functional analysis of the cpsa protein of streptococcus ... · gbs expressing cpsa-245, but not...

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Functional analysis of the CpsA protein of Streptococcus agalactiae 2

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Running title: GBS CpsA functional analysis 4

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Brett R. Hanson1, Donna L. Runft1, Cale Streeter1, Abhin Kumar1, Thomas W. Carion2, and 6

Melody N. Neely1* 7

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1Dept. of Immunology and Microbiology 9

Wayne State University School of Medicine 10

Detroit, MI 48201 11

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2 Department of Biology 13

Kalamazoo College 14

Kalamazoo, MI 49006 15

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*Corresponding author: 17

Dept. of Immunology & Microbiology 18

540 E. Canfield St. 19

Detroit, MI 48201 20

313-577-1314 21

313-577-1155 (Fax) 22

[email protected] 23

Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.06373-11 JB Accepts, published online ahead of print on 27 January 2012

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Abstract 24

Streptococcal pathogens such as Streptococcus agalactiae (GBS) are an important cause 25

of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The 26

CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution 27

to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and 28

two truncated forms of the protein were purified and analyzed for DNA binding ability. Assays 29

demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule 30

operon with similar affinity, and full-length protein is required for specificity. Functional 31

characterization of CpsA confirmed that the ΔcpsA strain produced less capsule than wild type, 32

and demonstrated that production of full length CpsA or the DNA-binding region of CpsA 33

resulted in increased capsule levels. In contrast, production of a truncated form of CpsA lacking 34

the extracellular LytR domain (CpsA-245) in the wild type background resulted in a dominant 35

negative decrease in capsule production. GBS expressing CpsA-245, but not the ΔcpsA strain, 36

were attenuated in human whole blood. However, the ΔcpsA strain showed significant 37

attenuation in a zebrafish infection model. Furthermore, chain length was observed to be 38

variable in a CpsA-dependent manner, but could be restored to wild type levels when grown with 39

lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing 40

multiple regulatory functions that may include not only capsule synthesis, but also cell wall 41

associated factors. 42

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Introduction 47

Streptococcal pathogens capable of causing systemic disease utilize a number of 48

strategies for survival in the host. The most important of these is the polysaccharide capsule that 49

is produced to shield the pathogens from clearance by components of the immune system, 50

including complement deposition (18) and phagocytosis (15). The production of a 51

polysaccharide capsule has proven to be a successful strategy for a number of human-specific 52

pathogens, including the Group B Streptococcus (GBS) Streptococcus agalactiae, as well as 53

Streptococcus pneumoniae. GBS has long been a significant cause of neonatal mortality (9), and 54

long term sequelae (10), with intrapartum antibiotic prophylaxis the recommended measure to 55

combat incidence of infection (32). GBS infection of neonates remains a disease of significant 56

import in developing countries, and the preemptive use of antibiotics in the colonized mother to 57

circumvent disease is not ideal, as the development of drug resistance is a major concern (32). 58

Though rare in adults, recent work has revealed an alarming trend of increased incidence of GBS 59

infection in the United States in non-pregnant adults (26, 29), particularly in elderly patients with 60

at least one underlying health issue, illustrating that GBS remains an important problem for 61

adults as well. These observations demonstrate the need for further characterization of targets 62

for antimicrobial therapy or vaccine generation, and the unequivocal importance of the 63

polysaccharide capsule during infection makes it a prime candidate for disruption and subsequent 64

alleviation or prevention of disease. 65

The production of a polysaccharide capsule by both GBS and S. pneumoniae rely on a 66

number of shared components, with the first four genes of the capsule operon having the highest 67

conservation between the two species with greater than 60% similarity (7). These genes are 68

annotated as cpsA, cpsB, cpsC, and cpsD for both species. The genes cpsB, cpsC, and cpsD, 69


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constitute a phospho-relay system that regulates polymerization and ligation of the capsular 70

polysaccharide to the cell wall peptidoglycan (3, 5, 21, 22, 34), with the encoded proteins CpsB 71

as a phospho-tyrosine protein phosphatase, CpsD as a tyrosine kinase, and CpsC as a membrane 72

tether and accessory protein for CpsD. The gene cpsA encodes a putative membrane-bound 73

transcriptional regulator of the capsule operon (11), and contains a small intracellular domain 74

and two conserved extracellular protein domains; the DNA Polymerase Processivity Factor 75

(DNA_PPF; Pfam accession no. PF02916) domain and the LytR_cpsA_psr (LytR; Pfam 76

accession no. PF03816) domain. 77

The presence of the DNA_PPF domain is curious for two reasons; first, although this 78

region of the protein is categorized as belonging to a family of sliding clamp proteins that bind 79

directly to DNA, the DNA_PPF domain of CpsA has been shown to reside extracellularly (12). 80

Second, despite this region of the protein being classified with the DNA_PPF designation, the 81

protein sequence of the DNA_PPF region of CpsA proteins diverges a great deal from traditional 82

DNA_PPF sliding clamp proteins, with a BLAST alignment of the GBS CpsA DNA_PPF 83

domain to the bacteriophage RB69 DNA_PPF domain giving no significant similarity. 84

Therefore, we propose that the DNA_PPF designation of this portion of the protein does not 85

correspond to a function consistent with sliding clamps, and that streptococcal species utilize the 86

DNA_PPF domain of CpsA for some other as yet unknown function, which has been suggested 87

previously (13). 88

In contrast to the DNA_PPF designation, the LytR designation of the CpsA protein is 89

much more robust, with a sequence alignment of the GBS CpsA LytR domain to the Bacillus 90

subtilis LytR protein’s LytR domain giving 38% identity and 58% similarity (E-value = 1e-25), 91

indicating possible functional conservation. LytR proteins have been associated with 92


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transcriptional attenuation of the lytRABC divergon (16), which encodes cell-wall modifying 93

enzymes. In contrast to the transcriptional attenuation observed for LytR, CpsA appears to have 94

a transcriptional activating function for the capsule locus (7). The mechanism by which the LytR 95

domain functions remains unclear, and it may act as an environmental sensor that modulates the 96

activity of the protein, thereby controlling the cytoplasmic domain, or in the case of CpsA 97

altering the function of the DNA_PPF domain. In addition to regulation of capsule expression, 98

CpsA may prove to exhibit roles associated with cell wall regulation as well, which would be 99

consistent with the function assigned to LytR proteins. Although GBS lacks the lytABC operon 100

of Bacillus and other species, CpsA may represent a regulatory module at the crossroads of 101

capsule and the cell wall, two of the major surface components of streptococcal cells. 102

