Using RNA-seq to determine the transcriptional landscape and the hypoxic response of the pathogenic yeast Candida parapsilosis

<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis </it>is one of the most common causes of <it>Candida </it>infection worldwide. However, the genome sequence annotation was made without experimental validation and little is known about the transcriptional landscape. The transcriptional response of <it>C. parapsilosis </it>to hypoxic (low oxygen) conditions, such as those encountered in the host, is also relatively unexplored.</p> <p>Results</p> <p>We used next generation sequencing (RNA-seq) to determine the transcriptional profile of <it>C. parapsilosis </it>growing in several conditions including different media, temperatures and oxygen concentrations. We identified 395 novel protein-coding sequences that had not previously been annotated. We removed > 300 unsupported gene models, and corrected approximately 900. We mapped the 5' and 3' UTR for thousands of genes. We also identified 422 introns, including two introns in the 3' UTR of one gene. This is the first report of 3' UTR introns in the Saccharomycotina. Comparing the introns in coding sequences with other species shows that small numbers have been gained and lost throughout evolution. Our analysis also identified a number of novel transcriptional active regions (nTARs). We used both RNA-seq and microarray analysis to determine the transcriptional profile of cells grown in normoxic and hypoxic conditions in rich media, and we showed that there was a high correlation between the approaches. We also generated a knockout of the <it>UPC2 </it>transcriptional regulator, and we found that similar to <it>C. albicans</it>, Upc2 is required for conferring resistance to azole drugs, and for regulation of expression of the ergosterol pathway in hypoxia.</p> <p>Conclusion</p> <p>We provide the first detailed annotation of the <it>C. parapsilosis </it>genome, based on gene predictions and transcriptional analysis. We identified a number of novel ORFs and other transcribed regions, and detected transcripts from approximately 90% of the annotated protein coding genes. We found that the transcription factor Upc2 role has a conserved role as a major regulator of the hypoxic response in <it>C. parapsilosis </it>and <it>C. albicans</it>.</p

Low Dosage of Histone H4 Leads to Growth Defects and Morphological Changes in Candida albicans

Chromatin function depends on adequate histone stoichiometry. Alterations in histone dosage affect transcription and chromosome segregation, leading to growth defects and aneuploidies. In the fungal pathogen Candida albicans, aneuploidy formation is associated with antifungal resistance and pathogenesis. Histone modifying enzymes and chromatin remodeling proteins are also required for pathogenesis. However, little is known about the mechanisms that generate aneuploidies or about the epigenetic mechanisms that shape the response of C. albicans to the host environment. Here, we determined the impact of histone H4 deficit in the growth and colony morphology of C. albicans. We found that C. albicans requires at least two of the four alleles that code for histone H4 (HHF1 and HHF22) to grow normally. Strains with only one histone H4 allele show a severe growth defect and unstable colony morphology, and produce faster-growing, morphologically stable suppressors. Segmental or whole chromosomal trisomies that increased wild-type histone H4 copy number were the preferred mechanism of suppression. This is the first study of a core nucleosomal histone in C. albicans, and constitutes the prelude to future, more detailed research on the function of histone H4 in this important fungal pathogen

Prediction of Phenotype-Associated Genes via a Cellular Network Approach: A Candida albicans Infection Case Study

Candida albicans is the most prevalent opportunistic fungal pathogen in humans causing superficial and serious systemic infections. The infection process can be divided into three stages: adhesion, invasion, and host cell damage. To enhance our understanding of these C. albicans infection stages, this study aimed to predict phenotype-associated genes involved during these three infection stages and their roles in C. albicans–host interactions. In light of the principles that proteins that lie closer to one another in a protein interaction network are more likely to have similar functions, and that genes regulated by the same transcription factors tend to have similar functions, a cellular network approach was proposed to predict the phenotype-associated genes in this study. A total of 4, 12, and 3 genes were predicted as adhesion-, invasion-, and damage-associated genes during C. albicans infection, respectively. These predicted genes highlight the facts that cell surface components are critical for cell adhesion, and that morphogenesis is crucial for cell invasion. In addition, they provide targets for further investigations into the mechanisms of the three C. albicans infection stages. These results give insights into the responses elicited in C. albicans during interaction with the host, possibly instrumental in identifying novel therapies to treat C. albicans infection

Pathway of Toll-Like Receptor 7/B Cell Activating Factor/B Cell Activating Factor Receptor Plays a Role in Immune Thrombocytopenia In Vivo

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by anti-platelet autoantibody-mediated platelet destruction. Antigen-presenting cell (APC) dysfunction is considered to play crucial roles in ITP. However, how APC affects autoreactive B cells in ITP is still unknown. Using a mouse model of immune thrombocytopenia, we demonstrated an increase in levels of TLR7 in splenic mononuclear cells (SMCs). Using both TLR7 agonist and TLR7 silencing lentivirus, we found stimulation of TLR7 decreased platelet counts and increased levels of platelet-associated IgG (PAIgG) in ITP mice, which correlates TLR7 with platelet destruction by autoantibodies. Levels of serum BAFF increased significantly in ITP mice and stimulation of TLR7 promoted secretion of BAFF. Among the three BAFF receptors, only BAFF receptor (BAFF-R) increased in ITP mice. However, activation of TLR7 showed no effect on the expression of BAFF receptors. These findings indicate that upregulation of TLR7 may augment BAFF secretion by APC and through ligation of BAFF-R promote autoreactive B cell survival and thus anti-platelet autoantibody production. The pathway of TLR7/BAFF/BAFF-R provides us with an explanation of how activation of APC affects autoantibody production by B cells in ITP and thus might provide a reasonable therapeutic strategy for ITP

