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VERSION 3 1

CovR regulates Streptococcus mutans susceptibility to 2

complement immunity and survival in blood 3

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Lívia A. Alves1, Ryota Nomura2, Flávia S. Mariano1, Erika N. Harth-Chu1, Rafael 5

N. Stipp1, Kazuhiko Nakano2 and Renata O. Mattos-Graner1* 6

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1Department of Oral Diagnosis, Piracicaba Dental School – State University of 8

Campinas, Piracicaba, SP, Brazil. 2 Department of Pediatric Dentistry, Osaka 9

University, Graduate School of Dentistry, Osaka, Japan. 10

11

Running title: SM susceptibility to complement immunity. 12

13

Corresponding author: 14

Renata O. Mattos-Graner 15

Department of Oral Diagnosis, Piracicaba Dental School, University of 16

Campinas (UNICAMP). Av. Limeira, 901. CEP 13414-903 Piracicaba, SP, 17

Brasil. 18

Phone: (19) 2106 5707. 19

Fax: (19) 2106 5218 20

e-mail: [email protected] 21

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IAI Accepted Manuscript Posted Online 29 August 2016Infect. Immun. doi:10.1128/IAI.00406-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 24

Streptococcus mutans (SM), a major pathogen of dental caries, may promote 25

systemic infections after accessing the bloodstream from oral niches. In this study, we 26

investigate pathways of complement immunity against SM, and show that the orphan 27

regulator CovRSm modulates susceptibility to complement opsonization and survival in 28

blood. SM blood isolates showed reduced susceptibility to C3b deposition compared to 29

oral isolates. Reduced expression of covRSm in blood strains was associated with 30

increased transcription of CovRSm-repressed genes required for SM interaction with 31

glucan (gbpC, gbpB and epsC), sucrose-derived exopolysaccharides (EPS). Consistently, 32

blood strains showed increased capacity to bind glucan in vitro. Deletion of covRSm in 33

strain UA159 (UAcov) impaired C3b deposition and binding to serum IgG and C-34

reactive protein (CRP) as well as phagocytosis through C3b/iC3b receptors and killing 35

by neutrophils. Opposite effects were observed in mutants of gbpC, epsC or gtfB/C/D 36

(required for glucan synthesis). C3b deposition on UA159 was abolished in C1q-37

depleted serum, implying that the classical pathway is essential for complement 38

activation on SM. Growth in sucrose-containing medium impaired binding of C3b and 39

IgG to UA159, UAcov and blood isolates, but had absent or reduced effects on C3b 40

deposition in gtfB/C/D, gbpC and epsC mutants. UAcov further showed increased ex 41

vivo survival in human blood in an EPS-dependent way. Consistently, reduced survival 42

was observed in the gbpC and epsC mutants. Finally, UAcov showed increased ability 43

to cause bacteremia in a rat model. These results reveal that CovRSm modulates 44

systemic virulence by regulating functions affecting SM susceptibility to complement 45

opsonization. 46

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INTRODUCTION 48

Streptococcus mutans (SM) is a common species of the oral cavity of humans 49

involved in the pathogenesis of dental caries, which can promote infective endocarditis 50

and other systemic infections after gaining access to the bloodstream (1,2-4). Factors 51

involved in SM survival in the bloodstream are however unknown, but likely include 52

mechanisms to evade host immunity. SM expresses the orphan response regulator 53

CovRSm (also known as GcrR) (5-8), which is an orthologue of the CovR protein of the 54

two-component regulatory system (TCS) CovRS (also known as CsrRS) of the 55

pathogenic species Streptococcus pyogenes (group A Streptococcus, GAS) and 56

Streptococcus agalactiae (group B Streptococcus, GBS). In GAS, CovRSpy typically 57

functions as a repressor of a panel of virulence genes involved in evasion of host 58

immunity and tissue invasiveness (9). In SM, CovRSm represses virulence factors 59

involved in the establishment of SM in dental biofilms (7,8,10,11) but its role in 60

systemic virulence is unknown. Genes directly repressed by CovRSm include gtfB and 61

gtfC, which encode glucosyltransferases B and C required for the extracellular 62

synthesis of glucan from sucrose (7), major structural exopolysaccharides (EPS) of 63

cariogenic biofilms (1,2). CovRSm also inhibits expression of several genes involved in 64

cell wall biogenesis and surface interactions with EPS, including GbpB (glucan-binding 65

protein B), GbpC (glucan-binding protein C), EpsC (enzyme for exopolysaccharide 66

synthesis: UDP-N_Acetylglucosamine 2-Epimerase), LysM (lysine motif protein) and 67

WapE (cell wall protein E) (8,10-12). 68

An isogenic mutant of covRSm obtained in UA159 (serotype c) shows impaired 69

susceptibility to phagocytosis by human PMN in a blood-dependent manner (13). 70

Among the four known SM serotypes (c, e, f, and k), serotype c is the most prominent 71


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in the oral cavity (approximately 70 to 80% of the strains), and is frequently associated 72

with systemic infections, being detected in 30.3 and 65.5% of SM-positive specimens 73

of cardiac valve and atheromatous plaque from patients subjected to cardiac surgeries 74

(14,15). The serotype c strain MT8148 survives during 1 to 2 days in the bloodstream 75

of rats (16), further suggesting mechanisms of evasion of blood immunity. In this 76

study, we investigated the roles of CovRSm in susceptibility of SM strains to 77

complement immunity mediated by C3b, a major opsonin present in blood and other 78

host fluids (17,18). Profiles of C3b deposition were compared between strains isolated 79

from blood of patients with bacteremia and/or infective endocarditis and from the oral 80

cavity, to assess diversity in susceptibility to complement immunity. Low susceptibility 81

to C3b deposition observed in blood isolates was then compared to transcript levels of 82

covRSm and of CovRSm-repressed genes. Effects of covRSm deletion in strain UA159 83

(serotype c) on binding of C3b, IgG antibodies and C-reactive protein (CRP), and on 84

phagocytosis mediated by C3b/iC3b or IgG receptors and killing by human neutrophils 85

(PMN) were determined. Mechanisms of CovRSm-regulation of SM susceptibility to 86

complement immunity were then investigated by assessing the effects of deletion of 87

CovRSm-regulated genes (gtfB, gtfC, gbpC, epsC, lysM and wapE) on C3b- and antibody-88

mediated immunity in the presence or absence of sucrose-derived EPS. Finally, strains 89

were compared regarding ex vivo survival in human blood and in a rat model of 90

bacteremia and infective endocarditis. 91

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MATERIAL AND METHODS 96

97

Studied strains and culture conditions. Strains used in this study are described in 98

Table 1.(11,13,19-22)Strains were grown (37°C; 10% CO2) from frozen stocks in Brain Heart 99

Infusion (BHI) agar (Difco). BHI or chemically defined medium (CDM) (10) with or 100

without sucrose (0.01 and 0.1%) were used in the experiments. Erythromycin (10 101

μg/ml), spectinomycin (200 μg/ml) and/or kanamycin (500 μg/ml) (Merck Labs, 102

Germany) were added to media for cultivation of deletion and complemented 103

mutants, respectively. 104

105

Construction of gbpC deletion and complemented mutants. The nonpolar gbpC 106

deletion mutant was obtained in strain UA159 (UAgbpC) by double cross-over 107

recombination with a null allele (of 2,315 pb) constructed by PCR-ligation (23). In the 108

recombinant allele, an internal sequence of 1,455 bp of gbpC encoding region was 109

replaced by an erythromycin resistance cassette (ermr) obtained from plasmid pVA838. 110

Complemented gbpC mutant (UAgbpC+) was obtained by transforming UAgbpC with 111

plasmid pDL278 containing the intact copy of gbpC and spectinomycin resistance gene. 112

Primers used for construction of mutants are depicted in Table 2. 113

114

RNA isolation, reverse transcription and qPCR. RNA was purified from strains at mid-115

log phase of growth (A550nm 0.3) using RNeasy kit (Qiagen, Germany) and treated with 116

Turbo DNase (Ambion, USA), as described elsewhere (11). The cDNA was obtained 117

from 1 μg of RNA using random primers (24) and SuperScript III (Life Technologies, 118

USA), according to the manufacturer's instruction. Quantitative PCR was performed in 119


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StepOne™ Real-Time PCR System (Life Technologies) with cDNA (10 ng), 10 μM of each 120

primer, and 1× Power SYBR® Green PCR Master Mix (Lifetech) in a total volume of 10 121

