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Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4\(^{+}\) T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4\(^{+}\) T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn\(^{2+}\) ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn\(^{2+}\). In vitro stimulation assays using highly purified mouse CD4\(^{+}\) T cells showed that Pra1 increased proliferation of CD4\(^{+}\) T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4\(^{+}\) T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4\(^{+}\) T cell responses as shown in our in vitro assays.
1. Summary Candida albicans is an opportunistic human fungal pathogen that causes a variety of infections, ranging from superficial mucosal to deep-seated systemic infections, especially in immunocompromised patients. Although the ability of C.albicans to cause disease largely depends on the immune status of the host, the fungus also exhibits specific characteristics that facilitate colonization, dissemination, and adaptation to different host niches and thereby turn C.albicans from a harmless commensal to an aggressive pathogen. In response to various environmental stimuli C.albicans switches from growth as a budding yeast to invasive filamentous growth, and this morphogenetic switch plays an important role in C.albicans pathogenesis. Nitrogen limitation is one of the signals that induce filamentous growth in C.albicans, and the control of the morphogenetic transition by nitrogen availability was studied in detail in the present work. Ammonium is a preferred nitrogen source for yeasts that is taken up into the cells by specific transporters. It was found in this study that C.albicans possesses two major ammonium transporters, encoded by the CaMEP1 and CaMEP2 genes, expression of which is induced by nitrogen starvation. Whereas mep1 or mep2 single mutants grew as well as the wild-type strain on limiting concentrations of ammonium, deletion of both transporters rendered C.albicans unable to grow at ammonium concentrations below 5 mM. In contrast to mep1 mutants, mep2 mutants failed to filament and grew only in the yeast form under nitrogen starvation conditions, indicating that in addition to its role as an ammonium transporter CaMep2p also has a signaling function in the induction of filamentous growth. CaMep2p was found to be a less efficient ammonium transporter than CaMep1p and to be expressed at much higher levels, a distinguishing feature important for its signaling function. By the construction and analysis of serially truncated versions of CaMep2p, the C-terminal cytoplasmic tail of the protein was shown to be essential for signaling but dispensable for ammonium transport, demonstrating that these two functions of CaMep2p are separable. In C.albicans at least two signal transduction pathways, a MAP kinase cascade and a cAMP-dependent pathway ending in the transcriptional regulators Cph1p and Efg1p, respectively, control filamentous growth, and mutants defective in either one of these pathways are defective for filamentation under nitrogen starvation conditions. A hyperactive CaMEP2 allele rescued the filamentation defect of a cph1 or a efg1 mutant, but not of a cph1 efg1 double mutant or a mutant deleted for RAS1, which acts upstream of and activates both signaling pathways. Conversely, a dominant active RAS1 allele or addition of exogenous cAMP rescued the filamentation defect of mep2 mutants. These results suggest that CaMep2p activates both the MAP kinase and the cAMP pathway in a Ras1p dependent manner to promote filamentous growth under nitrogen starvation conditions. At sufficiently high concentrations, ammonium repressed filamentous growth even when the signaling pathways were artificially activated. Therefore, C.albicans has established a regulatory circuit in which a preferred nitrogen source, ammonium, serves as an inhibitor of morphogenesis that is taken up into the cell by the same transporter that induces filamentous growth in response to nitrogen starvation. Although a detailed understanding of virulence mechanisms of C.albicans may ultimately lead to novel approaches to combat infections caused by this pathogen, the identification and characterization of essential genes as potential targets for the development of antifungal drugs is a strategy favoured by most pharmaceutical companies. Therefore, C.albicans homologs of three genes that are essential in other fungi were selected in collaboration with an industrial partner and functionally characterized in this work. RAP1 encodes the repressor/activator protein 1, a transcription factor and telomere binding protein that is essential for viability in the budding yeast Saccharomyces cerevisiae. However, deletion of the C.albicans RAP1 homolog did not affect viability or growth of the mutants, suggesting that it is not a promising target. CBF1 (centromere binding factor 1) is necessary for proper chromosome segregation and transcriptional activation of methionine biosynthesis genes in S.cerevisiae and is essential for viability in the related yeasts Kluyveromyces lactis and Candida glabrata. Deletion of CBF1 in C.albicans did not result in an increased frequency of chromosome loss, indicating that it has no role in chromosome segregation in this organism. However, the C.albicans cbf1 mutants exhibited severe growth impairment, temperature sensitivity at 42°C, and auxotrophy for sulphur amino acids, suggesting that Cbf1p is a transcription factor that is important for normal growth of C.albicans. YIL19 is an essential gene in S.cerevisiae that is involved in 18S rRNA maturation. YIL19 was found to be an essential gene also in C.albicans. Conditional mutants in which the YIL19 gene could be excised from the genome by inducible, FLP-mediated recombination were non-viable and accumulated rRNA precursors, demonstrating that YIL19 is essential for this important cellular process and for viability of C.albicans and could serve as a target for the development of antifungal drugs.
