysozyme, secretory leukocyte proteinase inhibitor (SLPI), and human α and β defensins (12, 13). Additionally, SIC is inhibitory to the antimicrobial activity of several chemokines which are secreted on the pharyngeal epithelial surface (14). DRS is a variant of SIC present in emm12 and emm55 GAS (15). The greatest homology between the two proteins is found in their proline-rich region (PRR) (16). While DRS is a ligand of C6 and C7, this binding does not inhibit complement function (15). DRS-SLPI interactions also do not prevent SLPI from killing M12 GAS. However, DRS binding to human β defensin 2 (hβD-2), hβD-3, and LL-37 abrogates their antibacterial activity (17).

DNA encoding an orthologue of the SIC gene, previously called sicG, has been described for S. dysgalactiae subsp. equisimilis (18). Indeed, microarray screening of several S. dysgalactiae subsp. equisimilis strains revealed the possible presence of a sic-like gene in some isolates (19). However, as the similarity between SicG and DRS is more pronounced than that between SicG and SIC, for this report we renamed the protein and its gene DrsG and drsG, respectively. Here we report that DrsG is a highly conserved secretory protein, with variation occurring principally due to the presence of one or more repeat domains. DrsG does not inhibit complement-mediated lysis of sheep red blood cells. However, DrsG does bind to LL-37, thereby inhibiting its bactericidal activity. The fact that DrsG, DRS, and SIC all have LL-37-inhibitory activity suggests that this is the primary function of this family of proteins. Finally, we show that many S. dysgalactiae subsp. equisimilis emm types contain isolates that harbor drsG. Unlike in GAS, not all isolates in these S. dysgalactiae subsp. equisimilis emm types possess drsG. The highly conserved nature of DrsG and its inconsistent distribution within isolates of an emm type suggest prolific acquisition of the gene through lateral gene transfers (LGT) in S. dysgalactiae subsp. equisimilis lineages.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The clinical isolates S. dysgalactiae subsp. equisimilis MD128 (stg93464), MD985 (stc1400), MD604 (stc1400), MD03 (stg2078), and GGS124 (stg480) were described previously (19, 20). An additional 188 S. dysgalactiae subsp. equisimilis isolates, collected primarily from Australia (n = 89), the United States (n = 66), Japan (n = 6), Portugal (n = 12), and India (n = 14) and representing 50 emm types, were used to examine the distribution of drsG (21–25). Unless otherwise specified, S. dysgalactiae subsp. equisimilis and S. pyogenes were grown in Todd-Hewitt broth (THB), Todd-Hewitt agar (Oxoid), or Columbia agar (CBA; Oxoid) supplemented with 5% defibrinated horse blood. Escherichia coli was grown on LB medium supplemented with ampicillin (100 μg ml−1) where appropriate.

Detection of drsG in S. dysgalactiae subsp. equisimilis.

The presence of drsG in the genomes of multiple S. dysgalactiae subsp. equisimilis isolates was determined using a combination of PCR, Southern hybridization, and interrogation of draft S. dysgalactiae subsp. equisimilis genome sequences by BLASTn. PCR was performed using GoTaq Green master mix (Promega) following the manufacturer's recommendations. The Sicn2F (5′-GGAGGTCACAAACTAAGCAA-3′) and Sicc3R (5′-TGCCTATAGAAGGCACAACT-3′) primer sequences are located upstream and downstream of drsG, respectively. The Sicn1F (5′-AGTAAAACACTACTATTTACA-3′) and Sicc1R (5′-AGTCATATGGCCAATCTT-3′) primer sequences are complementary to internal drsG sequences (20). For Southern hybridization, genomic DNA was digested to completion at 37°C for 3 h with KpnI (New England BioLabs), electrophoresed in a 0.8% agarose gel, transferred to a nylon membrane (Amersham Biosciences), and probed with a digoxigenin (DIG)-dUTP-labeled internal 250-bp fragment of drsG. Following hybridization, the membranes were washed, blocked, and incubated with anti-DIG alkaline phosphatase-conjugated antibody (Roche Diagnostics) (1:20,000) prior to immunodetection with CDP-Star detection reagent (Tropix) and development (26).

