fungi.docx

Fungi can quickly adapt to new and challenging environments in nature, equipping them with intrinsic defenses to overcome cellular stress induced by antifungal compounds ▪ Adaptation in this sense is described as overcoming environmental challenges by gaining beneficial mutations or adjusting cellul...

Fungi can quickly adapt to new and challenging environments in nature, equipping them with intrinsic defenses to overcome cellular stress induced by antifungal compounds ▪ Adaptation in this sense is described as overcoming environmental challenges by gaining beneficial mutations or adjusting cellular physiology depending on forces that drive evolution itself such as sexual reproduction, and genetic stability The antifungal compounds that are currently used in clinical therapy inhibit cell wall biosynthesis, interfere with sterol metabolism, or interfere with pyrimidine metabolism. ▪ They inhibit mechanisms uniquely present in fungi or target proteins that share little Many invasive fungal infections (IFIs) are a consequence of underlying health conditions associated with immunosuppression (opportunistic pathogens) Generally, these infections are associated with high mortality, and successful clinical outcome requires early diagnosis and effective antifungal therapy ▪ Yet, antifungal options are few, with chemical classes for invasive disease treatment limited to azoles, echinocandins, polyenes, and flucytosine. ▪ The emergence of drug resistance to any one drug class severely limits therapy because so few treatment options are available ▪ Multidrug resistance can eliminate treatment options entirely, which has a devastating effect on patient outcomes Intrinsic resistance When a microbial species is naturally resistant to certain antimicrobials, without the need for mutation or gain of further genes Acquired resistance It is the result of an evolutionary process by which microorganisms adapt to antibiotics through several mechanisms including alteration of drug target by mutations and horizontal transfer of novel/foreign genes, referred to as resistance genes Azole resistance ▪ Azole compounds (e.g., fluconazole, voriconazole, posaconazole) target the cytochrome P450 enzyme sterol 14α-demethylase, which converts lanosterol to ergosterol, and is encoded by ERG11 in yeast and Cyp51 in molds ▪ Inhibition of 14α-demethylase is fungistatic in yeasts and fungicidal in molds Epidemiological studies report substantial azole resistance among Candida and Aspergillus species, whereas azole resistance among Cryptococcus species remains low Azole resistance among Candida spp involves several well-defined mechanisms, including upregulation of drug transporters, overexpression or alteration of the drug target, and cellular changes caused, in some cases, by non-target effects induced by stress responses Azole drug resistance is primarily due to increased efflux of the drug from the fungal cell (particularly in Candida spp.) and modifications to the sterol biosynthesis pathway caused by point mutations and promoter insertions in CYP51A (Aspergillus fumigatus) In other fungal species, such as Cryptococcus neoformans, overexpression of the drug target and efflux pumps caused by chromosomal aneuploidy and hypermutation is common ▪ The induction of efflux pumps, which decrease drug concentration inside the cell, is the most common mechanism of drug resistance ▪ Drug pumps are encoded by genes of the ATP-binding cassette (ABC) superfamily or the major facilitator superfamily (MFS) Some amino acid substitutions cause structural changes in the active site of the demethylase, amino acid substitutions in Saccharomyces cerevisiae cause reduced target affinity and, thus, azole resistance Polyenes resistance ▪ The polyenes are the oldest antifungal drug class, and include amphotericin B and nystatin ▪ Amphotericin B was first approved in 1957 for the treatment of life-threatening IFIs ▪ Polyene drugs bind ergosterol, a fungal-specific sterol, in the plasma membrane of fungi, which causes the formation of concentration-dependent channels that kill cells by allowing ions and other cellular components to escape ▪ In extramembranous aggregates, amphotericin B is suggested to kill cells by extracting ergosterol from lipid bilayers Polyenes alter cell membrane permeability by forming a complex with ergosterol, and resistance is caused by loss-of-function mutations in ergosterol biosynthesis gene. Treatment with an azole antifungal that lowers cellular sterol concentrations can confer polyene Resistance. Echinocandins resistance ▪ The echinocandin drugs anidulafungin, caspofungin, and micafungin are lipopeptides that target fungal cell wall synthesis by inhibiting FKS1, a glucan synthase which is essential in the biosynthesis of 1,3 β-glucans in the fungal cell wall and are the recommended therapy for various