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The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea.

ABSTRACT: The pathogenic mechanisms of fungal infection during human keratomycosis were investigated in an ex vivo corneal model that used strains of Fusarium oxysporum differing in the production of a fungal transcription factor.A pacC loss-of-function mutant and a pacC dominant-activating mutant were constructed from a wild-type isolate of F. oxysporum, and the 3 strains were characterized by in vitro growth kinetics. Twenty-seven human donor corneas maintained in tissue culture were superficially scarified and topically inoculated with the wild-type, the pacC loss-of-function mutant, or the pacC dominant-activating strains. Relative hyphal invasion into the stroma was compared histopathologically in corneal sections.F. oxysporum strains demonstrated comparable exponential growth rates in vitro. Wild-type F. oxysporum invaded into the corneal tissue within 1 day and penetrated through the anterior stroma during the next 4 days. The pacC loss-of-function mutant invaded explanted corneas significantly less than the wild-type strain on day 1 (P < 0.0001) and on day 3 (P = 0.0003). The pacC dominant-activating strain adhered and penetrated explanted corneas similar to the wild-type strain.The PacC pathway regulating the transcription of fungal genes allows fungal adaptation to the ocular surface and enables invasion of the injured cornea by F. oxysporum.

SUBMITTER: Hua X

PROVIDER: S-EPMC2991523 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea.

Hua Xia X Yuan Xiaoyong X Di Pietro Antonio A Wilhelmus Kirk R KR

Cornea 20101201 12

<h4>Purpose</h4>The pathogenic mechanisms of fungal infection during human keratomycosis were investigated in an ex vivo corneal model that used strains of Fusarium oxysporum differing in the production of a fungal transcription factor.<h4>Methods</h4>A pacC loss-of-function mutant and a pacC dominant-activating mutant were constructed from a wild-type isolate of F. oxysporum, and the 3 strains were characterized by in vitro growth kinetics. Twenty-seven human donor corneas maintained in tissue ...[more]

PMID: 20856109

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Project description:Microbial keratitis is a major cause of blindness worldwide. An excessive host inflammatory response can occur even after adequate antimicrobial treatment. This results in tissue damage with corneal thinning and even perforation, which may require corneal transplantation. In this study we investigated the pathways involved in the pathophysiology of this disease by comparing the human transcriptome profile of tissue from culture-proven bacterial and fungal keratitis (n=7 and n=8 respectively) with normal non-infected cadaveric corneal tissue (C, n=12) using Illumina HT12 v4 microarrays. The causative organisms were Streptococcus pneumoniae (n=6) and Pseudomonas aeruginosa (n=1) for bacterial keratitis (BK). Fungal keratitis (FK) was caused by Fusarium sp. (n=5), Aspergillus sp. (n=2, A. flavus and terreus) and Lasiodiplodia sp. (n=1). Differential expression (DE) analysis revealed 2310 significantly altered probes in the BK v C comparison, and 1813 probes for FK v C. The most highly upregulated gene in both comparisons was MMP9 with fold changes (FC) of 64 (fdr-adjusted p<6 x10-11) for FK v C and 89 for BK v C (fdr-adjusted p<4 x10-11) respectively. Network co-expression analyses revealed the defense response, inflammatory response and extracellular matrix mechanisms to be the main functional pathways involved. Microarray results were validated by performing real-time quantitative PCR (RTqPCR) for 46 DE genes using RNA extracted from the same samples. There was a high correlation between log2 FC values from microarray and RTqPCR. Further studies are needed to evaluate the most highly differentially expressed genes as possible biomarkers of disease progression or therapeutic targets. Case - control study design. Corneal ulcer tissue from 8 bacterial and 9 fungal ulcers was excised at the time of corneal transplantation surgery and immediately preserved in RNALater. Non-infected corneal tissue from 13 cadaver corneas were the control tissue. Transcriptome profile generated using Illumina HT12 v4 beadchips. Differential expression analysis was performed with pairwise comparisons: bacterial ulcers versus controls, fungal ulcers versus controls and bacterial versus fungal ulcers. Microarray results validated with RTqPCR.

Project description:Hydrophobins (HPs) are small secreted fungal proteins possibly involved in several processes such as formation of fungal aerial structures, attachment to hydrophobic surfaces, interaction with the environment and protection against the host defense system. The genome of the necrotrophic plant pathogen Fusarium graminearum contains five genes encoding for HPs (FgHyd1-5). Single and triple FgHyd mutants were produced and characterized. A reduced growth was observed when the ΔFghyd2 and the three triple mutants including the deletion of FgHyd2 were grown in complete or minimal medium. Surprisingly, the growth of these mutants was similar to wild-type when grown under ionic, osmotic or oxidative stress conditions. All the mutant strains confirmed the ability to develop conidia and perithecia, suggesting that the FgHyds are not involved in normal development of asexual and sexual structures. A reduction in the ability of hyphae to penetrate through the water-air interface was observed for the single mutants ΔFghyd2 and ΔFghyd3 as well as for the triple mutants including the deletion of FgHyd2 and FgHyd3. Besides, ΔFghyd3 and the triple mutant ΔFghyd234 were also affected in the attachment to hydrophobic surface. Indeed, wheat infection experiments showed a reduction of symptomatic spikelets for ΔFghyd2 and ΔFghyd3 and the triple mutants only when spray inoculation was performed. This result could be ascribed to the affected ability of mutants deleted of FgHyd2 and FgHyd3 to penetrate through the water-air interface and to attach to hydrophobic surfaces such as the spike tissue. This hypothesis is strengthened by a histological analysis, performed by fluorescence microscopy, showing no defects in the morphology of infection structures produced by mutant strains. Interestingly, triple hydrophobin mutants were significantly more inhibited than wild-type by the treatment with a systemic triazole fungicide, while no defects at the cell wall level were observed.

Dataset Information

The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea.

Publications

The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea.

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