Discovery of isonucleotidic CDNs as potent STING agonists with immunomodulatory potential.

Stimulator of interferon genes (STING) is an adaptor protein of the cGAS-STING signaling pathway involved in the sensing of cytosolic DNA. It functions as a receptor for cyclic dinucleotides (CDNs) and, upon their binding, mediates cytokine expression and host immunity. Besides naturally occurring CDNs, various synthetic CDNs, such as ADU-S100, have been reported to effectively activate STING and are being evaluated in clinical trials for the treatment of cancer. Here, we describe the preparation of a unique new class of STING agonists: isonucleotidic cyclic dinucleotides and the synthesis of their prodrugs. The presented CDNs stimulate STING with comparable efficiency to ADU-S100, whereas their prodrugs demonstrate activity up to four orders of magnitude better due to the improved cellular uptake. The compounds are very potent inducers of inflammatory cytokines by peripheral blood mononuclear cells (PBMCs). We also report the X-ray crystal structure of the lead inhibitor bound to the wild-type (WT) STING.

Enzymatic Preparation of 2'-5',3'-5'-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB (IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling cascades. In this work, we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human, mouse, and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next, we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally, we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.

LC/MS analysis and deep sequencing reveal the accurate RNA composition in the HIV-1 virion.

The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.

Azole drug import into the pathogenic fungus Aspergillus fumigatus.

The fungal pathogen Aspergillus fumigatus causes serious illness and often death when it invades tissues, especially in immunocompromised individuals. The azole class of drugs is the most commonly prescribed treatment for many fungal infections and acts on the ergosterol biosynthesis pathway. One common mechanism of acquired azole drug resistance in fungi is the prevention of drug accumulation to toxic levels in the cell. While drug efflux is a well-known resistance strategy, reduced azole import would be another strategy to maintain low intracellular azole levels. Recently, azole uptake in Candida albicans and other yeasts was analyzed using [(3)H]fluconazole. Defective drug import was suggested to be a potential mechanism of drug resistance in several pathogenic fungi, including Cryptococcus neoformans, Candida krusei, and Saccharomyces cerevisiae. We have adapted and developed an assay to measure azole accumulation in A. fumigatus using radioactively labeled azole drugs, based on previous work done with C. albicans. We used this assay to study the differences in azole uptake in A. fumigatus isolates under a variety of drug treatment conditions, with different morphologies and with a select mutant strain with deficiencies in the sterol uptake and biosynthesis pathway. We conclude that azole drugs are specifically selected and imported into the fungal cell by a pH- and ATP-independent facilitated diffusion mechanism, not by passive diffusion. This method of drug transport is likely to be conserved across most fungal species.

Transcription factor Efg1 shows a haploinsufficiency phenotype in modulating the cell wall architecture and immunogenicity of Candida albicans.

The Candida albicans transcription factor Efg1 is known to be involved in many different cellular processes, including morphogenesis, general metabolism, and virulence. Here we show that besides its manifold roles, Efg1 also has a prominent effect on cell wall structure and composition, strongly affecting the structural glucan part. Deletion of only one allele of EFG1 already results in severe phenotypes for cell wall biogenesis, comparable to those with deletion of both alleles, indicative of a severe haploinsufficiency for EFG1. The observed defects in structural setup of the cell wall, together with previously reported alterations in expression of cell surface proteins, result in altered immunogenic properties of strains with compromised Efg1 function. This is shown by interaction studies with macrophages and primary dendritic cells. The structural changes in the cell wall carbohydrate meshwork presented here, together with the manifold changes in cell wall protein composition and metabolism reported in other studies, contribute to the altered immune response mounted by innate immune cells and to the altered virulence phenotypes observed for strains lacking EFG1.

Adaptation, adhesion and invasion during interaction of Candida albicans with the host--focus on the function of cell wall proteins.

Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review.

PHR1, a pH-regulated gene of Candida albicans encoding a glucan-remodelling enzyme, is required for adhesion and invasion.

The fungal cell wall plays a crucial role in host-pathogen interactions. Its formation is the result of the coordinated activity of several extracellular enzymes, which assemble the constituents, and remodel and hydrolyse them in the extracellular space. Candida albicans Phr1 and Phr2 proteins belong to family GH72 of the beta-(1,3)-glucanosyltransferases and play a crucial role in cell wall assembly. PHR1 and PHR2, homologues of Saccharomyces cerevisiae GAS1, are differently regulated by extracellular pH. PHR1 is expressed when ambient pH is 5.5 or higher, whereas PHR2 has the reverse expression pattern. Their deletion causes a pH-conditional defect in morphogenesis and virulence. In this work we explored whether PHR1 deletion affects the ability of C. albicans to adhere to and invade human epithelia. PHR1 null mutants exhibited a marked reduction in adhesion to both abiotic surfaces and epithelial cell monolayers. In addition, the mutant was unable to penetrate and invade reconstituted human epithelia. Transcription profiling of selected hyphal-specific and adhesin-encoding genes indicated that in the PHR1 null mutant, HWP1 and ECE1 transcript levels were similarly reduced in both adhesion and suspension conditions. These results, combined with microscopy analysis of the septum position, suggest that PHR1 is not required for the induction of hyphal development but plays a key role in the maintenance of hyphal growth. Thus, the beta-(1,3)-glucan processing catalysed by Phr1p is of fundamental importance in the maintenance of the morphological state on which the adhesive and invasive properties of C. albicans greatly depend.