Emergence Of Antifungal Resistance Research Articles

Candida Non-albicans and Non-auris Causing Invasive Candidiasis in a Fourth-Level Hospital in Colombia: Epidemiology, Antifungal Susceptibility, and Genetic Diversity.

Increasingly common and associated with healthcare settings, Candida infections are very important, since some species of this genus can develop antifungal resistance. We contribute data on the epidemiology, antifungal susceptibility, and genetic diversity of Candida non-albicans and non-auris affecting critically ill patients in a fourth-level hospital in Colombia. Ninety-seven isolates causing invasive infections, identified by conventional methods over 18 months, were studied. Data from patients affected by these yeasts, including sex, age, comorbidities, treatment, and outcome, were analysed. The antifungal susceptibility of the isolates was determined, and the ribosomal DNA was sequenced. Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida dubliniensis, and Candida guilliermondii caused 48.5% of all cases of invasive candidiasis. The species were mainly recovered from blood (50%). Patients were mostly men (53.4%), between 18 days and 93 years old, hospitalized in the ICU (70.7%). Overall mortality was 46.6%, but patients in the ICU, using antibiotics, with diabetes mellitus, or with C. glabrata infections were more likely to die. Resistant isolates were identified in C. parapsilosis, C. tropicalis, and C. glabrata. This study provides epidemiological data for the surveillance of emerging Candida species, highlighting their clinical impact, as well as the emergence of antifungal resistance and clonal dispersal.

New thymol-derived triazole exhibits promising activity against Trichophyton rubrum.

Fungal infections have emerged worldwide, and azole antifungals are widely used to control these infections. However, the emergence of antifungal resistance has been compromising the effectiveness of these drugs. Therefore, the objective of this study was to evaluate the antifungal and cytotoxic activities of the nine new 1,2,3 triazole compounds derived from thymol that were synthesized through Click chemistry. The binding mode prediction was carried out by docking studies using the crystallographic structure of Lanosterol 14α-demethylase G73E mutant from Saccharomyces cerevisiae. The new compounds showed potent antifungal activity against Trichophyton rubrum but did not show relevant action against Aspergillus fumigatus and Candida albicans. For T. rubrum, molecules nº 5 and 8 showed promising results, emphasizing nº 8, whose fungicidal and fungistatic effects were similar to fluconazole. In addition, molecule nº 8 showed low toxicity for keratinocytes and fibroblasts, concluding that this compound demonstrates promising characteristics for developing a new drug for dermatophytosis caused by T. rubrum, or serves as a structural basis for further research.

Time to Think Antifungal Resistance Increased Antifungal Resistance Exacerbates the Burden of Fungal Infections Including Resistant Dermatomycoses.

Increased antifungal resistance is exacerbating the burden of invasive fungal infections, as well as potentially contributing to the increase in resistant dermatomycoses. In this commentary, we focus on antifungal drug resistance, in contrast to antibacterial resistance. We provide a brief historical perspective on the emergence of antifungal resistance and propose measures for combating this growing health concern. The increase in the incidence of invasive and cutaneous fungal infections parallels advancements in medical interventions, such as immunosuppressive drugs, to manage cancer and reduce organ rejection following transplant. A disturbing relatively new trend in antifungal resistance is the observation of several fungal species that now exhibit multidrug resistance (eg, Candida auris, Trichophyton indotineae). Increasing awareness of these multidrug-resistant species is paramount. Therefore, increased education regarding potential fungus-associated infections is needed to address awareness in the general healthcare setting, which may result in a more realistic picture of the prevalence of antifungal-resistant infections. In addition to education, increased use of diagnostic tests (eg, micro and macro conventional assays or molecular testing) should be routine for healthcare providers facing an unknown fungal infection. Two critical barriers that affect the low rates for Antifungal Susceptibility Testing (AST) are low (or a lack of) sufficient insurance reimbursement rates and the low number of qualified laboratories with the capacity to perform AST. The ultimate aim is to improve the quality of patient care through fungal identification, diagnosis, and, where appropriate, susceptibility testing. Here we propose an all-encompassing call to action to address this emerging challenge.

Distribution and antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with HIV in the era of universal test and treat policy in Uganda.

Despite the increased frequency of oropharyngeal candidiasis among people living with human immunodeficiency virus (HIV), its management is no longer effective due to empirical treatment and emergence of antifungal resistance (AFR). This study sought to investigate the prevalence of oropharyngeal candidiasis and assess the antifungal susceptibility profile of oropharyngeal Candida species isolated from people living with human immunodeficiency virus. Additionally, we evaluated the correlation between oropharyngeal candidiasis and CD4 T cell as well as viral load counts. A descriptive cross-sectional study was carried out from April to October 2023 in which 384 people living with HIV underwent clinical examination for oral lesions. Oropharyngeal swabs were collected and cultured on Sabouraud Dextrose agar to isolate Candida species which were identified using the matrix assisted laser desorption ionization time of flight mass spectrometry. Additionally, the antifungal susceptibility profile of Candida isolates to six antifungal drugs was determined using VITEK® (Marcy-l'Étoile, France) compact system. Data on viral load were retrieved from records, and CD4 T cell count test was performed using Becton Dickinson Biosciences fluorescent antibody cell sorter presto. The prevalence of oropharyngeal candidiasis was 7.6%. Oropharyngeal candidiasis was significantly associated with low CD4 T cell count and high viral load. A total of 35 isolates were obtained out of which Candida albicans comprised of 20 (57.1%) while C. tropicalis and C. glabrata comprised 4 (11.4%) each. C. parapsilosis, C. dubliniensis and C. krusei accounted for 2 (5.7%) each. Additionally, 7 (20%) isolates were resistant to fluconazole, 1 (2.9%) to flucytocine and 0.2 (5.7%) isolates were intermediate to caspofungin. However, specific specie isolates like C. albicans showed 20% (4/20), C. glabrata 50% (2/4) and C. krusei 50% (1/2) resistance to fluconazole. Additionally, C. krusei showed 50% resistance to flucytosine. The prevalence of oropharyngeal candidiasis (OPC) among people living with HIV was low, and there was a significant association between OPC and CD4 T cell count as well as viral load. C. albicans was the most frequently isolated oropharyngeal Candida species. C. glabrata and C. krusei exhibited the highest AFR among the non-albicans Candida species. The highest resistance was demonstrated to fluconazole.

Epidemiology of Clinically Significant Aspergillus Species from a Large Tertiary Hospital in Shanghai, China, for the Period of Two Years.

Aspergillus species are becoming a major public health concern worldwide due to the increase in the incidence of aspergillosis and emergence of antifungal resistance. In this study, we surveyed all Aspergillus species isolated from aspergillosis patients in Zhongshan Hospital Fudan University, Shanghai, China, from 2019 to 2021. We characterized the susceptibility profiles of these Aspergillus species to medical azoles (voriconazole, itraconazole and posaconazole) using YeastOneTM broth microdilution system. To determine the underlying antifungal resistance mechanisms in azole-resistant A. fumigatus (ARAf) isolates, we characterized mutations in the cyp51A gene. Genotypic diversity of sampled A. fumigatus was investigated using CSP-typing. A total of 112 Aspergillus isolates (81 A. fumigatus, 17 A. flavus, 5 A. niger, 2 A. terreus, 2 A. lentulus, 2 A. oryzae, 1 A. nidulans, 1 A. versicolor and 1 A. sydowii) from 105 patients diagnosed with aspergillosis (including proven or probable invasive aspergillosis, chronic pulmonary aspergillosis, allergic bronchopulmonary aspergillosis and cutaneous aspergillosis) were obtained. Eight isolates (7 A. fumigatus and 1 A. niger) from seven patients were either azole non-susceptible or non-wild type. Azole non-susceptible or non-wild type rate was 7.1%/isolate and 6.7%/patient analysed. Four ARAf harbored TR34/L98H mutation, whereas one carried TR46/Y121F/T289A allele. The 81 A. fumigatus isolates were spread across 8 CSP types with t01 to be the predominant type (53.1%). ARAf isolates were distributed over CSP types t01, t02, t04A and t11. Results from this study provided us with an understanding of the antifungal resistance and related characteristics of Aspergillus species in Eastern China. Further comparisons of our results with those in other countries reflect potential clonal expansion of A. fumigatus in our region. Further surveillance study is warranted to guide antifungal therapy and for epidemiological purposes.

Evaluation of Systemic Antifungal Use in a Latin American General Care Hospital: A Retrospective Study.

Invasive fungal infections significantly contribute to mortality and morbidity rates. Despite the presence of all four major classes of antifungal medications, it is estimated that these infections result in the death of 1.5 million people each year, and death rates are increasing at an alarming rate. With increasing concerns about the emergence of antifungal resistance, there is a growing consideration in many countries to incorporate antifungal stewardship into existing antimicrobial stewardship programs. This approach aims to address issues hindering the appropriate use of antifungal drugs and to optimize their utilization. An analytical retrospective study of 48 hospitalized patients was conducted to assess factors related to the use of systemic antifungals and develop and implement an internal protocol to improve its use. All patients with severe comorbidity had SOFA scores linked with a mortality risk of more than 10%. Based on 48 evaluations of antifungal orders, 62.5% were considered appropriate, 14.6% were considered debatable, and 22.9% were considered inappropriate. Infectious disease physicians made most of the prescriptions considered appropriate in this study. Comorbidities and risk factors in patients receiving systemic antifungals can be associated with the development of more serious fungal infections; hence, the implementation of antifungal stewardship as a complement to antimicrobial stewardship programs can help facilitate decision-making when dealing with a suspected case of fungal infection.

High prevalence of fluconazole resistant Candida tropicalis among candiduria samples in China: An ignored matter of concern.

The rapid rise of azole resistance in Candida tropicalis causing invasive infections has become a public health concern; however, the prevalence of resistant isolates in urine samples was not well studied, because the clinical significance of candiduria was not unambiguous due to possible host colonization. We performed a 12-year laboratory-based surveillance study of C. tropicalis causing either invasive infection or candiduria and studied their susceptibility profiles to common antifungal drugs. The complete coding domain sequence of the ERG11 gene was amplified in all fluconazole resistant isolates, and aligned with the wild-type sequence to detect nucleotide mutations. A total of 519 unique C. tropicalis strains isolates, 69.9% of which were isolated from urine samples and remaining 30.1% were invasive strains. Overall, 16.5% isolates were confirmed to be resistant to fluconazole, of which 91.9% were cross-resistant voriconazole. Of note, at the beginning of surveillance (2010-2011), the fluconazole resistance rates were low in both candiduria and invasive groups (6.8% and 5.9%, respectively). However, the resistant rate in the candiduria group significantly increased to 29.5% since 2012-2013 (p = 0.001) and stayed high since then, whilst the resistance rate in the invasive group only showed a gradually increasing trends till 2021 (p > 0.05). Sequence analysis of ERG11 from fluconazole-resistant strains revealed the prevalence of A395T/W mutations were relatively low (16.7%) in the beginning but reached 87.5-100% after 2014. Moreover, the A395W heterozygous mutation isolates became predominant (>60% of resistant strains) after 2016, and indeed isolates carrying corresponding amino acid substitution (Y132F) was highly resistant to fluconazole with MIC50 exceeded 256 μg/ml. Our study revealed high azole resistant rate in candiduria with its increasing trends observed much earlier than stains causing invasive infections. Given antimicrobial resistance as a critical "One Health" issue, the emergence of antifungal resistance in Candida species that are common commensal colonizers in the human body should be concerned.

Aspergillus Section Terrei and Antifungals: From Broth to Agar-Based Susceptibility Testing Methods.

Providing timely antifungal treatment to patients suffering from life-threatening invasive fungal infections (IFIs) is essential. Due to the changing epidemiology and the emergence of antifungal resistance in Aspergillus, the most commonly responsible mold of IFIs, antifungal susceptibility testing (AFST) has become increasingly important to guide clinical decisions. This study assessed the essential agreement (EA) between broth microdilution methods (the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST)) and the Etest of amphotericin B (AmB), liposomal amphotericin B (L-AmB), and isavuconazole (ISA) against 112 Aspergillus section Terrei. An EA within ±2 dilutions of ≥90% between the two methods was considered acceptable. Excellent EA was found between EUCAST and CLSI of AmB and ISA (98.2% and 95.5%, respectively). The correlation of Etest results and EUCAST/CLSI was not acceptable (<90%) for any tested antifungal; however, Etest and CLSI for AmB (79.6%) and ISA (77.6%) showed a higher EA than Etest and EUCAST for AmB (49.5%) and ISA (46.4%). It was concluded that the Etest method requires its own clinical breakpoints (CBPs) and epidemiological cutoff values (ECVs), and interpreting Etest results using EUCAST and CLSI-adapted CBPs and ECVs could result in misinterpretation as Etest shows lower minimum inhibitory concentrations (MICs).

