Influence of the deletion of 𝘍𝘛𝘏2 on lipids and fluconazole susceptibility of 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴

Abstract

The opportunistic yeast 𝘊𝘢𝘯𝘥𝘪𝘥𝘢 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 resides harmlessly in the mucosal surfaces throughout the body but can become pathogenic under certain environmental conditions. Some of these mucosal surfaces that the yeast inhabits include the gastrointestinal tract, respiratory tract and even the oral cavity. With fungal infections becoming increasingly resistant to routinely used antifungals such as fluconazole, recent research has made efforts in exploring the role of iron availability on drug resistance. This arises from the fact that, the uptake of iron during infections is considered a virulence factor, and that colonisation and proliferation within the host are only possible when the pathogen has access to sufficient iron concentrations. As a result, the pathogen’s heightened demand for iron necessitates the host to restrict its availability. Moreover, in the host’s infected environment 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 can form biofilms fostering complex interactions that may also compete for micronutrients such as iron. This interspecies interaction is clearly demonstrated by the coexistence of 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 with the bacterium Pseudomonas aeruginosa which can persistently colonise airways or lungs of individuals with cystic fibrosis. In the realm of iron limitations, 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 resorts to its vacuolar stores where iron mobilisation into the cytoplasm is facilitated by vacuolar iron permeases such as fth2p. Thus, this study sought to explore the effect of 𝘍𝘛𝘏2 deletion on fluconazole susceptibility and lipids of 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴. The initial focus was to determine the effect of 𝘍𝘛𝘏2 deletion on 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 planktonic cells under varying fluconazole concentrations. Results indicated that both mutants (Δ/Δ𝘧𝘵𝘩2_II and Δ/Δ𝘧𝘵𝘩2) exhibited less susceptibility towards all fluconazole concentrations when compared to their C. albicans parental strains (SC5314 and BWP17) respectively. This was followed by evaluating the effect of 𝘍𝘛𝘏2 deletion on fluconazole susceptibility, in both mono and polymicrobial biofilms. Similar results were observed in monomicrobial biofilms. However, Δ/Δ𝘧𝘵𝘩2_II showed less metabolic activity when compared to its parental strain, 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 SC5314. As a result, these findings require further investigation. When evaluating the effect of fluconazole on polymicrobial biofilms, it became apparent that the combination of fluconazole and P. aeruginosa has a synergistic effect against Δ/Δ𝘧𝘵𝘩2. In contrast, the parental strain (𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 BWP17) remained less susceptible to fluconazole. We also evaluated the effect of FTH2 on iron localisation in both mono- and polymicrobial biofilms. Although we were able to visualise differences in iron localisation within the cells, it was rather difficult to pinpoint exactly, which structures of the cells contained more iron. Considerably, more work still needs to be done, to determine the exact localisation of both forms of iron (Fe²⁺ and Fe³⁺).

Moving forward, we evaluated the influence of 𝘍𝘛𝘏2 mediated iron transport on the lipidome of 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴. This was done both in the presence and absence of P. aeruginosa. The lipid content was compared between the 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 arental strain (BWP17) and its mutant (Δ/Δ𝘧𝘵𝘩2). It was found that lipid droplet size and lipid content differed between 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 BWP17 and the mutant strain Δ/Δ𝘧𝘵𝘩2. Competition for iron in polymicrobial biofilms also negatively influenced LD formation as well as the lipid content. However, the role and influence of 𝘗. 𝘢𝘦𝘳𝘶𝘨𝘪𝘯𝘰𝘴𝘢 n lipid droplet and lipid content production of 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 remains unclear and needs to be further explored. We have also demonstrated that there is a significant difference in the distribution of lipid species between 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 BWP17 and Δ/Δ𝘧𝘵𝘩2, and that these results may further point to the role of iron availability in lipid homeostasis and fluconazole susceptibility. Additionally, the results also showed that the presence of pyoverdine leads to a further decrease in lipid species in both 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 BWP17 and Δ/Δ𝘧𝘵𝘩2.

This study demonstrated the effect of 𝘍𝘛𝘏2 deletion on fluconazole activity against 𝘊. 𝘢𝘭𝘣𝘪𝘤𝘢𝘯𝘴 both in planktonic cells as well as mono-and polymicrobial biofilms. Furthermore, these findings also suggest that changes in lipid metabolism may influence membrane composition as well as fluconazole susceptibility. Although these initial findings provide insights on the influence of iron availability on fluconazole susceptibility and lipid homeostasis, more work is needed to establish a greater degree of accuracy in this matter.