Bioinformatic analyses.

Repeat sequences were identified using RADAR (27, 28). Pairwise and multiple-sequence alignments were conducted using NEEDLE and Clustal Omega (29), respectively. Putative signal sequences were detected using SignalP (30).

Detection of DrsG in culture supernatants.

Two milliliters of 100% trichloroacetic acid (TCA) was added to 20-ml overnight cultures of S. dysgalactiae subsp. equisimilis and left on ice for 30 min. The precipitated proteins were collected by centrifugation (10,000 × g for 5 min), washed in 10% TCA, and dissolved in 250 μl of 0.1 N NaOH. The total protein concentration was determined using a bicinchoninic acid (BCA) protein assay (Thermo Scientific). An aliquot of the protein solution was electrophoresed in an SDS-PAGE gel, transferred to nitrocellulose, and probed with murine anti-DrsGL or -DrsGS antiserum followed by secondary goat anti-mouse IgG conjugated to horseradish peroxidase (HRP). The membranes were then immersed in ECL Plus chemiluminescence substrate (Amersham Biosciences) and exposed to X-ray film.

Protein expression and production of antisera.

Codon-optimized DNAs encoding mature DrsGS and DrsGL were cloned into the expression vector pJ404 by DNA2.0 (Menlo Park, CA). The resulting plasmids were transformed into E. coli Top10 cells, and recombinant protein expression was induced by the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) to the mid-log-phase cultures. After a 3-h incubation, the pellets were lysed under native, nondenaturing conditions. Recombinant DrsG proteins were then purified using His-Trap HP 1-ml columns (GE Healthcare Life Sciences). Antibodies to the two DrsG variants were produced in Quackenbush mice by immunization with the proteins emulsified in complete Freund's adjuvant, followed by boosts without adjuvant on days 21 and 28 (31). Recombinant SIC from M1 GAS was expressed and purified as previously described (32). The use of mice in this project was approved by the QIMR Animal Ethics Committee.

Complement-mediated lysis.

Complement-mediated lysis of sheep erythrocytes was performed as previously described (15). Briefly, sheep erythrocytes (Applied Biological Products Management) were sensitized with hemolysin (Virion) in gelatin Veronal buffer (GVB++) for 30 min at 37°C, followed by 30 min on ice. Human serum from human male AB plasma (Sigma-Aldrich), used as the complement source in these experiments, was titrated with sensitized sheep erythrocytes to determine the dilution that caused approximately 50% hemolysis of sheep erythrocytes. For the assay, 200 pmol of recombinant protein (DrsGS, DrsGL, or SIC) or phosphate-buffered saline (PBS) was preincubated with diluted human serum that resulted in 50% hemolysis for 10 min at 37°C. Hemolysin-activated sheep erythrocytes (2.5 × 107 cells/well) were then added and incubated at 37°C for a further 15 min. Unlysed erythrocytes were removed by centrifugation at 400 × g for 10 min, and hemolysis was measured by reading the absorbance of the supernatant at 415 nm. Lysis of sheep erythrocytes with water was considered 100% lysis. Statistical significance was determined using the unpaired t test.

LL-37 binding to DrsG.

Microtiter plates (LinBro polyvinyl chloride enzyme immunoassay plates; MP Biomedicals) were coated with 100 μl of coating buffer (50 mM carbonate coating buffer, pH 9.6) containing 10 μg of DrsG or SIC, in triplicate. The plates were incubated overnight at 4°C, washed once with PBS-Tween 20 (PBST), and then blocked with PBST containing 5% skim milk powder for 1 h at 37°C. Plates were washed once using PBST and incubated (1 h at 37°C) with 10 μg of LL-37 (Peptide 2.0, Chantilly, VA) in 0.5% skim milk-PBST buffer. After washing, the binding of LL-37 was detected with primary rabbit anti-LL-37 IgG (1:5,000; Osenses) and horseradish peroxidase-labeled goat anti-rabbit secondary IgG (1:5,000; Sigma). Wells in which LL-37, primary antibody, or secondary antibody was omitted were used as controls. Reciprocal assays, in which LL-37 was used as the bound substrate and free recombinant DrsG or recombinant SIC was added, were also performed.

