Characterization of Candida albicans infection of an in vitro oral epithelial model using confocal laser scanning microscopy.
BACKGROUND
Oral candidosis presents as several distinct forms and one of these, chronic hyperplastic candidosis, is distinguished by penetration of the epithelium by Candida. The aim of this study was to use confocal laser scanning microscopy to examine invasion of the oral epithelium by Candida albicans from different oral conditions and to determine whether inherent strain differences exist that could relate to infection type. Reverse transcription-polymerase chain reaction was also used to detect products from virulence gene families.


METHODS
C. albicans (n = 19) was used to infect reconstituted human oral epithelium, which was incubated for 12 h. One half of the reconstituted human oral epithelium was then fixed and stained with concanavalin A-Alexa 594, pan-cytokeratin antibody-Alexa 488 and Hoechst nucleic acid dye. RNA was extracted from the remaining tissue for reverse transcription-polymerase chain reaction targeting secreted aspartyl proteinase, phospholipase and agglutinin-like sequence genes of C. albicans.


RESULTS
Confocal laser scanning microscopy revealed strain-dependent tissue invasion, with differences evident in surface colonization, C. albicans morphology and the extent and pattern of tissue penetration. Hyphae were seen to directly penetrate epithelial cells and migrate between keratinocytes with yeast budding also evident in the reconstituted human oral epithelium. A relationship between 'high tissue invasion' and expression of secreted aspartyl proteinase genes 4-6 was noted. Interestingly, four of the five 'high invaders' originated from chronic hyperplastic candidosis.


CONCLUSIONS
Confocal laser scanning microscopy permitted high resolution analysis of reconstituted human oral epithelium invasion by C. albicans and identified strain differences in the invasion process. Association between extensive hyphal morphology, direct epithelial penetration and high surface colonization were made with the 'highly invasive' strains.
Malic
S
S
Oral Surgery, Medicine & Pathology, School of Dentistry, Heath Park, Cardiff, UK.
Hill
K E
KE
Ralphs
J R
JR
Hayes
A
A
Thomas
D W
DW
Potts
A J
AJ
Williams
D W
DW
D016282:Aspartic Acid Endopeptidases / Q000235:genetics; D002176:Candida albicans / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D002180:Candidiasis, Oral / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D025301:Hyphae / Q000502:physiology; D015603:Keratinocytes / Q000382:microbiology*; D018613:Microscopy, Confocal; D008954:Models, Biological; D009061:Mouth Mucosa / Q000166:cytology / Q000382:microbiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D037521:Virulence Factors / Q000235:genetics
D005656:Fungal Proteins; D037521:Virulence Factors; D016282:Aspartic Acid Endopeptidases