A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules.

Trichoderma spp.-Related Pneumonia: A Case Report in Heart-Lung Transplantation Recipient and a Systematic Literature Review.

Opportunistic and hospital-acquired infections are common among recipients of solid organ transplantation. New pathogens are increasingly reported in the intensive care unit (ICU) population. We report a case of a patient who developed Trichoderma spp.-related pneumonia (TRP) after heart-lung transplantation. In the absence of antifungal susceptibility testing, TRP was confirmed by histological examination, and empirical therapy with voriconazole and caspofungin was swiftly initiated. Complete resolution of pneumonia was obtained after prolonged combination therapy. Given the lack of guidelines, we conducted a systematic review to elucidate the diagnostic and therapeutic strategies to apply during Trichoderma infection. After deduplication and selection of full texts, we found 42 articles eligible for the systematic review. Pneumonia seems to be the most common clinical manifestation (31.8%). The most used antifungal therapy was amphotericin B, while combination therapy was also reported (27.3%). All the patients were immunocompromised except for one case. Despite the rarity of Trichoderma spp. infection, the increase in invasive fungal infections is of growing importance in ICU, considering their impact on mortality and the emergence of antifungal resistance. In the absence of prospective and multicenter studies, a review can provide useful insight regarding the epidemiology, clinical manifestations, and management of these unexpected challenges.

Covid-19 and Fungal Infection: A literature review

Fungal infections have emerged as a significant and potentially lethal complication in critically ill patients with Covid-19. Infections caused by Aspergillus, Candida, and Mucormycetes, are a common cause of morbidity and mortality in critically ill patients, especially those with preexisting comorbidities. The underlying mechanisms that predispose fungal infections are not fully understood, but the use of corticosteroids, underlying conditions, prolonged hospitalization, and antibiotic are several factors that might be associated. They are associated with higher mortality rates, increased healthcare costs, delayed recovery, and prolonged hospitalization. Antifungal therapy for COVID-19-associated fungal infections can interact with other drugs used to treat COVID-19, which can complicate management. The emergence of antifungal resistance is a concern for the management of COVID-19-associated fungal infections.

Pulmonary aspergillosis: diagnosis and treatment.

Aspergillus species are the most frequent cause of fungal infections of the lungs with a broad spectrum of clinical presentations including invasive pulmonary aspergillosis (IPA) and chronic pulmonary aspergillosis (CPA). IPA affects immunocompromised populations, which are increasing in number and diversity with the advent of novel anti-cancer therapies. Moreover, IPA has emerged as a complication of severe influenza and coronavirus disease 2019 in apparently immunocompetent hosts. CPA mainly affects patients with pre-existing lung lesions and is recognised increasingly frequently among patients with long-term survival following cure of tuberculosis or lung cancer. The diagnosis of pulmonary aspergillosis is complex as it relies on the presence of clinical, radiological and microbiological criteria, which differ according to the type of pulmonary aspergillosis (IPA or CPA) and the type of patient population. The management of pulmonary aspergillosis is complicated by the limited number of treatment options, drug interactions, adverse events and the emergence of antifungal resistance.

Evaluation of Anti-Candida Potential of Piper nigrum Extract in Inhibiting Growth, Yeast-Hyphal Transition, Virulent Enzymes, and Biofilm Formation.