μl. The cycling conditions were: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 122

s, optimal temperature for primer annealing (Table 2) for 15 s, and 72 °C for 30 s. Ten-123

fold serial dilutions of genomic DNA (300 ng to 0.003 ng) were used to generate 124

standard curves for absolute quantification of RNA expression levels. Melting curves 125

were obtained for each primer set. Results were normalized against SM 16SrRNA gene 126

expression (24). Assays were performed in duplicate with at least two independent 127

RNA samples. 128

129

SM interaction with EPS. Cell aggregation mediated by sucrose-derived EPS was 130

assessed as described previously (25). Briefly, strains were grown in BHI (37°C; 10% 131

CO2, 18 h) and an equal number of cells was transferred to fresh BHI supplemented 132

with 0.1% sucrose and incubated for 24 h (37°C; 10% CO2). Cell aggregation was then 133

visually inspected. 134

Surface-associated EPS was analyzed by scanning electron microscopy (SEM) in 135

strains grown in BHI or CDM with or without 0.1% sucrose. Briefly, overnight cultures 136

obtained in BHI or CDM medium were 100-fold diluted with fresh medium containing 137

or not 0.1% sucrose, and incubated to reach A550nm 0.3. Cells were then harvested by 138

centrifugation (500 µl), washed with phosphate-buffered saline (PBS), and processed 139

for SEM analysis, as previously described (12). Samples were analyzed with a scanning 140

electron microscope (JEOL, JSM 5600LV, Japan). 141

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Volunteers, sera and blood samples. Blood samples were collected by 143

venipuncture in heparin vacuum tubes (BD Vacutainer®) from six healthy subjects 144

(three males, three females; mean age: 30 years, range 25 to 45 years), according to 145

standard protocols previously approved by the Ethical Committee of the Piracicaba 146

Dental School, State University of Campinas (proc. nº 031/2012). Serum samples were 147

stored in aliquots at -70 oC until use. Levels of C3 were determined in serum samples 148

as described below, and were within normal levels in all volunteers (mean 1.91 mg/ml; 149

SD 0.68; range 1.18 to 2.88 mg/ml) (26). Mean levels of IgG and IgM immunoglobulins 150

were also determined in the same samples and were, respectively, 11.68 mg/ml (± 151

1.82) and 1.35 mg/ml (± 0.64). Serum samples from one volunteer, which were 152

representative of C3, IgG and IgM levels were used as control. Commercial human 153

serum depleted of C1q, was obtained from Calbiochem, MA, U.S.A.. The Calbiochem 154

C1q-depleted serum is free of EDTA and retains alternative pathway activity (27). As a 155

control for integrity, C1q-depleted serum was supplemented with purified human C1q 156

(Calbiochem) to a physiological concentration range (75 µg/ml in 100% serum). Heat-157

inactivated sera (56 oC for 20 min) were also used as negative controls in preliminary 158

experiments, and showed minimal effects on comparative analyses of C3b deposition 159

between strains. 160

161

Determination of total levels of C3, IgG and IgM antibodies in serum. The serum 162

concentrations of C3, IgG and IgM antibodies were determined in enzyme-linked 163

immunosorbent assays (ELISA) using commercial systems for quantification of human 164

complement C3 (Molecular® Innovations, MI, U.S.A.), human IgG or human IgM (Bethyl 165

Laboratories, Inc., TX, U.S.A.), respectively. Briefly, 100 µl of serum samples diluted in 166


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dilution buffer (1:100,000; 1:500,000 and 1:10,000, respectively) were added to 96-167

well plates coated with anti-C3, anti-IgG or anti-IgM immunoglobulins, then incubated 168

for 30 min at room temperature (rt). After a series of three washes with wash buffer, 169

100 µl aliquots of antibodies specific to C3, human IgG, or human IgM were added per 170

well, and plates incubated (rt) for 1 h. After a new series of washes, 100 µl per well of 171

secondary horseradish peroxidase (HRP)-conjugated antibodies (1:50,000) were 172

added, and incubation continued for 30 min. After a new series of washes, 100 µl per 173

well of chromogenic HRP substrate (3,3',5,5'-tetramethylbenzidine) were added and 174

plates incubated for 30 min. Reactions were stopped by addition of 1 N H2SO4. 175

Absorbances (A450nm) were measured in a microtiter plate reader (Versa Max®), and 176

converted to mg/ml using standard curves of C3 (0.02 - 10 ng/ml), IgG (0.69 - 167 177

ng/ml) or IgM (1.03 - 250 ng/ml) antibodies. 178

179

C3b deposition on SM. Deposition of C3b on the surface of serum-treated SM strains 180

was determined as described elsewhere (27,28), with some modifications. Briefly, 181

approximately 107 cfu of strains at mid-log phase of growth (A550nm 0.3) were harvested 182

by centrifugation (10,000 x g, 4oC), washed two times with PBS (pH 7.4) and suspended 183

in 20 µl of 20% serum (diluted in PBS). Samples were then incubated (37oC; 30 min) 184

and washed twice with PBS-Tween 0.05% (PBST). Then, cells were incubated on ice (40 185

min) with fluorescein isothiocyanate (FITC)-conjugated polyclonal goat anti-human C3 186

IgG antibody (ICN, CA, U.S.A) (1:300 in PBST). After two washes with PBST, bacterial 187

cells were fixed in 3% paraformaldehyde in PBS and analyzed on a FACSCalibur flow 188

cytometer (BD Biosciences) using forward and side scatter parameters to gate at least 189

25,000 bacteria. Results were expressed as the geometric mean fluorescence intensity 190


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(MFI) of C3b-positive cells or as mean fluorescence index (FI; percentage of positive 191

cells multiplied by the MFI) (29,30). Control samples included bacteria treated only 192

with PBS instead of serum. 193

194

PMN isolation, opsonophagocytosis and killing assays. Human PMN were isolated 195

from fresh heparinized blood samples from one reference volunteer by centrifugation 196

over a double gradient composed of 1119 and 1083 density Histopaque (Sigma-197

Aldrich), as previously described, with modifications (31). Red blood cells were 198

eliminated by hypotonic lysis. Isolated PMN were suspended in RPMI 1640 (GIBCO®, 199

Life Technologies, NY, USA) medium supplemented with inactivated 10% fetal bovine 200

serum. Cell viability (>98%) was monitored by trypan blue exclusion. Cell purity (>95%) 201

was monitored by May-Grunwald Giemsa staining. 202

For the opsonophagocytosis assays, bacteria were previously labeled with FITC 203

as described elsewhere (32), with some modifications. Briefly, 500 µl of bacterial 204

strains (A550nm 0.3) were washed two times with PBS, suspended in 600 µl of carbonate 205

buffer (Na2CO3 0.15 M, 0.9% NaCl; pH 9) with 1.7 mg/ml of FITC (Sigma), and incubated 206

for 1 h (shaking at rt in the dark). Then, cells were harvested and washed three times 207

with PBST, and aliquots stored overnight in 10% glycerol at -70oC. Bacterial labeling 208

was analyzed in a fluorescent microscope (Leica DM LD), and by flow 209

cytometry (FACSCalibur, BD). 210

For C3b deposition, aliquots containing 107 cfu of FITC-labeled bacteria were 211

incubated with 20% serum and added to wells of 96-well plates containing 2 x 105 PMN 212

in 50 µl of RPMI medium to a multiplicity of infection (MOI) of 200 bacterial per PMN. 213

After incubation (37oC, 10% CO2, gentle shaking) for 5 or 30 min, reactions were fixed 214


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by addition of 100 µl of 3% of paraformaldehyde. PMNs were then analyzed using 215

FACSCalibur (BD Biosciences), and the frequency of phagocytosis expressed as the 216

number of PMN cells with intracellular bacteria, within a total of 10,000 PMN analyzed 217

(33). The MOI was determined in preliminary experiments testing MOIs of 20 to 200 218

bacteria per PMN, after 5 to 60 min of incubation. Flow cytometry analysis was 219

compared to light microscopy analysis of samples stained using May-Grunwald-220

Giemsa, as previously described (13). These comparisons confirmed that most of PMN-221

associated bacteria were internalized. 222

To assess phagocytosis by PMN through C3b/iC3b receptors, similar assays 223

were performed with PMN previously incubated (37°C, 30 min) with mouse 224

monoclonal antibodies (MAbs) anti-CD35 (Biolegend, CA, U.S.A.) or anti-CD11b/CD18 225

to block CR1 or CR3 receptors (34,35), respectively. As reference, PMN incubated with 226

MAbs anti-CD32 (FcγRII) (eBioscience,CA, U.S.A.) or anti-C16 (FcγRIII) (Biolegend, CA, 227