LL-37 survival assays.

Survival assays were performed to assess the capacity of DrsG to inhibit LL-37-mediated killing of S. dysgalactiae subsp. equisimilis MD985 (drsG negative). For these experiments, 25 μM LL-37 was preincubated with 25 μM DrsG or SIC for 30 min at 37°C in PBS. Mid-log-phase S. dysgalactiae subsp. equisimilis MD985 was harvested and diluted to an optical density at 600 nm (OD600) of 0.1. Ten microliters of the LL-37–protein mix was then added to 90 μl of the bacterial culture and incubated at 37°C for a further 2.5 h. Serially diluted mixes were plated in duplicate onto Todd-Hewitt agar and incubated overnight. The percentage of CFU recovered was determined by comparing recovery in the absence and presence of LL-37. Data presented are means for three independent experiments, each performed in duplicate.

RESULTS

Distribution and conservation of drsG in S. dysgalactiae subsp. equisimilis.

PCR (Fig. 1), Southern hybridization (Fig. 2), and examination of draft S. dysgalactiae subsp. equisimilis genome sequences resulted in identification of drsG in 30 of 193 S. dysgalactiae subsp. equisimilis isolates (Table 1). Analysis of these sequences revealed the presence of two major variants. DrsGL (DrsG-large) is a 25-kDa protein that contains a putative signal sequence and two repeat domains. The first repeat domain (RD1) comprises two repeat units, each of which also contains shorter internal repeat sequences (Fig. 3). The second repeat domain (RD2) includes two 12-amino-acid repeating units separated by a spacer sequence. In contrast, DrsGS (i.e., DrsG-small) lacks one of the repeat units in RD1, plus the amino acids between the first two repeat units. Otherwise, DrsGS is 100% identical to DrsGL at the amino acid level. In total, 23 isolates possessed drsGS, and 7 possessed drsGL. Of the 50 emm types examined, 14 contained isolates that were drsG positive (Table 2). However, for some emm types represented by multiple isolates, not all isolates within these emm types possessed drsG. Isolates of emm types stc839, stc1400, and stg480 may contain either of these variants.

FIG 1.

FIG 1

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PCR detection of drsG in S. dysgalactiae subsp. equisimilis. Representative gels demonstrate amplification of drsG from S. dysgalactiae subsp. equisimilis chromosomal DNA by use of internal (A) and external (B) primer sets. Markers (kb) are shown to the left of the gels. Isolates and their respective emm types are shown at the top of the figure.

FIG 2.

FIG 2

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Chromosomal detection of drsG in S. dysgalactiae subsp. equisimilis and GAS. KpnI-digested chromosomal DNAs from five S. dysgalactiae subsp. equisimilis isolates (MD10, ES61, TK01, MD128, and MD03) and two GAS isolates (M12 and M1) were probed with a DIG-dUTP-labeled internal 250-bp fragment of drsG. The emm type of each strain is also shown at the top of the figure. Positive bands at 4.3 kb were detected for MD10, TK01, and MD03, but not the other strains. Molecular size markers are shown to the left of the figure.

