Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum.

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

Structure-Guided Design of a Group†B Streptococcus Type†III Synthetic Glycan-Conjugate Vaccine.

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment-mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM197 , a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Crystal structure of the Streptococcus agalactiae CAMP factor provides insights into its membrane-permeabilizing activity.

Streptococcus agalactiae is an important human opportunistic pathogen that can cause serious health problems, particularly among newborns and older individuals. S. agalactiae contains the CAMP factor, a pore-forming toxin first identified in this bacterium. The CAMP reaction is based on the co-hemolytic activity of the CAMP factor and is commonly used to identify S. agalactiae in the clinic. Closely related proteins are present also in other Gram-positive pathogens. Although the CAMP toxin was discovered more than a half century ago, no structure from this toxin family has been reported, and the mechanism of action of this toxin remains unclear. Here, we report the first structure of this toxin family, revealing a structural fold composed of 5 + 3-helix bundles. Further analysis by protein truncation and site-directed mutagenesis indicated that the N-terminal 5-helix bundle is responsible for membrane permeabilization, whereas the C-terminal 3-helix bundle is likely responsible for host receptor binding. Interestingly, the C-terminal domain inhibited the activity of both full-length toxin and its N-terminal domain. Moreover, we observed that the linker region is highly conserved and has a conserved DLXXXDXAT sequence motif. Structurally, this linker region extensively interacted with both terminal CAMP factor domains, and mutagenesis disclosed that the conserved sequence motif is required for CAMP factor's co-hemolytic activity. In conclusion, our results reveal a unique structure of this bacterial toxin and help clarify the molecular mechanism of its co-hemolytic activity.

Chemical synthesis of the dimeric repeating unit of type Ia group B Streptococcus capsular polysaccharide.

The first chemical synthesis of the dimeric repeating unit of serotype Ia group B Streptococcus capsular polysaccharide was achieved. The on-site elongation and dual glycosylation strategies were utilized to construct two sialotrisaccharide branches based on a hexasaccharide containing adjacent 3,4-di-branched Gal units, which were synthesized via a preactivation-based one-pot glycosylation method.

Small-molecule inhibitors of nisin resistance protein NSR from the human pathogen Streptococcus agalactiae.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.

Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.).

Streptococcus agalactiae (Group B Streptococcus, GBS) is associated with diverse diseases in aquatic animals. The capsule polysaccharide (CPS) encoded by the cps gene cluster is the major virulence factor of S. agalactiae; however, limited information is available regarding the pathogenic role of the CPS of serotype Ia piscine GBS strains in fish. Here, a non-encapsulated mutant (Δcps) was constructed by insertional mutagenesis of the cps gene cluster. Mutant pathogenicity was evaluated in vitro based on the killing of whole blood from tilapia, in vivo infections, measuring mutant survival in tilapia spleen tissues and pathological analysis. Compared to wild-type (WT) GBS strain, the Δcps mutant had lower resistance to fresh tilapia whole blood in vitro (p < 0.01), and more easily cleared in tilapia spleen tissue, and was highly attenuated in tilapia and zebrafish. Additionally, compared to the Δcps mutant, numerous GBS strains and severe tissue necrosis were observed in the tilapia spleen tissue infected with WT strains. These results indicated that the CPS is essential for GBS pathogenicity and may serve as a target for attenuation in vaccine development. Gaining a better understanding of the role, the GBS pathogenicity in fish will provide insight into related pathogenesis and host-pathogen interactions.

Group B Streptococcus Biofilm Regulatory Protein A Contributes to Bacterial Physiology and Innate Immune Resistance.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) asymptomatically colonizes approximately 20% of adults; however, GBS causes severe disease in susceptible populations, including newborns, pregnant women, and elderly individuals. In shifting between commensal and pathogenic states, GBS reveals multiple mechanisms of virulence factor control. Here we describe a GBS protein that we named "biofilm regulatory protein A" (BrpA) on the basis of its homology with BrpA from Streptococcus mutans. Methods: We coupled phenotypic assays, RNA sequencing, human neutrophil and whole-blood killing assays, and murine infection models to investigate the contribution of BrpA to GBS physiology and virulence. Results: Sequence analysis identified BrpA as a LytR-CpsA-Psr enzyme. Targeted mutagenesis yielded a GBS mutant (ΔbrpA) with normal ultrastructural morphology but a 6-fold increase in chain length, a biofilm defect, and decreased acid tolerance. GBS ΔbrpA stimulated increased neutrophil reactive oxygen species and proved more susceptible to human and murine blood and neutrophil killing. Notably, the wild-type parent outcompeted ΔbrpA GBS in murine sepsis and vaginal colonization models. RNA sequencing of ΔbrpA uncovered multiple differences from the wild-type parent, including pathways of cell wall synthesis and cellular metabolism. Conclusions: We propose that BrpA is an important virulence regulator and potential target for design of novel antibacterial therapeutics against GBS.

Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems.