U.S.A.) were also tested. 228

Opsonophagocytic killing was assessed as previously described (36,37), with 229

modifications. Briefly, preopsonized bacteria (20% human serum for 30 min at 37°C) 230

were added to samples of human PMN in RPMI with 10% human serum at the MOI 231

200:1. After incubation (37°C; 10 and 30 min) under shaking, reactions were stopped 232

at 4°C. PMN were harvested by centrifugation (500 x g; 8 min, 4°C), washed twice with 233

PBS (pH 7.2), lysed with 2% saponin (12 min, 37°C) and viable counts of intracellular 234

bacteria determined by plating serial dilutions on BHI agar. Bacterial counts were also 235

determined in control wells with identically treated samples without PMN. Viable 236

bacteria were counted in culture supernatants of PMN samples to monitor the number 237


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of extracellular bacteria. Percentage of intracellular survival was calculated as follows: 238

(cfu ml-1 test well)/cfu ml-1 control well) x 100. 239

240

Determination of binding of serum IgG, IgM and CRP to SM. Binding of serum IgG, 241

IgM or CRP to SM was determined as previously described (27,38) with some 242

modifications. Briefly, bacterial strains were harvested from 500 µl of cultures of 243

UA159 (A550nm 0.3), washed twice with PBS (pH 7.0), incubated with 20% serum, and 244

washed with PBST. To assess surface levels of IgG, IgM or CRP, cells were then 245

incubated with polyclonal goat IgG anti-human IgG Fc conjugated with FITC (Novus 246

Biological, U.S.A.) (1:900), with polyclonal mouse IgG anti-human IgM Fc conjugated 247

with allophycocyanin (APC) (1:1000) (Biolegend, U.S.A.) or with goat IgG anti-human 248

CRP conjugated with FITC (GeneTex, U.S.A.) (1:100), respectively. Streptococcus 249

pneumoniae strains D39 and TIGR4 were used as controls for CRP binding, because S. 250

pneumoniae cell wall contains known CRP ligands (phosphorylcholine; PCho) (39). 251

After 40 min incubation on ice, bacterial cells were washed twice with 300 µl of PBST, 252

harvested (16,000 x g, 2 min) and suspended in 300 µl of 3% of paraformaldehyde. 253

Flow cytometry analyses were performed as described before, using forward and side 254

scatter parameters to gate at least 25,000 bacteria. Bacterial samples treated with PBS 255

instead of serum were used as negative controls. 256

257

Ex vivo survival of SM strains in human blood. Bacteria from BHI cultures (A550nm 0.3) 258

were harvested (11,000 x g, 2 min), washed twice in PBS, and resuspended in 1 ml of 259

fresh whole human blood. Samples were then incubated (37oC, 5% CO2, gentle 260

agitation), and aliquots collected at different time points (5, 30, 60, 120, 240 min), 261


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serially diluted and plated on BHI agar for determination of bacterial counts. Aliquots 262

collected just after bacterial suspension in blood were used for initial measure of 263

cfu/ml (time 0). Changes in numbers of viable bacteria were then calculated in relation 264

to counts at time 0, to reduce the influence of variations in blood-mediated 265

aggregation between strains on the numbers of cfu/ml. Three independent 266

experiments were performed in duplicate with blood samples from one volunteer with 267

reference levels of C3, IgG, and IgM (2.8, 14.6, 1.5 µg/ml of serum, respectively). 268

269

Survival of SM strains in a rat model of bacteremia and infective endocarditis. 270

Protocols of the animal experiments were approved by the institutional animal care 271

and use committee of Osaka University Graduate School of Dentistry (No. 24-019). 272

Animals were maintained and handled in accordance with guidelines for animal 273

research. The rat infective endocarditis model was performed according to methods 274

described previously, with some modifications (40). In brief, 21 Sprague-Dawley male 275

rats (400-500 g each) were anesthetized with a mixture of xylazine and midazolam (0.1 276

ml/100 g). A sterile polyethylene catheter with a guide wire was surgically placed 277

across the aortic valve of each animal via the right carotid artery, and the tip was 278

positioned and placed at the aortic valve in the left ventricle. Bacterial suspension (109 279

cells per body, from BHI cultures) in PBS was intravenously administered through the 280

jugular vein. Bacterial clearance was examined by measuring the numbers of bacteria 281

in blood samples from the jugular veins, which were taken at 1, 3, 6, and 24 h, and 7 282

days after the initial infection. The blood samples were placed on Mitis-salivarius agar 283

(Difco Laboratories, Detroit, MI, USA) plates containing bacitracin (0.2 U/ml; Sigma 284

Chemical Co., St. Louis, MO, USA) and 15% (wt/vol) sucrose (MSB agar) and incubated 285


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at 37°C for 48 h. Seven days after bacterial infection, the rats were sacrificed by an 286

overdose of anesthesia and the aortic valves were extirpated, transversely sectioned, 287

and subjected to Gram staining and to bacterial recovery for microbial counting. 288

289

Statistical analyses. Flow cytometry data (percentage of positive bacteria or PMN, and 290

MFI or FI values) were analyzed by comparing means of at least three independent 291

experiments, using nonparametric Kruskal-Wallis test with Dunns’s post hoc test. 292

Comparisons of the mean MFI or FI for surface-bound C3b between oral and blood 293

isolates were performed using Mann-Whitney U-test. Spearman’s rank correlation was 294

applied to analyze associations between MFI of surface C3b and surface IgG. Ex vivo 295

survival in blood was compared between strains by testing differences in relative 296

values of bacterial counts (log of cfu/ml) at each time point of incubation in relation to 297

initial counts in blood suspensions. Bacterial counts in the rat bloodstream were also 298

compared between strains. Relative or absolute measures of bacterial counts were 299

compared between strains at each time-point using Kruskal-Wallis with post hoc 300

Dunns’s test, using correction for repeated measures. (30) 301

302

RESULTS 303

304

SM strains isolated from blood show reduced susceptibility to C3b deposition 305

compared to oral isolates. Although complement immunity is recognized as an 306

important blood defense against streptococcal species (18,28,41), the profiles of SM 307

susceptibility to complement-mediated opsonization were unknown. Thus, we 308

compared patterns of C3b deposition on strains isolated from the bloodstream of 309


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patients with bacteremia associated or not with infective endocarditis (n = 7), and 310

isolates of the oral cavity (n = 13), including the reference strain UA159. As shown in 311

Fig. 1, blood isolates showed reduced levels of C3b deposition compared to oral 312

isolates. Two blood serotype c strains (SA13 and SA18) showed the lowest MFI for C3b 313

(mean MFI: 148.9 ± 65.8 and 215.7 ± 112.0, respectively), and low FI (mean FI: 2,069.0 314

± 802.8 and 3,019.9 ± 1,056.4, respectively). Means of MFI and FI of strain UA159 were 315

623.2 (± 100.3) and 17,678.4 (± 2,908.7), respectively. 316

317

SM blood strains show reduced activity of covRSm and increased 318

transcription of CovRSm-target genes required for surface interaction with EPS. To 319

investigate the role of CovRSm on strain susceptibilities to C3b deposition, transcript 320

levels of covRSm and downstream genes were compared between four blood strains 321

showing the lowest levels of C3b deposition (SA13, SA15, SA16 and SA18) with four 322

oral isolates with the highest levels of binding to C3b (2VS1, 11A1, 11SSST2 and 8ID3), 323

and the reference strain UA159. The CovRSm-repressed genes selected were those 324

affecting SM cell surface properties, including lysM, wapE, epsC, gbpB, gbpC, gtfB, 325

gtfC, and gtfD (glucosyltransferase D encoding gene) (7,11,42). As shown in Fig. 2, 326

blood strains showed lower levels of covRSm transcripts than the oral isolates. 327

Consistently, blood isolates showed increased transcription of epsC, gbpC, and gbpB, 328

genes involved in SM surface interaction with EPS (11,12,43). No significant differences 329

in transcript levels of lysM, wapE, gtfB, gtfC or gtfD were detected (Fig. 2). Levels of 330

C3b deposition in the analyzed strains negatively correlated with transcript levels of 331

epsC (Spearman correlation: r: -0.45, p<0.05), gbpB (r: -0.21, p<0.05) and gbpC (r: -332

0.35, p<0.05). Thus, diversity in transcriptional activities of covRSm and of CovRSm-333


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repressed genes are associated with differences in levels of C3b deposition in SM 334

strains. 335

336

SM blood strains show increased capacity to bind EPS produced in the 337

presence of sucrose, similarly to the covRSm isogenic mutant. Because gbpB, gbpC and 338

epsC encode proteins for SM binding to sucrose-derived EPS (glucan) (11,12,43), we 339

compared the capacities of aggregation in the presence of sucrose between blood and 340

oral isolates. Isogenic mutants of covRSm and of CovRSm-repressed genes (gbpC, epsC, 341

gtfB, gtfC, lysM, wapE) were also tested, except for gbpB, which is essential for SM 342

viability (12). As shown in Fig. 3A, blood isolates showed higher capacity to aggregate 343

in 0.1% sucrose BHI medium, compared to oral isolates. The blood strains SA13 and 344