The presence of a small N-terminal cytoplasmic domain is common to both GBS CpsA 103

and B. subtilis LytR proteins with 26 and 11 amino acids respectively. We have previously 104

shown that this small cytoplasmic region attached to the transmembrane domains is sufficient for 105

S. iniae CpsA to bind to the promoter upstream of cpsA (12), and it may be that LytR proteins 106

function similarly. Both CpsA and LytR proteins contain a high density of positively charged 107

amino acids in the intracellular domains with 11/26 amino acids for CpsA and 7/11 amino acids 108

for LytR which may help facilitate interaction with specific DNA sequences. Additionally, the 109

CpsA proteins of GBS and S. pneumoniae have putative leucine zipper domains extending from 110

the cytoplasmic region into the first transmembrane region, which could also help facilitate DNA 111

binding through dimerization. LytR proteins lack this property. Although the leucine repeat is 112

present, there is no predicted coiled-coil sequence for CpsA, so the presence of a functional 113

leucine zipper domain requires validation. In addition to the operon promoter upstream of the 114

cpsA gene, a second promoter element may also exist upstream of the cpsE gene in GBS, as a 115


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secondary transcription initiation site was identified in this region with upstream A-T rich 116

repeats (33). 117

This study focuses on the function of each of the CpsA protein domains as they relate to 118

the capsule locus promoters, actual capsule levels, and preliminarily, to cell wall stability. We 119

demonstrate that the GBS CpsA protein is able to bind specifically to both the GBS cpsA and 120

cpsE promoter elements, as well as define the regions of CpsA that facilitate binding to DNA 121

and contribute to the specificity of the interaction. We also demonstrate that expression of these 122

different domains in either the WT or a ΔcpsA strain of GBS alters capsule level and the capacity 123

of the bacteria to survive in human whole blood. Additionally, we present data implicating 124

CpsA in modulating the cell wall. 125

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Materials and Methods 127

Bacterial strains and growth conditions: Plasmids were maintained in Escherichia coli 128

electro-competent Top 10 cells (Invitrogen). Luria-Bertani (LB) medium (BD) was used to 129

culture E. coli strains. Antibiotics were added as necessary to LB medium at the following 130

concentrations: chloramphenicol, 20 μg/ml, and ampicillin, 100 μg/ml for E. coli strains. E. coli 131

cultures were grown at 37°C with shaking. When growing E. coli cultures for protein 132

purification, LB media was supplemented with 0.2% glucose (w/v). Solid media was generated 133

by supplementing the liquid media with 1.4% agar (Acumedia). The streptococcal strain 134

Streptococcus agalactiae Group B Strep (GBS) 515, a human clinical isolate from the blood of a 135

patient with neonatal septicemia and GBS 515 ΔcpsA were generously provided by M. R. 136

Wessels (7). GBS 515 was cultured in Todd-Hewitt medium (BD) supplemented with 0.2% yeast 137

extract (THYB) (BD) in airtight conical tubes without agitation at 37°C. When transforming 138


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GBS 515 by electroporation, bacteria were grown on solid media supplemented with 1.4% agar 139

(BD) and incubated in GasPak jars (BBL) with GasPak anaerobic system envelopes (BD). 140

Transformation of GBS 515. GBS 515 cultures were grown statically in THYB 141

supplemented with 80 mM glycine overnight, diluted 1:20 in 25 mL of THYB supplemented 142

with 80 mM glycine the following day, and grown with shaking to an OD600 of 0.4. The cells 143

were then harvested by centrifugation, washed 3 times with 10 mL of ice-cold 10% glycerol, and 144

resuspended in 1 mL of ice-cold 10% glycerol. Plasmid DNA was mixed with 200 μl of cells, 145

placed into an electroporation cuvette (DOT Scientific), and electroporated with a BIO-RAD 146

Gene Pulser II at 25 μF, 2.0 kV, and 400 Ω. Cells were immediately transferred to 10 mL of 147

fresh THYB medium, and allowed to recover for 90 minutes at 37°C prior to plating on selective 148

media. 149

Cloning of maltose-binding-protein (MBP)-CpsA fusions. The full-length cpsA gene 150

was amplified from GBS 515 genomic DNA using the primers 5’ GBS-cpsA-SmaI and 3’ GBS-151

cpsA-full-stop-PstI. Truncations of the 3’ end of cpsA were amplified from GBS 515 genomic 152

DNA using the primer 5’GBS-cpsA-SmaI in conjunction with the primers 3’GBS-cpsA-245-stop-153

PstI, 3’GBS-cpsA-117-stop-PstI or 3’GBS-cpsA-39-stop-PstI. These products were digested with 154

SmaI and PstI and cloned into the corresponding SmaI and PstI sites of pMAL-c2x (NEB) 155

leading to in-frame fusions of cpsA fragments downstream of the malE gene. This generated the 156

following plasmids: pMAL-GBS-cpsA-full, pMAL-GBS-cpsA-245, pMAL-GBS-cpsA-117, and 157

pMAL-GBS-cpsA-39. These constructs were transformed into E. coli Top10 cells (Invitrogen). 158

Protein purification: Overnight pMAL-cpsA expressing E. coli strains were sub-159

cultured 1:40 into 300 mL new LB medium supplemented with 0.2% glucose and grown at 37°C 160

with shaking until reaching an OD600 of approximately 0.5, and protein expression was induced 161


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by addition of 0.3 mM isopropyl-β-D-1-thiogalactopyranoside, followed by incubation for 3 162

hours. Cells were then harvested by centrifugation at 6500 × g for 10 minutes, the supernatant 163

discarded, and the cells resuspended in 30 ml of Column Buffer (20 mM Tris-HCl, 200 mM 164

NaCl, and 1mM EDTA) and stored at -20°C overnight. The frozen cultures were thawed on ice, 165

and 10 mL of lysis buffer was added (50 mM Tris-HCl, 150 mM NaCl, 1% Sarkosyl (w/v), 1% 166

Triton-X 100 (v/v), 10 mM CHAPS, pH 7.4) along with 40 μl of 100X ProteoBlock protease 167

inhibitor cocktail (Fermentas). The mixture was then sonicated in 30 second bursts to lyse cells. 168

The lysate was centrifuged at 10,000 x g for 30 minutes and the clarified lysate diluted to a total 169

volume of 100 ml using Column Buffer. The lysate was added to the top of a glass column 170

containing amylose beads (NEB) and eluted according to the manufacturer’s specifications. 171

