Isogenic strain construction and gene mapping in Candida albicans.

Virulence factors of Candida albicans

The incidence of fungal infections has increased significantly, so contributing to morbidity and mortality. This is caused by an increase in antimicrobial resistance and the restricted number of antifungal drugs, which retain many side effects. Candida species are major human fungal pathogens that cause both mucosal and deep tissue infections. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drug-resistant biofilms is an important factor in their contribution to human disease. The study of plants as an alternative to other forms of drug discovery has attracted great attention because, according to the World Health Organization, these would be the best sources for obtaining a wide variety of drugs and could benefit a large population. Furthermore, silver nanoparticles, antibodies and photodynamic inactivation have also been used with good results. This article presents a brief review of the literature regarding the epidemiology of Candida species, as well as their pathogenicity and ability to form biofilms, the antifungal activity of natural products and other therapeutic options.

Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options

The fungus Candida albicans is often a benign member of the mucosal flora; however, it commonly causes mucosal disease with substantial morbidity and in vulnerable patients it causes life-threatening bloodstream infections. A striking feature of its biology is its ability to grow in yeast, pseudohyphal and hyphal forms. The hyphal form has an important role in causing disease by invading epithelial cells and causing tissue damage. This Review describes our current understanding of the network of signal transduction pathways that monitors environmental cues to activate a programme of hypha-specific gene transcription, and the molecular processes that drive the highly polarized growth of hyphae.

Growth of Candida albicans hyphae

Farnesol is a quorum-sensing molecule that inhibits filamentation in Candida albicans. Both filamentation and quorum sensing are deemed to be important factors in C. albicans biofilm development. Here we examined the effect of farnesol on C. albicans biofilm formation. C. albicans adherent cell populations (after 0, 1, 2, and 4 h of adherence) and preformed biofilms (24 h) were treated with various concentrations of farnesol (0, 3, 30, and 300 μM) and incubated at 37°C for 24 h. The extent and characteristics of biofilm formation were then assessed microscopically and with a semiquantitative colorimetric technique based on the use of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The results indicated that the effect of farnesol was dependent on the concentration of this compound and the initial adherence time, and preincubation with 300 μM farnesol completely inhibited biofilm formation. Supernatant media recovered from mature biofilms inhibited the ability of planktonic C. albicans to form filaments, indicating that a morphogenetic autoregulatory compound is produced in situ in biofilms. Northern blot analysis of RNA extracted from cells in biofilms indicated that the levels of expression of HWP1, encoding a hypha-specific wall protein, were decreased in farnesol-treated biofilms compared to the levels in controls. Our results indicate that farnesol acts as a naturally occurring quorum-sensing molecule which inhibits biofilm formation, and we discuss its potential for further development and use as a novel therapeutic agent.

Inhibition of Candida albicans Biofilm Formation by Farnesol, a Quorum-Sensing Molecule

Hyphal growth in the opportunistic fungal pathogen Candida albicans is believed to contribute to the virulence of the organism by promoting penetration of fungal cells into host tissue. In this study, stimulation of hyphal growth by a feature of the physical environment was demonstrated. Specifically, growth of cells embedded within a matrix promoted the formation of hyphae. The CZF1 gene, encoding a putative transcription factor, was shown to be involved in the regulation of hyphal growth under certain conditions, including embedded conditions. Ectopic expression of CZF1 in embedded cells promoted the rapid formation of hyphae. Elimination of CZF1 and CPH1, encoding a homologue of the Saccharomyces cerevisiae Ste12p transcription factor, led to a pronounced defect in filamentous growth of embedded cells. Elimination of CZF1 alone led to a moderate defect in hyphal growth under some conditions, including embedded conditions. Hyphal morphogenesis in response to matrix embedding may occur in the opportunistic pathogen, C. albicans, to promote invasion of fungal cells into host tissue.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.1999.01619.x

Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene.

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 Trp→Leu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3′ end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance.

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC albicans MAL2 gene To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC albicans URA3 gene This plasmid was introduced into a Ura− strain ofC albicans using the process of restriction enzyme-mediated integration (REMI) This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity The Ura+ phenotype of the transformants was stable during growth under nonselective conditions This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC albicans

Douglas Brown

Papers

Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene.

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance.

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

Stable transformation and regulated expression of an inducible reporter construct in