Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.     

Resistance to antifungals that target CYP51

Fungal diseases are an increasing global burden. Fungi are now recognised to kill more people annually than malaria, whilst in agriculture, fungi threaten crop yields and food security. Azole resistance, mediated by several mechanisms including point mutations in the target enzyme (CYP51), is increasing through selection pressure as a result of widespread use of triazole fungicides in agriculture and triazole antifungal drugs in the clinic. Mutations similar to those seen in clinical isolates as long ago as the 1990s in Candida albicans and later in Aspergillus fumigatus have been identified in agriculturally important fungal species and also wider combinations of point mutations. Recently, evidence that mutations originate in the field and now appear in clinical infections has been suggested. This situation is likely to increase in prevalence as triazole fungicide use continues to rise. Here, we review the progress made in understanding azole resistance found amongst clinically and agriculturally important fungal species focussing on resistance mechanisms associated with CYP51. Biochemical characterisation of wild-type and mutant CYP51 enzymes through ligand binding studies and azole IC₅₀ determinations is an important tool for understanding azole susceptibility and can be used in conjunction with microbiological methods (MIC₅₀ values), molecular biological studies (site-directed mutagenesis) and protein modelling studies to inform future antifungal development with increased specificity for the target enzyme over the host homologue.     

Antifungal Pharmacokinetics and Dosing Considerations in Burn Patients

Patients with burn injuries are at high risk of developing invasive fungal infections leading to increased morbidity and mortality. Burn patients undergo major physiologic changes, which produce significant alterations in the pharmacokinetics and pharmacodynamics of antimicrobial agents. These changes result from the breakdown of the body’s natural barriers to infection and the systemic responses that subsequently ensue after burn injury, including systemic inflammatory responses, third spacing, and development of a hypermetabolic state. Severe burn injuries often lead to larger volumes of distribution and increased drug clearance. Limited data are available to guide the clinician in optimizing the dosing regimen of antifungals in patients with burn injuries. We present a review of antifungal pharmacokinetics and describe how these properties can be used to design rational therapeutic regimens tailored to the pharmacodynamic alterations characteristic of burn patients.     

Case-control study to evaluate the link between immunosuppression and Candida spp. infection

Candidiasis is one of the fungal infections with the highest incidence in the immunosuppressed host. The evolution of infection and the increase of antifungal medical drugs resistance could both contribute to the mortality attributable to Candida infection in the immunosuppressed host. Even though the data from international studies are well known, few studies have been published in Romania on this subject. In the case-control study we demonstrated the link between the immunosuppression and the presence of Candida infection. Further studies are to be carried out in order to identify more accurately this link and to extend the study to other fungi. There is a need to increase the microbiological diagnosis use at least at the hospital laboratory level in order to better identify the real situation of fungal infections and the link between them and the concrete status of different hosts. Continued surveillance for infections caused by C. albicans and other species of Candida among hospitalized patients is recommended. Control of antimicrobial resistance among nosocomial infections caused by C. albicans and other species of Candida requires rational policies for use of both antifungal and antibacterial agents and appropriate surveillance for the emergence of resistant strains and species.     

Heterologous expression and biochemical characterisation of the recombinant β-carbonic anhydrase (MpaCA) from the warm-blooded vertebrate pathogen malassezia pachydermatis

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO₂/HCO₃^-/pH-sensing in fungi is triggered by HCO₃^- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the β-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.     

Sphingolipids: Regulators of azole drug resistance and fungal pathogenicity

In recent years, the role of sphingolipids in pathogenic fungi, in terms of pathogenicity and resistance to azole drugs, has been a rapidly growing field. This review describes evidence about the roles of sphingolipids in azole resistance and fungal virulence. Sphingolipids can serve as signaling molecules that contribute to azole resistance through modulation of the expression of drug efflux pumps. They also contribute to azole resistance by participating in various microbial pathways such as the unfolded protein response (UPR), pH-responsive Rim pathway, and pleiotropic drug resistance (PDR) pathway. In addition, sphingolipid signaling and eisosomes also coordinately regulate sphingolipid biosynthesis in response to azole-induced membrane stress. Sphingolipids are important for fungal virulence, playing roles during growth in hosts under stressful conditions, maintenance of cell wall integrity, biofilm formation, and production of various virulence factors. Finally, we discuss the possibility of exploiting fungal sphingolipids for the development of new therapeutic strategies to treat infections caused by pathogenic fungi.     

