Sara Tamminga

Antibiotics have saved countless lives by treating infections and enabling modern medical procedures. However, the rise of antibiotic resistance threatens a return to the pre-antibiotic era, and antimicrobial resistance-related mortality is expected to increase by 70% over the next 25 years globally.

Vaccination is a promising strategy to prevent severe bacterial infections, yet eective vaccines for opportunistic pathogens Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) are still lacking. e world health organization (WHO) has classied these bacteria as priority pathogens for vaccine research. Alternative therapies, such as monoclonal antibodies and phages, are also being explored.

S. aureus and S. pyogenes asymptomatically colonize 10-30% of the population. However, they can also cause skin and soft tissue infections (SSTIs) ranging from mild (e.g. impetigo, folliculitis) to severe (necrotizing fasciitis, surgical wound infection). Studying local host defenses in the skin can help identify why some individuals develop infections while others remain asymptomatic. is knowledge can aid in the development of eective therapies that combat these opportunistic pathogens.

In this thesis we focused specically on the interaction between the human immune system and the surface glycans of S. aureus and S. pyogenes. Chapter 2 and chapter 3 of this thesis focus on S. aureus. In chapter 2 we focused on wall teichoic acids (WTAs), which are glycopolymers anchored in the cell wall of S. aureus. ey play a role in S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTA is composed of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three enzymes (glycosyltransferases): TarS, TarM and TarP. TarS and TarP add β-linked GlcNAc at the C-4 (β1,4-GlcNAc) and C-3 (β1,3-GlcNAc) positions of the RboP subunit, respectively. TarM adds α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). ese glycosylation patterns or “glyco-coats” aect immune recognition of S. aureus. In this chapter we focused on the distribution and genetic diversity of the three glycosyltransferases. We analyzed 25,652 S. aureus genomes and found that over 99% contained tarS, with 37% and 7% co-containing tarM and tarP, respectively. Furthermore, we observed alleles of these genes that contained amino acid substitutions in critical residues of the enzyme, or premature stop codons. eir expression in a tar-negative strain showed severely attenuated or absent WTA glycosylation, and thereby abrogated recognition by specic monoclonal antibodies and human langerin. Overall chapter 2 provides a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

In chapter 3 we focused on the role of Langerhans cells (LCs) in eczema, also known as atopic dermatitis (AD). AD is a chronic inammatory skin disorder that aects 230 million people world-wide. AD is a multifactorial disease characterized by dysregulated T cell immunity and a dysbiotic skin microbiota composition with predominant presence of S. aureus. LCs are sentinel immune cells located in the epidermis of the skin. Specic glycosylation patterns of S. aureus WTA amplify skin inammation through interaction with LCs. In this study we performed single-cell RNA sequencing of primary epidermal LCs and dermal T cells from AD patients and healthy controls. Additionally, we analyzed the glyco-coat of S. aureus isolated from the AD patients’ lesional sites. We found four LC subpopulations: two steady-state clusters (LC1 and LC1H) and two proinammatory/matured subsets (LC2 and migratory LCs). e latter two subsets were observed to be enriched in AD skin. Furthermore, we found increased expression of C-type lectin receptors, the high-anity IgE receptor and activation of prostaglandin and leukotriene biosynthesis pathways in AD LCs. Furthermore, we identied upregulated transcriptional pathways related to T cell activation in AD LCs, this corroborated with the increased T helper 2 and T regulatory cell populations in AD lesions. Finally, we stimulated primary LCs with the S. aureus strains isolated from the AD lesions and performed bulk RNA sequencing. is showed upregulation of T helper 2-related pathways in the stimulated LCs. All together, these ndings provide proof-of-concept for a role for LCs in connecting S. aureus colonization in the epidermis, and aberrant T helper 2 cell responses in the dermis of AD lesions.

Chapters 4 and 5 focus on S. pyogenes, which is a major contributor to infection-related deaths worldwide. In chapter 4 we focused on the distinctive cell wall-anchored Group A Carbohydrate (GAC), present in all S. pyogenes strains. GAC comprises of a polyrhamnose backbone with alternating GlcNAc sidechains, of which 25% are decorated with glycerol phosphate (GroP). e genes in the gacA-L cluster are essential for GAC biosynthesis, with gacI-L being responsible for the GlcNAc-GroP decoration that facilitates agglutination in rapid diagnostic tests and enhances S. pyogenes virulence. Historical research back in the ‘40s and ‘50s identied S. pyogenes strains that lost the GlcNAc side chain after serial animal passage, and termed these “A-variant” strains. We analyzed the genome sequences of a single viable historic parent/A-variant strain pair, revealing a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analyzed the genetic diversity of the 12 gacA-L genes in a collection of 2,044 S. pyogenes genome sequences. We identied 31 isolates (1.5%) with a premature stop codon in one of the gac genes. Nearly 40% of these isolates had a premature stop codon in gacH. Expressing these variants in a gacH deletion mutant conrmed the loss-of-function through a signicant reduction of GroP using cell wall analysis. Additionally, we demonstrated that strains expressing these gacH loss-of-function variants rendered to be completely resistant against the human bactericidal enzyme group IIA-secreted phospholipase. Overall, these ndings provide a broad overview of the genetic variation in the gacA-L gene cluster, within a global population of S. pyogenes strains and the functional consequences of GacH variation in immune recognition and clearance.

In chapter 5 we studied the interactions between S. pyogenes and LCs through transcriptional analysis, and langerin as an interacting receptor. Langerin is a C-type lectin receptor that has anity for glycan structures including mannose, β-GlcNAc and β-glucan. We found that langerin binds to dierent S. pyogenes strains with varying intensities. Using a medium-binding strain, we stimulated human primary LCs for 8 hours. Upon S. pyogenes stimulation we observed that LCs upregulated the expression of CCL3, CCL4, MMP12, and IL10. Indicating that exposure to S. pyogenes triggers multiple functions in LCs. Subsequently, we used a Krmit transposon library screen to identify streptococcal genes involved in langerin binding. Validation experiments conrmed that deletion of emm or mga in dierent S. pyogenes backgrounds signicantly reduced langerin binding, and complementation with emm3 restored binding in S. pyogenes but not Lactobacillus lactis. ese ndings suggest that, besides M protein, additional factors in S. pyogenes are necessary for langerin binding. Overall these ndings enhance our understanding of the interaction between this opportunistic pathogen and the human host, which is important for new strategies to prevent and treat S. pyogenes infections.

Collectively, the research described in this thesis provides a comprehensive overview of genetic diversity in the enzymes responsible for WTA and GAC biosynthesis, in S. aureus and S. pyogenes respectively. Additionally it describes the impact of glycan diversity on the interaction with the human host, specically human LCs. Since the rise in antimicrobial resistance is a worldwide threat to public health to date, it is important that research focuses on alternative strategies such as vaccines, phages and antibodies. In order to successfully design such therapies, comprehensive understanding of the interactions with the human immune system is needed. is thesis highlights the importance of studying genetic diversity of surface glycans and their role in immune recognition, providing valuable insights for future research and potential treatments.