Plasminogen-Dependent Collagenolytic Properties ofStaphylococcus aureusin Collagen Gel Cultures of Human Corneal Fibroblasts

Abstract

PURPOSE. Staphylococcus aureus is a common cause of corneal ulceration, and staphylokinase (SAK) produced by this bacterium is a plasminogen activator. To investigate the pathogenesis of corneal ulceration induced by S. aureus, we examined the effects of bacterial culture broth and SAK on collagen degradation in a culture model in which human corneal fibroblasts are embedded in a collagen gel. METHODS. Corneal fibroblasts embedded in collagen were exposed to S. aureus culture broth or SAK. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Expression of pro-matrix metalloproteinase-1 (pro-MMP-1) was detected by immunoblot analysis as well as reverse transcription and real-time polymerase chain reaction analysis. RESULTS. Both S. aureus culture broth and SAK markedly increased collagen degradation in the presence of corneal fibroblasts and plasminogen. This effect of the culture broth was dependent on cell number to a greater extent than was that of SAK. Whereas the culture broth also increased the expression of pro-MMP-1 in corneal fibroblasts at both mRNA and protein levels, SAK did not. CONCLUSIONS. Our results suggest that S. aureus may promote collagen degradation both by upregulating pro-MMP1 expression in corneal fibroblasts, with pro-MMP-1 then being converted to active MMP-1 by plasmin, and by directing plasmin activity toward collagen in a SAK-dependent manner.

Stimulation of Phagocytic Activity in Cultured Human Corneal Fibroblasts by Plasminogen

“…In addition, plasminogen is thought to contribute to the pathophysiology of conditions that are associated with the excessive degradation of stromal collagen, such as corneal ulceration, by activating proteolytic enzymes, such as MMPs, secreted from corneal fibroblasts stimulated by IL-1 or bacterial pathogens. 31,32,41 In summary, we showed that plasminogen promotes phagocytic activity of corneal fibroblasts. Although plasminogen has been shown previously to contribute to ECM degradation by corneal fibroblasts under inflammatory conditions, 31,32,39 plasminogen-induced phagocytosis of microparticles did not affect the expression of uPA or MMPs by corneal fibroblasts.…”

“…31,32,41 In summary, we showed that plasminogen promotes phagocytic activity of corneal fibroblasts. Although plasminogen has been shown previously to contribute to ECM degradation by corneal fibroblasts under inflammatory conditions, 31,32,39 plasminogen-induced phagocytosis of microparticles did not affect the expression of uPA or MMPs by corneal fibroblasts. Plasminogen likely has a regulatory role in the clearance of infectious agents or damaged tissue components by corneal fibroblasts during the early phase of inflammation or wound healing before the arrival of professional phagocytes at corneal stromal lesions.…”

“…38 In the case of bacterial infection of the cornea, factors released from bacteria also promote collagen degradation by keratocytes. 32,39 We found that exposure of corneal fibroblasts to plasminogen did not affect the intracellular activity of cathepsin B, whereas the presence of microparticles per se increased cathepsin B activity in a manner independent of prior exposure of the cells to plasminogen. These results suggested that lysosomal enzyme activity is regulated by contact of the cells with microparticles or by phagocytosis in an all-or-none fashion, rather than being dependent on the extent of phagocytosis.…”

“…Our previous studies provided support for the notion that corneal fibroblasts have a key role in such collagen degradation through the synthesis and secretion of the collagenolytic enzyme MMP-1. 31,32 Infiltrating immune cells also have been shown to positively modulate this function of the stromal resident cells. 38 In the case of bacterial infection of the cornea, factors released from bacteria also promote collagen degradation by keratocytes.…”

“…Reverse transcription (RT) and real-time polymerase chain reaction (PCR) analysis was performed as described previously. 32 In brief, total RNA was isolated from corneal fibroblasts with the use of an RNeasy Kit (Qiagen, Valencia, CA, USA) and then was subjected to RT with random primers and a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The resulting cDNA was subjected to real-time PCR analysis with SYBR Premix Ex Taq (Takara Bio, Shiga, Japan) and an ABI 7900HT Sequence Detection System (Applied Biosystems).…”