Due to the increased incidence of fungal infections and the emergence of antifungal resistance mainly by Candida species, the need for safe and effective novel therapies is imperative. Consequently, plants and herbs are a powerful source to combat infections. Here, we evaluated the anti-Candida potential of an ethanolic extract from Piper nigrum. The phytochemical analysis of P. nigrum revealed bioactive compounds such as alkaloids, terpenoids, and tannis. Our results showed that P. nigrum extract suppressed the virulence factors of C. albicans strains, including hyphae formation in both liquid and solid media, reduced secretion of phospholipases/proteinases, and affected biofilm formation. Furthermore, the P. nigrum extract showed no hemolytic effect in vitro and exhibited reduced cytotoxicity on Vero cells and G. mellonella larvae at concentrations that inhibited hyphae and biofilm in C. albicans. Moreover, the extract demonstrated antifungal activity against C. auris strains. In conclusion, the P. nigrum extract affected the growth and morphogenesis of Candida (even in resistant strains), demonstrating that this plant has an anti-candida activity and represents a promising resource for discovering novel antifungal compounds.

Antifungal Activity of NP20 Derived from Amphioxus Midkine/Pleiotrophin Homolog Against Aspergillus niger and Aspergillus fumigatus.

With the emergence of antifungal resistance, systematic infections with Aspergillus are becoming the major cause of the clinical morbidity. The development of novel antifungal agents with high efficacy, low drug tolerance, and few side effects is urgent. In response to that need, we have identified NP20. Here we demonstrate clearly that NP20 has antifungal activity, capable of killing the spores of Aspergillus niger and Aspergillus fumigatus as well as causing direct damage to the surface, membrane, cytoplasm, organelle, and nucleus of the fungal spores. Interestingly, NP20 is active under temperature stress and a wide range of pH. Subsequently, MTT assay, assay for binding of NP20 to fungal cell wall components, membrane depolarization assay, confocal microscopy, ROS assay, DNA replication, and protein synthesis assay are performed to clarify the mechanisms underlying NP20 against Aspergillus. The results show that NP20 can bind with and pass through the fungal cell wall, and then interfere with the lipid membrane. Moreover, NP20 can induce intracellular ROS production, DNA fragmentation, and protein synthesis inhibition of the fungal cells. These together indicate that NP20 is a novel antifungal peptide, which has considerable potential for future development as novel peptide antibiotics against Aspergillus.

CRISPR-Cas9 approach confirms Calcineurin-responsive zinc finger 1 (Crz1) transcription factor as a promising therapeutic target in echinocandin-resistant Candida glabrata

Invasive fungal infections, which kill more than 1.6 million patients each year worldwide, are difficult to treat due to the limited number of antifungal drugs (azoles, echinocandins, and polyenes) and the emergence of antifungal resistance. The transcription factor Crz1, a key regulator of cellular stress responses and virulence, is an attractive therapeutic target because this protein is absent in human cells. Here, we used a CRISPR-Cas9 approach to generate isogenic crz1Δ strains in two clinical isolates of caspofungin-resistant C. glabrata to analyze the role of this transcription factor in susceptibility to echinocandins, stress tolerance, biofilm formation, and pathogenicity in both non-vertebrate (Galleria mellonella) and vertebrate (mice) models of candidiasis. In these clinical isolates, CRZ1 disruption restores the susceptibility to echinocandins in both in vitro and in vivo models, and affects their oxidative stress response, biofilm formation, cell size, and pathogenicity. These results strongly suggest that Crz1 inhibitors may play an important role in the development of novel therapeutic agents against fungal infections considering the emergence of antifungal resistance and the low number of available antifungal drugs.

Bioisosteric modification on benzylidene-carbonyl compounds improved the drug-likeness and maintained the antifungal activity against Sporothrix brasiliensis.

Considering the emergence of antifungal resistance on Sporothrix brasiliensis, we aimed to assess new benzylidene-carbonyl compounds against feline-borne S. brasiliensis isolates. The compounds were designed as bioisosteres from previously reported benzylidene-ketones generating the p-coumaric (1), cinnamic (2), p-methoxycinnamic (3) and caffeic acid (4) analogues. The corresponding compounds were tested against feline isolates of S. brasiliensis with sensitivity (n=4) and resistance (n=5) to itraconazole (ITZ), following the M38-A2 protocol (CLSI, Reference method for broth dilution antifungal susceptibility testing of filamentous fungi M38-A2 Guideline, 2008). Eleven analogues showed activity against all fungal strains with minimum inhibitory concentrations (MIC) ≤1mg/ml (1a-d, 2e, 3b, 3e, 4, 4a and 5e) and fungicidal concentrations (MFC) ≤1mg/ml (1b, 1d, 3e and 4a), whereas 3 was the less active with both MIC and MFC values above 1mg/ml. Compound 3e (4-methoxy-N-butylcinnamamide) was the most potent (MICrange 0.08-0.16mg/ml; MFCrange 0.32-0.64mg/ml) from the set, suggesting a different role of the substituents in ester and amide derivatives. The designed compounds proved to be important prototypes with improved drug-likeness to achieve compounds with higher activity against ITZ-resistant S. brasiliensis.