Purified protein concentration was determined using the BCA protein assay kit (Thermo 172

Scientific) according to the manufacturer’s instructions. Protein purity was assessed by SDS-173

PAGE. 174

Generating digoxigenin-labeled DNA probe and competitor DNA probes: Probes 175

consisting of the GBS 515 cpsA promoter (217 bp) and GBS 515 cpsE promoter (221 bp) were 176

amplified from GBS 515 genomic DNA using the primers 5’ GBS-cpsA-pro with 3’ GBS-cpsA-177

pro, and 5’ GBS-cpsE-pro with 3’ GBS-cpsE-pro, respectively. The S. iniae cpsA promoter (182 178

bp) was amplified from S. iniae 9117 genomic DNA using the primers 5’iniae-cpsA-pro and 179

3’iniae-cpsA-pro. The 515 GBS promoter products were then labeled with digoxigenin using the 180

DIG Gel Shift Kit, 2nd Generation (Roche) according to manufacturer’s instructions. 181

Electromobility Shift Assays: To conduct the EMSA, constant amounts of the MBP–182

CpsA protein fusions were incubated with a constant amount of labeled probe (12 fmol) in a 183

binding buffer containing 100 mM HEPES pH 7.2, 1 mM EDTA, 50 mM KCl, 50 mM MgCl2, 1 184


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mM DTT, and 30% (v/v) glycerol for 30 minutes at room temperature. For reactions with 185

competitor DNA, an excess of unlabeled GBS 515 probe DNA (either cpsA-pro or cpsE-pro) was 186

used as a specific competitor, and unlabeled S. iniae cpsA-pro was used as a non-specific 187

competitor. The samples were loaded onto a 6% polyacrylamide native gel consisting of 6% 188

(v/v) polyacrylamide, 44.5 mM tris base, 44.5 mM boric acid, and 1mM EDTA. Electrophoresis 189

was performed at 4˚C. The gel was then transferred to a nylon membrane (Santa Cruz) using a 190

semi-dry transfer apparatus (Hoefer). Chemiluminescent detection of DIG-labeled DNA on 191

membranes was accomplished with CDP-Star (Roche) according to manufacturer instructions, 192

followed by exposure to X-ray film. Each EMSA was repeated at least twice, and was also 193

repeated by using sheared salmon sperm DNA as a non-specific competitor to confirm results 194

with the S. iniae cpsA-pro non-specific competitor. Only EMSAs using the S. iniae cpsA-pro as 195

a non-specific competitor are reported in the results. 196

Cloning of MBP-cpsA fusions for complementation. The MBP-cpsA fusions MBP-197

cpsA-full, MBP-cpsA-246, and MBP-cpsA-117 were amplified from the plasmids generated 198

above using the primer 5’ MBP-RBS-BamHI in conjunction with the primers 3’ GBS-cpsA-full-199

stop-PstI, 3’ GBS-cpsA-245-stop-PstI, and 3’GBS-cpsA-117-stop-PstI respectively. These 200

fragments were then digested with BamHI and PstI and ligated into the corresponding BamHI 201

and PstI sites on the plasmid pLZ12-rofA-pro (23) behind the rofA promoter (12) creating the 202

following plasmids: pGBS-cpsA-full, pGBS-cpsA-245, and pGBS-cpsA-117. These constructs 203

were transformed into E. coli Top10 cells, propagated, and then transformed into GBS 515 as 204

described above. These GBS 515 strains are listed in Table 2. 205

Measurement of GBS 515 capsule levels using buoyant density centrifugation. 206

Buoyant density centrifugation was performed similarly to previous work (8, 17), but with 207


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modification. Linear density gradients of Percoll (GE Healthcare) were generated by diluting 208

Percoll to a high density limit (1.120 g/mL) and low density limit (1.085 g/mL) with a final 209

concentration of 0.15 M NaCl according to the manufacturer’s instructions, and carefully 210

layering 2 mL of the low density solution on top of 2 mL of the high density solution in a 5 mL 211

Falcon tube (BD). These tubes were then set horizontally at a 15° angle and left overnight. The 212

next morning tubes were set upright and allowed to settle for 30 minutes prior to use. Bacterial 213

cultures were grown overnight as described above, and cultures were normalized to an OD600 of 214

0.6 in 1 mL of THYB medium, pelleted by centrifugation, resuspended in 50 μl of PBS, and 215

added directly to the top of the Percoll gradients. Tubes were then centrifuged for 30 minutes at 216

5000 rpm in a swinging bucket Eppendorf Centrifuge 5403 with Rotor 16A4-44 at room 217

temperature. Measurements were then taken from the meniscus to the bottom of the cell band in 218

each tube, and compared to a set of colored beads of known density (GE Healthcare) to 219

determine bacterial cell density. Results are reported as an average of three separate 220

experiments. When appropriate, the Student’s t-test was used to evaluate the difference in means 221

between groups. 222

Incubation of GBS 515 in human whole blood. Human blood was obtained from 223

healthy volunteers and collected in heparinized vacuum tubes. Bacteria were grown overnight 224

and normalized to an OD600 of 0.3 in PBS, corresponding to ~1 x 108 CFU/ml. Bacteria were 225

serially diluted in PBS, and 2.5 x 105 CFU were added to 1 mL of blood and incubated with 226

rotation at 37°C for 3 hours. After incubation, 100 μl of the inoculated blood was plated onto 227

selective media, and incubated overnight at 37°C in a CO2 incubator to determine colony-228

forming units (CFU). Initial inoculum was serially diluted and plated as above to determine 229


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actual starting CFU. Whole blood assays were repeated a minimum of three times with results 230

reported using a representative experiment. 231

Zebrafish survival assays with GBS 515. Bacteria were grown overnight, diluted 1:50 232

the following day in fresh medium, and grown to an OD600 of approximately 0.3. Cultures were 233

then normalized to 1 x 108 CFU/mL and 10 μl of culture or media alone was injected into 234

zebrafish intramuscularly, resulting in an infectious dose of 1 x 106 CFU. A total of 25 fish were 235

infected with each strain over three experiments, and zebrafish survival monitored over a 6-day 236

period, after which all surviving fish were euthanized. 237

Quantification of GBS 515 chain length. Cultures were grown overnight and 6 μl of 238

culture was directly placed onto a glass slide with a coverslip (Fisher) and viewed at 1000X 239

magnification on a Zeiss AxioSkop 40. Images were captured of randomly selected visual fields 240

using an attached Zeiss AxioCam MRc. Images were captured from two separate experiments 241

and at least 250 chains were counted from each set, for a total of 500 or more chains counted for 242

each strain. Chain length values were distributed between arbitrarily set numerical categories 243

and calculated as a percentage of all counted chains. For analysis of lysozyme treated strains, 244

cultures were grown overnight in the presence of a sub-inhibitory concentration of lysozyme 245

