Bi-fluorescent Staphylococcus aureus infection enables single-cell analysis of intracellular killing in vivo.

Techniques for studying the clearance of bacterial infections are critical for advances in understanding disease states, immune cell effector functions, and novel antimicrobial therapeutics. Intracellular killing of Staphylococcus aureus by neutrophils can be monitored using a S. aureus strain stably expressing GFP, a fluorophore that is quenched when exposed to the reactive oxygen species (ROS) present in the phagolysosome. Here, we expand upon this method by developing a bi-fluorescent S. aureus killing assay for use in vivo. Conjugating S. aureus with a stable secondary fluorescent marker enables the separation of infected cell samples into three populations: cells that have not engaged in phagocytosis, cells that have engulfed and killed S. aureus, and cells that have viable internalized S. aureus. We identified ATTO647N-NHS Ester as a favorable dye conjugate for generating bi-fluorescent S. aureus due to its stability over time and invariant signal within the neutrophil phagolysosome. To resolve the in vivo utility of ATTO647N/GFP bi-fluorescent S. aureus, we evaluated neutrophil function in a murine model of chronic granulomatous disease (CGD) known to have impaired clearance of S. aureus infection. Analysis of bronchoalveolar lavage (BAL) from animals subjected to pulmonary infection with bi-fluorescent S. aureus demonstrated differences in neutrophil antimicrobial function consistent with the established phenotype of CGD.

Inhibition of Candida albicans adhesion by recombinant human antibody single-chain variable fragment specific for Als3p.

The Candida albicans adhesin, Als3p, was identified as a potential cognate antigen for previously described human antibody fragments [single-chain variable fragment (scFv)] based on similarity of the binding pattern of the scFv to the distribution of this protein on the hyphal surface. Although all scFv bound avidly to wild type, scFv3 showed no detectable binding via immunofluorescence assay to strain 1843, containing a homozygous deletion of ALS3. Binding to the ALS3 reintegrant strain, 2322, was preserved, and scFv3 also bound to Saccharomyces cerevisiae expressing ALS3. Other scFv retained binding to 1843, but with a markedly altered pattern. To determine if scFv3 could interfere with Als3p function, adhesion assays were conducted using human epithelial or endothelial cells as target. Treatment of wild-type C. albicans with scFv3 reduced adhesion of the fungus to both cell types to levels comparable to the als3Delta/als3Delta mutant. These experiments confirm that phage display is a viable method to isolate human scFv specific to an antigen implicated in C. albicans virulence, and that the scFv interfere with adhesion to human cells. The altered pattern of immunostaining with other scFv that retain binding to the als3Delta/als3Delta mutant suggest that Als3p may also have a role in structural organization of the C. albicans cell surface.