The human body is laden with trillions of microorganisms that belong to all three domains of life. Some species of this microbiota subsist as harmless commensals in healthy adults, but under certain circumstances, they can cause mucosal disease or even systemic, life-threatening infections. While the bacterial members of our microbiota are heavily studied today, much less attention is afforded to eukaryotic species that colonize different mucocutaneous surfaces of the human body. This dissertation focuses on identifying regulatory circuits that enable a prominent member of these eukaryotes, C. albicans, to, on the one hand, live on a specific mammalian mucosal surface as a harmless commensal and, on the other hand, proliferate as a pathogen. Since the ultimate source of many fatal Candida infections is the gastrointestinal (GI) tract of the infected individual, this organism is particularly suited to distinguishing traits essential for the gut colonization of commensal fungi and their ability to cause disease. Sequence-specific DNA-binding proteins that regulate transcription are important to most biological processes; I thus used these proteins as starting points to gain insights into 1) how a specific transcription regulator promotes virulence in C. albicans; 2) which traits C. albicans requires to inhabit the GI tract of a specific, well-defined mouse model as a harmless commensal; and 3) how three previously undescribed transcriptional regulators contribute to the commensal colonization of the digestive tract of this mouse model. Altogether, this work advances the knowledge concerning the biology of commensal fungi in the mammalian gut and genetic determinants of fungal commensalism, as well as pathogenicity.
The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice
(2017)
Many microorganisms that cause systemic, life-threatening infections in humans reside as harmless commensals in our digestive tract. Yet little is known about the biology of these microbes in the gut. Here, we visualize the interface between the human commensal and pathogenic fungus Candida albicans and the intestine of mice, a surrogate host. Because the indigenous mouse microbiota restricts C. albicans settlement, we compared the patterns of colonization in the gut of germ free and antibiotic-treated conventionally raised mice. In contrast to the heterogeneous morphologies found in the latter, we establish that in germ free animals the fungus almost uniformly adopts the yeast cell form, a proxy of its commensal state. By screening a collection of C. albicans transcription regulator deletion mutants in gnotobiotic mice, we identify several genes previously unknown to contribute to in vivo fitness. We investigate three of these regulators—ZCF8, ZFU2 and TRY4—and show that indeed they favor the yeast form over other morphologies. Consistent with this finding, we demonstrate that genetically inducing non-yeast cell morphologies is detrimental to the fitness of C. albicans in the gut. Furthermore, the identified regulators promote adherence of the fungus to a surface covered with mucin and to mucus-producing intestinal epithelial cells. In agreement with this result, histology sections indicate that C. albicans dwells in the murine gut in close proximity to the mucus layer. Thus, our findings reveal a set of regulators that endows C. albicans with the ability to endure in the intestine through multiple mechanisms.
