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dc.contributor.advisorCannon, Richard
dc.contributor.advisorHolmes, Ann
dc.contributor.advisorSchmidt, J
dc.contributor.authorRodrigues, Ely Montes
dc.date.available2013-09-03T04:04:45Z
dc.date.copyright2013
dc.identifier.citationRodrigues, E. M. (2013). Candida albicans: sex and survival in a rat model of oral colonisation (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/4271en
dc.identifier.urihttp://hdl.handle.net/10523/4271
dc.description.abstractUnlike for most animal species, sex is not an obligatory reproductive mechanism for fungi, and for some fungi the complex diversity of sexual and asexual reproductive mechanisms has yet to be unravelled. A classic example of this diversity is Candida albicans, an opportunistic, commensal yeast found in normal oral and gastrointestinal flora, which is capable of causing infections in immune compromised hosts. It was long believed that C. albicans is asexual existing exclusively as a diploid yeast. However, with the identification of a mating type-like locus homologous to those in sexually reproducing fungi, demonstration of mating in vitro, and the discovery that a morphology switch, from white to opaque, promotes mating, it is now hypothesised that C. albicans may possess a cryptic sexual cycle. No evidence of meiosis has been found, however, and thus any matants are probably produced via a parasexual cycle. The question is: can such a cycle confer advantages, or does it simply represent the "remains" of a sexual mode of reproduction that is no longer effective for diversity generation but may have acquired other functions? In this study I developed a rat model of oral colonisation in order to investigate whether sex confers an advantage to the survival of C. albicans, and how likely sex is to occur, in vivo. Specific pathogen free Sprague Dawley rats were used and treated with immunoesupressant (dexamethasone) and antibiotic (doxycycline) throughout the course of the study. In this model, colonisation of oral surfaces was followed for 4 weeks post inoculation. Initially, colonisation increased until reaching a plateau 7 days post-inoculation which remained constant to the end of the sampling period. The commensal nature of C. albicans in this model was evidenced by: (i) healthy growth of the rats; (ii) lack of visible signs of oral disease; and (iii) no sub-epithelial penetration detected in histology of rat tongues following euthanasia. Seven genetically manipulated C. albicans clinical isolates were used in this study. Strain pairs which had previously been demonstrated to be mating competent in vitro were selected for co-inoculation into the rat model. Strains of α mating type were marked with the NAT (nourseothricin) resistance cassette and strains of the compatible a mating type were marked with the MPA (mycophenolic acid) resistance cassette. Matants were detected in both oral and fecal samples from two in vivo co-inoculation experiments, inoculated with the same numbers of white phase cells. Two combinations of the C. albicans strains, namely C. albicans OD8916 with C. albicans W43 and C. albicans W17 with C. albicans W43, displayed mating in vivo in all experiments conducted. Detection of matants could be enhanced (~10 fold) by changing the inoculum from white to opaque cells. For example, when rats were co inoculated with either white or opaque cells of the strain pair C. albicans W17α and W43a a total of 30 CFU and 520 CFU respectively matants were detected at day 7. However, after an initial increase, the number of matants observed in samples declined over time whereas overall yeast counts remained constant. The decline was probably not because of a lower growth rate of matants as the in vitro growth rates of recombinants recovered from in vivo experiments and their parents did not vary greatly. These growth curves were done two times to measure if growth rates varied considerably between the strains employed, as this will affect the out-competition by the co-inoculant. Neither was the decline in matant recovery because of the genetic modification of the strains, or marker loss, as co-inoculation in the rat model of strains containing one or both genetic markers did not affect the survivability of the strains in vivo and no marker loss in recovered isolates was observed. One explanation for the reduced detection of matants during an in vivo experiment is that the opaque (mating competent) cells switched rapidly to white, as demonstrated by plating of sequentially obtained samples on YPD agar plates containing Phloxine B, a dye that stains opaque cells pink. Another possible contributing factor is that one parent outcompeted the other over time, as indicated by a decrease in the number of the other parent recovered, thus reducing the possibility for mating to occur. To test if matants were less fit than their parents, rats were co-inoculated with a matant (obtained via an in vivo mating event) and each of its respective parents. By day 28 i.e. the last sampling point, very few recombinants were detected. The parent strain contributed as much as 95 % of the total yeast count and had clearly outcompeted the matant strain. Four other strain combinations tested did not give matants. These strains did not switch to the opaque form readily as no or very few opaque cells were observed in samples post inoculum. Furthermore, one parent quickly outcompeted the other but, despite adjusting the numbers of the parents to give the less fit parent a better chance of survival, no mating was detected. In conclusion, this study has demonstrated that mating of C. albicans does occur in the oral cavity in vivo, but that it appears to be a rare event. This possibly reflects both the inherent instability of mating-competent opaque cells and/or dynamic changes in the commensal populations of each parent, due to differences in colonisation abilities, thus reducing the chances of meeting and mating. The results also demonstrated that when mating was detectable in vivo, the resultant matants were not as fit as the parents, as they were quickly outcompeted by parental strains. This provides evidence to confirm the hypothesis that, within the experimental systems used, there appears to be no advantage conferred on C. albicans by sex.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectCandida
dc.subjectalbicans
dc.subjectsex
dc.subjectmating
dc.subjectrat
dc.subjectoral
dc.subjectmodel
dc.titleCandida albicans: sex and survival in a rat model of colonisation
dc.typeThesis
dc.date.updated2013-09-03T02:47:52Z
dc.language.rfc3066en
thesis.degree.disciplineOral Sciences
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.openaccessOpen
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