Group B streptococcus (GBS) is a Gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence, and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or nonspecific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescent protein (GFP)-variant-expressing plasmid, pGU2664, which can be applied as a marker to visualize GBS in experimental studies. The synthetic constitutively active CP25 promoter drives strong and stable expression of the GFPmut3 biomarker in GBS strains carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. The application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the autofluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking CovR (the control of virulence regulator) in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS.IMPORTANCE Group B streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed GFPmut3. We show that this biomarker can be successfully applied to track the growth of bacteria in liquid medium, and it enables the detailed visualization of GBS in the context of live human cells in real-time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans Complementary crystallographic and biophysical characterization of BspA reveal a novel ß-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections.

Antibody Kinetics and Response to Routine Vaccinations in Infants Born to Women Who Received an Investigational Trivalent Group B Streptococcus Polysaccharide CRM197-Conjugate Vaccine During Pregnancy.

BACKGROUND: Maternal vaccination against group B Streptococcus (GBS) might provide protection against invasive GBS disease in infants. We investigated the kinetics of transplacentally transferred GBS serotype-specific capsular antibodies in the infants and their immune response to diphtheria toxoid and pneumococcal vaccination. METHODS: This phase 1b/2, observer-blind, single-center study (NCT01193920) enrolled infants born to women previously randomized (1:1:1:1) to receive either GBS vaccine at dosages of 0.5, 2.5, or 5.0 µg of each of 3 CRM197-glycoconjugates (serotypes Ia, Ib, and III), or placebo. Infants received routine immunization: combination diphtheria vaccine (diphtheria-tetanus-acellular pertussis-inactivated poliovirus/Haemophilus influenzae type b vaccine; age 6/10/ 14 weeks) and 13-valent pneumococcal CRM197-conjugate vaccine (PCV13; age 6/14 weeks and 9 months). Antibody levels were assessed at birth, day (D) 43, and D91 for GBS serotypes; 1 month postdose 3 (D127) for diphtheria; and 1 month postprimary (D127) and postbooster (D301) doses for pneumococcal serotypes. RESULTS: Of 317 infants enrolled, 295 completed the study. In infants of GBS vaccine recipients, GBS serotype-specific antibody geometric mean concentrations were significantly higher than in the placebo group at all timepoints and predictably decreased to 41%-61% and 26%-76% of birth levels by D43 and D91, respectively. Across all groups, ≥95% of infants were seroprotected against diphtheria at D127 and ≥91% of infants had seroprotective antibody levels against each PCV13 pneumococcal serotype at D301. CONCLUSIONS: Maternal vaccination with an investigational CRM197-glycoconjugate GBS vaccine elicited higher GBS serotype-specific antibody levels in infants until 90 days of age, compared with a placebo group, and did not affect infant immune responses to diphtheria toxoid and pneumococcal vaccination. CLINICAL TRIALS REGISTRATION: NCT01193920.

Metabolic fate of unsaturated glucuronic/iduronic acids from glycosaminoglycans: molecular identification and structure determination of streptococcal isomerase and dehydrogenase.

Glycosaminoglycans in mammalian extracellular matrices are degraded to their constituents, unsaturated uronic (glucuronic/iduronic) acids and amino sugars, through successive reactions of bacterial polysaccharide lyase and unsaturated glucuronyl hydrolase. Genes coding for glycosaminoglycan-acting lyase, unsaturated glucuronyl hydrolase, and the phosphotransferase system are assembled into a cluster in the genome of pathogenic bacteria, such as streptococci and clostridia. Here, we studied the streptococcal metabolic pathway of unsaturated uronic acids and the structure/function relationship of its relevant isomerase and dehydrogenase. Two proteins (gbs1892 and gbs1891) of Streptococcus agalactiae strain NEM316 were overexpressed in Escherichia coli, purified, and characterized. 4-Deoxy-l-threo-5-hexosulose-uronate (Dhu) nonenzymatically generated from unsaturated uronic acids was converted to 2-keto-3-deoxy-d-gluconate via 3-deoxy-d-glycero-2,5-hexodiulosonate through successive reactions of gbs1892 isomerase (DhuI) and gbs1891 NADH-dependent reductase/dehydrogenase (DhuD). DhuI and DhuD enzymatically corresponded to 4-deoxy-l-threo-5-hexosulose-uronate ketol-isomerase (KduI) and 2-keto-3-deoxy-d-gluconate dehydrogenase (KduD), respectively, involved in pectin metabolism, although no or low sequence identity was observed between DhuI and KduI or between DhuD and KduD, respectively. Genes for DhuI and DhuD were found to be included in the streptococcal genetic cluster, whereas KduI and KduD are encoded in clostridia. Tertiary and quaternary structures of DhuI and DhuD were determined by x-ray crystallography. Distinct from KduI ß-barrels, DhuI adopts an α/ß/α-barrel structure as a basic scaffold similar to that of ribose 5-phosphate isomerase. The structure of DhuD is unable to accommodate the substrate/cofactor, suggesting that conformational changes are essential to trigger enzyme catalysis. This is the first report on the bacterial metabolism of glycosaminoglycan-derived unsaturated uronic acids by isomerase and dehydrogenase.

