Abstract
Neutrophils are a key component of the innate immune system, functioning to locate, phagocytose and destroy invading pathogens. Engulfed bacteria are internalized into a vacuole called the phagosome, which is subsequently filled with microbicidal oxidants and enzymes. Neutrophils utilize a combination of both oxygen-dependent and oxygen-independent mechanisms to kill invading pathogens, the most potent of which is thought to be hypochlorous acid (HOCl). Staphylococcus aureus is an important human pathogen, causing a range of diseases, and is difficult to treat effectively due to the many evasion strategies it employs. Although susceptible to oxidative killing, a small proportion of phagocytosed S. aureus are able to survive within the phagosome. This latent population are protected from antimicrobial drugs, and may emerge later to exacerbate the infection.
It was hypothesized that S. aureus phagosomal survivors are more resistant to oxidative stress, and that targeting their antioxidant defences may render the bacteria more susceptible to neutrophil killing.
An assay was developed to monitor bacterial survival up to three hours after phagocytosis by human neutrophils isolated from peripheral blood. Differential centrifugation and lysostaphin were implemented into the assay to remove all extracellular bacteria and enable quantification of intraphagosomal survivors. It was consistently found that around 1% of the phagocytosed S. aureus were viable after three hours. Past this point bacterial numbers began to increase. Bacteria may have either been dividing inside neutrophils or had been released from dead neutrophils into the surrounding media. Mutant strains lacking antioxidant genes were compared against isogenic wildtype strains to determine if the mutants were more susceptible to neutrophil killing. Wildtype strains were COL and USA300 with the knockout mutants: ΔhypR, ΔmerA, ΔbshA, Δcdr and ΔaldA. Contrary to the hypothesis, none of the mutants tested exhibited any increased susceptibility to neutrophil killing.
These mutants were exposed to hypochlorous acid and compared against their isogenic wildtype parent strains. The LD50 for COL wildtype was 37.7 nmol HOCl/109 bacteria and 45.5 nmol HOCl/109 bacteria for USA300 wildtype. No increase in sensitivity to HOCl was seen. A bacillithiol knockout mutant was analysed by mass spectrometry to determine whether any low molecular weight thiols were being upregulated to compensate for the gene knockout. Out of four thiols tested, only Coenzyme A was slightly more abundant in the USA ΔbshA mutant compared to the wildtype. This suggested that there was no significant compensatory upregulation of known thiols. Preliminary work focussed on pre-treating bacteria with a sub-lethal dose of HOCl to examine whether this could confer a survival advantage when exposed to either subsequent doses of reagent HOCl or neutrophils. More experimental repeats need to be conducted before proper conclusions can be drawn.
It is apparent that a single knockout of the five antioxidant genes assessed in this study was not sufficient to sensitise S. aureus to HOCl or killing by human neutrophils. Other attempts to sensitise the bacteria are warranted.