(200 μg/mL) and images captured as above. 246

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Results 248

GBS CpsA binds to two separate putative promoters located in the capsule operon. 249

All members of the LytR_cpsA_psr protein family have been connected to transcriptional 250

regulation, but the precise method in which these proteins contribute to regulation has not yet 251

been elucidated. As a member of this protein family, CpsA has been identified as a potential 252


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transcriptional activator of the capsule operon (7). Previous work in our lab demonstrated that 253

purified CpsA protein from S. iniae was capable of binding to the promoter region of the S. iniae 254

capsule operon upstream of the cpsA gene with specificity (12), suggesting that S. iniae CpsA 255

may modulate transcription by directly binding to promoter sequences. An alignment of the 256

cpsA promoter DNA sequences of S. iniae and GBS using BLAST reveals that these promoters 257

share no significant similarity. Additionally, despite the CpsA proteins of S. iniae and GBS 258

sharing 57 % amino acid identity and 76 % amino acid similarity, the intracellular regions of 259

CpsA responsible for binding to DNA in S. iniae share no significant similarity with the same 260

regions on the GBS form of the protein. These differences in sequence at the DNA and protein 261

level necessitate confirmation of DNA binding by the GBS form of CpsA. Therefore, 262

electromobility shift assays (EMSA) were performed using labeled probes with the sequence of 263

two putative promoter regions within the GBS capsule operon, upstream of the transcriptional 264

start sites of the cpsA gene and cpsE gene (Fig. 1A) as described previously (33). To determine 265

the importance of different regions of the GBS CpsA protein in binding to DNA, multiple 266

maltose binding protein (MBP) fusions of the CpsA protein were constructed and purified 267

including the full-length protein (MBP-CpsA-full) as well as two truncated forms of the protein, 268

MBP-CpsA-117 and MBP-CpsA-39 (Fig. 1C). 269

Purified MBP-CpsA-full was incubated with either the DIG-labeled GBS cpsA promoter 270

or the DIG-labeled GBS cpsE promoter. MBP-CpsA-full demonstrated the ability to bind to the 271

labeled cpsA promoter with specificity (Fig. 2A). Lane 1 demonstrates the migration of unbound 272

labeled cpsA-pro probe, lanes 2 and 3 demonstrate labeled probe bound by the protein in the 273

presence of 25 fold specific or non-specific unlabeled competitor DNA, lanes 4 and 5 274

demonstrate labeled probe bound by the protein in the presence of 50 fold specific or non-275


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specific unlabeled competitor DNA, and lanes 6 and 7 demonstrate labeled probe bound by the 276

protein in the presence of 75 fold specific or non-specific unlabeled competitor DNA. In the 277

same manner, using the same concentration of protein and probe, the full length GBS CpsA 278

protein demonstrated specific binding to the labeled cpsE promoter as well (Fig. 2B). 279

After observing specific binding of MBP-CpsA-full to both the labeled GBS cpsA and 280

GBS cpsE promoters, determination of binding preference to one of these promoters over the 281

other was analyzed by cross-competing each labeled probe with unlabeled probe (Fig 3A). As 282

shown above, full-length CpsA protein binds to the cspA-pro and cpsE pro probes (Fig 3A, lanes 283

2 and 7). Competition with an excess of unlabeled probe of either cpsA-pro (lanes 3 and 9) or 284

cpsE-pro (lanes 4 and 8), revealed that the full length CpsA protein was able to bind both labeled 285

promoters and did not demonstrate a clear preference for either cpsA or cpsE probe to the 286

exclusion of the other. Again, these interactions were specific as a 50-fold excess of unlabeled S. 287

iniae cpsA-pro (nonspecific competitor) showed no competition (Fig 3A, lanes 6 and 10). 288

To determine what regions of the protein were required for DNA binding, EMSAs were 289

performed using both truncated forms of the GBS CpsA protein, MBP-CpsA-117 and MBP-290

CpsA-39. MBP-CpsA-117 is a truncation of CpsA after the third transmembrane domain (see 291

Fig 1C), thereby removing the large extracellular region to assess its contribution to binding or 292

specificity. When full length GBS CpsA was replaced with the truncation MBP-CpsA-117 (Fig. 293

3B), the protein was still able to bind both labeled probes, and no clear preference for either the 294

labeled cpsA-pro or cpsE-pro probes was observed when cross-competed, as seen for the full 295

length CpsA. However, a reduction in specificity was observed for both the cpsA and cpsE 296

labeled probes when comparing competition with unlabeled specific and non-specific DNA, 297

though some level of specificity was still present. This indicated that the large extracellular 298


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portion of CpsA is not required for binding to DNA, but does affect the specificity with which 299

CpsA is able to interact with a specific DNA sequence. 300

The MBP-CpsA-39 construct produces the CpsA protein truncated at the end of the first 301

transmembrane domain, leaving the putative leucine zipper domain intact, but removing the 302

cytoplasmic loop between the second and third transmembrane domains (see Fig 1C). Thus, an 303

EMSA using this protein fusion assessed the contribution of the cytoplasmic loop to binding 304

ability and specificity. When the MBP-CpsA-39 truncation was used with the same parameters 305

(Fig. 3C), the protein retained the ability to bind to both labeled probes, but lost all semblance of 306

specificity for either the cpsA-pro or cpsE-pro when comparing competition for unlabeled 307

specific and non-specific DNA. This confirmed that the cytoplasmic loop contributes to 308

specificity, but is not required for binding ability. Importantly, previous work with the CpsA 309

protein from S. iniae demonstrated that the MBP domain does not contribute to DNA binding 310

(12). Taken together, these results demonstrate that only the cytoplasmic N-terminus of GBS 311

CpsA is required for binding to DNA, but that the cytoplasmic loop and extracellular region of 312

the protein both contribute to specificity of the interaction. 313

Ectopic expression of CpsA affects GBS capsule level. Deletion of the cpsA gene has 314

previously been associated with decreased capsule production (7). To assess the contribution of 315

each domain of the CpsA protein to capsule production, full length and truncated forms of CpsA 316