Filamentous Basidiomycetes in the Clinical Laboratory

Filamentous basidiomycetes are difficult to identify in the clinical laboratory, mostly due to lack of sporulation, and their role as agents of fungal infection can be difficult to assess. More cases of infection with these agents are being reported as more laboratories gain proficiency with the recognition of their subtle morphologic features and the use of DNA-based methods for identification. Most infections occur in the respiratory tract and sinuses, although brain infection has been reported. Susceptibility testing suggests that these agents will respond well to azole drugs other than fluconazole.     

Fungal Lanosterol 14α-demethylase: A target for next-generation antifungal design

The cytochrome P450 enzyme lanosterol 14α-demethylase (LDM) is the target of the azole antifungals used widely in medicine and agriculture as prophylaxis or treatments of infections or diseases caused by fungal pathogens. These drugs and agrochemicals contain an imidazole, triazole or tetrazole substituent, with one of the nitrogens in the azole ring coordinating as the sixth axial ligand to the LDM heme iron. Structural studies show that this membrane bound enzyme contains a relatively rigid ligand binding pocket comprised of a deeply buried heme-containing active site together with a substrate entry channel and putative product exit channel that reach to the membrane. Within the ligand binding pocket the azole antifungals have additional affinity determining interactions with hydrophobic side-chains, the polypeptide backbone and via water-mediated hydrogen bond networks. This review will describe the tools that can be used to identify and characterise the next generation of antifungals targeting LDM, with the goal of obtaining highly potent broad-spectrum fungicides that will be able to avoid target and drug efflux mediated antifungal resistance.     

Evaluation of antifungal pharmacodynamic characteristics of AmBisome against Candida albicans

A liposomal formulation of Amphotericin B (AmBisome), with small unilamellar vesicles containing amphotericin B, shows characteristic pharmacokinetics as liposomes, and in consequence, has different pharmacological activity and toxicity from amphotericin B deoxycholate (Fungizone). In this study, we evaluated the antifungal pharmacodynamic characteristics of AmBisome against Candida albicans using the in vitro time-kill method and murine systemic infection model. A time-kill study indicated that the in vitro fungicidal activities of AmBisome and Fungizone against C. albicans ATCC 90029 increased with increasing drug concentration. For in vivo experiments, leucopenic mice were infected intravenously with the isolate 4 hr prior to the start of therapy. The infected mice were treated for 24 hr with twelve dosing regimens of AmBisome administered at 8-, 12-, 24-hr dosing intervals. Correlation analysis between the fungal burden in the kidney after 24 hr of therapy and each pharmacokinetic/pharmacodynamic parameter showed that the peak level/MIC ratio was the best predictive parameter of the in vivo outcome of AmBisome. These results suggest that AmBisome, as well as Fungizone, has concentration-dependent antifungal activity. Furthermore, since AmBisome can safely achieve higher concentrations in serum than Fungizone, AmBisome is thought to have superior potency to Fungizone against fungal infections.     

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H⁺-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma ⁻ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca²⁺ and H⁺ surges triggered by the antimicrobial agent amiodarone, and impaired Ca²⁺ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.     

Antifungal Therapy in Pediatric Patients

Invasive fungal infections still play a major role in morbidity and mortality in pediatric patients, especially in children undergoing therapy for an underlying malignancy and in preterm infants. Relative to the adult population, pediatric age groups display important differences not only in host biology, predisposing conditions, epidemiology, and presentation of fungal infections, but also in the disposition and clearance of antifungal compounds. During the past decade, several new antifungal agents have been developed. Although not all of these agents are yet approved for children, the pediatric development of antifungal agents has moved forward in an exemplary manner, which is essential for the successful management of the individual patient. This article reviews the current data on pharmacokinetics, safety, and dosing of antifungal agents in pediatric patients.     