patients with candidiasis ▪ The echinocandins are highly active against most Candida species, but are less active against Candida parapsilosis, and are inactive against Cryptococcus ▪ The mechanism of echinocandin resistance in Candida species involves the genetic acquisition of mutations in FKS genes, which encode the catalytic subunits of glucan synthase ▪ Echinocandin resistance is associated with amino acid substitutions in two narrow hot spot regions of Fks for all Candida species and Fks in C. glabrata ▪ This substitution is the only mechanism to produce clinical breakthrough infection during therapy ▪ The echinocandins are not substrates for multidrug transporters, and other mechanisms causing azole resistance are not cross-resistant with echinocandins Biofilm formation ▪ Many pathogenic yeasts and molds have the ability to form a biofilm, which works as a shield against host defense and antifungal compounds and increases adherence to the host surface ▪ A biofilm consists of a network of cells that are associated with each other or a surface, which produces an extracellular matrix (ECM) that provides protection against a stressful environment ▪ The ECM formed is thought to prevent the diffusion of antifungals to the cells ▪ Furthermore, a high amount of extracellular DNA was found in the EMC in C. albicans biofilms, which is thought to contribute to the structure and formation of the biofilm Structural target site alterations ▪ The acquisition of mutations in genes encoding antifungal targets has resulted in resistance in many pathogenic fungi ▪ Non-synonymous SNPs lead to amino-acid alterations, an altered structure and reduced binding affinity of the antifungal to the target, thus the enzyme can remain functional Metabolic bypass ▪ A lesser observed mechanism to gain antifungal resistance encompasses the prevention of accumulating toxic metabolites by the inactivation of another enzyme in the pathway ▪ In C. albicans, this mechanism has proven to confer resistance against azole compounds ▪ Furthermore, this inactivation of accumulating toxic does not always lead to a decrease in virulence. Overexpression of efflux-pumps ▪ The ability of fungal pathogens to excrete antifungal compounds works through overexpression of specific efflux pumps and is a resistance mechanism found in many fungal species ▪ Although this mechanism is widespread, it is not found in echinocandin or polyene-resistant fungal isolates, as these are not substrates of efflux pumps and exercise fungicidal activity on the outside of the cell ▪ Increased gene expression feeds the synthesis of more efflux pump protein complexes resulting in increased antifungal transportation outside the cell ▪ These transporters usually belong to the ABC or MFS transporter family and blocking these transporters reduces the MICs significantly Azole resistance through up-regulated efflux pump expression was reported for the MDR1 Mitochondrial alterations possibly facilitate resistance ▪ Various studies have elucidated potential roles for the mitochondria to be involved in antifungal drug resistance ▪ C. glabrata isolate with loss of mitochondrial function showed increased fluconazole resistance up to 50 μg/ml, an effect that was appointed to the differential expression of azole-resistance-related genes due to loss of mitochondrial function ▪ The authors suggest that this comes from a rebalancing of the hypoxic response ▪ Upon addition of azoles, the hypoxic response is ‘unintentionally’ activated by dysfunctional oxygen sensing because of sterol biosynthesis inhibition, although oxygen levels are normal. Activation of stress pathways Cellular stress signaling is essential to survive stressful conditions in the environment, such as high salt concentrations or high temperatures ▪ The mechanisms that help cells cope with stress, can also provide protection against drug-induced stress conditions ▪ One of the conserved key regulators in stress signaling is the Hsp90 chaperone, which is involved in protein folding and stabilizing many proteins involved in signal transduction Adjustment of membrane homeostasis ▪ Eukaryotic membranes are dynamic structures that contain many lipid species that contribute to membrane integrity and maintenance ▪ Azoles and amphotericin B have an impact on membrane homeostasis by disturbing cellular ergosterol content, which leads to membrane destabilization and subsequent lysis of the cell ▪ Therefore, it is suggested that the cell adjust its membrane homeostasis to make it more rigid, or more fluid ▪ This can for instance be achieved by changing the ratio of phospholipid species (PLs), as all these PLs have different chemico-physical properties that contribute to the nature of the membrane ▪ Additionally, sterols and sphingolipids also contribute to membrane integrity and are thought to function together in complexes