Different Group A Streptococcus pili lead to varying proinflammatory cytokine responses and virulence.

The human pathogen Streptococcus pyogenes, or Group A Streptococcus (GAS), is associated with a variety of diseases ranging from mild skin and soft tissue infections to invasive diseases and immune sequelae such as rheumatic heart disease. We have recently reported that one of the virulence factors of this pathogen, the pilus, has inflammatory properties and strongly stimulates the innate immune system. Here we used a range of nonpathogenic Lactococcus lactis gain-of-function mutants, each expressing one of the major pilus types of GAS, to compare the immune responses generated by various types of fully assembled pili. In vitro assays indicated variability in the inflammatory response induced by different pili, with the fibronectin-binding, collagen-binding, T antigen (FCT)-1-type pilus from GAS serotype M6/T6 inducing significantly stronger cytokine secretion than other pili. Furthermore, we established that the same trend of pili-mediated immune response could be modeled in Galleria mellonella larvae, which possess a similar innate immune system to vertebrates. Counterintuitively, across the panel of pili types examined in this study, we observed a negative correlation between the intensity of the immune response demonstrated in our experiments and the disease severity observed clinically in the GAS strains associated with each pilus type. This observation suggests that pili-mediated inflammation is more likely to promote bacterial clearance instead of causing disruptive damages that intensify pathogenesis. This also indicates that pili may not be the main contributor to the inflammatory symptoms seen in GAS diseases. Rather, the immune-potentiating properties of the pilus components could potentially be exploited as a vaccine adjuvant.

Expanding strain coverage of a group A Streptococcus pilus-expressing Lactococcus lactis mucosal vaccine.

Group A Streptococcus (GAS) is a human pathogenic bacterium that can trigger a wide range of diseases, including the autoimmune diseases acute rheumatic fever and rheumatic heart disease, causing major morbidity and mortality in many low- and middle-income countries. Primary intervention programs have had limited success thus far, and a licensed vaccine has yet to be developed. The pilus of GAS is known to be involved in host cell adhesion, biofilm formation and immune evasion. We have a mucosal vaccine in development that expresses the pilus of GAS on the surface of the nonpathogenic bacterium Lactococcus lactis. To expand strain coverage, we combined seven L. lactis constructs, each expressing a different GAS pilus variant, and investigated the systemic and mucosal immune responses following immunization. Mice immunized with this combination showed specific immunoglobin G and immunoglobin A responses to the GAS pilus proteins of vaccine strains, at levels comparable to mice immunized with a single construct. Cross-reactivity to pilus proteins of nonvaccine strains was also evident. Furthermore, protective efficacy against a homologous strain of GAS in a murine nasopharyngeal colonization model was observed. Overall, this study provides further evidence for using pilus-expressing lactic acid bacteria as a vaccine to prevent upper respiratory tract GAS infections.

Pilus proteins from Streptococcus pyogenes stimulate innate immune responses through Toll-like receptor 2.

The group A Streptococcus (GAS) pilus is a long, flexible, hair-like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin-8. We further show that the surface-exposed backbone pilin and ancillary pilin 1 subunits are Toll-like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid-phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune-modulation agent.

Cell wall-anchored 5'-nucleotidases in Gram-positive cocci.

5'-nucleotidases (5'-NTs) are enzymes that catalyze the hydrolysis of nucleoside monophosphates to produce nucleosides and phosphate. Since the identification of adenosine synthase A (AdsA) in Staphylococcus aureus in 2009, several other 5'-NTs have been discovered in Gram-positive cocci, mainly in streptococci. Despite some differences in substrate specificity, pH range and metal ion requirements, all characterized 5'-NTs use AMP and ADP, and in some cases ATP, to produce the immunosuppressive adenosine, which dampens pro-inflammatory immune responses. Several 5'-NTs are also able to use dAMP as substrate to generate deoxy-adenosine which is cytotoxic for macrophages. A synergy between 5'-NTs and exonucleases which are commonly expressed in Gram-positive cocci has been described, where the nucleases provide dAMP as a cleavage product from DNA. Some of these nucleases produce dAMP by degrading the DNA backbone of neutrophil extracellular traps (NETs) resulting in a "double hit" strategy of immune evasion. This Micro Review provides an overview of the biochemical properties of Gram-positive cell wall-anchored 5'-NTs and their role as virulence factors. A potential use of 5'-NTs for vaccine development is also briefly discussed.