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90‐dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.     

Protease expression by microorganisms and its relevance to crucial physiological/pathological events

The treatment of infections caused by fungi and trypanosomatids is difficult due to the eukaryotic nature of these microbial cells,which are similar in several biochemical and genetic aspects to host cells.Aggravating this scenario,very few antifungal and anti-trypanosomatidal agents are in clinical use and,therefore,therapy is limited by drug safety considerations and their narrow spectrum of activity,efficacy and resistance. The search for new bioactive agents against fungi and trypanosomatids has been expanded because progress in biochemistry and molecular biology has led to a better understanding of important and essential pathways in these microorganisms including nutrition,growth, proliferation,signaling,differentiation and death.In this context,proteolytic enzymes produced by these eukaryotic microorganisms are appointed and,in some cases,proven to be excellent targets for searching novel natural and/or synthetic pharmacological compounds,in order to cure or prevent invasive fungal/trypanosomatid diseases.With this task in mind,our research group and others have focused on aspartic-type proteases,since the activity of this class of hydrolytic enzymes is directly implicated in several facets of basic biological processes of both fungal and trypanosomatid cells as well as due to the participation in numerous events of interaction between these microorganisms and host structures.In the present paper,a concise revision of the beneficial effects of aspartic protease inhibitors,with emphasis on the aspartic protease inhibitors used in the anti-human immunodeficiency virus therapy,will be presented and discussed using our experience with the following microbial models:the yeast Candida albicans,the filamentous fungus Fonsecaea pedrosoi and the protozoan trypanosomatid Leishmania amazonensis.     

Design stars: how small DNA viruses remodel the host nucleus

Numerous host components are encountered by viruses during the infection process. While some of these host structures are left unchanged, others may go through dramatic remodeling processes. In this review, we summarize these host changes that occur during small DNA virus infections, with a focus on host nuclear components and pathways. Although these viruses differ significantly in their genome structures and infectious pathways, there are common nuclear targets that are altered by various viral factors. Accumulating evidence suggests that these nuclear remodeling processes are often essential for productive viral infections and/or viral-induced transformation. Understanding the complex interactions between viruses and these host structures and pathways will help to build a more integrated network of how the virus completes its life cycle and point toward the design of novel therapeutic regimens that either prevent harmful viral infections or employ viruses as nontraditional treatment options or molecular tools.     

Multiple infections by the anther smut pathogen are frequent and involve related strains

Population models of host-parasite interactions predict that when different parasite genotypes compete within a host for limited resources, those that exploit the host faster will be selected, leading to an increase in parasite virulence. When parasites sharing a host are related, however, kin selection should lead to more cooperative host exploitation that may involve slower rates of parasite reproduction. Despite their potential importance, studies that assess the prevalence of multiple genotype infections in natural populations remain rare, and studies quantifying the relatedness of parasites occurring together as natural multiple infections are particularly scarce. We investigated multiple infections in natural populations of the systemic fungal plant parasite Microbotryum violaceum, the anther smut of Caryophyllaceae, on its host, Silene latifolia. We found that multiple infections can be extremely frequent, with different fungal genotypes found in different stems of single plants. Multiple infections involved parasite genotypes more closely related than would be expected based upon their genetic diversity or due to spatial substructuring within the parasite populations. Together with previous sequential inoculation experiments, our results suggest that M. violaceum actively excludes divergent competitors while tolerating closely related genotypes. Such an exclusion mechanism might explain why multiple infections were less frequent in populations with the highest genetic diversity, which is at odds with intuitive expectations. Thus, these results demonstrate that genetic diversity can influence the prevalence of multiple infections in nature, which will have important consequences for their optimal levels of virulence. Measuring the occurrence of multiple infections and the relatedness among parasites within hosts in natural populations may be important for understanding the evolutionary dynamics of disease, the consequences of vaccine use, and forces driving the population genetic structure of parasites.     