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 yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions. The yeast Candida albicans is a member of the normal microflora on the mucosal surfaces of the gastrointestinal and urogenital tract in healthy persons. However, it is an opportunistic pathogen that can cause a range of infections from superficial to disseminated, in response to perturbation of the normal microflora or alterations in the host immunity. C. albicans exhibits a variety of characteristics such as adhesion, morphogenetic switching and secreted aspartic protease production that contribute to its virulence. Expression of many of these virulence factors is controlled by the availability of essential element, nitrogen. C. albicans undergoes morphogenetic transition to form filaments under nitrogen starvation conditions and this switch is controlled by the ammonium permease Mep2p. However, little is known about how this signaling function of Mep2p is regulated. Mutational analysis of Mep2p was carried out to identify the residues that confer signaling activity to this permease. The C-terminal cytoplasmic tail of Mep2p contains a signaling domain that is dispensable for ammonium transport but essential for the signaling activity of Mep2p. In this work, progressive C-terminal truncations analysis demonstrated that a MEP2DC433 allele was still able to induce filamentation while nitrogen starvation-induced filamentous growth was abolished in cells expressing a MEP2DC432 allele. Therefore, tyrosine at position 433 (Y433) is the last amino acid in Mep2p that is essential for signaling. To gain insights into how the signaling activity of Mep2p is regulated by ammonium availability and transport, conserved residues that have been implicated in ammonium binding or uptake were mutated. Mutation of D180, which has been proposed to mediate initial contact with extracellular ammonium, or the pore-lining residues H188 and H342 abolished Mep2p expression, indicating that these residues are important for protein stability. Mutation of F239, which together with F126 is predicted to form an extracytosolic gate to the conductance channel, abolished both ammonium uptake and Mep2p-dependent filamentation, despite proper localization of the protein. On the other hand, mutation of W167, which is assumed to participate along with Y122, F126, and S243 in the recruitment and coordination of the ammonium ion at the extracytosolic side of the cell membrane, also abolished filamentation without having a strong impact on ammonium transport, demonstrating that extracellular alterations in Mep2p can affect intracellular signaling. Mutation of Y122 reduced ammonium uptake much more strongly than mutation of W167 but still allowed efficient filamentation, indicating that the signaling activity of Mep2p is not directly correlated with its transport activity. An important aspect in the ability of Mep2p to stimulate filamentation in response to nitrogen limitation is its high expression levels. The cis-acting sequences and trans-acting regulators that mediate MEP2 induction in response to nitrogen limitation were identified. Promoter analysis revealed that two putative binding sites for GATA transcription factors have a central role in MEP2 expression, as deletion of the region containing these sites or mutation of the GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To elucidate the roles of the GATA transcription factors GLN3 and GAT1 in regulating MEP2 expression, mutants lacking one or both of these transcription factors were constructed. Mep2p expression was strongly reduced in gln3D and gat1D single mutants and virtually abolished in gln3D gat1D double mutants. Deletion of GLN3 strongly inhibited filamentous growth under limiting nitrogen conditions, which could be rescued by constitutive expression of MEP2 from the ADH1 promoter. In contrast, inactivation of GAT1 had no effect on filamentation. Surprisingly, filamentation became partially independent of the presence of a functional MEP2 gene in the gat1D mutants, indicating that the loss of GAT1 function results in the activation of other pathways that induce filamentous growth. These findings demonstrated that the GATA transcription factors Gln3p and Gat1p control expression of the MEP2 ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced filamentous growth in C. albicans. C. albicans mutants lacking both the GATA transcription factors Gln3p and Gat1p were unable to grow in a medium containing an alternative nitrogen source, bovine serum albumin (BSA) as the sole nitrogen source. The ability to utilize proteins as sole source of nitrogen for growth of C. albicans is conferred by the secreted aspartic protease Sap2p, which degrades the proteins, and oligopeptide transporters that mediate uptake of the proteolytic products into cell. The growth defect of gln3D gat1D mutants was mainly caused by their inability to express the SAP2 gene, as SAP2 expression from the constitutive ADH1 promoter restored the ability of the mutants to grow on BSA. Expression of STP1, which encodes a transcription factor that is required for SAP2 induction in the presence of proteins, was regulated by Gln3p and Gat1p. Forced expression of STP1 from a tetracycline-inducible promoter bypassed the requirement of the GATA transcription factors for growth of C. albicans on proteins. When preferred nitrogen sources are available, SAP2 is repressed and this nitrogen catabolite repression of SAP2 was correlated with downregulation of STP1 under these conditions. Tetracycline-induced STP1 expression abolished nitrogen catabolite repression of SAP2, demonstrating that regulation of STP1 expression levels by the GATA transcription factors is a key aspect of both positive and negative regulation of SAP2 expression. Therefore, by using a regulatory cascade in which expression of the specific transcription factor Stp1p is controlled by the general regulators Gln3p and Gat1p, C. albicans places SAP2 expression under nitrogen control and ensures proper expression of this virulence determinant. In summary, the present study illustrated how GATA factors, Gln3p and Gat1p, play partially overlapping, but distinct roles, in mediating the appropriate responses of C. albicans to the availability of different nitrogen sources. These responses are also determinants of pathogenicity of the fungus. The relative contributions of Gln3p and Gat1p vary with their target genes and the availability of nitrogen source. Overall, these findings provide us with a better understanding of the molecular basis of some of the important processes that help in adaptation of C. albicans to various environmental conditions.
