Characterization of the cytopathic effect of Haemophilus ducreyi.
OBJECTIVE
Haemophilus ducreyi is the etiologic agent of chancroid, which is a genital ulcer disease that increases the risk of acquiring and transmitting HIV. The pathogenesis of H. ducreyi is not well understood.


OBJECTIVE
The goal of this study was to use a quantitative tetrazolium-based XTT assay to characterize the cytopathic effect of H. ducreyi on human foreskin fibroblasts.


METHODS
Haemophilus ducreyi strains 35000, R018, A77 and CIP542 were evaluated using the XTT assay. The role of attachment on resultant CPE was assessed using a wash step 2 hours post-infection. Internalization was evaluated by the gentamicin kill assay. Secreted exotoxin was studied using permeable inserts to separate the bacteria from the HFF monolayer.


RESULTS
HFF cell damage did not appear to be mediated by a secreted H. ducreyi cytotoxin. Direct contact of viable H. ducreyi with HFF cells was required for cell damage. H. ducreyi strains that attached poorly could be readily removed by a wash step. This reduced their capacity to damage HFF cells significantly. Although some H. ducreyi strains attach to high levels within 4 hours, no HFF cell damage was detected by the XTT assay. However, once HFF cell damage was detected by 24 hours, it was not easily reversible, despite antibiotic treatment that eradicated H. ducreyi. Internalization of H. ducreyi by HFF cells apparently did not occur to a significant degree.


CONCLUSIONS
This study indicates that classic "soluble exotoxins" are not likely the key component in H. ducreyi pathogenesis. Attachment or direct contact with HFF cells are required for H. ducreyi to cause a CPE.
Hollyer
T T
TT
Department of Medical Microbiology, University of Manitoba, Winnipeg, Canada.
DeGagne
P A
PA
Alfa
M J
MJ
D001422:Bacterial Adhesion; D001681:Biological Assay; D002460:Cell Line; D002478:Cells, Cultured; D005069:Evaluation Studies as Topic; D005098:Exotoxins / Q000378:metabolism*; D005347:Fibroblasts / Q000382:microbiology*; D006191:Haemophilus ducreyi / Q000145:classification / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D010413:Penis / Q000166:cytology; D012703:Serotyping; D013778:Tetrazolium Salts
D005098:Exotoxins; D007202:Indicators and Reagents; D013778:Tetrazolium Salts; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide