Differential possession of type 6 secretion system effector genes in exoU and exoS Pseudomonas aeruginosa in microbial keratitis

Abstract

Corneal infection (microbial keratitis; MK) is most frequently caused by Pseudomonas aeruginosa, which can utilize an array of secreted virulence factors for pathogenesis. Invasive strains of P. aeruginosa possessing the exoS gene, invade mammalian cells, while cytotoxic strains with the exoU gene rapidly kill host cells. This study investigated the presence of the type six secretion system (T6SS) effector genes in exoU and exoS strains isolated from MK. Fourteen different T6SS effector genes within 20 exoU and 19 exoS P. aeruginosa were explored using whole genome sequence data by BLAST search. To confirm the BLAST search result, PCR was used to detect exoU, exoS and those genes significantly different in the BLAST search, in a separate set of 56 MK isolates from India (24) and Australia (32). The phospholipase D (PLD) activity was measured using the Amplex Red Phospholipase D Assay kit. Three effector genes, tse7, tle1, and pldA, were differentially possessed in the exoU and exoS strains in the BLAST search (p < 0.05). When combining the BLAST search and PCR results, pldA was significantly more common in exoU (81.8%) than the exoS strains (37.3%) (p < 0.01) and trends were similar in Indian (81% exoU vs 45.5% exoS) and Australian (82.6% exoU vs 23% exoS) isolates. PldA expression was associated with detectable PLD activity. Irrespective of geographical region, pldA was more commonly found in exoU P. aeruginosa. While MK due to exoU is generally more severe than those due to exoS, the association between expression of pldA and its function requires further investigation.