Intranasal immunization with Ag85B peptide 25 displayed on Lactococcus lactis using the PilVax platform induces antigen-specific B- and T-cell responses.

Mycobacterium tuberculosis (Mtb) remains a global epidemic despite the widespread use of Bacillus Calmette-Guérin (BCG). Consequently, novel vaccines are required to facilitate a reduction in Mtb morbidity and mortality. PilVax is a peptide delivery strategy for the generation of highly specific mucosal immune responses and is based on the food-grade bacterium Lactococcus lactis that is used to express selected peptides engineered within the Streptococcus pyogenes M1T1 pilus, allowing for peptide amplification, stabilization and enhanced immunogenicity. In the present study, the dominant T-cell epitope from the Mtb protein Ag85B was genetically engineered into the pilus backbone subunit and expressed on the surface of L. lactis. Western blot and flow cytometry confirmed formation of pilus containing the peptide DNA sequence. B-cell responses in intranasally vaccinated mice were analyzed by ELISA while T-cell responses were analyzed by flow cytometry. Serum titers of peptide-specific immunoglobulin (Ig) G and IgA were detected, confirming that vaccination produced antibodies against the cognate peptide. Peptide-specific IgA was also detected across several mucosal sites sampled. Peptide-specific CD4+ T cells were detected at levels similar to those of mice immunized with BCG. PilVax immunization resulted in an unexpected increase in the numbers of CD3+ CD4- CD8- [double negative (DN)] T cells in the lungs of vaccinated mice. Analysis of cytokine production following stimulation with the cognate peptide showed the major cytokine producing cells to be CD4+ T cells and DN T cells. This study provides insight into the antibody and peptide-specific cellular immune responses generated by PilVax vaccination and demonstrates the suitability of this vaccine for conducting a protection study.

Preformulation studies of thymopentin: analytical method development, physicochemical properties, kinetic degradation investigations and formulation perspective.

Thymopentin (TP5) is a synthetic pentapeptide with immunomodulatory properties. Given the previously described poor absorption of TP5, preformulation data is required to support effective formulation development. In this manuscript, an analytical method of TP5 was developed and validated to determine the aqueous solubility, stability, and Log P of TP5. Thermal properties were investigated, and chemical, physical and enzymatic degradation were evaluated. TP5 was informed to load in a microemulsion (ME) system according to the preformulation parameters and characterized for rheological behavior, droplet size, morphology and in vitro drug release. TP5 displayed high aqueous solubility (294.3 mg/mL), low Log P (-4.2) and 2% water content with a melting temperature of 193 °C. TP5 degraded rapidly in alkaline conditions, at elevated temperature, in oxidizing agents, and with UV exposure, however TP5 had a longer half-life in acidic conditions. The fastest enzymatic degradation was with Trypsin (half-life 6.3 h) compared with other digestive enzymes. The different degradation pathways followed first-order kinetics, and half-lives were obtained from the kinetic studies. The TP5 loaded ME exhibited a droplet size of 143 ± 35 nm with a Higuchi-model fitted sustained release profile for 24 h. These data justify and support the design of formulations to stabilize and enhance the absorption of TP5, with a ME formulation demonstrated.

PilVax, a novel Lactococcus lactis-based mucosal vaccine platform, stimulates systemic and mucosal immune responses to Staphylococcus aureus.

Most pathogens initiate infection via the mucosa, therefore delivery of vaccines directly to the mucosa is likely to be advantageous for stimulating protective immunity at the site of entry. PilVax is a novel mucosal vaccine platform that harnesses Lactococcus lactis bacteria engineered to stably express multiple copies of vaccine peptide antigens within pili, hair-like structures which extend from the cell wall. This strategy elicited systemic and mucosal antibody responses to a model antigen after intranasal immunization, but has not been tested for its capacity to stimulate protective mucosal immunity. A well-characterized linear B-cell epitope, D3(22-33) , from the fibronectin-binding protein A of Staphylococcus aureus was successfully introduced into PilVax and delivered intranasally to mice. Specific antipeptide immunoglobulin (Ig) G and IgA antibodies were detected in the serum and respiratory mucosa of vaccinated mice. Responses to the major pilus backbone protein Spy0128 were also assessed; robust antibody responses to this antigen were generated both systemically and in the respiratory and intestinal mucosa. Mice were challenged intranasally with the mouse-adapted S. aureus JSNZ strain and the S. aureus load quantified 7 days after challenge. Unexpectedly, exposure to PilVax, irrespective of the presence of the peptide, resulted in a significant reduction in S. aureus load in both the intestine and nasal mucosa (both P < 0.05) when compared with unvaccinated control mice. The mechanism(s) of protection are unclear, but merit further investigation to determine whether PilVax is a suitable platform for delivery of vaccine candidate antigens to the mucosa.

Development and Evaluation of a New Triplex Immunoassay That Detects Group A Streptococcus Antibodies for the Diagnosis of Rheumatic Fever.

Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a postinfectious sequela associated with group A Streptococcus infection. Current tests that measure anti-streptolysin O (ASO) and anti-DNaseB (ADB) titers require parallel processing, with their predictive value limited by the low rate of decay in antibody response. Accordingly, our objective was to develop and assess the diagnostic potential of a triplex bead-based assay, which simultaneously quantifies ASO and ADB together with titers for a third antigen, SpnA. Our previous cytometric bead assay was transferred to the clinically appropriate Luminex platform by coupling streptolysin O, DNaseB, and SpnA to spectrally unique magnetic beads. Sera from more than 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Operating parameters demonstrate that the triplex assay produces accurate and reproducible antibody titers which, for ASO and ADB, are highly correlative with existing assay methodology. When ARF patients were stratified by time (days following hospital admission), there was no difference in ASO and ADB between <28 and 28+ day groups. However, for anti-SpnA, there was a significant decrease (P < 0.05) in the 28+ day group, indicative of faster anti-SpnA antibody decay. Anti-SpnA immunokinetics support very recent group A Streptococcus infection and may assist in diagnostic classification of ARF. Further, bead-based assays enable streptococcal serology to be performed efficiently in a high-throughput manner.

Group A Streptococcus T Antigens Have a Highly Conserved Structure Concealed under a Heterogeneous Surface That Has Implications for Vaccine Design.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

The Group A Streptococcus serotype M2 pilus plays a role in host cell adhesion and immune evasion.

Group A Streptococcus (GAS), or Streptococcus pyogenes, is a human pathogen that causes diseases ranging from skin and soft tissue infections to severe invasive diseases, such as toxic shock syndrome. Each GAS strain carries a particular pilus type encoded in the variable fibronectin-binding, collagen-binding, T antigen (FCT) genomic region. Here, we describe the functional analysis of the serotype M2 pilus encoded in the FCT-6 region. We found that, in contrast to other investigated GAS pili, the ancillary pilin 1 lacks adhesive properties. Instead, the backbone pilin is important for host cell adhesion and binds several host factors, including fibronectin and fibrinogen. Using a panel of recombinant pilus proteins, GAS gene deletion mutants and Lactococcus lactis gain-of-function mutants we show that, unlike other GAS pili, the FCT-6 pilus also contributes to immune evasion. This was demonstrated by a delay in blood clotting, increased intracellular survival of the bacteria in macrophages, higher bacterial survival rates in human whole blood and greater virulence in a Galleria mellonella infection model in the presence of fully assembled FCT-6 pili.

A potential role for staphylococcal and streptococcal superantigens in driving skewing of TCR Vß subsets in tonsillar hyperplasia.

The TCR Vß repertoire from patients with recurrent tonsillitis and/or tonsillar hyperplasia was examined to determine whether the TCR Vß composition is suggestive of local superantigen activity and if so, whether it is associated with the presence of superantigen producing bacteria. Tonsil specimens were cultured aerobically to allow identification and isolation of the bacterial pathogens Staphylococcus aureus and Group A Streptococcus. TCR Vß subset analysis of tonsil leucocytes was performed by flow cytometry. The superantigenic potential of tonsil S. aureus isolates was determined by multiplex PCR and a T-cell mitogenicity assay. Tonsils were collected from 40 patients who were predominantly pre-school-aged children undergoing surgery for either recurrent tonsillitis or tonsillar hyperplasia causing obstructive sleep apnoea. S. aureus was cultured from 23/40 and Group A Streptococcus from 5/40 patients. Both CD4+ and CD8+ TCR Vß populations were skewed in 17/40 patients. Twelve of these had recurrent tonsillitis of whom 9 also harboured S. aureus. Characterisation of tonsillar S. aureus isolates revealed that many contained genes for one or more potent superantigens and detection of these genes was associated with in vitro mitogenic activity. Skewing of the tonsillar TCR Vß repertoire was observed at high frequency and was most commonly associated with the presence of S. aureus. Many S. aureus isolates were mitogenic suggesting that they have a potential for local impact on the function of tonsil T cell populations. These results suggest the possibility that anti-staphylococcal antibiotics may be an effective treatment option for some patients.

Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 µm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.

Stabilized plasmid-based system for bioluminescent labeling of multiple streptococcal species.

OBJECTIVE: To determine if multiple streptococcal species can be easily labeled for biophotonic imaging using a toxin-antitoxin stabilized reporter plasmid containing the native firefly luciferase gene, originally developed for use in Group A Streptococcus. RESULTS: A number of streptococcal species including Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, S. iniae, S. vestibularis, and S. salivarius were successfully transformed with the reporter plasmid. In absence of antibiotic selection, the plasmid had variable stability amongst the six strains. The expression of firefly luciferase was highest in Group B Streptococcus and S. iniae, as observed by the brightest signal and lowest detection limits in vitro. CONCLUSION: Multiple streptococcal species can be easily transformed with our toxin-antitoxin stabilized bioluminescent reporter plasmid. However, this plasmid shows variable stability and signal in different species, restricting its use for certain applications.

M-Protein Analysis of Streptococcus pyogenes Isolates Associated with Acute Rheumatic Fever in New Zealand.

We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.

Vaccination with Streptococcus pyogenes nuclease A stimulates a high antibody response but no protective immunity in a mouse model of infection.

Streptococcus pyogenes is a human pathogen which causes a spectrum of diseases ranging from pharyngitis to rheumatic fever, necrotising fasciitis and toxic shock syndrome. Development of a vaccine for S. pyogenes has been confounded both by the diversity of the disease-causing serotypes and the spectre of inadvertently stimulating autoimmunity. The S. pyogenes nuclease A (SpnA) is a recently characterised virulence factor that is highly conserved across strains and expressed during human disease. Deletion of spnA from S. pyogenes results in reduced survival of bacteria in whole human blood and attenuated virulence in a mouse model of infection. Collectively these features suggest that SpnA has potential as a vaccine candidate for S. pyogenes. Mice vaccinated subcutaneously with single or multiple doses of recombinant SpnA emulsified in Incomplete Freund's Adjuvant developed a robust and durable IgG response, including neutralising activity, to this protein. However, vaccination with rSpnA conferred no advantage in terms of lesion development, disease symptoms or colonisation levels after a sub-lethal subcutaneous challenge with S. pyogenes. Anti-SpnA serum IgG responses and neutralising activity were increased in response to challenge, indicating that SpnA is expressed in vivo. SpnA is unlikely to be a suitable antigen for a vaccine against S. pyogenes.

Structural conservation, variability, and immunogenicity of the T6 backbone pilin of serotype M6 Streptococcus pyogenes.

Group A streptococcus (GAS; Streptococcus pyogenes) is a Gram-positive human pathogen that causes a broad range of diseases ranging from acute pharyngitis to the poststreptococcal sequelae of acute rheumatic fever. GAS pili are highly diverse, long protein polymers that extend from the cell surface. They have multiple roles in infection and are promising candidates for vaccine development. This study describes the structure of the T6 backbone pilin (BP; Lancefield T-antigen) from the important M6 serotype. The structure reveals a modular arrangement of three tandem immunoglobulin-like domains, two with internal isopeptide bonds. The T6 pilin lysine, essential for polymerization, is located in a novel VAKS motif that is structurally homologous to the canonical YPKN pilin lysine in other three- and four-domain Gram-positive pilins. The T6 structure also highlights a conserved pilin core whose surface is decorated with highly variable loops and extensions. Comparison to other Gram-positive BPs shows that many of the largest variable extensions are found in conserved locations. Studies with sera from patients diagnosed with GAS-associated acute rheumatic fever showed that each of the three T6 domains, and the largest of the variable extensions (V8), are targeted by IgG during infection in vivo. Although the GAS BP show large variations in size and sequence, the modular nature of the pilus proteins revealed by the T6 structure may aid the future design of a pilus-based vaccine.

Comparison of firefly luciferase and NanoLuc luciferase for biophotonic labeling of group A Streptococcus.

NanoLuc luciferase (Nluc) is an engineered enzyme that catalyses the substrate, furimazine, to produce light. Nluc has higher sensitivity than the commonly used bioluminescent reporter, firefly luciferase (FFluc). We have introduced Nluc into a toxin-antitoxin stabilised plasmid for the efficient labeling of group A Streptococcus. Comparison of signal strength and kinetic properties between Nluc-labeled bacteria and similarly previously-labeled FFluc bacteria, showed that the bioluminescent signal produced by Nluc-labeled bacteria is up to 15-times higher than FFluc-labeled bacteria during the logarithmic phase. However, with Nluc we were unable to differentiate between bacteria that are metabolically active and inactive because of its ATP-independence. Nluc therefore offers a more sensitive reporter but, perhaps, one more restricted for downstream applications.

Cellular Uptake and Transport Mechanism Investigations of PEGylated Niosomes for Improving the Oral Delivery of Thymopentin.

BACKGROUND: Although its immunomodulatory properties make thymopentin (TP5) appealing, its rapid metabolism and inactivation in the digestive system pose significant challenges for global scientists. PEGylated niosomal nanocarriers are hypothesized to improve the physicochemical stability of TP5, and to enhance its intestinal permeability for oral administration. METHODS: TP5-loaded PEGylated niosomes were fabricated using the thin film hydration method. Co-cultured Caco-2 and HT29 cells with different ratios were screened as in vitro intestinal models. The cytotoxicity of TP5 and its formulations were evaluated using an MTT assay. The cellular uptake and transport studies were investigated in the absence or presence of variable inhibitors or enhancers, and their mechanisms were explored. RESULTS AND DISCUSSION: All TP5 solutions and their niosomal formulations were nontoxic to Caco-2 and HT-29 cells. The uptake of TP5-PEG-niosomes by cells relied on active endocytosis, exhibiting dependence on time, energy, and concentration, which has the potential to significantly enhance its cellular uptake compared to TP5 in solution. Nevertheless, cellular transport rates were similar between TP5 in solution and its niosomal groups. The cellular transport of TP5 in solution was carried out mainly through MRP5 endocytosis and a passive pathway and effluxed by MRP5 transporters, while that of TP5-niosomes and TP5-PEG-niosomes was carried out through adsorptive- and clathrin-mediated endocytosis requiring energy. The permeability and transport rate was further enhanced when EDTA and sodium taurocholate were used as the penetration enhancers. CONCLUSIONS: This research has illustrated that PEG-niosomes were able to enhance the cellular uptake and maintain the cellular transport of TP5. This study also shows this formulation's potential to serve as an effective carrier for improving the oral delivery of peptides.

Group A Streptococcus Pili-Roles in Pathogenesis and Potential for Vaccine Development.

The Gram-positive human pathogen Group A Streptococcus (GAS, Streptococcus pyogenes) employs an arsenal of virulence factors that contribute to its pathogenesis. The pilus is an important factor that enables the pathogen to adhere to and colonize host tissues. Emerging research in pilus function shows that pili's involvement in establishing infection extends beyond host adhesion. The diversity of GAS pilus types reflect the varying characteristics identified in different pili. With the development of new experimental systems and animal models, a wider range of biological functions have been explored. This brief review summarizes recent reports of new functions in different GAS pilus types and the methodologies that contributed to the findings. The established importance of the pilus in GAS pathogenesis makes this surface structure a promising vaccine target. This article also reviews recent advancements in pilus-based vaccine strategies and discusses certain aspects that should be considered in vaccine development according to the newly defined properties of pili.

The Emergence and Impact of the M1UK Lineage on Invasive Group A Streptococcus Disease in Aotearoa New Zealand.

M1UK is associated with current surges in invasive infection globally, partly due to increased production of superantigen streptococcal pyrogenic exotoxin A. We show that M1UK is now the dominant invasive emm1 lineage in Aotearoa New Zealand and is genomically related to community infections, suggesting that measures that effectively prevent group A Streptococcus pharyngitis in children could reduce invasive disease.