Towards a Standardized Procedure for the Production of Infective Spores to Study the Pathogenesis of Dermatophytosis.

Dermatophytoses are superficial infections of human and animal keratinized tissues caused by filamentous fungi named dermatophytes. Because of a high and increasing incidence, as well as the emergence of antifungal resistance, a better understanding of mechanisms involved in adhesion and invasion by dermatophytes is required for the further development of new therapeutic strategies. In the last years, several in vitro and in vivo models have emerged to study dermatophytosis pathogenesis. However, the procedures used for the growth of fungi are quite different, leading to a highly variable composition of inoculum for these models (microconidia, arthroconidia, hyphae), thus rendering difficult the global interpretation of observations. We hereby optimized growth conditions, including medium, temperature, atmosphere, and duration of culture, to improve the sporulation and viability and to favour the production of arthroconidia of several dermatophyte species, including Trichophyton rubrum and Trichophyton benhamiae. The resulting suspensions were then used as inoculum to infect reconstructed human epidermis in order to validate their ability to adhere to and to invade host tissues. By this way, this paper provides recommendations for dermatophytes culture and paves the way towards a standardized procedure for the production of infective spores usable in in vitro and in vivo experimental models.

Consensus guidelines for antifungal stewardship, surveillance and infection prevention, 2021.

Invasive fungal diseases (IFD) are serious infections associated with high mortality, particularly in immunocompromised patients. The prescribing of antifungal agents to prevent and treat IFD is associated with substantial economic burden on the health system, high rates of adverse drug reactions, significant drug-drug interactions and the emergence of antifungal resistance. As the population at risk of IFD continues to grow due to the increased burden of cancer and related factors, the need for hospitals to employ antifungal stewardship (AFS) programmes and measures to monitor and prevent infection has become increasingly important. These guidelines outline the essential components, key interventions and metrics, which can help guide implementation of an AFS programme in order to optimise antifungal prescribing and IFD management. Specific recommendations are provided for quality processes for the prevention of IFD in the setting of outbreaks, during hospital building works, and in the context of Candida auris infection. Recommendations are detailed for the implementation of IFD surveillance to enhance detection of outbreaks, evaluate infection prevention and prophylaxis interventions and to allow benchmarking between hospitals. Areas in which information is still lacking and further research is required are also highlighted.

Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance.

Invasive pulmonary aspergillosis is a life-threatening fungal infection especially in the immunocompromised patients. The low diversity of available antifungal drugs coupled with the emergence of antifungal resistance has become a worldwide clinical concern. The echinocandin Caspofungin (CSP) is recommended as a second-line therapy but resistance and tolerance mechanisms have been reported. However, how the fungal cell articulates the response to CSP is not completely understood. This work provides a detailed characterization of ZnfA, a transcription factor (TF) identified in previous screening studies that is involved in the A. fumigatus responses to calcium and CSP. This TF plays an important role in the regulation of iron homeostasis and cell wall organization in response to high CSP concentrations as revealed by Chromatin Immunoprecipitation coupled to DNA sequencing (ChIP-seq) analysis. Furthermore, ZnfA acts collaboratively with the key TF CrzA in modulating the response to calcium as well as cell wall and osmotic stresses. This study therefore describes the existence of an additional, previously unknown TF that bridges calcium signaling and the CSP cellular response and further exposes the complex connections that exist among different pathways which govern stress sensing and signaling in A. fumigatus.

Analysis of putative quadruplex-forming sequences in fungal genomes: novel antifungal targets?

Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.