Histoplasma capsulatum Encodes a Dipeptidyl Peptidase Active against the Mammalian Immunoregulatory Peptide, Substance P

The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42°C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P

A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of DiMIQ, a Potent Indolo[2,3-b]Quinoline Agent, against Candida albicans Biofilms

Candida albicans forms extremely drug-resistant biofilms, which present a serious threat to public health globally. Biofilm-based infections are difficult to treat due to the lack of efficient antifungal therapeutics, resulting in an urgent demand for the development of novel antibiofilm strategies. In this study, the antibiofilm activity of DiMIQ (5,11-dimethyl-5H-indolo[2,3-b]quinoline) was evaluated against C. albicans biofilms. DiMIQ is a synthetic derivative of indoquinoline alkaloid neocryptolepine isolated from a medicinal African plant, Cryptolepis sanguinolenta. Antifungal activity of DiMIQ was determined using the XTT assay, followed by cell wall and extracellular matrix profiling and cellular proteomes. Here, we demonstrated that DiMIQ inhibited C. albicans biofilm formation and altered fungal cell walls and the extracellular matrix. Cellular proteomics revealed inhibitory action against numerous translation-involved ribosomal proteins, enzymes involved in general energy producing processes and select amino acid metabolic pathways including alanine, aspartate, glutamate, valine, leucine and isoleucine. DiMIQ also stimulated pathways of cellular oxidation, metabolism of carbohydrates, amino acids (glycine, serine, threonine, arginine, phenylalanine, tyrosine, tryptophan) and nucleic acids (aminoacyl-tRNA biosynthesis, RNA transport, nucleotide metabolism). Our findings suggest that DiMIQ inhibits C. albicans biofilms by arresting translation and multidirectional pathway reshaping of cellular metabolism. Overall, this agent may provide a potent alternative to treating biofilm-associated Candida infections

Two Δ9-Stearic Acid Desaturases Are Required for \u3ci\u3eAspergillus nidulans\u3c/i\u3e Growth and Development

Unsaturated fatty acids are important constituents of all cell membranes and are required for normal growth. In the filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives also influence asexual (conidial) and sexual (ascospore) sporulation processes. To investigate the relationship between fatty acid metabolism and fungal development, we disrupted the A. nidulans sdeA and sdeB genes, both encoding Δ9-stearic acid desaturases responsible for the conversion of palmitic acid (16:0) and stearic acid (18:0) to palmitoleic acid (16:1) and oleic acid (18:1). The effects of sdeA deletion on development were profound, such that growth, conidial and ascospore production were all reduced at 22 and 37°C. Total fatty acid content was increased over 3-fold in the ΔsdeA strain, reflected in up-regulation of the expression of the fasA gene encoding the a chain of the fatty acid synthase, compared to wild type. Stearic acid accumulated approximately 3-fold compared to wild type in the ΔsdeA strain, while unsaturated fatty acid production was decreased. In contrast, disruption of sdeB reduced fungal growth and conidiation at 22°C, but did not affect these processes at 37°C compared to wild type. Interestingly, ascospore production was increased at 37°C for ΔsdeB compared to wild type. Total fatty acid content was not increased in this strain, although stearic acid accumulated 2-fold compared to wild type, and unsaturated fatty acid production was decreased. Combining the ΔsdeA and ΔsdeB alleles created a synthetic lethal strain requiring the addition of oleic acid to the medium for a modicum of growth. Taken together, our results suggest a role for sdeA in growth and development at all temperatures, while sdeB is involved in growth and development at lower temperatures

Activity and stability of purified recombinant HcDppIVA.

<p>A range of temperature (A) and pH (B) conditions were examined using substrate Gly-pro-AMC. To determine activity at different temperatures and pH, enzyme activity assays were performed at the indicated temperatures or pH values (squares). To determine stability, purified enzyme was incubated at the indicated condition for 30 minutes and subsequently assayed for remaining activity at pH 7, 37°C (triangles). The values in conditions with the highest activity were set at 100%. Error bars represent the variability between two experiments.</p