Fungal infections in the immunocompromised host

In recent years many remarkable changes occurred in our way of life, producing opportunities for microbes. All these changes are related to the recent emergence of previously unrecognized diseases, or the resurgence of diseases that, at least in developed countries, were thought to be under control. This concept is reviewed regarding fungal infections and their agents in the immunocompromised host. The changing pattern of these infections, the portals of entry of fungi into the human host, fungal pathogenicity and the main predisposing factors are analyzed. Opportunistic fungal infections in cancer, organ transplant and acquired immunodeficiency syndrome patients are reviewed, specially candidiasis and aspergillosis.     

Antifungal Dose Adjustment in Renal and Hepatic Dysfunction: Pharmacokinetic and Pharmacodynamic Considerations

Adjusting the dose of antifungal agents for renal and hepatic impairment can be challenging given that clinicians must rely on limited pharmacokinetic data to derive specific regimens. These pharmacokinetic studies are typically performed in a small number of patients without invasive fungal infection, and results are not often reported in concert with accepted pharmacodynamic indices. This article aims to review pertinent pharmacokinetic studies of antifungal drugs in patients with renal or hepatic dysfunction. The impact of novel continuous renal replacement therapy techniques on the pharmacokinetic disposition of antifungal agents will also be described where data are available. Subsequently, this review provides recommendations for antifungal drug dosing in patients with kidney or liver dysfunction after accounting for established or emerging pharmacokinetic-pharmacodynamic relationships as they relate to antifungal drug efficacy in vivo.     

Clotrimazole as a pharmaceutical: past,present and future.

Clotrimazole is a broad‐spectrum antimycotic drug mainly used for the treatment of Candida albicans and other fungal infections. A synthetic, azole antimycotic, clotrimazole is widely used as a topical treatment for tinea pedis (athlete's foot), as well as vulvovaginal and oropharyngeal candidiasis. It displays fungistatic antimycotic activity by targeting the biosynthesis of ergosterol, thereby inhibiting fungal growth. As well as its antimycotic activity, clotrimazole has become a drug of interest against several other diseases such as sickle cell disease, malaria and some cancers. It has also been combined with other molecules, such as the metals, to produce clotrimazole complexes that show improved pharmacological efficacy. Moreover, several new, modified‐release pharmaceutical formulations are also undergoing development. Clotrimazole is a very well‐tolerated product with few side effects, although there is some drug resistance appearing among immunocompromised patients. Here, we review the pharmaceutical chemistry, application and pharmacology of clotrimazole and discuss future prospects for its further development as a chemotherapeutic agent.     

真菌对唑类抗真菌药物的耐受机制

唑类抗真菌药物广泛用于临床和农业。唑类药物通过与羊毛甾醇14α-去甲基化酶（Erg11p/Cyp51）结合,抑制麦角甾醇合成,同时导致有毒甾醇积累。真菌可快速在转录水平上对唑类药物胁迫作出响应而导致耐药性,尤其是唑类药物外排泵基因和麦角甾醇合成相关基因表达的上调。农业和临床上绝大多数唑类药物耐药菌株的形成都是由麦角甾醇合成基因和唑类药物外排泵表达的变化或是突变所致。一些转录因子（如Pdr1p、Pdr3p、Upc2p、Yap1p、Tac1p、Mrr1p、CCG-8）和信号通路（如cAMP途径、PKC-MAPK途径、HOG MAPK途径、钙调磷酸酶途径）均参与对药物外排泵基因和麦角甾醇合成基因等的调控,影响唑类药物耐药性。针对于这些调控因子设计的抑制剂将有助于提高唑类药物的治疗效果。本文概述了唑类药物的抑菌机制、真菌对唑类药物耐药性形成的原因、真菌对唑类药物适应性响应机理,并对未来此领域的热点和方向进行了展望。