(Fig. 1C) were constructed and placed on the plasmid pLZ12-rofA-pro (23), providing 317

constitutive expression. These plasmids were then transformed into either the WT GBS 515 318

strain or a GBS 515 cpsA in-frame deletion strain (ΔcpsA) (7). A growth curve of all strains 319

grown in THYB demonstrated that all strains grew similarly with no significant differences (S1). 320

Buoyant density centrifugation was used to determine relative differences in the level of capsule 321


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produced by the strains created as described above. Measurement of capsule demonstrated that 322

the ΔcpsA strain produced less capsule than the WT strain when both strains harbored the vector 323

alone (Fig. 4). Ectopic expression of MBP-CpsA-full in both the WT and ΔcpsA background led 324

to an increase in capsule over that produced normally by the WT strain, (Fig. 4), demonstrating 325

that expression of the full length form of CpsA was able to complement the ΔcpsA strain. When 326

a truncated form of the protein lacking the LytR domain (MBP-CpsA-245) was expressed in 327

either background a loss of capsule was experienced to levels below that of the ΔcpsA strain 328

(Fig. 4), indicative of a possible dominant negative or repression mechanism. The addition of 329

just the N-terminal DNA-binding domain of CpsA (MBP-CpsA-117) resulted in an increase in 330

capsule for the WT strain to the same level as expression of MBP-CpsA-full and an increase in 331

capsule for the ΔcpsA strain to a slightly lesser degree (Fig. 4), showing complementation of the 332

ΔcpsA mutant with a truncated form of CpsA missing the entire extracellular domain. Overall, 333

these data suggest that specific domains of CpsA contribute to regulation of capsule production 334

in different ways. 335

GBS survival in whole blood is altered by ectopic expression of CpsA. GBS 336

virulence entails dissemination through the bloodstream, an ability that relies on inhibiting 337

phagocytic clearance, which is primarily dependent on the presence of capsule (27). The 338

variations in capsule production observed when different domains of CpsA are ectopically 339

expressed in GBS led us to question whether these variations corresponded to changes in 340

survival in human blood. Surprisingly, when incubated in human whole blood, the ΔcpsA strain 341

of GBS shows no major difference in the number of bacteria killed compared to the WT strain of 342

GBS under the conditions tested (Fig. 5), despite the presence of less capsule as measured by 343

buoyant density. However, the amount of capsule production observed when grown in laboratory 344


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medium may not correlate to that produced in whole blood. Expression of MBP-CpsA-full or 345

MBP-CpsA-117 in the WT background did not significantly alter the number of bacteria killed 346

(data not shown), despite the presence of more capsule than the WT strain as measured by 347

buoyant density (Fig. 4). In contrast, expression of MBP-CpsA-full or MBP-CpsA-117 in the 348

ΔcpsA background caused an approximately 0.4 log increase in bacterial killing compared to the 349

parent strain (Fig. 5), despite the production of more capsule (Fig. 4). Expression of the LytR 350

deletion (MBP-CpsA-245) in both the WT and ΔcpsA background resulted in an approximately 351

0.6 log increase in the number of bacteria killed compared to the respective parent strain (Fig. 5), 352

which may correspond to the loss of capsule these strains exhibit as measured by buoyant density 353

shown above. 354

In the absence of CpsA, GBS virulence is attenuated in a zebrafish model of 355

infectious disease. The unexpected result that the GBS ΔcpsA strain was not attenuated in 356

human whole blood, despite the production of less capsule, led to an in-vivo assessment of 357

virulence for the GBS 515 strains using a zebrafish model of infectious disease. When zebrafish 358

were inoculated with the WT strain of GBS, only 8% of zebrafish survived to day 6 (Fig. 6). In 359

contrast, when zebrafish were inoculated with a ΔcpsA strain of GBS, 68% of fish were viable at 360

day 6 (Fig. 6). The observed decrease in virulence of the ΔcpsA strain in an in-vivo model of 361

pathogenesis, when compared to a lack of attenuation in human whole blood (Fig. 5), suggests 362

that disruption of cpsA may lead to deficiencies that are only observable in the context of 363

systemic disease. These deficiencies could be associated with synthesis of capsular 364

polysaccharide, cell wall integrity, or a combination of both. 365

CpsA affects chain length. The chain length of cocci adopted by streptococcal species 366

depends on a number of conditions, not all of which have been determined. Chain length is often 367


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controlled by production of cell wall amidases or other autolysins (6), but the presence of capsule 368

has also been shown to affect chain length as well (1, 2, 19). CpsA is a member of the same 369

family of proteins as LytR, a group of proteins associated with attenuating expression of cell wall 370

modifying enzymes (16), and various domains of CpsA also influence capsule levels (Fig. 4). 371

Therefore, the effect of these associations on chain length in GBS was analyzed using 372

microscopy. Despite a small relative difference in capsule level, the ΔcpsA mutant produced 373

considerably longer chains than the WT GBS strain (Fig. 7). Complementation of the ΔcpsA 374

strain with either MBP-CpsA-full or MBP-CpsA-117, both of which increased capsule, showed a 375

shift to shorter chains, but did not fully restore the WT shorter chain phenotype (Fig. 7). 376

Furthermore, the expression of the LytR deletion construct, MBP-CpsA-245, in the WT strain, 377

which greatly decreased capsule, led to markedly longer chains than the WT strain (Fig. 7), while 378

the ΔcpsA strain expressing MBP-CpsA-245 maintained the long chain phenotype (Fig. 7). 379

To confirm the microscopy observations, the number of cells per chain for each strain 380

was calculated as described in the Materials and Methods. The results verified the prevalence of 381

short chains in the WT/vector strain with 1-2 cells per chain predominating at 70% of chains 382

(Fig. 8A) as well as a preponderance of long chains for the ΔcpsA/vector strain with greater than 383

10 cells per chain making up 45% of chains (Fig. 8B). Addition of MBP-CpsA-full to the WT 384

background did not substantially alter chain length (Fig. 8A). Addition of MBP-CpsA-full to the 385

ΔcpsA background did not fully alleviate the long chain phenotype, with greater than 10 cells and 386

3-4 cells per chain representing the largest populations at 27% and 26% respectively (Fig. 8B). 387

When the LytR deletion strain (MBP-CpsA-245) was expressed in the WT background, the long 388

chain phenotype predominated with the majority of chains showing greater than 10 cells per 389

chain (Fig. 8A), while the presence of MBP-CpsA-245 in the ΔcpsA parent strain seemed to 390


