Jacelyn M. S. Loh

Structural Conservation, Variability, and Immunogenicity of the T6 Backbone Pilin of Serotype M6 Streptococcus pyogenes

ABSTRACT 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.

Metal-derivatized Major Histocompatibility Complex: Zeroing in on Contact Hypersensitivity

One of the prices we pay for our love of jewelry is allergic contact hypersensitivity to metal, an autoimmune condition that variably afflicts around 10% of all Caucasians (1). Contact hypersensitivity is a classic type IV DTH (delayed type hypersensitivity) response, involving primed T cells that are specific for metal modified antigens generated at a local site in the body (most commonly the epidermal layer of the skin). The T cells respond by producing proinflammatory mediators that result in local redness, swelling, and itching (2). In many cases nickel is found to be the culprit and unfortunately for sensitive people, nickel is one of the most common metals in the environment, which makes it particularly difficult to avoid. Being a ubiquitous component of metal alloys, nickel is found not only in catheters, needles, dental braces, and many other medical devices, but also in everyday items such as jewelry, watches, coins, and even in some foods. Cases can range in severity from a mild localized swelling, redness, and itchiness to a much more debilitating reaction involving larger areas (e.g., the entire mouth in some patients with hypersensitivity to the metal of their dental braces). Ni2+ reactive T cell clones have been isolated from patients and found to display varying degree of MHC class II restriction (some are more promiscuous than others) and two models have been advanced to explain what might be happening at the molecular level (3). The first model (Fig. 1) proposes that Ni2+ derivatized self-proteins are naturally processed and presented as Ni2+/peptides by the APC resident in the skin (e.g., Langerhan cells). The second model proposes a processing independent pathway where the metal directly derivatizes MHC peptide complexes on the surface of the APC. While both mechanisms seem feasible, firm evidence for either has been elusive … until now. In this issue, Lu et al. (4), present direct evidence for the formation of a preformed HLA-DR/Ni2+ complex that stimulates a Ni2+ reactive human T cell clone called ANi-2.3. ANi-2.3 is a CD4+ T cell with a Vβ17/Vα1 T cell receptor and is restricted by HLA-DR52c pretreated with soluble Ni2+. Using mutagenesis, they localize the Ni2+ binding site to histidine 81 on top of MHC class II β-chain. His81 is familiar to anyone who has worked with bacterial superantigens because its the same residue targeted by a subset of bacterial superantigens using a Zn2+ atom to bind tightly to MHC class II (5). His81 is one of only a few conserved residues on the top of the MHC class II and plays an important role in stabilizing bound peptide through a hydrogen bond to the peptide backbone (6). The important feature of Zn2+ in superantigen binding is the stability it provides the MHC class II/Sag complex, so the analogy between stable superantigen binding and metal-mediated TCR binding is clear and important. The Lu paper reveals for the first time, a possible molecular structure that might explain how normally tolerant, self-reactive T cells are stimulated into action through the addition of a single metal atom on the top of the MHC class II molecule. Figure 1. Two hypothetical pathways for nickel presentation. (1) Processing-dependent pathway. Nickel ions bind to self proteins which are then processed and presented by MHC on Langerhan cells. (2) Processing-independent pathway. Nickel ions enter through the ...

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.

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.

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

Background: 5′-Nucleotidases are important virulence factors found in several bacterial pathogens. Results: Streptococcal 5′-nucleotidase A (S5nA) generated immunomodulatory molecules adenosine and deoxyadenosine and rescued Lactococcus lactis in a blood killing assay. Conclusion: S5nA is a novel Streptococcus pyogenes virulence factor that facilitates immune evasion from the host. Significance: S5nA might be a target for developing new therapeutics or vaccines. 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 Mg2+, Ca2+, or Mn2+. However, Zn2+ 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.

Serological Evidence of Immune Priming by Group A Streptococci in Patients with Acute Rheumatic Fever.

Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to ‘prime’ the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host.

Mucosal vaccination with pili from Group A Streptococcus expressed on Lactococcus lactis generates protective immune responses

The human pathogen Group A Streptococcus (GAS) produces pili that are involved in adhesion and colonisation of the host. These surface-exposed pili are immunogenic and therefore represent an attractive target for vaccine development. The pilus is encoded in the genomic region known as the fibronectin-collagen-T-antigen (FCT)-region, of which at least nine different types have been identified. In this study we investigate expressing two of the most common FCT-types (FCT-3 and FCT-4) in the food-grade bacteria Lactococcus lactis for use as a mucosal vaccine. We show that mucosally delivered L. lactis expressing GAS pili generates specific antibody responses in rabbits. Rabbit anti-pilus antibodies were shown to have both a neutralising effect on bacterial adhesion, and immunised rabbit antiserum was able to facilitate immune-mediated killing of bacteria via opsonophagocytosis. Furthermore, intranasal immunisation of mice improved clearance rates of GAS after nasopharyngeal challenge. These results demonstrate the potential for a novel, pilus-based vaccine to protect against GAS infections.

Development of a high-throughput opsonophagocytic assay for the determination of functional antibody activity against Streptococcus pyogenes using bioluminescence

The lack of standardised protocols for the assessment of functional antibodies has hindered Streptococcus pyogenes research and the development of vaccines. A robust, high throughput opsonophagocytic bactericidal assay to determine protective antibodies in human and rabbit serum has been developed that utilises bioluminescence as a rapid read out.

Streptococcus pyogenes nuclease A (SpnA) mediated virulence does not exclusively depend on nuclease activity

BACKGROUND Streptococcus pyogenes, or Group A Streptococcus (GAS), is a human pathogen that causes a wide range of diseases, including pharyngitis, necrotizing fasciitis and toxic shock syndrome. The bacterium produces a large arsenal of virulence factors, including the cell wall-anchored Streptococcus pyogenes nuclease A (SpnA), which facilitates immune evasion by degrading the DNA backbone of neutrophil extracellular traps. SpnA consists of a C-terminal endo/exonuclease domain and a N-terminal domain of unknown function. METHODS Recombinant SpnA mutants were generated by alanine conversion of selected residues that were predicted to play a role in the enzymatic activity and tested for their ability to degrade DNA. A GAS spnA deletion mutant was complemented with a plasmid-borne catalytic site mutant and analyzed for virulence in a Galleria mellonella (wax moth) infection model. RESULTS Several predicted residues were experimentally confirmed to play a role in SpnA enzymatic activity. These include Glu592, Arg696, His716, Asp767, Asn769, Asp810 and Asp842. Complementation of a GAS spnA deletion mutant with a spnA H716A mutant gene partially restored virulence in wax moth larvae, whereas complementation with the spnA wt gene completely restored activity. Furthermore, complementation with a secreted form of SpnA showed reduced virulence. CONCLUSION Our results show that abolishing the enzymatic activity of SpnA only partially reduces virulence suggesting that SpnA has an additional virulence function, which might be located on the N-terminal domain. Furthermore, cell wall-anchoring of SpnA results in higher virulence compared to secreted SpnA, probably due to a higher local density of the enzyme.

Antigen targeting to major histocompatibility complex class II with streptococcal mitogenic exotoxin Z-2 M1, a superantigen-based vaccine carrier.

ABSTRACT Streptococcal mitogenic exotoxin Z-2 (SMEZ-2) is a streptococcal superantigen that primarily stimulates human T cells bearing Vβ8 and mouse T cells bearing Vβ11. Mutagenesis of T cell receptor (TCR)-binding residues (W75L, K182Q, D42C) produced a mutant called M1 that was >105-fold less active toward human peripheral blood lymphocytes and splenocytes from transgenic mice that express human CD4 and either human HLA-DR3-DQ2 or HLA-DR4-DQ8. Similarly, cytokine production in response to M1 in lymphocyte culture was rendered undetectable, and no change in the frequency of Vβ11-bearing T cells in mice receiving M1 was observed. M1 toxoid was tested as a potential vaccine conjugate. Vaccination with 1 to 10 μg M1 conjugated to ovalbumin (M1-ovalbumin) resulted in more rapid and quantitatively higher levels of anti-ovalbumin IgG, with endpoint titers being 1,000- to 10,000-fold greater than those in animals immunized with unconjugated ovalbumin. Substantially higher levels of anti-ovalbumin IgG were observed in mice transgenic for human major histocompatibility complex (MHC) class II. Substitution of M1 with an MHC class II binding mutant (DM) eliminated enhanced immunity, suggesting that M1 enhanced the delivery of antigen via MHC class II-positive antigen-presenting cells that predominate within lymphoid tissue. Immunization of animals with a conjugate consisting of M1 and ovalbumin peptide from positions 323 to 339 generated levels of anti-peptide IgG 100-fold higher than those in animals immunized with peptide alone. Coupling of a TCR-defective superantigen toxoid presents a new strategy for conjugate vaccines with the additional benefit of targeted delivery to MHC class II-bearing cells.