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.

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.

The Cell Wall Deacetylases Spy1094 and Spy1370 Contribute to Streptococcus pyogenes Virulence.

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a strictly human pathogen that causes a wide range of diseases, including skin and soft tissue infections, toxic shock syndrome and acute rheumatic fever. We have recently reported that Spy1094 and Spy1370 of S. pyogenes serotype M1 are N-acetylglucosamine (GlcNAc) deacetylases. We have generated spy1094 and spy1370 gene deletion mutants in S. pyogenes and gain-of-function mutants in Lactococcus lactis. Similar to other cell wall deacetylases, our results show that Spy1094 and Spy1370 confer lysozyme-resistance. Furthermore, deletion of the genes decreased S. pyogenes virulence in a human whole blood killing assay and a Galleria mellonella (Greater wax moth) larvae infection model. Expression of the two genes in L. lactis resulted in increased lysozyme resistance and survival in whole human blood, and reduced survival of infected G. mellonella larvae. Deletion of the spy1370, but not the spy1094 gene, decreased resistance to the cationic antimicrobial peptide cecropin B, whereas both enzymes increased biofilm formation, probably resulting from the increase in positive charges due to deacetylation of the cell wall. In conclusion, Spy1094 and Spy1370 are important S. pyogenes virulence factors and might represent attractive targets for the development of antibacterial agents.

PilVax: A Novel Platform for the Development of Mucosal Vaccines.

Peptide vaccines offer an attractive strategy to induce highly specific immune responses while reducing potential side effects. However, peptides are often poorly immunogenic and unstable on their own, requiring the need for potentially toxic adjuvants or expensive chemical coupling. The novel peptide delivery platform PilVax utilizes the rigid pilus structure from Group A Streptococcus (GAS) to stabilize and amplify the peptide, and present it on the surface of the non-pathogenic food-grade bacterium Lactococcus lactis. Upon intranasal immunization, PilVax vaccines have proven to induce peptide-specific systemic and mucosal responses. PilVax provides an alternative method to develop mucosal vaccines that are inexpensive to produce and easy to administer.

Downregulation of Ribosomal Contents and Kinase Activities Is Associated with the Inhibitive Effect on the Growth of Group B Streptococcus Induced by Placental Extracellular Vesicles.

BACKGROUND: Like many other cell types, the human placenta produces large amounts of extracellular vesicles (EVs). Increasing evidence has shown that placental EVs contribute to the regulation of maternal immune and vascular systems during pregnancy via the transfer of their cargos. In this study, we investigated the effect of placental EVs on the growth of opportunistic pathogens that commonly colonise the female reproductive tract. METHODS: Gram-positive bacterium Group B Streptococcus (GBS) and Gram-negative bacterium Escherichia coli (E. coli) were treated with placental EVs that were collected from placental explant cultures, and the growth, susceptibility, and resistance to antibiotics of the bacteria were measured. In addition, comparative proteomics analysis was also performed for the GBS with or without exposure to placental EVs. RESULTS: When treated with placental micro-EVs or nano-EVs, the GBS growth curve entered the stationary phase earlier, compared to untreated GBS. Treatment with placental EVs also inhibited the growth of GBS on solid medium, compared to untreated GBS. However, these biological activities were not seen in E. coli. This attenuative effect required interaction of placental EVs with GBS but not phagocytosis. In addition, the susceptibility or resistance to antibiotics of GBS or E. coli was not directly affected by treatment with placental EVs. The proteomic and Western blotting analysis of GBS that had been treated with placental EVs suggested that the downregulation of cellular components and proteins associated with phosphorylation and cell energy in GBS may contribute to these attenuative effects. CONCLUSION: We demonstrated the attenuative effect of the growth of GBS treated with placental EVs. Downregulation of cellular components and proteins associated with phosphorylation and cell energy may contribute to the physiological changes in GBS treated with placental EVs.

Using Lactococcus lactis as Surrogate Organism to Study Group A Streptococcus Surface Proteins.

The isolation of a single Group A Streptococcus (GAS) virulence determinant in functional investigations is challenging, as GAS employs a multitude of virulence factors. The redundancy between many surface proteins such as adhesins also adds complexity and difficulty. Lactococcus lactis is a non-pathogenic Gram-positive species related to GAS that can be an ideal surrogate organism to circumvent this problem. Genetic manipulation in L. lactis is easy, and the mechanisms for processing and cell wall-anchoring of surface proteins are similar to GAS. Lactococci have been extensively used to express heterologous surface proteins from other bacterial species, and modern molecular cloning tools and protocols have been developed. This chapter describes the workflow of generating recombinant L. lactis strains expressing GAS surface proteins and the validation and quantification of their surface expression.

The Use of Galleria mellonella (Wax Moth) as an Infection Model for Group A Streptococcus.

Recently, the use of Galleria mellonella larvae as a nonmammalian model to simulate bacterial infectious diseases has shown to be a rapid, simple, and cost-effective alternative. The insect's innate immune response is remarkably similar to that of the vertebrates, and consists of both the cellular and the humoral immune response. Here, we provide a protocol for using G. mellonella larvae to study virulence of GAS, including the use of a health score system for quantitative analysis and the methods for assessing post-infection bacterial burden in vivo.

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.

Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing.

Galleria mellonella (greater wax moth or honeycomb moth) has been introduced as an alternative model to study microbial infections. G. mellonella larvae can be easily and inexpensively obtained in large numbers and are simple to use as they don't require special lab equipment. There are no ethical constraints and their short life cycle makes them ideal for large-scale studies. Although insects lack an adaptive immune response, their innate immune response shows remarkable similarities with the immune response in vertebrates. This review gives a current update of what is known about the immune system of G. mellonella and provides an extensive overview of how G. mellonella is used to study the virulence of Gram-positive and Gram-negative bacteria. In addition, the use of G. mellonella to evaluate the efficacy of antimicrobial agents and experimental phage therapy are also discussed. The review concludes with a critical assessment of the current limitatons of G. mellonella infection models.