Synovitis in osteoarthritis: current understanding with therapeutic implications

Modern concepts of osteoarthritis (OA) have been forever changed by modern imaging phenotypes demonstrating complex and multi-tissue pathologies involving cartilage, subchondral bone and (increasingly recognized) inflammation of the synovium. The synovium may show significant changes, even before visible cartilage degeneration has occurred, with infiltration of mononuclear cells, thickening of the synovial lining layer and production of inflammatory cytokines. The combination of sensitive imaging modalities and tissue examination has confirmed a high prevalence of synovial inflammation in all stages of OA, with a number of studies demonstrating that synovitis is related to pain, poor function and may even be an independent driver of radiographic OA onset and structural progression. Treating key aspects of synovial inflammation therefore holds great promise for analgesia and also for structure modification. This article will review current knowledge on the prevalence of synovitis in OA and its role in symptoms and structural progression, and explore lessons learnt from targeting synovitis therapeutically

Quantitative and Qualitative Stem Rust Resistance Factors in Barley Are Associated with Transcriptional Suppression of Defense Regulons

Stem rust (Puccinia graminis f. sp. tritici; Pgt) is a devastating fungal disease of wheat and barley. Pgt race TTKSK (isolate Ug99) is a serious threat to these Triticeae grain crops because resistance is rare. In barley, the complex Rpg-TTKSK locus on chromosome 5H is presently the only known source of qualitative resistance to this aggressive Pgt race. Segregation for resistance observed on seedlings of the Q21861 × SM89010 (QSM) doubled-haploid (DH) population was found to be predominantly qualitative, with little of the remaining variance explained by loci other than Rpg-TTKSK. In contrast, analysis of adult QSM DH plants infected by field inoculum of Pgt race TTKSK in Njoro, Kenya, revealed several additional quantitative trait loci that contribute to resistance. To molecularly characterize these loci, Barley1 GeneChips were used to measure the expression of 22,792 genes in the QSM population after inoculation with Pgt race TTKSK or mock-inoculation. Comparison of expression Quantitative Trait Loci (eQTL) between treatments revealed an inoculation-dependent expression polymorphism implicating Actin depolymerizing factor3 (within the Rpg-TTKSK locus) as a candidate susceptibility gene. In parallel, we identified a chromosome 2H trans-eQTL hotspot that co-segregates with an enhancer of Rpg-TTKSK-mediated, adult plant resistance discovered through the Njoro field trials. Our genome-wide eQTL studies demonstrate that transcript accumulation of 25% of barley genes is altered following challenge by Pgt race TTKSK, but that few of these genes are regulated by the qualitative Rpg-TTKSK on chromosome 5H. It is instead the chromosome 2H trans-eQTL hotspot that orchestrates the largest inoculation-specific responses, where enhanced resistance is associated with transcriptional suppression of hundreds of genes scattered throughout the genome. Hence, the present study associates the early suppression of genes expressed in this host–pathogen interaction with enhancement of R-gene mediated resistance

Cytokine preconditioning of engineered cartilage provides protection against interleukin-1 insult

Research reported in this publication was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R01AR60361, R01AR061988, P41EB002520). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. ART was supported by a National Science Foundation Graduate Fellowship

In Vivo Systematic Analysis of Candida albicans Zn2-Cys6 Transcription Factors Mutants for Mice Organ Colonization

The incidence of fungal infections in immuno-compromised patients increased considerably over the last 30 years. New treatments are therefore needed against pathogenic fungi. With Candida albicans as a model, study of host-fungal pathogen interactions might reveal new sources of therapies. Transcription factors (TF) are of interest since they integrate signals from the host environment and participate in an adapted microbial response. TFs of the Zn2-Cys6 class are specific to fungi and are important regulators of fungal metabolism. This work analyzed the importance of the C. albicans Zn2-Cys6 TF for mice kidney colonization. For this purpose, 77 Zn2-Cys6 TF mutants were screened in a systemic mice model of infection by pools of 10 mutants. We developed a simple barcoding strategy to specifically detect each mutant DNA from mice kidney by quantitative PCR. Among the 77 TF mutant strains tested, eight showed a decreased colonization including mutants for orf19.3405, orf19.255, orf19.5133, RGT1, UGA3, orf19.6182, SEF1 and orf19.2646, and four an increased colonization including mutants for orf19.4166, ZFU2, orf19.1685 and UPC2 as compared to the isogenic wild type strain. Our approach was validated by comparable results obtained with the same animal model using a single mutant and the revertant for an ORF (orf19.2646) with still unknown functions. In an attempt to identify putative involvement of such TFs in already known C. albicans virulence mechanisms, we determined their in vitro susceptibility to pH, heat and oxidative stresses, as well as ability to produce hyphae and invade agar. A poor correlation was found between in vitro and in vivo assays, thus suggesting that TFs needed for mice kidney colonization may involve still unknown mechanisms. This large-scale analysis of mice organ colonization by C. albicans can now be extended to other mutant libraries since our in vivo screening strategy can be adapted to any preexisting mutants

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis