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Azobenzene derivatives with activity against drug‐resistant Candida albicans and Candida auris
(2023)
Increasing resistance against antimycotic drugs challenges anti‐infective therapies today and contributes to the mortality of infections by drug‐resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'‐dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole‐susceptible and fluconazole‐resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.
Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.
The fungal cell wall is essential for the maintenance of cellular integrity and mediates interactions of the cells with the environment. It is a highly flexible organelle whose composition and organization is modulated in response to changing growth conditions. In the pathogenic yeast Candida albicans, a network of signaling pathways regulates the structure of the cell wall, and mutants with defects in these pathways are hypersensitive to cell wall stress. By harnessing a library of genetically activated forms of all C. albicans zinc cluster transcription factors, we found that a hyperactive Czf1 rescued the hypersensitivity to cell wall stress of different protein kinase deletion mutants. The hyperactive Czf1 induced the expression of many genes with cell wall-related functions and caused visible changes in the cell wall structure. C. albicans czf1Δ mutants were hypersensitive to the antifungal drug caspofungin, which inhibits cell wall biosynthesis. The changes in cell wall architecture caused by hyperactivity or absence of Czf1 resulted in an increased recognition of C. albicans by human neutrophils. Our results show that Czf1, which is known as a regulator of filamentous growth and white-opaque switching, controls the expression of cell wall genes and modulates the architecture of the cell wall.
Microorganisms that colonize the human body face large fluctuations in their surroundings. Therefore, those microbes developed sophisticated mechanisms that allow them to adapt their cell biology and maintain cellular homeostasis. One organelle vital to preserve cell physiology is the vacuole. The vacuole exhibits a wide range of functions and is able to adjust itself in response to both external and internal stimuli. Moreover, it plays an important role in host interaction and virulence in fungi such as Candida albicans. Despite this connection, only a few regulatory proteins have been described to modulate vacuolar biology in fungal pathogens. Furthermore, whether such regulation alters fungus-host interplay remains largely unknown.
This thesis focuses on the characterization of ZCF8, a fungus-specific transcription regulator in the human-associated yeast C. albicans. To this end, I combined genome-wide protein-DNA interaction assays and gene expression analysis that identified genes regulated by Zcf8p. Fluorescence microscopy uncovered that several top targets of Zcf8p localize to the fungal vacuole. Moreover, deletion and overexpression of ZCF8 resulted in alterations in vacuolar morphology and in luminal pH and rendered the fungus resistant or susceptible to a vacuole-disturbing drug. Finally, in vitro adherence assays showed that Zcf8p modulates the attachment of C. albicans to human epithelial cells in a vacuole-dependent manner.
Given those findings, I posit that the previously uncharacterized transcription regulator Zcf8p modulates fungal attachment to epithelial cells in a manner that depends on the status of the fungal vacuole. Furthermore, the results highlight that vacuolar physiology is a substantial factor influencing the physical interaction between Candida cells and mammalian mucosal surfaces.
Candida albicans is ubiquitously present, and colonization in the nose and oral cavity is common. In healthy patients, it usually does not act as a pathogen, but in some cases can cause diseases. The influence of C. albicans as a trigger of T cell activation on the pathogenesis of chronic rhinosinusitis (CRS) is controversial, and its exact role is not clear to date. The aim of the present study was to detect and characterize C. albicans-specific CD4+ and CD8+ T cells in patients with CRS, with and without nasal polyps. Tissue and blood samples were collected from patients suffering from chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP), and from healthy controls. A peptide pool derived from C. albicans antigen was added to tissue and blood samples. After 6 days, lymphocytes were analyzed by multicolor flow cytometry. Activation was assessed by the intracellular marker Ki-67, and the cytokine secretion was measured. Tissue CD8+ T cells of CRSsNP patients showed a significantly higher proportion of Ki-67+ cells after activation with C. albicans antigen compared to peripheral blood CD8+ T cells. Cytokine secretion in response to C. albicans antigen was similar for all study groups. In this study, C. albicans-specific CD4+ and CD8+ T cells were detected in peripheral blood and mucosal tissue in all study groups. In patients suffering from CRSsNP, C. albicans-specific CD8+ T cells were relatively enriched in the nasal mucosa, suggesting that they might play a role in the pathogenesis of CRSsNP.