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exacerbate the long chain phenotype with greater than 10 cells per chain predominating (Fig. 391

8B). Expression of MBP-CpsA-117 in the WT strain did not change the WT chain phenotype 392

(Fig. 8A). Similar to MBP-CpsA-full, expression of MBP-CpsA-117 did not fully alleviate the 393

long chain phenotype of the ΔcpsA strain, with approximately equal chains at greater than 10 394

cells per chain (27%) and 3-4 cells per chain (28%) (Fig. 8B). These results suggest that CpsA 395

either directly or indirectly influences chain length and that capsule level alone is not sufficient 396

to explain chain length variance in these circumstances. 397

To determine if cell wall related factors were primarily responsible for the observed 398

variances in chain lengths, each GBS strain above was cultured in sub-inhibitory concentrations 399

of lysozyme. Lysozyme has muramidase activity and cleaves N-acetyl-D-glucosamine residues 400

of the peptidoglycan cell wall. When grown in the presence of lysozyme, all strains existed 401

almost exclusively as diplococci or single cells (Fig. 9), indicating that CpsA-dependent changes 402

to the cell wall may be responsible for the observed chain length variances. 403

404

Discussion 405

Streptococcal pathogens capable of causing systemic disease remain a major health 406

concern worldwide, and new strategies are currently being utilized to identify and exploit novel 407

vaccine and antimicrobial targets (25, 30). The streptococcal CpsA protein is part of the 408

LytR_cpsA_psr family of proteins associated with regulatory control over cell surface 409

physiology, including polysaccharide synthesis (7), cell wall processing (6, 14, 16), and response 410

to antimicrobial stress (31). The involvement of this protein family with these important 411

virulence determinants highlights its potential as a possible target for virulence reduction, 412

increased clearance by host immune function or antimicrobial therapy. Therefore, our aim was 413


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to characterize the functional properties of CpsA to better understand the role it may play during 414

initiation and perpetuation of disease. 415

The streptococcal CpsA protein has been identified as a putative regulatory activator of 416

capsule, with an in-frame deletion of cpsA resulting in reduced levels of transcript from the 417

capsule operon as well as a concomitant loss in capsule level in GBS (7). Work on a strain of the 418

aquatic pathogen S. iniae in which the cpsA gene was insertionally inactivated demonstrated that 419

interruption of cpsA led to significant attenuation in a zebrafish model of infectious disease when 420

compared to the WT S. iniae strain, similar to what was demonstrated here with the GBS 515 421

strains (17). This evidence suggests that CpsA is required for full virulence as a positive 422

regulator of capsule, and that this may occur through direct interaction with DNA to facilitate 423

transcriptional changes. 424

CpsA proteins contain three discrete domains: a cytoplasmic N-terminal DNA-binding 425

domain, an extracellular DNA_PPF domain, and an extracellular C-terminal LytR domain (12). 426

The N-terminal DNA-binding domain is conserved in other streptococcal CpsA proteins and 427

contains a possible lecuine zipper motif, which may facilitate homo- or hetero-dimerization and 428

DNA binding ability (4). The DNA_PPF domain function is canonically ascribed to sliding 429

clamp structures that enhance the rate of DNA replication through association with DNA 430

polymerases (20, 28). However, the protein sequence of the DNA_PPF domain of CpsA 431

diverges considerably from that of traditional sliding clamp DNA_PPF domains, suggesting a 432

different function for the DNA_PPF domain in the CpsA protein. This is also supported by the 433

observation that the DNA_PPF domain of CpsA resides extracellularly where it would be unable 434

to participate in DNA replication (6). Sliding clamps that contain the DNA_PPF domain 435

typically participate in a number of protein-protein interactions that contribute to their function 436


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(28), and although it appears that the DNA_PPF domain of CpsA has been functionally 437

redirected from traditional sliding clamps, it may be that the ability for facilitating protein-438

protein interactions has been retained. 439

The LytR domain of CpsA demonstrates a relatively high level of homology to traditional 440

LytR proteins of Gram-positive species with a comparison of the GBS CpsA LytR domain and 441

the B. subtilis LytR protein showing 38% identity and 58% similarity at the amino acid level. 442

LytR proteins of Gram-positive species are generally associated with regulation of cell wall 443

maintenance through transcriptional attenuation of autolysin genes, as well as their own 444

promoter (16). Analysis of the LytR protein from Streptococcus pneumoniae demonstrated that 445

LytR is required for normal septum formation during cell division (7). lytR null mutants divide 446

non-symmetrically and have highly variable cell shape and size, and with lytR mutants 447

sometimes demonstrating much larger cell size (14). Similar results were reported for the LytR 448

protein of Streptococcus mutans, with a lytR null mutant exhibiting cell division defects, 449

including the production of significantly longer chains of bacteria (6). These reports are 450

consistent with our results with the CpsA truncation in which the LytR domain has been 451

removed (MBP-CpsA-245). When this construct is expressed alone (in the ΔcpsA strain) or 452

along with the WT CpsA, consistently longer chain lengths are observed. In addition, this 453

construct produces cocci that are noticeably larger in size than the WT strain expressing full 454

length CpsA (data not shown). Increased autolysin production was also observed for the S. 455

mutans lytR mutant, suggestive of a transcriptional attenuator role for LytR over autolysin genes 456

(6). The homology between CpsA and LytR proteins indicates that some functional overlap may 457

be present, and that in addition to regulation of capsule, CpsA may also contribute to regulation 458


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of cell wall maintenance. Evidence to support this hypothesis is found in Staphylococcus aureus 459

where LytR_cpsA_psR family members have been shown to exhibit functional redundancy (24). 460

Members of the LytR_cpsA_psr family of proteins have generally been associated with a 461

regulatory role at the transcriptional level. Using a series of truncated CpsA proteins to analyze 462

the contribution of different domains of the protein to DNA binding ability and specificity, we 463

found that the full length GBS CpsA protein was capable of binding to both the cpsA and cpsE 464

promoters with specificity. Furthermore, we determined that the CpsA protein had the same 465

apparent affinity for both promoters. These results suggest that direct binding of GBS CpsA to 466

both the cpsA and cpsE promoters of the capsule operon may provide a mechanism for the 467

transcriptional changes associated with deletion or interruption of the cpsA gene. The 468

observation that specificity of DNA binding decreases with sequential truncation from the C-469

terminus of the protein suggests that both the large extracellular region of the protein and the 470

cytoplasmic loop between transmembrane domains 2 and 3 likely contribute to specificity either 471

structurally or through direct interaction, much like what has been demonstrated with S. iniae 472

