Immunology

During infection, complement plays a critical role in inflammation, opsonisation, and destruction of microorganisms. This presents a challenge for pathogens such as Staphylococcus aureus to overcome when invading the host. Our current knowledge on the mechanisms that evolved to counteract and disable this system is limited by the molecular tools available. Present techniques utilise labelled complement-specific antibodies to detect deposition upon the bacterial surface, a method not compatible with pathogens such as S. aureus, which are equipped with immunoglobulin-binding proteins, Protein A and Sbi. This protocol uses a novel antibody-independent probe, derived from the C3 binding domain of staphylococcal protein Sbi, in combination with flow cytometry, to quantify complement deposition. Sbi-IV is biotinylated, and deposition is quantified with fluorophore-labelled streptavidin. This novel method allows observation of wild-type cells without the need to disrupt key immune modulating proteins, presenting the opportunity to analyse the complement evasion mechanism used by clinical isolates. Here, we describe a step-by-step protocol for the expression and purification of Sbi-IV protein, quantification and biotinylation of the probe, and finally, optimisation of flow cytometry to detect complement deposition using normal human serum (NHS) and both Lactococcus lactis and S. aureus.

The complement system is a central component of innate immunity, responsible for recognition and killing of bacteria by tagging invaders through opsonisation, thereby promoting phagocytosis, and by direct lysis. Complement activity is routinely measured using functional assays that utilise erythrocytes as targets. The classical pathway haemolytic assay (CH50) with antibody sensitised sheep erythrocytes as target is used worldwide in clinical and research laboratories to measure complement activity in human and rodent sera. While there are no particular limitations in the human assay, measuring complement in mouse serum is more difficult and usually requires large amounts of serum, which is challenging to collect in experiments. In particular, it is challenging to measure the activities of individual mouse complement proteins. To overcome this hurdle, we have developed protocols that employ human sera depleted of single complement proteins as the source of the other complement proteins and test mouse serum to restore the relevant component. This simple haemolytic assay is a useful tool for confirming natural or engineered complement deficiencies and complement dysregulation in mouse models.

Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the outer blood retinal barrier. RPE cells are highly polarized with apical microvilli and basolateral contact with Bruch’s membrane. In order to obtain biologically relevant polarized RPE cultures in vitro, RPE cells are seeded onto the apical side of a transwell filter and cultured for 4 weeks in low serum media. MAC formation on RPE cells has been reported to be sub-lytic. MAC formation can be achieved in vitro by introduction of normal human serum (NHS) to media following serum starvation for 24 h. NHS contains all serum complement proteins required to initiate complement activation and MAC formation. We combined in vitro RPE polarization and complement activation to visualize MAC formation in vitro utilizing confocal microscopy allowing for high resolution MAC imaging.