Regulation of gene expression by the control of transcription is essential for any cell to adapt to the environment and survive. Transcription regulators, i.e. sequence-specific DNA binding proteins that regulate gene expression, are central elements within the gene networks of most organisms. Transcription regulators are grouped into distinct families based on structural features that determine, to a large extent, the DNA sequence(s) that they can recognise and bind. Less is known, however, about how the DNA binding preferences can diversify within transcription regulator families during evolutionary timescales, and how such diversification can affect the biology of the organism.
In this dissertation I study the SREBP (sterol regulatory element binding protein) family of transcriptional regulators in yeasts, and in Candida albicans in particular, as an experimental system to address these questions. The SREBPs are conserved from fungi to humans and represent a subgroup of basic helix-loop-helix DNA binding proteins. Early chromatin immunoprecipitation experiments with SREBPs from humans and yeasts showed that these proteins bound in vivo to the canonical DNA sequence, termed E-box, most basic helix-loop-helix proteins bind to. By contrast, most recent analysis carried out with less-studied fungal SREBPs revealed a non-canonical DNA motif to be the most overrepresented sequence in the bound regions.
This study aims to establish the intrinsic DNA binding preferences of key branches of this family and to determine how the divergence in DNA binding affinities originated. To this end, I combined phylogenetic and ancestral reconstruction with extensive biochemical characterisation of key SREBP proteins. The results indicated that while the most-studied SREBPs (in mammals) indeed show preference for the E-box, a second branch of the family preferentially binds the non-E-box, and a third one is able to bind both sequences with similar affinity. The preference for one or the other DNA sequence is an intrinsic property of each protein because their purified DNA binding domain was sufficient to recapitulate their in vivo binding preference. The ancestor that gave rise to these two different types of SREBPs (the branch that binds E-box and the one that binds non-E-box DNA) appears to be a protein with a broader DNA binding capability that had a slight preference for the non-canonical motif. Thus, the results imply these two branches originated by either enhancing the original ancestral preference for non-E-box or tilting it towards the E-box DNA and flipping the preference for this sequence.
The main function associated with members of the SREBP family in most eukaryotes is the control of lipid biosynthesis. I have further studied the function of these proteins in the lineage that encompasses the human associated yeast C. albicans. Strikingly, the three SREBPs present in the fungus’ genome contribute to the colonisation of the mammalian gut by regulating cellular processes unrelated to lipid metabolism. Here I describe that two of the three C. albicans SREBPs form a regulatory cascade that regulates morphology and cell wall modifications under anaerobic conditions, whereas the third SREBP has been shown to be involved in the regulation of glycolysis genes.
Therefore, I posit that the described diversification in DNA binding specificity in these proteins and the concomitant expansion of targets of regulation were key in enabling this fungal lineage to associate with animals.