Evolution of antifungal drug resistance of the human-pathogenic fungus \(Candida\) \(albicans\)
(2021)
Infections with the opportunistic yeast Candida albicans are frequently treated with the first-line drug fluconazole, which inhibits ergosterol biosynthesis. An alarming problem in clinics is the development of resistances against this azole, especially during long-term treatment of patients. Well-known resistance mechanisms include mutations in the zinc cluster transcription factors (ZnTFs) Mrr1 and Tac1, which cause an overexpression of efflux pump genes, and Upc2, which results in an overexpression of the drug target. C. albicans strains with such gain-of-function mutations (GOF) have an increased drug resistance conferring a selective advantage in the presence of the drug. It was previously shown that this advantage comes with a fitness defect in the absence of the drug. This was observed in different conditions and is presumably caused by a deregulated gene expression.
One aim of the present study was to examine whether C. albicans can overcome the costs of drug resistance by further evolution. Therefore, the relative fitness of clinical isolates with one or a combination of different resistance mutations in Mrr1, Tac1 and/or Upc2 was analyzed in competition with the matched fluconazole-susceptible partner. Most fluconazole-resistant isolates had a decreased fitness in competition with their susceptible partner in vitro in rich medium. In contrast, three fluconazole-resistant strains with Mrr1 resistance mutations did not show a fitness defect in competition with their susceptible partner. In addition, the fitness of four selected clinical isolate pairs was examined in vivo in mouse models of gastrointestinal colonization (GI) and disseminated infection (IV). In the GI model all four fluconazole-resistant strains were outcompeted by their respective susceptible partner. In contrast, in the IV model only one out of four fluconazole-resistant isolates did show a slight fitness defect in competition with its susceptible partner during infection of the kidneys. It can be stated, that in the present work the in vitro fitness did not reflect the in vivo fitness and that the overall fitness was dependent on the tested conditions. In conclusion, C. albicans cannot easily overcome the costs of drug resistance caused by a deregulated gene expression.
In addition to GOFs in Mrr1, Tac1 and Upc2, resistance mutations in the drug target Erg11 are a further key fluconazole resistance mechanism of C. albicans. Clinical isolates often harbor several resistance mechanisms, as the fluconazole resistance level is further increased in strains with a combination of different resistance mutations. In this regard, the question arises of how strains with multiple resistance mechanisms evolve. One possibility is that strains acquire mutations successively. In the present study it was examined whether highly drug-resistant C. albicans strains with multiple resistance mechanisms can evolve by parasexual recombination as another possibility. In a clonal population, cells with individually acquired resistance mutations could combine these advantageous traits by mating. Thereupon selection could act on the mating progeny resulting in even better adapted derivatives.
Therefore, strains heterozygous for a resistance mutation and the mating type locus (MTL) were grown in the presence of fluconazole. Derivatives were isolated, which had become homozygous for the resistance mutation and at the same time for the MTL. This loss of heterozygosity was accompanied by increased drug resistance. In general, strains which are homozygous for one of both MTL configurations (MTLa and MTLα) can switch to the opaque phenotype, which is the mating-competent form of the yeast, and mate with cells of the opposite MTL. In the following, MTLa and MTLα homozygous strains in the opaque phenotype were mated in all possible combinations. The resulting mating products with combined genetic material from both parents did not show an increased drug resistance. Selected products of each mating cross were passaged with stepwise increasing concentrations of fluconazole. The isolated progeny showed high levels of drug resistance and loss of wild-type alleles of resistance-associated genes. In conclusion, selective pressure caused by fluconazole exposure selects for resistance mutations and at the same time induces genomic rearrangements, resulting in mating competence. Therefore, in a clonal population, cells with individually acquired resistance mutations can mate with each other and generate mating products with combined genetic backgrounds. Selection can act on these mating products and highly drug-resistant und thus highly adapted derivatives can evolve as a result.
In summary, the present study contributes to the current understanding of the evolution of antifungal drug resistance by elucidating the effect of resistance mutations on the fitness of the strains in the absence of the drug selection pressure and investigates how highly drug-resistant strains could evolve within a mammalian host.
Oligopeptides incorporating \(N3\)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), an inhibitor of glucosamine-6-phosphate synthase, exhibited growth inhibitory activity against \(Candida\) \(albicans\), with minimal inhibitory concentration values in the 0.05–50 μg mL\(^{-1}\) range. Uptake by the peptide permeases was found to be the main factor limiting an anticandidal activity of these compounds. Di- and tripeptide containing FMDP (F2 and F3) were transported by Ptr2p/Ptr22p peptide transporters (PTR) and FMDP-containing hexa-, hepta-, and undecapeptide (F6, F7, and F11) were taken up by the oligopeptide transporters (OPT) oligopeptide permeases, preferably by Opt2p/Opt3p. A phenotypic, apparent resistance of \(C. albicans\) to FMDP-oligopeptides transported by OPT permeases was triggered by the environmental factors, whereas resistance to those taken up by the PTR system had a genetic basis. Anticandidal activity of longer FMDP-oligopeptides was strongly diminished in minimal media containing easily assimilated ammonium sulfate or L-glutamine as the nitrogen source, both known to downregulate expression of the OPT genes. All FMDP-oligopeptides tested were more active at lower pH and this effect was slightly more remarkable for peptides F6, F7, and F11, compared to F2 and F3. Formation of isolated colonies was observed inside the growth inhibitory zones induced by F2 and F3 but not inside those induced by F6, F7, and F11. The vast majority (98%) of those colonies did not originate from truly resistant cells. The true resistance of 2% of isolates was due to the impaired transport of di- and to a lower extent, tripeptides. The resistant cells did not exhibit a lower expression of \(PTR2\), \(PTR22\), or \(OPT1–3\) genes, but mutations in the \(PTR2\) gene resulting in T422H, A320S, D119V, and A320S substitutions in the amino acid sequence of Ptr2p were found.
The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1 Delta mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1 Delta null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1 Delta mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37 degrees C instead of 30 degrees C, and this phenotype enabled us to isolate homozygous snf1 Delta mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1 Delta mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.
IMPORTANCE
Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1 Delta mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.
Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation.
The human body is colonized by trillions of microbes from all three domains of life – eukaryotes, bacteria and archaea. The lower gastrointestinal tract is the most densely colonized part of the body, harbouring a diverse and dynamic community of microbes. While the importance of bacteria in this so-called microbiota is well acknowledged, the role of commensal fungi remains underexplored. The most prominent fungus of the human gastrointestinal microbiota is Candida albicans. This fungus occasionally causes life-threatening disseminated infections in individuals with debilitated immune defences. It is this “pathogenic” facet that has received the most attention from researchers in the past, leaving many aspects of its “commensal” lifestyle understudied. Using gnotobiotic mice as a model system to explore the biology of C. albicans in the mammalian gut, in this dissertation I establish the global response of the host to C. albicans monocolonization as well as the spatial distribution of the fungus in the intestine in the context of co-colonization with single gut bacterial species. The fungus elicited transcriptome changes in murine intestinal tissue, which included the activation of a reactive oxygen species-related defence mechanism and the induction of regulators of the circadian clock circuitry. Both responses have previously been described in the context of a complete bacterial microbiota. Imaging the intestine of animals monocolonized with the fungus or co-colonized with C. albicans and the gut bacteria Bacteroides thetaiotaomicron or Lactobacillus reuteri revealed that the fungus was embedded in a B. thetaiotaomicron-promoted outer mucus layer in the murine colon. The gel-like outer mucus constitutes a unique microhabitat, distinct in microbial composition from the adjacent intestinal lumen. This finding indicates that bacteria can shape the specific microhabitat occupied by the fungus in the intestine. Overall, the results described in this dissertation suggest that gnotobiotic mice constitute a valuable tool to dissect multiple aspects of the interactions among host, commensal fungi and cohabiting bacteria.