Investigating the therapeutic potential of signalling molecules to control the expression of Candida albicans virulence factors

Candida albicans infections are common and can be severe in the immunocompromised. However, there are a limited number of chemotherapeutic agents that can be used to treat these infections, resistance to these agents is rising and these agents may have side effects. Phytotherapy with garlic extracts has been reported to be a possible alternative form of treatment, however the reasons why some women do not respond to this therapy is unknown. It is possible that efficacy of garlic treatment is strain dependent. In vitro results of susceptibility to garlic may also be dependent on the testing milieu as it is known that serum influences whether C. albicans transitions to the more invasive hyphal form.

C. albicans has several virulence genes that play an essential role in its pathogenicity. It has previously been reported that fresh garlic extract (FGE) down-regulates the SIR2 gene in C. albicans ATCC 14053. Our study aimed to see if this observation was reproducible in the ATCC strain and to pilot investigations in two clinical strains from two patients with reported variable efficacy of using garlic for treatment of VVC.

Since the report of garlic down-regulation in SIR2, an additional virulence gene ECE1 (extent of cell elongation) has been shown to be important in one of the crucial steps to the commencement of infection (attachment) and results in the production of a cytotoxin called Candidalysin. Candidalysin has been demonstrated to be responsible for the immunopathogenesis of C. albicans vaginitis. These results suggest that ECE1 is an essential virulence gene of C. albicans expressed during the invasion of host cells.

Garlic extract was used to test its effect on two genes (SIR2 and ECE1) using Real-time PCR. in the absence and presence of foetal bovine serum (FBS), which is known to enhance the switching of blastospores to the more invasive hyphal form. In addition, the isolates were investigated to see if differences in response to garlic therapy could be attributed to biofilm formation or fluconazole susceptibility. The isolates were not resistant to fluconazole and biofilm measurements indicated that the clinical isolates were similar to ATCC strain 14053 regarding biofilm formation (P < 0.05). Treatment of cells with FGE and pure allicin (the active ingredient of garlic) resulted in a decrease in SIR2 expression in all strains. In contrast, ECE1 expression was up-regulated in the ATCC strain 14053 and clinical isolate 1358 (isolate from patient unresponsive to garlic therapy) in the presence of FGE or pure allicin, while in clinical isolate 0861 (isolate from the patient responsive to garlic therapy) there was down-regulation of ECE1. This variation in ECE1 expression among these isolates may indicate that treatment with garlic is strain dependent and may explain differing response of patients to garlic therapy. Further examination of the genetic background of these two isolates as well as examination of additional clinical isolates should be performed in further studies to support this association.

To further investigate the pathogenic potential of ECE1, the gene was cloned, expressed and purified. For ECE1 cloning, RNA of C. albicans ATCC 14053 was extracted, cDNA synthesised and ECE1 amplified. ECE1 was transformed and expressed in an E. coli expression system to produce a recombinant ECE1 (rECE1) protein. Also, native proteins from C. albicans ATCC 14053 were extracted and analysed using SDS-PAGE and also confirmed by immunoblotting assay using rabbit anti-ECE1 polyclonal antibodies. An immunoblotting assay of rECE1 indicated the presence of a 32.51 kDa molecular weight protein that was in agreement with the size of native ECE1 protein that had been extracted from C. albicans ATCC strain 14053. Sequencing of ECE1 from C. albicans ATCC strain 14053 revealed high sequence homology with published ECE1 of other C. albicans isolates.  This study is the first study to show in full, a methodology for cloning of ECE1 protein with expression at high levels in an E. coli expression system.

Inflammation is the primary symptom of local mucosal infection caused by C. albicans. Candidiasis also includes invasive candidiasis. As inflammation is the primary symptom suffered by patients with candidiasis, the inflammatory effects of rECE1 were investigated in cell culture assay by measuring interleukin production.  This work was to support the hypothesis that complementary medicines such as garlic have a biological effect in reducing the pro-inflammatory effects of C. albicans infections. The pro-inflammatory effect of rECE1 was investigated and demonstrated in a tissue culture assay using the macrophage cell line J774 and by ELISA measurement of three cytokines (TNF-alpha, IL-6 and IL-1 beta). Next, the role of ECE1 protein in participation in the morphological switch of yeast to hyphal form was investigated parallel with the investigation of the effect of FGE on the expression of ECE1 protein during the transformation process, in a cell culture system. A polyclonal ECE1-antibody was developed through predictive sequence analysis and used to investigate in situ production of native ECE1 by confocal microscopy.

Interestingly, we were unable to see any ECE1 associated fluorescence with 20 µg/ml of FGE, yet we were able to demonstrate a small amount of ECE1 production, even in the presence of 20 µg/ml of FGE, indicating that fluorescence was not as sensitive as immunoblotting for detection of proteins. The overall findings of the last part of this work support recent publications that ECE1 protein induces a pro-inflammatory response and that treatment with FGE inhibited the expression of the ECE1 protein.

In summary, FGE showed a significant role in the inhibition of expression of one of the virulence genes (ECE1) that is responsible for the pathogenicity of C. albicans. The response to the FGE treatment was strain dependent however this work should be confirmed in further clinical trials of the efficacy of garlic.  Secondly, this thesis presents the full methodology for the cloning, expression and purification of the ECE1 protein from C. albicans ATCC 14053 in an E. coli expression system. Thirdly, this study reported for the first time the ability of the rECE1 as a whole protein to produce pro-inflammatory effects in cell culture assay when tested in vitro. This thesis provides a model of prokaryotic expression of eukaryotic proteins that may help other researchers investigate essential virulence genes. The high level of protein expression was able to demonstrate subtle antimicrobial effects by immunoblotting that could not be seen through immunofluorescence. Finally, the investigation into published ECE1 shows that there is a significant variation in the sequence of this gene in isolates examined. This finding supports the hypothesis that the efficacy of complementary medicine may be strain dependent and thus a personalised medicine approach may be needed to determine when garlic can be effectively used in the management of VVC. This finding should be investigated further in larger clinical trials. This finding also has broader implications as antibodies to ECE1 have previously been demonstrated in the serum of patients with invasive candidiasis, and thus complementary medicine such as garlic may have usefulness beyond the treatment of mucosal candidiasis.