Carlos Tiago Alves1, Xiao-Qing Wei2, Sónia Silva1, Joana Azeredo1, Mariana Henriques3, David W Williams4. 1. CEB - Centre of Biological Engineering, LIBRO - Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, 4710-057 Braga, Portugal. 2. Tissue Engineering & Reparative Dentistry, School of Dentistry, Cardiff University, Cardiff CF14 4XY, United Kingdom. 3. CEB - Centre of Biological Engineering, LIBRO - Laboratório de Investigação em Biofilmes Rosário Oliveira, University of Minho, 4710-057 Braga, Portugal. Electronic address: mcrh@deb.uminho.pt. 4. Tissue Engineering & Reparative Dentistry, School of Dentistry, Cardiff University, Cardiff CF14 4XY, United Kingdom. Electronic address: williamsdd@cf.ac.uk.
Abstract
OBJECTIVE: The principal aim of this study was to investigate the in vitro co-infection of a reconstituted human vaginal epithelium (RHVE) by Candida albicans and Candida glabrata. METHODS: The ability of both species to invade and colonise the RHVE was examined using species-specific peptide nucleic acid (PNA) probe hybridisation, confocal laser scanning microscopy (CLSM) and a novel qRT-PCR protocol for Candida quantification in the tissues. RHVE damage was evaluated by measuring lactate dehydrogenase (LDH) activity. Candida virulence gene expression (HWP1, ALS, EPA, PLB, PLD and SAP) was evaluated by quantitative RT-PCR. RESULTS: The results showed that whilst both species induced damage to the RHVE, this was notably less with C. glabrata. Interestingly, there was a significant increase in C. glabrata RHVE colonisation and invasiveness when it was added to the tissue with C. albicans. The extent of RHVE damage caused by the two species appeared to be primarily dependent on the process of invasion. Of the virulence genes assayed, HWP1, PLD1 and ALS3 were deemed to be most associated with pathogenicity in the model. CONCLUSIONS: For the first time, we have demonstrated that the RHVE model coupled with specific tools of analysis, allows assessment of Candida colonisation and invasion in single and co-infection. Using this model we have demonstrated that C. albicans enhanced C. glabrata colonisation, invasion and tissue damage, which was also evidenced by the expression of virulence genes.
OBJECTIVE: The principal aim of this study was to investigate the in vitro co-infection of a reconstituted human vaginal epithelium (RHVE) by Candida albicans and Candida glabrata. METHODS: The ability of both species to invade and colonise the RHVE was examined using species-specific peptide nucleic acid (PNA) probe hybridisation, confocal laser scanning microscopy (CLSM) and a novel qRT-PCR protocol for Candida quantification in the tissues. RHVE damage was evaluated by measuring lactate dehydrogenase (LDH) activity. Candida virulence gene expression (HWP1, ALS, EPA, PLB, PLD and SAP) was evaluated by quantitative RT-PCR. RESULTS: The results showed that whilst both species induced damage to the RHVE, this was notably less with C. glabrata. Interestingly, there was a significant increase in C. glabrata RHVE colonisation and invasiveness when it was added to the tissue with C. albicans. The extent of RHVE damage caused by the two species appeared to be primarily dependent on the process of invasion. Of the virulence genes assayed, HWP1, PLD1 and ALS3 were deemed to be most associated with pathogenicity in the model. CONCLUSIONS: For the first time, we have demonstrated that the RHVE model coupled with specific tools of analysis, allows assessment of Candida colonisation and invasion in single and co-infection. Using this model we have demonstrated that C. albicans enhanced C. glabrata colonisation, invasion and tissue damage, which was also evidenced by the expression of virulence genes.
Authors: Megan L Falsetta; David C Foster; Collynn F Woeller; Stephen J Pollock; Adrienne D Bonham; Constantine G Haidaris; Christopher J Stodgell; Richard P Phipps Journal: Am J Obstet Gynecol Date: 2015-02-12 Impact factor: 8.661
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