SA18 showed aggregation phenotypes similar to that of UAcov strain (Fig 3B). As 345

anticipated, the gbpC isogenic mutant did not aggregate, while only weak aggregation 346

was detected in the epsC mutant (Fig. 3C). In addition, because gtfB, gtfC and gtfD are 347

required for the synthesis of glucan from sucrose, mutants of these genes obtained in 348

strain MT8148 did not aggregate (Fig. 3C). The aggregation phenotypes of the gbpC 349

mutants obtained in MT8148 (Fig. 3C) and in UA159 (Fig. 3B) were similar. SEM 350

analysis supported previous report (11) on the increased interaction of UAcov with 351

sucrose-derived EPS in biofilms, and confirmed strain capacities to bind sucrose-352

derived EPS under the growth conditions applied in the C3b binding assays (data not 353

shown). 354

355

Inactivation of covRSm impairs deposition of C3b, phagocytosis mediated by 356

C3b/iC3b receptors and opsonophagocytic killing by human PMN. UAcov shows low 357


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susceptibility to phagocytosis by human PMN in a serum-dependent way (13), 358

suggesting that CovRSm regulates surface components affecting serum opsonization. As 359

shown in Fig. 4A, deposition of C3b was impaired in UAcov. This phenotype was 360

completely restored in the complemented mutant UAcov+. Significant reductions in 361

the frequencies of phagocytosis of UAcov in the presence of 20% serum were also 362

observed in comparison to parent UA159 or UAcov+ (Fig. 4B). Importantly, blocking of 363

CR1 and/or CR3 receptors of PMN with anti-CD35 (CR1) or anti-CD11b/CD18 (CR3) 364

antibodies reduced differences in the frequencies of phagocytosis between UA159 and 365

UAcov (Fig. 4B). Additionally, simultaneous blockage of CR1 and CR3 receptors 366

abolished differences in the frequencies of phagocytosis between these strains (Fig. 367

4B), reflecting the multiple and cooperative functions of CR1 and CR3 on bacterial 368

phagocytosis mediated by C3b/iC3b (44). PMN treatment with anti-CD32 (FcɤRII) or 369

anti-CD16 (FcɤRIII) antibodies also reduced phagocytosis of UA159, although blockage 370

of both Fcɤ receptors did not eliminate differences in the frequencies of phagocytosis 371

between UA159 and UAcov (Fig. 4B). 372

To examine if reduced phagocytosis of UAcov was associated with reduced 373

killing by PMN, strains were compared in opsonophagocytic killing assays. As shown in 374

Fig. 4C, UAcov showed increased survival to PMN killing during 10 min of incubation. 375

Similar results were obtained after PMN exposure to the tested strains during 30 min 376

(data not shown). Viable bacteria in PMN culture supernatants were monitored, 377

confirming reduced phagocytosis of UAcov compared to UA159; mean counts of UAcov 378

in culture fluids were significantly higher than UA159 counts (p<0.05). These data 379

establish the strong influence of C3b/iC3b deposition on SM phagocytosis and imply 380


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that reduced deposition of C3b/iC3b on UAcov not only impairs phagocytosis mediated 381

by C3b/iC3b receptors, but is associated with reduced killing by PMN. 382

383

C3b deposition on SM is strongly dependent of C1q of the classical pathway 384

of complement activation. We hypothesized that EPS bound to SM surface could 385

compromise antibody recognition of immunogenic surface proteins, thus affecting the 386

classical pathway of complement activation. In this pathway, proteolytic cascade 387

initiates with C1q binding to different host components bound to the bacterial surface, 388

most prominently IgG or IgM antibodies, but also acute-phase proteins of the innate 389

immunity, e.g. CRP (17,18). Therefore, we analyzed the effect of covRSm inactivation on 390

levels of antibodies bound to SM (from pools of sera from six volunteers), and 391

investigated the effect of the classical pathway on binding of C3b to SM. Because the 392

classical pathway can be also activated by CRP bound to the surface of streptococcal 393

species containing CRP ligands (27,45), we additionally assessed binding of this acute-394

phase protein to SM. As shown in Fig. 5A, significant reductions in binding of IgG 395

antibodies were observed in UAcov, compared to parent strain. In addition, although 396

levels of CRP bound to SM UA159 were low, compared to the S. pneumoniae control 397

strains, UAcov showed reduced binding to CRP compared to UA159 (Fig. 5B). 398

Importantly, levels of surface-bound IgG and CRP were restored in the complemented 399

mutant UAcov+ (Fig. 5A,B). No significant changes in binding of IgM antibodies to SM 400

were observed (data not shown). 401

C3b deposition was minimal when strains were treated with C1q-depleted 402

serum compared to normal serum. Supplementation of C1q-depleted serum with 403

purified C1q (to physiological levels) restored C3b deposition (Fig. 5C). These data 404


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indicate that most of the C3b bound to SM surface resulted from activation of C1 405

component of the classical pathway. Thus, covRSm inactivation impairs SM-surface 406

binding of serum IgG antibodies and CRP, serum components that trigger the classical 407

pathway of complement activation, a major pathway involved in C3b deposition on 408

SM. 409

410

C3b deposition on SM is affected by interaction with sucrose-derived EPS. 411

SM accesses the bloodstream from oral niches, where it is exposed to dietary sucrose 412

to synthesize EPS, including glucan. CovRSm inactivation in serotype c strains not only 413

up-regulates genes for the synthesis of sucrose-derived glucan (gtfB, gtfC, gtfD), but 414

genes encoding glucan-binding proteins (gbpB, gbpC) and EpsC (epsC), which are 415

involved in surface binding to these polymers (7,8,10,11,43). Therefore, to address 416

whether sucrose-derived EPS on SM surface influences on C3b deposition, we 417

compared levels of C3b binding between parent and isogenic mutants of covRSm, 418

gtfB/C/D, gbpC and epsC previously grown in media with different concentrations of 419

sucrose, which were then harvested, washed and exposed to serum. As observed in 420

Fig. 6A, parent and covRSm grown in the presence of sucrose showed reduced levels of 421

C3b compared to the same strain grown in medium without sucrose. C3b deposition 422

was more intensely reduced in the UAcov in the presence of sucrose. When grown in 423

CDM supplemented with 0.1% sucrose, mean levels of C3b (MFI) on UAcov were 4-fold 424

lower than in the parent UA159 (82.3 versus 326.4). In sucrose-free CDM, levels of C3b 425

on UAcov were only 1.9-fold lower than C3b levels of parent UA159 (507.4 versus 426

991.5). In BHI, UAcov showed a 4-fold reduction in C3b deposition, but in BHI with 427

0.1% sucrose, the UAcov showed 9.4-fold reduction in C3b deposition compared to 428


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parent strain (33.4 versus 315.4). Although C3b binding was higher on strains grown in 429

sucrose-free CDM compared to BHI (Fig 6A), addition of 0.01% sucrose to CDM was 430

sufficient to eliminate these differences. This result suggests trace amounts of sucrose 431

in the complex BHI medium, although we can not rule out that unknown BHI 432

components adsorbed to the SM surface could also influence on C3b deposition. 433

Because sucrose-derived glucan are synthesized by GtfB, GtfC and GtfD, we 434

further confirmed that deletion of multiple gtf genes (double gtfB/C and triple 435

gtfB/C/D mutants) would eliminate the effect of previous exposure to sucrose on SM 436

susceptibility to C3b deposition. GtfB synthesizes insoluble glucan (rich in α1-3 437

linkages), while GtfC a mixture of insoluble and soluble (rich in α1-6 linkages) glucan, 438

and GtfD synthesizes only soluble glucan (1). As expected, significant increases in C3b 439

deposition were observed in the gtfB/C and gtfB/C/D mutants compared to parent 440

strain MT8148 in all culture conditions (Kruskal-Wallis with post hoc Dunn’s test; 441

p<0.05). Supplementation of growth medium with sucrose, did not significantly affect 442

C3b deposition on these mutants (Fig. 6B). Therefore, sucrose-derived EPS impacts C3b 443

deposition on SM surface. Of note, analyses of the effects of gtfB/C/D on C3b 444

opsonization were performed with mutants previously obtained in strain MT8148, 445

because gbpC inactivation affected C3b deposition on MT8148 in a similar fashion to 446

that observed in UA159 (see below). 447

Because levels of sucrose in the bloodstream seems to be minimal (46), we 448

assessed whether the SM capacity to bind sucrose-derived EPS influences C3b 449

deposition. Thus, levels of C3b were compared between the gbpC mutant (UAgbpC) 450

and parent strain UA159 previously grown in the presence or absence of sucrose. As 451

shown in Fig. 6C, deletion of gbpC promoted significant increase in C3b deposition 452


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when compared to the parent strain, especially when strains were recovered from 453

sucrose-containing medium: 0.1% sucrose BHI (mean 4.6-fold increase in C3b MFI) and 454

0.1% sucrose CDM (mean 3.2-fold increase in C3b MFI) (Kruskal-Wallis with post hoc 455

test, p<0.05). These results are compatible with the UAgbpC inability to bind EPS 456

produced from sucrose (Fig. 3B; Fig. S2). Similar results were observed in the gbpC 457

mutant obtained in strain MT8148 (data not shown). Influence of sucrose-derived 458

products on C3b deposition was further analyzed in the epsC mutant. Previous growth 459

of the epsC mutant in the presence of sucrose reduced C3b deposition (Fig. 6C), which 460

is compatible with the finding that UAepsC retained some capacity to bind EPS (Fig. 461

3B). Thus, expression of proteins which bind sucrose-derived EPS significantly affects 462

SM susceptibility to C3b deposition. Of note, the wild type strain MT8148 shows lower 463

binding to C3b than UA159 strain (Fig. 6A,B), consistently with its higher capacity to 464

interact with sucrose-derived EPS compared to UA159 (Fig. 3C). 465

Finally, to confirm that reduced susceptibility of blood isolates to C3b 466

deposition was promoted by sucrose-derived EPS, we compared levels of C3b 467

deposition on the blood strains SA13 and SA18 grown in the four growth media. As 468

reference, the oral strain 2VS1 (with reduced binding to sucrose-derived EPS; Fig. 3A) 469

was also tested. As expected, levels of C3b deposition on SA13 and SA18 increased 470

when strains were grown in sucrose-free CDM (Fig. 6D). Levels of C3b deposition on 471

2VS1 were significantly higher than that observed in the blood strains in all growth 472

conditions (Kruskal-Wallis; p<0.05), but addition of sucrose to media did not 473

significantly affect C3b binding to this strain (Fig. 6D). Thus, low susceptibility of blood 474

strains to C3b deposition is influenced by sucrose-derived EPS in a fashion similar to 475

that observed in the covR mutant. 476


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Sucrose-derived EPS influence on binding of serum IgG to the SM surface, 477

and on frequencies of opsonophagocytosis and killing by human PMN. Because 478

complement activation on SM was found to be strongly dependent on the classical 479

pathway, we investigated whether changes in C3b deposition in SM promoted by 480

previous growth in the presence of sucrose could be associated with reduced binding 481

to serum IgG. As observed in Fig. 7A, levels of IgG bound to UA159 and UAcov were 482

impaired when these strains were grown in medium added of sucrose. In addition, 483

exposure to sucrose significantly reduced phagocytosis and killing by PMN, specially in 484

UAcov (Fig. 7B,C). Consistently with flow cytometry analysis of phagocytosis (Fig. 7B), 485

mean counts of extracellular UAcov in the supernatants of PMN analyzed in killing 486

assays were 1.6 and 6.3-fold higher than UA159 extracellular counts, when strains 487

were respectively grown in CDM and CDM with 0.1% sucrose (data not shown). In 488

addition, similar to UAcov, grow of the blood strains SA13 and SA18 in medium 489

supplemented with sucrose impaired binding of serum IgG (Fig. 7D), reduced the 490

frequency of phagocytosis (Fig. 7E) and increased resistance to killing by PMN (Fig. 7F). 491

Differently, media supplementation with sucrose promoted limited effects on IgG 492

binding and phagocytosis in 2VS1 (Fig. 7D, E). These results support the role of surface-493

associated EPS on evasion to opsonophagocytosis and killing by PMN. 494

Binding of serum IgG to SM surface and rates of serum-mediated phagocytosis 495

were also assessed in mutants of CovRSm-repressed genes involved in the synthesis 496

and/or interaction with EPS (Fig. 8). Significant increases in IgG binding to SM surface 497

were promoted by inactivation of epsC, gbpC (Fig. 8A) and gtfB/C/D (Fig. 8C). 498

Inactivation of lysM and wapE had modest effects on IgG binding (Fig. 8A), compatible 499

with limited effects of these genes on binding to sucrose-derived EPS (Fig. 3B). 500


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Increases in phagocytosis were also observed in epsC, gbpC and gtfB/C/D (Fig. 8B, D). 501

As observed for C3b deposition, complementation of gbpC and epsC mutants restored 502

levels of IgG binding and phagocytosis (Fig. 8A, B). These findings strengthen the 503

influence of SM interactions with sucrose-derived EPS on strain susceptibilities to 504

opsonic phagocytosis by human PMN. 505

506

Inactivation of covRSm and of CovRSm-repressed gbpC and epsC affects 507

survival of SM in human blood and systemic virulence. Because complement 508

immunity is an important mechanism of blood clearance of streptococcal pathogens 509

(28,30,47), we investigated the effects of inactivation of covRSm and downstream genes 510

on ex vivo survival of SM in human blood. The UAcov mutant showed increased 511

capacity to survive in human blood, which was completely restored in the UAcov+ 512

complemented mutant (Fig. 9A). Consistent with the role of CovRSm as a direct 513

repressor of gbpC and epsC, the UAgbpC and UAepsC mutants showed reduced 514

survival in blood, when compared to parent and respective complemented mutants 515

(Fig. 9B, C). To further confirm the effects of EPS on the increased ex vivo survival of 516

UAcov in blood, assays were performed with strains grown in sucrose-free CDM and in 517

CDM with 0.1% sucrose. As shown in Fig. 9D, differences in survival in blood between 518

UAcov and UA159 were eliminated when strains were grown in CDM, whereas CDM 519

supplementation with sucrose increased differences between UAcov and parent 520

strains (Fig. 9E). Thus, sucrose-derived EPS are involved in the increased survival of 521

UAcov in human blood. 522

Comparisons of bacterial counts in the bloodstream of rats, confirmed 523

findings of ex vivo survival in human blood for UAcov. As shown in Table 3, UAcov 524


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mutant survives for longer periods and at higher counts in the rat bloodstream, when 525

compared to parent or complemented strains. Two rats infected with UAcov died 526

during the experiment, while no deaths occurred in the UA159-infected group. Higher 527

SM counts were found in valves of UAcov-infected rats (cfu/ml; mean: 20,090 ± 528

44,467; median: 40) compared to the valves of UA159-infected animals (mean: 1,352 ± 529

2,723; median: 0), although differences between strains did not reach statistical 530

significance (Kruskal-Wallis with post hoc Dunn’s test; p>0.05). 531

532

DISCUSSION 533

534

The complement system plays multiple roles in elimination of 535

microorganisms, both as part of the innate immune system and by augmenting 536

antibody-mediated immunity (17,18). Reduced susceptibilities to C3b deposition found 537

in SM blood strains (Fig. 1) indicate that evasion to complement immunity is important 538

for systemic virulence of SM. In GAS, natural mutations in covRSpy were detected in 539

strains involved in human infections, and inactivation of the TCS CovRSSpy enhanced 540

strain virulence in murine models (48-50). Virulence genes repressed by CovRSpy 541

include genes involved in complement evasion (e.g. has operon for hyaluronic acid 542

capsule synthesis) (50-52), which are not present in the SM genomes (53,54). Reduced 543

transcript levels of covRSm in blood isolates associated with increased transcription of 544

CovRSm-repressed genes (gbpC, gbpB, epsC), suggests that diversity in covRSm activities 545

influences on SM strain capacity to survive in the bloodstream. 546

In GAS and GBS, CovRSSpy/Sag regulons show strain-specificity (55,56).The 547

CovRSm regulon was assessed in the serotype c strain UA159 (8,11), but its diversity 548


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remains to be investigated in strains associated with systemic infections. SM serotype 549

c was detected with higher frequencies in SM-positive specimens of heart valve and 550

atheromatous plaque from patients subjected to cardiovascular surgeries (30.3 and 551

65.5% of specimens respectively), compared to serotype k (detected in 9.1 and 25% of 552

these specimens, respectively) (14), previously implicated in systemic infections (57). 553

Interestingly, 77% of serotype k-positive specimens were also positive for serotype c 554

(41), suggesting synergy of SM serotypes for systemic virulence. Systemic virulence of 555

serotype k is associated with expression of the collagen-binding proteins Cnm and 556

Cbm, which are involved in SM capacity to invade endothelial cells in vitro (40,58-60) 557

and to form vegetations on injured heart valves in a rat model of infective endocarditis 558

(40). However, serotype c strains rarely harbour these genes (19) and there is no 559

report that CovRSm regulates cnm or cbm. 560

A major function of complement immunity against Gram-positive bacteria is 561

to covalent bind C3b/iC3b opsonins on the bacterial surface through the activity of C3-562

convertases on C3 (17,18). C3-convertases result from proteolytic cascades initiated by 563

different mechanisms, known as the classical, mannan-binding lectin and alternative 564

pathways (17). Functions of each pathway in complement immunity against 565

streptococci seem to be species-specific (27,28,30,45), and are usually circumvented 566

by multiple evasion mechanisms (41,61,62). Here, we show that the classical pathway 567

plays a major role in complement deposition on SM (Fig. 5C), which is consistent with 568

reduced binding to IgG antibodies to UAcov (Fig. 5A). Because C1q is activated through 569

its binding to IgG on bacterial surface, assessing individual roles of complement and 570

IgG to S. mutans opsonization is difficult. In addition, although SM seems to not have 571

prototypical CRP-ligands (63), covRSm inactivation also reduced binding to CRP (Fig. 5B). 572


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CRP levels increase in the bloodstream of subjects with biofilm-dependent oral 573

diseases, e.g. gingivitis and periodontitis (64), thus the role of acute-phase proteins on 574

SM blood clearance need to be investigated. 575

The major known role of CovRSm in SM biology is to regulate the expression of 576

secreted enzymes for the synthesis of EPS from sucrose, and cell surface components 577

involved in interaction with EPS (5,6,10,11). Some of these genes, e.g. gtfBC, ftf and 578

wapE, are controlled by a complex regulatory circuit (11,65-67), which might explain 579

the lack of associations between gtfB/C transcription and profiles of covRSm expression 580

among the tested strains (Fig. 2). Because secreted GtfB/C are stable in saliva and bind 581

to several oral bacteria (2), strains with increased binding to EPS could benefit from 582

Gtf-producing members of the same ecological niche. Thus, increased capacity to bind 583

EPS would be more significant for systemic virulence than the ability to produce Gtfs 584

itself. In the serotype c strain V403, deletion of multiple genes required for the 585

synthesis of EPS from sucrose (gtfB, gtfC, ftf), increased SM phagocytosis by human 586

granulocytes and reduced virulence in an animal model of infectious endocarditis (68). 587

Consistently, our gtfB/C and gtfB/C/D mutants showed high susceptibility to C3b-588

opsonization even when grown in the presence of sucrose (Fig. 6). However, 589

production of sucrose-derived EPS only impaired C3b deposition in strains capable to 590

bind these polymers; the gbpC mutant was susceptible to C3b deposition even when 591

grown in the presence of sucrose (Fig. 6). Thus, expression of gbpC, and perhaps other 592

glucan-binding proteins upregulated in blood isolates, e.g. GbpB (Fig. 2), might be 593

critical for EPS-mediated complement evasion. 594

Increased binding of the gbpC mutant to serum IgG (Fig. 6), further indicates 595

that surface EPS may impair antibody-mediated activation of complement in a way 596


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analogous to capsules of S. pneumoniae (45,47,69). Besides GbpC, EpsC, showed 597

prominent influence in complement opsonization (Fig. 6,8). In Gram-positive bacteria 598

EpsC is required for the production of UDP-ManNAc, an intermediate for the synthesis 599

of EPS, which is also required for the attachment of teichoic acids to the cell wall (70-600

73). The epsC mutant retained some degree of binding to sucrose-derived EPS (Fig. 3B), 601

which might explain, at least in part, the remaining influence of sucrose on binding of 602

C3b to this mutant (Fig. 6). Alternatively, EpsC could also affect sucrose-independent 603

mechanisms of SM evasion to complement immunity. Functional analyses of EpsC 604

might shed new light on its roles in complement susceptibility. 605

Although the effects of covR deletion on C3b opsonization were more clearly 606

observed when strains were grown in sucrose-containing media, reductions in levels of 607

C3b binding to UAcov were still observed in sucrose-free CDM (Fig. 6), which suggests 608

that CovRSm regulates additional functions of complement evasion. Lower levels of C3b 609

binding to blood strains grown in sucrose-free CDM (especially in SA13; Fig. 6), account 610

for the hypothesis that SM strains apply multiple mechanisms of complement evasion. 611

In GAS strains, CovRSpy plays multiple roles in complement evasion besides regulating 612

capsule production (50,52,74). Studies are in course to identify additional factors 613

affecting SM susceptibility to complement immunity. 614

The increased persistence of UAcov in human blood mediated by sucrose-615

derived EPS (Fig. 9) and its ability to cause bacteremia in rats (Table 3), further 616

strengthen the role of CovRSm in systemic virulence. Reduced C3b/IgG-opsonization of 617

UAcov is, at least in part, explained by up-regulation of epsC and gbpC, because 618

inactivation of these genes reduced survival in human blood (Fig. 9A,B). Different from 619

this study, no reduction in survival in the bloodstream of rats was observed in the gbpC 620


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mutant (C1) obtained in MT8148, compared to a streptomycin-resistant MT8148 621

variant (MT8148R) (75). Although we found that C1 has increased susceptibility to C3b 622

deposition compared to MT8148 parent strain (data not shown), C3b deposition in the 623

MT8148R variant is unknown. As shown in this study, differences in growth media can 624

affect SM susceptibility to complement opsonization. Furthermore, bacterial 625

aggregation mediated by blood components could affect bacterial counts in blood 626

suspensions. UAcov shows increased aggregation in blood compared to UA159 (data 627

not shown), which could explain the initial reductions in UAcov counts in the ex vivo 628

assays, although bacterial loads were normalized by initial blood counts (Fig. 9). 629

Increased aggregation of UAcov could occur in the rat bloodstream, thus survival rates 630

of UAcov (Table 3) might be underestimated. 631

Differences in complement activation on SM between the human and rat 632

blood may also exist. SM is an exclusive species of humans, thus levels and epitope 633

specificities of IgG antibodies to SM may differ between human and rat sera. Further 634

differences exist in production and structure of CRP between rats and humans (76). In 635

addition, CR1, shown to be important for SM opsonophagocytosis, is also involved in 636

blood clearance by human erythrocytes through immune adherence (77). Because 637

rodent erythrocytes do not express CR1 (77), a more complete analysis of the influence 638

of C3b deposition on blood clearance of SM would require animal models designed to 639

assess CR1-mediated immune adherence (78). Apart from the limitations of our model, 640

significant increases were detected in viable counts of UAcov in the rat bloodstream, 641

compared to UA159 (Table 3). At 6 h post-infection, there was 6.3-fold higher counts 642

of UAcov mutant (detected in 85.7% of the animals) compared to parent strain 643

(detected in 57.1% of animals). Although UAcov counts in heart valves were higher 644


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compared to UA159 counts, these differences did not reach significance. Because only 645

the numbers of viable bacteria were assessed, we cannot exclude the possibility that 646

increased differences on tissue infection might have been observed between strains if 647

total levels of bacteria in heart valve specimens were measured using culture-648

independent methods. In addition, survival of UAcov in the rat bloodstream would 649

likely increase if strains were previously grown in medium added of 0.1% sucrose. 650

Therefore, studies are required to improve in vivo models for assessing the influence of 651

complement evasion on systemic virulence of SM. 652

In summary, this study provides evidence that systemic virulence of SM 653

strains involves reduced susceptibility to complement-mediated opsonization. Roles of 654

CovRSm in resistance to complement immunity involves the regulation of SM capacity 655

to interact EPS, which in turn affects complement activation. Two CovRSm-repressed 656

genes, gbpC and epsC were identified as playing important roles in resistance to 657

complement immunity and survival in blood, as revealed by transcriptional profiles of 658

these genes in isolates from systemic infections and by molecular analyses of isogenic 659

mutants. 660

661

ACKNOWLEDGEMENTS 662

We would like to thank Dr. Satu Alaluusua for providing the blood isolates 663

analyzed in this study. We thank Dr. Daniel J. Smith for the critical reading of this 664

manuscript. This study was supported by Fundação de Amparo à Pesquisa do Estado 665

de São Paulo (FAPESP, proc. 2012/50966-6 and 2015/12940-3). LAA was supported by 666

FAPESP (proc. 2012/04222-5; 2015/07237-1). EHC was supported by FAPESP (proc 667


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2009/50547-0) and CAPES-PNPD (2013). This work was also supported by KAKENHI 668