The work deals with the synthesis and characterization of cerulenin analogues as inhibitors of efflux pump mediated resistance of Candida albicans isolates and as inhibitors of the fatty acid synthesis enzyme KasA of Mycobacterium tuberculosis. Cerulenin was chosen as the lead structure, being a substrate of the efflux pumps in Candida albicans on one hand and therefore variations on the structure could lead to a blocking of the efflux pumps as in the case of tetracycline and inhibitor 13-CPTC of the TetB efflux pump. On the other hand, cerulenin is a known inhibitor of the FAS system but inhibition is unselective in type I and II FAS. Therefore, analogues could result in increased selectivity towards the type II FAS system in M. tuberculosis. The first cerulenin derivatives were prepared by coupling 2,3-dihydrofuran to the before synthesized 1-octaniodide, followed by ring opening and oxidation in one step by chromic acid and transfer of the resulting 4-keto acid to amides to give analogues 4a-d, 4e was prepared in analogy. To include the epoxide function especially with regard to the mechanism of action of cerulenin in the FAS system (considering known crystal structures of cerulenin and the KasA analogue of E. coli) tetrahydro- and dihydrocerulenin analogues were synthesized. Starting from the corresponding aldehyde, lactone 5 (tetrahydrocerulenin analogues) was obtained via two different routes A and B. Route A included the coupling of the aldehyde 1-nonanal to propiolic acid via a Grignard reaction with subsequent hydrogenation with the Lindlar catalyst under hydrogen pressure to give 5. Via Route B 1-nonanal was coupled to methyl propiolate by n-BuLi with subsequent hydrogenation under reflux with the catalytic system Lindlar cat./NH4HCO2 to yield 5. These hydrogenations were also executed in a microwave oven resulting in better yields and/or reaction times. The lactone 5 was then epoxidized, the ring opened by amidation and the remaining alcohol was oxidized via Collins oxidation to result in tetrahydrocerulenin analogues 8a-e. The same procedure was used for dihydrocerulenin analogues 10a-c except that to obtain the corresponding lactone 9a only route A was used and a further step had to be executed for ring closure. To obtain analogues with all structural features of cerulenin including two double bonds and the epoxide function, a third pathway was chosen. To obtain the future side chain, aldehyde 12 was synthesized by coupling protected 4-pentyn-1-ol to either crotyl bromide or crotyl chloride, which then was deprotected, hydrogenated with Lindlar catalyst under hydrogen pressure and oxidized via a Swern oxidation. The following synthesis sequence starting from 12 was executed similar to that of dihydrocerulenins via the corresponding lactone (51) with the major exception of the oxidation procedure in the last step via TPAP/NMO to result in (4Z,7E)-cerulenin analogues 15a-b. A fourth class of cerulenin analogues was synthesized with the aromatic analogues 17a-e. This synthesis pathway started with the formation of the benzoyl acrylamides 16a-e from benzoylacrylic acid via a mixed anhydride which was prepared with isobutylchloroformate followed by the addition of the corresponding amine. Subsequent epoxidation with H2O2 in basic EtOH gave the aromatic cerulenin analogues 17a-e. Pharmacological testings for the synthesized substances were executed on efflux pump-resistant and -sensitive Candida albicans isolates, on the fatty acid synthesis enzyme KasA of Mycobacterium tuberculosis and on other organisms such as Leishmania major, Trypanosoma brucei brucei, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa within the Sonderforschungsbereich 630.
Background
Invasive fungal infections with Candida albicans (C. albicans) occur frequently in extremely low birthweight (ELBW) infants and are associated with poor outcome. Phagocytosis of C.albicans initializes apoptosis in monocytes (phagocytosis induced cell death, PICD). PICD is reduced in neonatal cord blood monocytes (CBMO).
Hypothesis
Phagocytosis of C. albicans causes PICD which differs between neonatal monocytes (CBMO) and adult peripheral blood monocytes (PBMO) due to lower stimulation of TLR-mediated immune responses.
Methods
The ability to phagocytose C. albicans, expression of TLRs, the induction of apoptosis (assessment of sub-G1 and nick-strand breaks) were analyzed by FACS. TLR signalling was induced by agonists such as lipopolysaccharide (LPS), Pam3Cys, FSL-1 and Zymosan and blocked (neutralizing TLR2 antibodies and MYD88 inhibitor).
Results
Phagocytic indices of PBMO and CBMO were similar. Following stimulation with agonists and C. albicans induced up-regulation of TLR2 and consecutive phosphorylation of MAP kinase P38 and expression of TNF-α, which were stronger on PBMO compared to CBMO (p < 0.005). Downstream, TLR2 signalling initiated caspase-3-dependent PICD which was found reduced in CBMO (p < 0.05 vs PBMO).
Conclusion
Our data suggest direct involvement of TLR2-signalling in C. albicans-induced PICD in monocytes and an alteration of this pathway in CBMO.
Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.