TABLE 1.

drsG-positive isolates are geographically distributed

Strain emm type Geographical origin drsG variant
NS4186 stc6979 Australia drsGS
NS4760 stc6979 Australia drsGS
NS3874 stc74a Australia drsGS
MR363962 stg2078 Portugal drsGS
MR394314 stg2078 Portugal drsGS
TK01 stg2078 Japan drsGS
NCU133 stg2078 Japan drsGS
6458-05 stg2078 USA drsGS
3993-06 stg2078 USA drsGS
3537-05 stg2078 USA drsGS
MD03 stg2078 Australia drsGS
GCS11ny stc839 India drsGL
MR223754 stc839 Portugal drsGL
MD504 stc839 Australia drsGS
NS3975 stc839 Australia drsGS
NS4722 stc839 Australia drsGS
NS4599 stc839 Australia drsGS
3878–06 emm57 USA drsGL
8139–05 emm57 USA drsGL
NCU21 stg62647 Japan drsGS
GGS124 stg480 Japan drsGL
KTS-1 stg480 Japan drsGS
81-4 stg643 Japan drsGS
6037-03 stg245 USA drsGS
3442-04 stg3442 USA drsGS
3122-05 stg485 USA drsGS
3836-05 stg5063 USA drsGL
8983-04 stc1400 USA drsGL
MD604 stg6 Australia drsGS
MD10 stg1400 Australia drsGS
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FIG 3.

FIG 3

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Schematic diagram showing the conserved repeat domain structure of DrsGL, DrsGS, DRS, and SIC AP1. For DrsGL and DrsGS, the proline-rich sequences are found within RD2. Abbreviations: SS, signal sequence; RD1, repeat domain 1; RD2, repeat domain 2; SRR, short repeat region; PRR, proline-rich region; LRR, long repeat region; C-term, C-terminal region.

TABLE 2.

Distribution of drsGL and drsGS in S. dysgalactiae subsp. equisimilis

emm typea No. of drsG-positive isolates No. of drsG-negative isolates DrsG variant(s)b
emm57 2 1 drsGL
stc74a 1 10 drsGL
stc839 6 3 drsGL (2), drsGS (4)
stc1400 2 12 drsGL, drsGS
stc6979 2 12 drsGS
stg6 1 12 drsGS
stg245 1 1 drsGL
stg480 2 12 drsGL, drsGS
stg485 1 3 drsGS
stg643 1 7 drsGS
stg2078 8 3 drsGS
stg3442 1 0 drsGS
stg5063 1 1 drsGL
stg62647 1 3 drsGS
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a

Only results for emm types with isolates possessing drsG are presented in the table.

b

Numerals in parentheses represent the numbers of isolates possessing drsGS or drsGL in the emm type specified.

Similarity between DrsG, DRS, and SIC.

DRS and SIC from GAS possess a signal sequence, a short repeat region (SRR), a proline-rich region (PRR), and a C-terminal domain (C-term). SIC additionally possesses a long repeat region (LRR). To identify the regions of identity between these proteins and DrsGS from S. dysgalactiae subsp. equisimilis, pairwise alignments using full-length proteins were performed. Relatively low overall sequence identities of 34% and 22% were observed in comparing DrsGS with DRS from GAS NS488 and with SIC from GAS AP1, respectively, with the greatest identity in the signal sequence. When only amino acid-represented overlapping sequences for the mature proteins were used, the identities increased to 42% and 33%, respectively. On the basis of these observations, we use the terminology DrsG to describe this protein of S. dysgalactiae subsp. equisimilis. Similar to DRS and SIC, the C-terminal region of DrsG contains an overrepresentation of proline residues.

DrsG is a secretory protein.

DRS and SIC in GAS are secretory products. Since DrsGS and DrsGL possess similar signal sequences, we predicted that they would also be secreted by S. dysgalactiae subsp. equisimilis isolates positive for these genes. This was confirmed by Western blotting of TCA-precipitated culture supernatants from both stationary- and log-phase S. dysgalactiae subsp. equisimilis cultures (Fig. 4). After probing with DrsGL and DrsGS antibodies, bands at ∼27 kDa and ∼24 kDa were observed in the culture supernatants of GGS124 (drsGL positive) and MD604 (drsGS positive), respectively, but were absent in those of the drsG-negative isolates, MD985 and MD128.

FIG 4.