Grant Number 15K11363 from Japan Society for the Promotion of Science. 669

670

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FIGURE LEGENDS 960

Fig. 1. A. Box plot comparisons of C3b deposition between S. mutans (SM) strains 961

isolated from blood samples and from the oral cavity. C3b bound to serum-treated 962

strains was probed with anti-C3 antibody conjugated with FITC for flow cytometry 963

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Fluorescence Intensity (MFI). B) Fluorescence Index (FI) values were obtained by 965

multiplying the percentage of C3b-positive cells by MFI for C3b. MFI and FI data are 966

means of three independent experiments. Asterisks indicate significant differences 967

between groups (Mann-Whitney U-test; p< 0.05). 968

969

Fig. 2. RT-qPCR comparisons of transcript levels of covRSm and CovRSm-regulated 970

genes (lysM, wapE, epsC, gbpC, gbpB, gtfB, gtfC) between blood (n=4) and oral (n=5) 971

strains of S. mutans. Gene gtfD, which is not regulated by CovRSm was tested as a 972

control. Asterisks indicate significant differences in mean levels of transcripts between 973

groups (ANOVA post hoc Tukey´s test; p< 0.05). 974

975

Fig. 3. Comparisons of S. mutans capacities to aggregate in the presence of sucrose-976

derived EPS. Strains were incubated in BHI supplemented with 0.1% sucrose during 24 977

h for visual analysis of clump formation. Intensities of cell aggregation were 978

determined by a blind examiner and indicated below the respective images. A) 979

Comparisons between blood and oral strains. B) Comparisons between parent strain 980

UA159 with the respective knockout mutants. C) Comparisons of parent strain MT8148 981

with the respective knockout mutants. 982

983


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Fig. 4. Comparisons of C3b deposition, opsonophagocytosis and killing by PMN 984

between covR mutant (UAcov) with parent (UA159) and complemented strains (+). A) 985

Intensities of C3b deposition (MFI) were determined by flow cytometry in strains 986

treated with a reference serum or pools of sera obtained from six volunteers. B) 987

Percentages of PMN with associated bacteria were assessed after 5 min of PMN 988

exposure to FITC-labeled bacteria in the presence of 20% serum. Untreated PMN and 989

PMN treated with MAbs to block CR1 (CD35), CR3 (CD11b/CD18), FcɤRII (CD32) and/or 990

FcɤRIII (CD16) receptors were tested. C) Percentages of intracellular survival in PMN 991

after 10 min of incubation with preopsonized bacteria were calculated in relation to 992

bacterial counts of no PMN control samples. Columns represent means of three 993

independent experiments. Bars indicate standard deviations. Asterisks indicate 994

significant differences in relation to UA159 within the same condition (Kruskal-Wallis 995

with post hoc Dunn’s test ; p< 0.05). 996

997

Fig. 5. Binding of serum IgG, CRP and C3b to S. mutans strains in the presence of 998

serum. A,B) Strains were treated with 20% human serum and levels of surface IgG or 999

CRP determined by flow cytometry (MFI). S. pneumoniae strains D39 and TIGR4 were 1000

used as control for CRP binding. C) Levels of C3b binding were measured after bacterial 1001

treatment with a reference serum, serum depleted of C1q (C1q-) or C1q- serum 1002

supplemented with purified C1q (C1q+). Columns represent means of three 1003

independent experiments; bars represent standard deviations. Strains were compared 1004

using Kruskal-Wallis with post hoc Dunn’s test. Asterisks indicate significant differences 1005

in relation to UA159 within the same condition (p< 0.05). 1006

1007


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44

Fig. 6. Effects of previous growth in medium supplemented with sucrose in strain 1008

susceptibilities to C3b deposition. Strains grown in BHI or CDM supplemented or not 1009

with 0.01 or 0.1% sucrose were harvested, washed with PBS and treated with human 1010

serum for C3b deposition. Columns represent means of MFI for C3b determined by 1011

flow cytometry in three independent experiments. Bars represent standard deviations. 1012

Asterisks indicate significant differences between groups (Kruskal-Wallis with post hoc 1013

Dunn’s test: * p< 0.05). 1014

1015

Fig. 7. Effects of previous growth of S. mutans strains in medium supplemented with 1016

sucrose in binding to serum IgG, in susceptibility to phagocytosis by PMN and in killing 1017

by PMN. A,D) Strains grown in BHI or CDM supplemented or not with 0.01 or 0.1% 1018

sucrose were harvested, washed with PBS and treated with human serum for IgG 1019

binding. Levels of IgG binding were determined by flow cytometry and expressed as 1020

MFI. B,E) FITC-labeled strains grown in different media were incubated with PMN 1021

isolated from human peripheral blood in the presence of 20% serum during 5 min. C, F) 1022

Strains grown in CDM supplemented or not with 0.1% sucrose were preopsonized and 1023

incubated with PMN (10 min). Percentages of intracellular survival were calculated in 1024

relation to viable counts determined in the no PMN control samples. Columns 1025

represent means of three independent experiments. Bars indicate standard deviations. 1026

Asterisks indicate significant differences in comparison to parent strain (Kruskal-Wallis 1027

with post hoc Dunn’ test ; * p<0.05). 1028

1029

Fig. 8. Comparisons of binging to serum IgG (A,C) and of phagocytosis by human PMN 1030

(B, D) between mutants of genes regulated by CovRSm. Mutant (gray columns) or 1031


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complemented (dashed columns) strains were compared with the respective parent 1032

strain (UA159 or MT8148). Columns represent means of three independent 1033

experiments; bars indicate standard deviations. Asterisks indicate significant 1034

differences in relation to parent strain (Kruskal-Wallis with post hoc Dunn’s test ; * 1035

p<0.05). 1036

1037

Fig. 9. Ex vivo viability in human blood. Numbers of viable bacteria (log cfu/ml) were 1038

expressed in relation to initial counts of blood suspensions (time 0). A, B, C) Strains 1039

were grown in BHI, D) CDM or E) CDM supplemented with 0.1% sucrose. Data 1040

represent means of triplicate of one representative experiment. Bars indicate standard 1041

deviations. Differences in relation to parent strain at each time-point were tested by 1042

Kruskal-Wallis with post hoc Dunn’s test (*p<0.05). 1043

1044

TABLE FOOTNOTES 1045

Table 3. *Significant differences in relation to parent strain at the same time period 1046

(Kruskal-Wallis with post hoc Dunn’s test; p < 0.05). aNumber of rats with recovered 1047

bacteria are indicated within parentheses. 1048


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A *

C3

b d

ep

osi

tio

n (

MF

I)

(n=7) (n=13)

Blood isolates Oral isolates

B

C3

b d

ep

osi

tio

n (

FI)

(n=7) (n=13)


*

Fig. 1


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Fig. 2

Re

lati

ve

le

ve

ls o

f wapE

tra

nsc

rip

ts



Re

lati

ve

le

ve

ls o

f covR

tra

nsc

rip

ts


Re

lati

ve

le

ve

ls o

f lysM

tra

nsc

rip

ts

Re

lati

ve

le

ve

ls o

f epsC

tra

nsc

rip

ts


Re

lati

ve

le

ve

ls o

f gbpC

tra

nsc

rip

ts


Re

lati

ve

le

ve

ls o

f gtfB

tra

nsc

rip

ts

Blood isolates Oral isolates Blood isolates Oral isolates

Re

lati

ve

le

ve

ls o

f gtfC

tra

nsc

rip

ts

Blood isolates Oral isolates R

ela

tive

le

ve

ls o

f gtfD

tra

nsc

rip

ts

*

Re

lati

ve

le

ve

ls o

f gbpB

tra

nsc

rip

ts


*

* *


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UA159 UAcov

+ + + + +

MT8148 gbpC gtfBC gtfBCD

+ + + _ _ _

+ + + +

UA159 wapE lysM epsC gbpC

_ _

Oral isolates

SA13 SA15 SA16 8ID3 11SSST2 SA18

Blood isolates

2VS1 11A1

+ + + + + + + _ _ + _ + _ + + + + +

A

B

C

Fig. 3


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0

10

20

30

40

50

60

70

80

No

blocking

CR1 CR3 CR1 + CR3 FcγRII FcγRIII FcγRII +

FcγRIII

CR1 + CR3

+ FcγRII +

FcγRIII

% P

MN

s w

ith

in

tra

cell

ula

r b

act

eri

a

UA159 UAcov UAcov+

0

100

200

300

400

500

600

700

800

UA159 UAcov UAcov+

C3

b d

ep

osi

tio

n (

MF

I)

Strains

Reference serum Serum Pool

A

* *

B

*

* *

*

*

*

C

0

5

10

15

20

UA159 UAcov UAcov+

% I

ntr

ace

lull

ar

surv

iva

l

Strains

*

Fig. 4


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A

0

50

100

150

200

250

UA159 UAcov UAcov+ Bin

din

g o

f se

rum

Ig

G (

MF

I)

