Bioinformatically predicted, stress-associated mitogen-activated protein kinase 7 (PsMPK7) of Pythium insidiosum is significantly associated with infection progression in human cadaveric cornea

Abstract

Purpose : Keratitis caused by Pythium insidiosum is rare but severe and can lead to vision loss. To study its pathogenicity and determine its putative virulence genes, we investigated the genome of ocular P. insidiosum isolate using bioinformatics tools. The selected virulence genes of P. insidiosum were studied for mRNA expression in infected human cadaveric cornea (HCC)

Methods : The whole genome sequencing of P. insidiosum obtained from a patient with keratitis was performed for gene prediction (GeneMark-ES) and virulence genes identification (PHI base). Primers were designed for the identified genes [Aspartate aminotransferase 3 (PsAAT3), Stress associated mitogen activated protein kinase 7 (PsMPK7), ADP-ribosylation factor 2 (ARF2)] and a housekeeping gene (GAPDH) using Primer3. Ex-vivo cultures (n=6) of HCC were infected (intrastromal injection) with zoospores of a clinical isolate from a patient with severe P. insidiosum keratitis. Postinfection (PI) the HCC were incubated at 37^oC, harvested (PI- 24hrs, n=3; 48hrs n=3) and preserved in RNAlater. The culture of P. insidiosum was used as control. RNA extraction, cDNA synthesis and RT-qPCR (QuantStudio 3) were performed for gene expression analysis. Data was analyzed using one-way ANOVA with Post-Hoc Tukey HSD test (p-value ≤0.05-significant).

Results : The gene prediction in P. insidiosum genome resulted in the identification of 27706 coding sequences. These coding sequences showed a total of 6812 distinct virulence genes. Among these, we selected 3 genes based on the least e-value (0 to 3.5e-56), >90% similarity matches and their function. The mRNA analysis, showed significant elevation of PsMPK7 expression after 48hrs PI [mean±SD (9.09±2.29)] compared to 24hrs (1.66±1.32), p-value ≤0.008. Although, the expressions of PsAAT3 [24hrs vs 48hrs (0.42±0.063 vs 0.55±0.26, p-value≤ 0.42)] and ARF2 (0.34±0.09 vs 0.74±0.31, p-value≤ 0.10) were not significantly different they were higher at 48 hrs.

Conclusions : This study demonstrates the expression of PsMPK7 gene in P. insidiosum in ex-vivo human corneal culture. This gene, known to play a crucial role in response to various stresses and reactive oxygen species detoxification, seems to play an important role in the pathogenicity of severe P. insidiosum keratitis.

This is a 2021 ARVO Annual Meeting abstract.