FIG 4

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DrsG is expressed and secreted by S. dysgalactiae subsp. equisimilis. (A) Western blot of concentrated culture supernatants from mid-log- and stationary-phase cultures of drsGL-positive GGS124 (lanes 1 and 2, respectively) and drsG-negative MD985 (lanes 3 and 4, respectively) and of recombinant DrsGL (lane 5) probed with anti-DrsGL antibodies. (B) Western blot of concentrated culture supernatants from mid-log- and stationary-phase cultures of drsGS-positive MD604 (lanes 1 and 2, respectively) and drsG-negative MD128 (lanes 3 and 4, respectively) and of recombinant DrsGS (lane 5) probed with anti-DrsGS antibodies. Molecular size markers are indicated on the left.

DrsG does not inhibit complement-mediated lysis.

DRS and SIC are known to bind to complement proteins, but only SIC inhibits complement-mediated lysis. We therefore examined whether DrsGS and DrsGL had any inhibitory effect on complement function. To do so, sheep erythrocytes sensitized with hemolysin were incubated with equimolar concentrations of the recombinant proteins and with human serum as a source of complement. Recombinant SIC showed significant inhibition of lysis of erythrocytes (37.3% ± 1.5%; P = 0.0005) compared to PBS controls (no inhibition). However, neither DrsGL nor DrsGS showed inhibitory activity (Fig. 5).

FIG 5.

FIG 5

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DrsG does not inhibit complement-mediated lysis of erythrocytes. Complement-mediated lysis assays were performed using DrsGL, DrsGS, SIC, or PBS preincubated with human serum. Results are presented as % inhibition of complement-mediated lysis, where incubation of sheep erythrocytes with water was used as the reference point for 100% lysis. Data represent the means for three independent experiments. Statistical significance (***) was determined using the unpaired t test.

DrsG is a ligand of LL-37.

Indirect enzyme-linked immunosorbent assays (ELISAs) were used to determine whether LL-37 was a ligand of DrsGS and DrsGL. Wells coated with the recombinant proteins reacted with LL-37, and the binding was detected with anti-LL-37 antibodies and labeled secondary antibody. Compared to the negative controls, which lacked individual assay components, the optical densities observed in experimental wells were significantly greater (Fig. 6). In fact, binding between LL-37 and DrsGS was the same as that observed for LL-37 and SIC. However, when the reciprocal assays were performed, with LL-37 bound to the plate and free DrsG and anti-DrsG antibodies used, no binding between DrsG and LL-37 was observed. This equivocal result on physical binding required further experiments using biological activity, as described below.

FIG 6.

FIG 6

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LL-37 interactions with DrsGL (A), DrsGS (B), and SIC (C). Graphs on the left show the results when DrsGL, DrsGS, or SIC was used to coat the wells of a 96-well plate. LL-37 was added, followed by anti-LL-37 antibody and HRP-labeled secondary antibody. Results for assays in which LL-37 (C1), LL-37 and primary antibody (C2), or secondary antibody (C3) was not added are included as controls. The graphs on the right represent the reciprocal assays, in which LL-37 was used as the bound substrate, and free DrsGL, DrsG, or SIC was added.

Addition of DrsG increases the viability of S. dysgalactiae subsp. equisimilis in the presence of LL-37.

To investigate whether the interactions between DrsG and LL-37 observed above inhibited the bactericidal activity of LL-37, S. dysgalactiae subsp. equisimilis MD985 (drsG negative) was incubated in the presence of LL-37, DrsG, or LL-37 preincubated with DrsG (Fig. 7). LL-37 inhibited the growth of MD985 by 61% compared to controls. Preincubation of LL-37 with DrsGL, DrsGS, or SIC restored growth to 80% or more. These results suggest that DrsG is capable of inhibiting the bactericidal activity of LL-37.

FIG 7.

FIG 7

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Growth of S. dysgalactiae subsp. equisimilis in the presence of LL-37 and DrsG. S. dysgalactiae subsp. equisimilis MD985 (drsG negative) was grown in the presence of LL-37 and/or DrsGL (A), DrsGS (B), or SIC (C). After a 2.5-h incubation, the bacteria were recovered, plated onto Todd-Hewitt agar, and incubated overnight. The percentage of growth compared to that of controls was determined for each individual assay. The results presented are the means for three independent experiments.