Strains

*

C

0

400

800

1200

1600

UA159 UAcov UAcov+

C3

b d

ep

osi

tio

n (

MF

I)

Strains

Reference serum C1q- C1q+

*

*

B

0

20

40

60

80

100

120

140

160

UA159 UAcov UAcov+ D39 TIGR4B

ind

ing

of

CR

P (M

FI)

Strains

*

*

Fig. 5


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Fig. 6

C

A

0

500

1000

1500

2000

2500

UA159 UAgbpC UAgbpC+ UAepsC UAepsC+

C3

b d

ep

osi

tio

n (

MF

I)

Strains

*

*

*

*

*

*

*

*

* *

*

B

BHI BHI 0.1% sucrose CDM CDM 0.01% sucrose CDM 0.1% sucrose

D

0

500

1000

1500

2000

2500

3000

SA13 SA18 2VS1

C3

b d

ep

osi

tio

n (

MF

I)

Strains

*

*

*

*

*

0

500

1000

1500

2000

2500

MT8148 gtfBC gtfBCD

C3

b d

ep

osi

tio

n (

MF

I)

Strains

* *

gtfBC gtfBCD

0

200

400

600

800

1000

1200

1400

1600

UA159 UAcov UAcov+

C3

b d

ep

osi

tio

n (

MF

I)

Strains

*

*

*

*

*

*

*

*


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Fig. 7

A

0

100

200

300

400

500

UA159 UAcov UAcov+

Bin

din

g t

o s

eru

m I

gG

(M

FI)

Strains

*

*

*

*

*

*

B

0

20

40

60

80

100

120

UA159 UAcov UAcov+

% P

MN

s w

ith

in

tra

cell

ula

r

ba

cte

ria

Strains

*

*

*

*

* *

D

0

100

200

300

400

500

SA13 SA18 2VS1

Bin

din

g t

o s

eru

m I

gG

(M

FI)

Strains

*

*

*

*

0

20

40

60

80

100

SA13 SA18 2VS1

% P

MN

s w

ith

in

tra

cell

ula

r

ba

cte

ria

Strains

*

* *

*

* *

E

BHI BHI 0.1% sucrose CDM CDM 0.1% sucrose

C

F

0

20

40

60

80

100

120

SA13 SA18

% I

ntr

ace

lull

ar

surv

iva

l

Strains

*

*

0

20

40

60

80

100

120

140

UA159 UAcov UAcov+

% I

ntr

ace

lull

ar

surv

iva

l

Strains

*


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Fig. 8

0

100

200

300

400

500

600

700

800

MT8148 gtfBC gtfBCD

Re

lati

ve

bin

din

g t

o

se

rum

Ig

G (

MF

I)

Strains

C

* *

MT8148 gtfBC gtfBCD 0

50

100

150

200

250

UA159 lysM wapE epsC gbpC

Re

lati

ve

bin

din

g t

o

seru

m I

gG

(M

FI)

Strains

Mutant Complemented

*

* *

A

lysM wapE epsC gbpC

0

20

40

60

80

100

120

140

UA159 lysM wapE epsC gbpC

Re

lati

ve

% o

f P

MN

s w

ith

intr

ace

llu

lar

ba

cte

ria

Strains

Mutant Complemented

B

*

* *

lysM wapE epsC gbpC

0

20

40

60

80

100

120

140

160

180

200

MT8148 gtfBC gtfBCD

Re

lati

ve

% o

f P

MN

s w

ith

intr

ace

llu

lar

ba

cte

ria

Strains

* *

D

MT8148 gtfBC gtfBCD


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0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 0.5 1 2 4 24

Re

lati

ve

nu

mb

ers

of

via

ble

ba

cte

ria

time (h)

UA159 UAcov UAcov+

* *

*

A

1.20

1.00

0.80

0.60

0.40

0.20

0.00 0 0.5 1 2 4 24

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 0.5 1 2 4 24

Re

lati

ve

nu

mb

ers

of

via

ble

ba

cte

ria

time (h)

UA159 UAepsC UAepsC+

*

* *

B 1.20

1.00

0.80

0.60

0.40

0.20

0.00 0 0.5 1 2 4 24

C

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 0.5 1 2 4 24

Re

lati

ve

nu

mb

ers

of

via

ble

ba

cte

ria

time (h)

UA159 UAgbpC UAgbpC+

* *

* *

1.20

1.00

0.80

0.60

0.40

0.20

0.00 0 0.5 1 2 4 24

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 0.5 1 2 4 24

Re

lati

ve

nu

mb

ers

of

via

ble

ba

cte

ria

time (h)

UA159 UAcov UAcov+

D 1.20

1.00

0.80

0.60

0.40

0.20

0.00 0 0.5 1 2 4 24

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 0.5 1 2 4 24

Re

lati

ve

nu

mb

ers

of

via

ble

ba

cte

ria

time (h)

UA159 UAcov UAcov+

* * *

E 1.20

1.00

0.80

0.60

0.40

0.20

0.00 0 0.5 1 2 4 24

Fig. 9


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Ta le . Strai s used i this study.

Strai Rele a t hara teristi s Sour e or refere eUA Oral isolate, aries-affe ted hild; er s, spe s, ka s ATCC UA o R ∆ o R::Er r UA apE ∆ apE::Er r UAlysM ∆lysM::Er r UAepsC ∆epsC::Er r UAg pC ∆g pC::Er r This studyUA o R+ r ∆ o R::Er r; pDL ::SMU. 9 ; Spe r UA apE+ ∆ apE::Er r; pDL ::SMU. 9 ; Spe r UAlysM+ ∆lysM::Er r; pDL ::SMU. ; Spe r UAepsC+ ∆epsC::Er r; pDL ::SMU. ; Spe r UAg pC+ ∆g pC::Er r; pDL ::SMU. 9 ; Spe r This studyMT Oral isolate; healthy Japa ese hild C ∆g pC::Ka r, uta t of MT S ∆gtfBC::Er r, dou le uta t of MT BC s ∆gtfD::Er r, ∆gtfBC::Ka r, triple uta t of MT

ST Oral Isolate, aries-affe ted hild VS Oral Isolate, aries-affe ted hild SN Oral Isolate, aries-free hild SM Oral Isolate, aries-free hild VF Oral Isolate, aries-affe ted hild SM Oral Isolate, aries-free hild ID Oral Isolate, aries-free hild

A Oral Isolate, aries-free hild SSST Oral Isolate, aries-free hild VS Oral Isolate, aries-free hild JP Oral Isolate, aries-free hild VF Oral Isolate, aries-affe ted hild

SA Blood, i fe ti e e do arditis SA Blood, a tere ia SA Blood, i fe ti e e do arditis SA Blood, a tere ia SA Blood, i fe ti e e do arditis SA Blood, a tere ia SA Blood, i fe ti e e do arditis D Strepto o us p eu o iae serotype NCTC NCTC TIGR Strepto o us p eu o iae serotype ATCC BAA- ATCC


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Ta le . Oligo u leotides used i this study. Na e Se ue e ’ – ’ a

P odu t size; positio o ele a t ha a te isti

e E -As I e E -XhoI

TTGGCGCGCCTGGCGGAAACGTAAAAGAAG TTCTCGAGGGCTCCTTGGAAGCTGTCAGT

p; a pli o o tai i g the ermr ge e f o pVA .

g pCP g pCP -As I

CCCTCAACACACTCTGCTAA TTGGCGCGCCCGGTTCTGATGCTTGTGTAT

p; p upst ea to p do st ea of gbpC ORF.

g pCP -XhoI g pCP

TTCTCGAGGGAGAAATGCGTGTTAGAGA CTTACCCATCACAAAAACCA

p; , p upst ea to p do st ea of gbpC e odi g egio .

C - Sa I C - SphI

GGGAGCTCCCCTCAACACACTCTGCTAA GGGCATGCAACAAGAACTGCTGCTCAAG

, p; a pli o o tai i g gbpC e odi g egio fo uta t o ple e tatio .

a U de li ed se ue es i di ate est i tio e zy e li ke s.


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Ta le . Ba terial ou ts fu/ l re o ered fro lood of rats = .

I fe ted strai Mea fu/ l of lood ± SD a at differe t post-i fe tio periods h h h h days

UA , ± ,

±

±

UA o , ± ,

±

± *

± *

UA o + , ± ,

± *

±

±

*Sig ifi a t differe es i relatio to pare t strai at the sa e ti e period Kruskal-Wallis ith post hoc Du ’s test; p < . . aNu er of rats ith re o ered a teria are i di ated ithi pare theses.


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