CpsA (12). The observed DNA binding ability for GBS MBP-CpsA-39 is in contrast to what 473

was found with S. iniae CpsA, in which truncation to the cytoplasmic N-terminus abolished 474

DNA binding ability (12). This discrepancy could be explained by the inclusion of the first 475

transmembrane domain for GBS MBP-CpsA-39 (including the putative leucine zipper), which 476

was removed from the comparable S. iniae form of the protein. 477

Functional analysis of different CpsA domains when expressed in either a WT or ΔcpsA 478

GBS background was assayed by determining capsule level with percoll buoyant density 479

gradients. While the ΔcpsA strain produced less capsule than WT, both parent strains could be 480

induced to produce higher levels of capsule when either full length CpsA (MBP-CpsA-full) or 481


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just the DNA-binding domain of CpsA (MBP-CpsA-117) was ectopically expressed. These 482

results support the hypothesis that CpsA is an activator of capsule, and that it does this by 483

binding to the capsule operon promoter, as the DNA-binding domain of CpsA was sufficient for 484

complementation of capsule levels in a ΔcpsA background. In contrast, the production of CpsA 485

lacking the LytR domain (MBP-CpsA-245) resulted in a decrease in capsule for both the WT and 486

the ΔcpsA parent strains. The presence of the DNA-binding domain was not the cause of 487

decreased capsule, because as mentioned above, expression of this domain alone had an 488

activating effect. This indicated that the DNA_PPF domain was responsible for the decrease in 489

capsule levels, which could be due to a dominant negative function that is adopted by CpsA in 490

the absence of the LytR domain, perhaps through inappropriate protein-protein interactions. 491

Another possibility is that the DNA_PPF normally induces a repressing effect on capsule 492

through the DNA-binding domain by a change in protein conformation, and that the LytR 493

domain controls this repressive mechanism. With either option, because of the extracellular 494

location of the DNA_PPF domain, the repression of capsule expression is likely facilitated 495

through either a protein-protein interaction or an induced conformational change. This negative 496

regulation appears to be enacted at the transcriptional level as preliminary analysis of GBS cpsD 497

and cpsG via real time qRT-PCR revealed a ~2.5 fold decrease in transcript for the WT strain 498

ectopically expressing MBP-CpsA-245 compared to WT with vector alone (data not shown). 499

However, further analysis of these strains is necessary to fully characterize the association 500

between CpsA truncation and transcription from the capsule operon. 501

The production of a polysaccharide capsule allows GBS to evade immune clearance upon 502

introduction to the host bloodstream during infection. Therefore, the above strains were 503

incubated in human whole blood to assess how differing levels of capsule, due to manipulation 504


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of CpsA, affect the ability of the bacteria to survive. Despite the production of less capsule in 505

the ΔcpsA background, we observed no major difference in survival when compared to the WT 506

strain. While production of the full length CpsA (MBP-CpsA-full) or the DNA-binding domain 507

(MBP-CpsA-117) in the WT background did not alter the ability of the WT parent to survive in 508

human blood (data not shown), when these constructs were expressed in the ΔcpsA background, 509

a decrease in survival was observed compared to the parent strain alone, despite the presence of 510

more capsule as measured by buoyant density centrifugation. Production of CpsA lacking the 511

LytR domain (MBP-CpsA-245) also led to a decrease in survival in both parent strains, which 512

may be due to reduced capsule levels or some other property of the bacteria, perhaps associated 513

with the cell wall, or a combination thereof. These results demonstrate that survival in human 514

whole blood does not always correlate with levels of capsule, and suggest that there is another 515

CpsA-dependent mechanism. Supporting this hypothesis is the observation that the ΔcpsA strain 516

was attenuated for virulence in the zebrafish model of infectious disease when compared to the 517

WT strain. Although the ΔcpsA strain was not attenuated in human whole blood, a regulatory 518

role for CpsA appears to exist in the context of systemic disease. The discrepancy between these 519

two observations may be due to an amplification of the capsule defect phenotype during systemic 520

disease and its absolute requirement, or it may be due to pleiotropic effects that are not apparent 521

during incubation in blood, but affect the ability of GBS to survive or disseminate within a host 522

organism. 523

Streptococcal LytR proteins have been associated with regulating cell division, including 524

septum formation, cell size, and chain length (6, 14). The homology of CpsA to LytR proteins 525

led us to investigate if similar regulatory events could be observed with the expression of 526

different domains of CpsA. Results from these assays indicate that capsule level does not 527


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necessarily correlate with chain length, at least in GBS, as ΔcpsA strains harboring MBP-CpsA-528

full and MBP-CpsA-117 produce more capsule than WT, but retain a higher proportion of long 529

chains than the WT strain. Additionally, the observation that encapsulation leads to longer 530

chains in S. pneumoniae (1, 2), is not consistent with our observation that the ΔcpsA strain, as 531

well as both parent strains producing MBP-CpsA-245, produce less capsule than WT, yet 532

produce primarily much longer chains than the WT strain. This phenomenon was not due to 533

differences in growth, as all strains grew similarly (see S1). Therefore, CpsA appears to exert 534

direct or indirect control over chain length, suggesting a possible dual role for this protein that is 535

separate from the control of capsule expression. Consistent with this hypothesis, is the 536

observation that the WT strain expressing MBP-CpsA-245 (LytR deletion) appeared to produce 537

larger sized cells compared to other strains. However, further characterization of these strains at 538

higher magnification levels using electron microscopy would be required to better discern 539

differences in septum placement and actual cell size and shape. We propose that CpsA-540

dependent changes in cell wall maintenance are responsible for the observed differences in chain 541

length, as growth in medium with a sub-inhibitory concentration of lysozyme, which selectively 542

cleaves peptidoglycan of the cell wall, results in eradication of long chains for all strains and a 543

switch of nearly all bacteria to single cells or diplococci. 544

Taken together, the results of this study suggest that GBS CpsA is a modular protein 545

containing three functionally distinct domains. The N-terminal region of GBS CpsA is able to 546

bind to promoters within the capsule operon upstream of both the cpsA and cpsE genes, and 547

expression of the N-terminal region alone (MBP-CpsA-117) in both the WT and ΔcpsA strains is 548

sufficient to increase capsule levels, and to partially complement the ΔcpsA strain chain length 549

distribution. This seems to indicate that despite the reduction in DNA-binding specificity of 550