Structure of the type IX group B Streptococcus capsular polysaccharide and its evolutionary relationship with types V and VII.

The Group B Streptococcus capsular polysaccharide type IX was isolated and purified, and the structure of its repeating unit was determined. Type IX capsule → 4)[NeupNAc-α-(2 → 3)-Galp-ß-(1 → 4)-GlcpNAc-ß-(1 → 6)]-ß-GlcpNAc-(1 → 4)-ß-Galp-(1 → 4)-ß-Glcp-(1 → appears most similar to types VII and V, although it contains two GlcpNAc residues. Genetic analysis identified differences in cpsM, cpsO, and cpsI gene sequences as responsible for the differentiation between the three capsular polysaccharide types, leading us to hypothesize that type V emerged from a recombination event in a type IX background.

Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae.

The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

Role of the Group B antigen of Streptococcus agalactiae: a peptidoglycan-anchored polysaccharide involved in cell wall biogenesis.

Streptococcus agalactiae (Group B streptococcus, GBS) is a leading cause of infections in neonates and an emerging pathogen in adults. The Lancefield Group B carbohydrate (GBC) is a peptidoglycan-anchored antigen that defines this species as a Group B Streptococcus. Despite earlier immunological and biochemical characterizations, the function of this abundant glycopolymer has never been addressed experimentally. Here, we inactivated the gene gbcO encoding a putative UDP-N-acetylglucosamine-1-phosphate:lipid phosphate transferase thought to catalyze the first step of GBC synthesis. Indeed, the gbcO mutant was unable to synthesize the GBC polymer, and displayed an important growth defect in vitro. Electron microscopy study of the GBC-depleted strain of S. agalactiae revealed a series of growth-related abnormalities: random placement of septa, defective cell division and separation processes, and aberrant cell morphology. Furthermore, vancomycin labeling and peptidoglycan structure analysis demonstrated that, in the absence of GBC, cells failed to initiate normal PG synthesis and cannot complete polymerization of the murein sacculus. Finally, the subcellular localization of the PG hydrolase PcsB, which has a critical role in cell division of streptococci, was altered in the gbcO mutant. Collectively, these findings show that GBC is an essential component of the cell wall of S. agalactiae whose function is reminiscent of that of conventional wall teichoic acids found in Staphylococcus aureus or Bacillus subtilis. Furthermore, our findings raise the possibility that GBC-like molecules play a major role in the growth of most if not all beta-hemolytic streptococci.

Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.

We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.

Expression of group B protective surface protein (BPS) by invasive and colonizing isolates of group B streptococci.

Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (ß) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia-VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + ß),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles.

Structure of Streptococcus agalactiae tip pilin GBS104: a model for GBS pili assembly and host interactions.

The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. In addition, a homology model of the remaining two domains of GBS104 was built and a model of full-length GBS104 was generated by combining the homology model (the N1 and N4 domains) and the crystal structure of the 75 kDa fragment (the N2 and N3 domains). This rod-shaped GBS104 model is constructed of three IgG-like domains (the N1, N2 and N4 domains) and one vWFA-like domain (the N3 domain). The N1 and N2 domains of GBS104 are assembled with distinct and remote segments contributed by the N- and C-termini. The metal-binding site in the N3 domain of GBS104 is in the closed/low-affinity conformation. Interestingly, this domain hosts two long arms that project away from the metal-binding site. Using site-directed mutagenesis, two cysteine residues that lock the N3 domain of GBS104 into the open/high-affinity conformation were introduced. Both wild-type and disulfide-locked recombinant proteins were tested for binding to extracellular matrix proteins such as collagen, fibronectin, fibrinogen and laminin, and an increase in fibronectin binding affinity was identified for the disulfide-locked N3 domain, suggesting that induced conformational changes may play a possible role in receptor binding.

NSR from Streptococcus agalactiae confers resistance against nisin and is encoded by a conserved nsr operon.

Nisin is a lantibiotic produced by Lactococcus lactis (L. lactis), which is active against many Gram-positive bacteria. However, in various pathogenic and nonpathogenic bacteria, the presence of a nisin resistance protein (NSR) confers resistance against nisin. Here, we show that NSR from Streptococcus agalactiae (SaNSR) confers 20-fold resistance when expressed in L. lactis. We also show that SaNSR is encoded by an operon structure comprising of a lipoprotein and an ATP-binding cassette transporter as well as a two-component system that is putatively involved in expression and regulation. This organization of the operon is conserved in several (non)pathogenic strains that do not produce nisin themselves.

Functional analysis of the CpsA protein of Streptococcus agalactiae.

Streptococcal pathogens, such as the group B streptococcus (GBS) Streptococcus agalactiae, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the ΔcpsA strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the ΔcpsA strain, was attenuated in human whole blood. However, the ΔcpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.