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The TCR Vβ repertoire from patients with recurrent tonsillitis and/or tonsillar hyperplasia was examined to determine whether the TCR Vβ 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 Vβ 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 Vβ 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 Vβ 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.

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.

Commensal and pathogenic bacteria express adhesive proteins on their cell surface, which are important for colonisation of the host. In Gram-positive bacteria, these adhesins are often covalently anchored to the cell wall by a sortase enzyme. A recent bioinformatic study has revealed a total of 860 predicted cell wall-anchored proteins in 94 completely sequenced genomes. The interaction of adhesins with host cells can be analysed with the use of adhesin-coated microbeads. Here we show that sortase-mediated ligation can be used for the site-specific immobilisation of adhesins to red-fluorescence microspheres. This coupling method allows for the native orientation of the adhesins on the beads. Furthermore, the high substrate specificity of the sortase enzyme allows the use of only partially purified recombinant proteins, which reduces preparation time and costs, and also prevents coupling of any contaminants that might interfere with cell binding.

Sortases are transpeptidases produced by Gram-positive bacteria to anchor cell surface proteins covalently to the cell wall. The Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG) on the target protein followed by the formation of an amide bond with the pentaglycine cross-bridge in the cell wall. Over recent years, several researchers have exploited this specific reaction for a range of biotechnology applications, including the incorporation of non-native peptides and non-peptidic molecules into proteins, the generation of nucleic acid-peptide conjugates and neoglycoconjugates, protein circularisation, and labelling of cell surface proteins on living cells.

Many bacterial species possess long filamentous structures known as pili or fimbriae extending from their surfaces. Despite the diversity in pilus structure and biogenesis, pili in Gram-negative bacteria are typically formed by non-covalent homopolymerization of major pilus subunit proteins (pilins), which generates the pilus shaft. Additional pilins may be added to the fiber and often function as host cell adhesins. Some pili are also involved in biofilm formation, phage transduction, DNA uptake and a special form of bacterial cell movement, known as 'twitching motility'. In contrast, the more recently discovered pili in Gram-positive bacteria are formed by covalent polymerization of pilin subunits in a process that requires a dedicated sortase enzyme. Minor pilins are added to the fiber and play a major role in host cell colonization.This review gives an overview of the structure, assembly and function of the best-characterized pili of both Gram-negative and Gram-positive bacteria.

The immobilization of proteins to surfaces is an active area of research due to strong interest in protein-based sensors. Here, we describe a novel method for immobilizing ligand proteins onto Biacore sensor chips using the transpeptidase activity of Staphylococcus aureus sortase A (SrtA). This method provides a robust and gentle approach for the site-directed, covalent coupling of proteins to biosensor chips. Notably, the high specificity of the sortase allows immobilization of proteins from less than pure protein samples allowing short cuts in protein purification protocols.

Superantigens have been implicated in a wide variety of human diseases. Yet, solid evidence for their role in pathogenesis is available only for Toxic Shock Syndrome and a few other conditions. This evidence is critically reviewed herein.