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MBP-CpsA-117 when used alone in EMSAs, a tangible effect can still be incurred in-vivo, 551

possibly through binding of the capsule operon promoters and potentially other gene targets that 552

regulate chain length. The second module of CpsA, the DNA_PPF domain, may be responsible 553

for facilitating protein-protein interactions, a function that could be regulated by the LytR 554

domain as removal of the LytR domain results in a dominant-negative or repressive function for 555

both capsule level and chain length. To date, no mechanistic function has been applied to the 556

LytR domain in either CpsA or LytR proteins, but it appears that the LytR domain may play a 557

similar role in both proteins as similar phenotypes of increased chain length and size are 558

observed when it is removed from CpsA, both of which are seemingly independent of capsule 559

level. This suggests that CpsA may also have a role in regulation of autolysin genes, which 560

would give it a unique position at the interface of regulating the two major components of the 561

bacterial cell surface, capsular polysaccharide and the cell wall peptidoglycan. 562

Acknowledgements: 563

B.R.H. was supported by Wayne State University’s UGRD Fellowship. 564

The authors wish to thank Jennifer Dittmer for phlebotomy services and Daniel Ortiz for 565

technical assistance. 566

567

568

569

570

571

572

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682

683

684

685

686

687


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688

689

690

691

692

693


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Table 1. Primers used in this study.

Primer Sequence (5’ - 3’) EMSA: 5’ GBS-cpsA-pro CGC GGA TCC GTT GAA TTC TCA TAA CTC TAG 3’ GBS-cpsA-pro CCG GAA TTC GCG AAT GAT TAG ACA TTG 5’ GBS-cpsE-pro GAA AAA GGA AGT AAG GGG CTC TTG 3’ GBS-cpsE-pro GCC ACG ACT CCA AAA GTC TC 5’ iniae-cpsA-pro CTC ATA ATG ACA GTC TAT C 3’ iniae-cpsA-pro CCA TCA ATA TCA TTT AAG TC Protein purification: 5’ GBS-cpsA-SmaI TCC CCC GGG TCT AAT CAT TCG CGC CGT C 3’ GBS-cpsA-full-stop-PstI AAA ACT GCA GTT ATT CCT CCA TTG TGT TC 3’ GBS-cpsA-117-stop-PstI AAA ACT GCA GTT ACT CAA TTT CAG AGT ATG AAG C 3’ GBS-cpsA-39-stop-PstI AAA ACT GCA GTT ACA TAA GAA ATA ATG AGA CTA C 3’ GBS-cpsA-245-stop-PstI AAA ACT GCA GTT ATG TTG ATA TAG AGC CAA AAG 5’ MBP-RBS-BamHI CGC GGA TCC GCG GAT AAC AAT TTC ACA CAG G


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Table 2: GBS strains used in this study.

Parent strain Plasmid Protein Construct 515 WT Vector None 515 WT pGBS-cpsA-full Full length CpsA 515 WT pGBS-cpsA-245 CpsA with LytR domain removed 515 WT pGBS-cpsA-117 CpsA with LytR and DNA_PPF domains removed 515 ΔcpsA Vector None 515 ΔcpsA pGBS-cpsA-full Full length CpsA 515 ΔcpsA pGBS-cpsA-245 CpsA with LytR domain removed 515 ΔcpsA pGBS-cpsA-117 CpsA with LytR and DNA_PPF domains removed


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Figure Legends 1

Figure 1. GBS 515 CpsA protein. (A) Genes in the capsule operon of GBS 515. Putative 2

promoter sequences within the capsule operon are indicated by bent arrows. (B) Membrane 3

topology of CpsA. (C) Arrangement of the CpsA protein where domains are shown as TM 4

(Transmembrane domains), DNA_PPF (DNA Polymerase Processivity Factor domain), and 5

LytR (LytR_cpsA_psr family domain). Below are truncations made to MBP fusions of CpsA 6

representing the full protein, a truncation at amino acid 245, a truncation at amino acid 117, and 7

a truncation at amino acid 39. 8

Figure 2. Electromobility shift assay demonstrating binding of GBS MBP-CpsA-full (10 pmol) 9

to either (A) the labeled GBS cpsA-pro probe or (B) the labeled GBS cpsE-pro probe. 10

Figure 3. Electromobility shift assays showing binding of (A) MBP-CpsA-full (10 pmol), or (B) 11

MBP-CpsA-117 (52 pmol), or (C) MBP-CpsA-39 (7 pmol), to either the labeled GBS cpsA-pro 12

or labeled GBS cpsE-pro probes in the absence or presence of competitor DNA representing 13

unlabeled GBS cpsA-pro, GBS cpsE-pro, or S. iniae cpsA-pro (unlabeled nonspecific). Unbound 14

labeled probe is indicated by “U” and bound labeled probe is indicated by “B.” 15

Figure 4. Percoll buoyant density assay reflecting capsule levels of the GBS WT or ΔcpsA 16

strains harboring the vector plasmid, or a plasmid containing MBP-CpsA-full, MBP-CpsA-245, 17

or MBP-CpsA-117. Error bars represent SEM. Significance between GBS WT/vector and 18

ΔcpsA/vector is * p < 0.01. 19

Figure 5. Whole blood assay measuring the Log10 level of CFU killed for bacterial strains 20

incubated in human whole blood for 3 hours. Error bars represent the standard deviation. 21


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Figure 6. Zebrafish infection study tracking survival over time of zebrafish infected 22

intramuscularly with either the GBS 515 WT or ΔcpsA strain, compared to a media only mock 23

infection. 24

Figure 7. Visualization of chain length at 1000X magnification for GBS 515 WT and ΔcpsA 25

strains, carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as 26

indicated on the top of the panel. 27

Figure 8. Quantification of chain length for parent strains of GBS 515, (A) WT and (B) ΔcpsA, 28

carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as indicated 29

on the bottom of the panel. Error bars represent the standard deviation. 30

Figure 9. Visualization of chain length at 1000X magnification for GBS 515 WT and ΔcpsA 31

strains, carrying the plasmid vector, MBP-CpsA-full, MBP-CpsA-245, or MBP-CpsA-117 as 32

indicated on the top of the panel when grown in the presence of a sub-inhibitory concentration of 33

lysozyme. 34

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functional analysis of the cpsa protein of streptococcus ... · gbs expressing cpsa-245, but not...

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