Lecture 8: Microbial Genetics & Antifungal Resistance PDF

MICROBIAL GENETICS Lecture 8 Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Prepared by: Dr. Abeer Aloufi Assistant professor in Microbiology MDR More than 4 Clasis b Kiten and Glucan in the cell wall offing Orgesterol cell membrane ...

MICROBIAL GENETICS Lecture 8 Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Prepared by: Dr. Abeer Aloufi Assistant professor in Microbiology MDR More than 4 Clasis b Kiten and Glucan in the cell wall offing Orgesterol cell membrane Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi The adaptive potential of fungi ▪ 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 ▪ These processes greatly influence the ability to develop and pass on resistance mechanisms, allowing resistance to spread through the population, regionally and globally Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Antifungal compounds ▪ The antifungal compounds that are currently used in clinical therapy inhibit cell wall biosynthesis, interfere with sterol metabolism, or interfere with pyrimidine metabolism (Perfect, 2018) ▪ They inhibit mechanisms uniquely present in fungi or target proteins that share little homology Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Antifungal compounds membran wall (Review) Facilitators of adaptation and antifungal resistance mechanisms in clinically relevant fungi | Volume 132 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Antifungal compounds Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Epidemiology of antifungal resistance ▪ Fungal pathogens cause life-threatening invasive diseases (e.g., fungaemia, meningitis, pneumonia), severe chronic conditions (e.g., chronic pulmonary aspergillosis, allergic bronchopulmonary aspergillosis), and complex chronic respiratory conditions (e.g., asthma, chronic obstructive pulmonary disease) ▪ These pathogens also cause recurrent infections, such as oral and vaginal candidiasis (mouth and vagina thrush) ▪ Many invasive fungal infections (IFIs) are a consequence of underlying health conditions associated with immunosuppression (opportunistic pathogens) (Brown GD, et al., 2012) Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Epidemiology of antifungal resistance ▪ 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 (Odds FC, Brown AJ, et al., 2003) ▪ 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 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance 1. Intrinsic resistance ▪ When a microbial species is naturally resistant to certain antimicrobials, without the need for mutation or gain of further genes 2. Acquired resistance mutation ▪ It is the result of an evolutionary process by which microorganisms adapt to Antimicrobialantibiotics through several mechanisms including alteration of drug target by mutations and horizontal transfer of ▪ This means that these antimicrobials can never be used to treat infections caused by that species of microbes novel/foreign genes, resistance genes referred to as Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance natural Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi membrane cell Assessment of antifungal resistance - Azole resistance gf ▪ 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 jfrgulatechroma and Cyp51 in molds sa ▪ Inhibition of 14α-demethylase is fungistatic in yeasts and fungicidal in molds ▪ Triazoles are recommended for the treatment of aspergillosis and are widely used for the treatment of candidiasis (Pappas PG, et al., 2016) ▪ Epidemiological studies report substantial azole resistance among Candida and Aspergillus species (Pfaller MA, 2012; Howard SJ, Arendrup MC, 2011), remains low whereas azole resistance among Cryptococcus species Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Azole resistance ▪ 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 ▪ These mechanisms can occur either alone or concurrently in a single isolate and can produce additive effects or lead to cross-resistance among azole drugs Up regulation Down regulation ex 2 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Azole resistance ▪ 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) why point mutation e inced b delect ▪ 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 transport mechanism of drug resistance ATP 9 ▪ Drug pumps are encoded by genes of the ATP-binding cassette (ABC) superfamily or the major facilitator superfamily (MFS) IN A Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Azole resistance tolorent BE ▪ The transcription factors regulating the expression of these pumps, Tac1 and Mrr1 in Candida TT albicans and CgPdr1 in C. glabrata are well characterized x yeast Jex Candida X ▪ Numerous point mutations in ERG11 have been reported in response to fluconazole MÉnrom y acid substitutions in Saccharomyces cerevisiae cause reduced target affinity and, thus, azole resistance ▪ Some amino acid substitutions cause structural changes in the active site of the demethylase, amino point mutation Casset protein in ergo gene ifflux pump led to regulation of over expression of pump Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Azole resistance targetcell membra e my ne y Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal 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, including invasive aspergillosis, cryptococcosis, blastomycosis, candidaemia, coccidioidomycosis, histoplasmosis, and mucormycosis ▪ 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 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Polyenes resistance gregulate membuan Permepility Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Polyenes resistance ▪ Polyenes alter cell membrane permeability by forming a complex with ergosterol, and resistance is caused by loss-of-function mutations in ergosterol biosynthesis genes (particularly in Aspergillus and Candida spp.) yeast ▪ In Candida albicans in particular, double loss of ERG3 confers resistance ▪ However, drug tolerance is common, via up-regulation of ERG5, ERG6, and ERG25 in C. albicans ▪ Treatment with an azole antifungal that lowers cellular sterol concentrations can confer polyene resistance Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Polyenes resistance Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Echinocandins resistance if resestanc to azal will it be also resest echinocandian No Diffrent target location ▪ 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 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance - Echinocandins resistance ▪ 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 Fks1I for all Candida species and Fks2 in C. glabrata just fks O Azo and Eceno ▪ This substitution is the only mechanism to produce clinical breakthrough infection during therapy while polyn ▪ The echinocandins are not substrates for multidrug transporters, and other mechanisms causing azole resistance are not cross-resistant with echinocandins Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Assessment of antifungal resistance The global problem of antifungal resistance: prevalence, mechanisms, and management| Volume 17, Issue 12 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - 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 ▪ Biofilms formed by C. albicans exhibit very high MICs against multiple antifungals after only 72 h of growth ▪ Biofilms also conferred high levels of resistance against antifungals of various classes in Trichosporon asahii, A. fumigatus and C. neoformans Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - Structural target site alterations ▪ The acquisition of mutations in genes encoding antifungal targets has resulted in resistance in many pathogenic fungi new AA ▪ 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 ▪ This mechanism has evolved in isolates that were exposed to echinocandins, allylamines, and is the resistance mechanism found in pan-azole resistant A. fumigatus isolates that have spread across all continents Polyn of Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - 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 cell membrane wall ▪ In C. albicans, this mechanism has proven to confer resistance against azole compounds ▪ Furthermore, this inactivation does not always lead to a decrease in virulence b accumulation Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - 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 (Multi-Drug Resistant), CDR1, and CDR2 (Candida-Drug Resistant) genes in many Candida spp. Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - Mitochondrial alterations possibly facilitate resistance not mode of action ▪ 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 Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - 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 protein Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance - Adjustment of membrane homeostasis ▪ Eukaryotic membranes are dynamic structures that contain many lipid species that contribute to membrane integrity and maintenance ▪ Azoles and polynx 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 Y 4000 active lipid Lecture 8 - Epidemiology and molecular mechanisms of antifungal resistance in Candida and other infectious fungi Mechanisms of antifungal resistance