DISCUSSION

LL-37 is an important AMP found in many parts of the human body. With respect to the preferred tissue sites of colonization of S. dysgalactiae subsp. equisimilis, LL-37 is expressed in both respiratory secretions and skin (3, 33). Inhibition of the activity of LL-37 would therefore be beneficial for S. dysgalactiae subsp. equisimilis colonization at these sites. Here we demonstrated binding of DrsG and LL-37 when DrsG was used as the bound substrate in ligand assays. However, when the reciprocal experiment was performed, with LL-37 as the bound substrate, no interaction was observed. A similar result was reported by Fernie-King et al. (12) for interactions between SIC and hβD-2. They suggested that the difference between the two configurations of the assay was attributable to the masking of binding sites when a small molecule (i.e., hβD-2; 64 amino acids) was bound to a plate. Given the small size of LL-37 (37 amino acids), a similar explanation could be applied. Notwithstanding this, the biological assay showed that DrsG counteracted the bactericidal activity of LL-37 and provides clear support for DrsG–LL-37 interactions.

In S. pyogenes, drs and sic are found in a restricted number of emm types (emm12 and -55 and emm1 and -57, respectively). Within these emm types, drs and sic are universally found in all isolates. In contrast, we found that although drsG was present in 14 of 50 different S. dysgalactiae subsp. equisimilis emm types tested, not all isolates from a drsG-positive emm type possessed the gene. These results are in concordance with a recent publication by Oppegaard et al. (34). When the results from this study are combined with results from two other distribution studies (20, 34), a total of 19 S. dysgalactiae subsp. equisimilis emm types have now been identified as possessing isolates that harbor drsG. Thus, the distribution of drsG is widespread in S. dysgalactiae subsp. equisimilis isolates as defined by emm type. Oppegaard et al. also identified a third variant of DrsG. The drsGS variant from our study corresponds to the sicG3 to -5 alleles, whereas drsGL corresponds to sicG6 in their study. The smaller alleles in their study, sicG1 and sicG2, were not found in our study. sicG1 is shorter than both drsGS and drsGL. With the exception of the signal sequence, the identity between the sicG1 and sicG2 alleles and all other sicG/drsG alleles is extremely low. The mature SicG1 protein also has a single repeat region, with no similarity to the repeat regions in other DrsG isoforms.

The major difference between DrsGL and DrsGS is the presence of one additional repeat unit in DrsGL. Thus, within each variant, conservation is very high both within an emm type and across multiple emm types. In some instances, drsGS and drsGL were present in discrete isolates of the same emm type. There are three possible explanations for these observations. First, once a particular isoform is acquired by an emm type, a duplication event resulting in conversion of drsGS to drsGL or a deletion event resulting in conversion of drsGL to drsGS may occur. Such recombination events may occur multiple times, giving rise to both drsGS-positive and drsGL-positive lineages within an emm type. Second, drsGS and drsGL may have been acquired multiple times by different isolates of the same emm type. A third, highly improbable hypothesis is that drsG was ubiquitously present in multiple emm types and that, over time, deletions within the alleles (full gene or regions within the gene) have been occurring.

Given the high degree of identity within DrsGL and DrsGS, irrespective of emm type, the inconsistent presence of drsG within emm types, and the fact that drsGS or drsGL can be found within the same emm types, multiple acquisitions of drsG via lateral gene transfer (LGT) seem the most likely explanation for the observed distribution of drsG observed in this study. Inter- and intraspecies LGT within the beta-hemolytic streptococci have been described in several studies (35, 36). Whereas interspecies transfers between GAS and S. dysgalactiae subsp. equisimilis appear to be mediated by integrative conjugative elements (ICE) (37–39), both ICEs and bacteriophages contribute to intraspecies LGT (40). Importantly, many mobile genetic elements (MGEs) in GAS also carry virulence genes that may alter the pathogenesis of individual strains (41–43). Analysis of the DNA surrounding drsG in the draft genomic sequences used in this study found drsG to be located between scpG and SDEG_0931 (18). sic and drs are also found adjacent to scpA in GAS. This region has previously been shown to include MGE-associated genes in both GAS and S. dysgalactiae subsp. equisimilis (44, 45). A gene encoding a putative transposase was also identified between drsG and scpG in one of the draft assemblies in this study. Since no other bacteriophage-related genes are found in this or surrounding regions, it is likely that nonbacteriophage MGEs are responsible for distribution of drsG throughout the S. dysgalactiae subsp. equisimilis population.

Within the DrsGS variants, conservation at the amino acid level is >95%. Similarly high levels of amino acid conservation are also present in DrsGL. Differences in the number of repeat units in RD1 therefore represent the greatest level of diversity in DrsG. Conservation of DRS has also been reported for S. pyogenes (46). The conserved nature of DRS in the two species suggests that for isolates that acquire this gene, it plays an important biological role. This sequence conservation is in stark contrast to the case for sic, where variation can be observed in epidemic waves (47), and suggests that although SIC and DRS/DrsG have similar ligands, their role in respective species may be different. Indeed, this is evident from the wide range of ligands and functions for SIC and the much-restricted properties of DrsG.

Proline-rich C-terminal regions are a feature of SIC and DRS. Here we have extended this observation to DrsG. While the proline-rich regions of SIC and DRS are known to bind C6, C7, hβD-2, and hβD-3, the specific amino acids involved in the binding have not been elucidated. Proline-rich motifs have been identified as being important in many signaling and binding proteins (48), and there is a growing field of research investigating the binding capabilities and structural properties of proline-rich domains. One such motif is the peptide ligand motif PxxP (where “x” denotes any amino acid), which usually sits at the core of a proline-rich domain sequence (49, 50). These domains are usually positioned toward the ends of a protein, where they form extended structures that have been described as “sticky arms” (51). Four such motifs are present in DrsG. Despite the limited identity, four PxxP motifs are also found in SicG1. DRS contains four PxxP motifs, whereas SIC contains six. The prolines involved in these motifs are also conserved between the three proteins. Given the relatively low identity across the rest of the molecules, it is tempting to suggest that the PxxP motif plays a critical role in the binding of LL-37.

Both commensals and pathogens have to overcome the antimicrobial properties of AMPs if they are to successfully colonize and persist on mucosal surfaces. Here we have shown that DrsG is conserved and protects S. dysgalactiae subsp. equisimilis from the antimicrobial activity of LL-37. Our results suggest that direct inhibition of the antimicrobial activity of LL-37 is an intrinsic property of the SIC/DRS group of proteins. Given the multifunctional nature of both SIC and DRS, it is likely that DrsG inhibits other AMPs. In doing so, we speculate that DrsG may assist S. dysgalactiae subsp. equisimilis in colonizing and persisting in the throat and on the skin. However, the low prevalence of drsG-positive strains in the population suggests that the selective advantages of such activity may be limited.

ACKNOWLEDGMENTS

This work was supported by funding provided by the National Health and Medical Research Council of Australia, the Australian Infectious Diseases Research Centre, and the University of the Sunshine Coast. The ongoing S. dysgalactiae subsp. equisimilis genome sequencing project is supported by The Wellcome Trust, United Kingdom.

Bacterial strains and/or genomic DNAs used for the genomic analyses were kindly provided by Rebecca Towers (Menzies School of Health Research), Tadao Hasegawa (Nagoya City University Graduate School of Medicine, Japan), Bernard Beall (U.S. Centers for Disease Control and Prevention), Debra Bessen (New York Medical College), Mario Ramirez (University of Lisbon, Portugal), and Michael Batzloff (Griffith University, Australia).

Footnotes

Published ahead of print 24 March 2014

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