Abstract
Purpose :
Transition metals such as Zn, Co, Ni, Fe contribute to metabolism, DNA synthesis, regulating virulence factors, and defense against oxidative stress in SA infection. Hosts sequester these metals to generate an immune response called “nutritional immunity”. To infect, SA must overcome the sequestration of these essential nutrients. In the present study, we aim to find these virulence factors contributing to endophthalmitis.
Methods :
USA300 JE2 wild-type and its isogenic mutant strains encoding for Zn-family metallophores were ordered from BEI resources. Growth curves were compared between strains. In vitro studies were done to assess adhesion, invasion, and intracellular replication of the strains in cultured human retinal pigment epithelial cells(ARPE-19), human Müller glial cells(MIO-M1) and human leukocyte monocyte cells, differentiated to macrophages(THP1). Endophthalmitis was induced intravitreally with the wild-type and mutant strains in BL6 mice. Disease progression was monitored by eye exam, bacterial burden enumeration, and assessment of inflammation. The expression of inflammatory mediators was also evaluated in MIO-M1 and ARPE-19 cells via qPCR and ELISA post bacterial challenge. LDH assay was done to determine the cytotoxic effects of the strains in the cell lines.
Results :
Our data showed similar growth rates for the wild-type and mutant strains (aur, cntA, cntD, cntM, and cntL). Studies revealed that aur adhered significantly less to ARPE-19 and THP1. However, cntD, and cntL encoding for the metallophore staphylopine showed significantly reduced adhesion to THP-1. Although, the intracellular replication assay in ARPE-19 did not show any change among the strains, aur and cntL had drastic reductions in their cytotoxic levels as compared to the wild-type. All the mutants exhibited significant replication defect with no changes in cytotoxicity for aur in MIO-M1. Inflammatory mediators in retinal cells exhibited significant reduction in the mutant infected cells. In vivo studies indicated reduced corneal haze in the eyes infected with aur and cntL, with lowered bacterial burden for cntL. Both mutant infected eyes had attenuated inflammation as compared to wild-type.
Conclusions :
Our study revealed that metallophores in SA are essential for its virulence and contribute to endophthalmitis and hence, could further be explored to develop therapeutic targets against bacterial ocular infections.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.