Cryptococcus at work: Gene expression during human infection

Meningitis is a frequent manifestation of infection due to Cryptococcus neoformans and a major cause of increased morbidity in patients with AIDS. Numerous in vitro gene expression and genetic studies of the fungus have predicted a myriad of genes, pathways, and biological processes that may be critical for pathogenesis, and many studies using animal models have supported the role of these processes during infection. However, the relevance of these hypotheses based on in vitro and animal models has often been questioned. A recent study by Chen et al. [Y. Chen, D. L. Toffaletti, J. L. Tenor, A. P. Litvintseva, C. Fang, T. G. Mitchell, T. R. McDonald, K. Nielsen, D. R. Boulware, T. Bicanic, and J. R. Perfect, mBio 5(1):e01087-13, 2014] represents an important step in understanding the cryptococcal response during human infection

Comparing the Similarity of Different Groups of Bacteria to the Human Proteome

Numerous aspects of the relationship between bacteria and human have been investigated. One aspect that has recently received attention is sequence overlap at the proteomic level. However, there has not yet been a study that comprehensively characterizes the level of sequence overlap between bacteria and human, especially as it relates to bacterial characteristics like pathogenicity, G-C content, and proteome size. In this study, we began by performing a general characterization of the range of bacteria-human similarity at the proteomic level, and identified characteristics of the most- and least-similar bacterial species. We then examined the relationship between proteomic similarity and numerous other variables. While pathogens and nonpathogens had comparable similarity to the human proteome, pathogens causing chronic infections were found to be more similar to the human proteome than those causing acute infections. Although no general correspondence between a bacterium’s proteome size and its similarity to the human proteome was noted, no bacteria with small proteomes had high similarity to the human proteome. Finally, we discovered an interesting relationship between similarity and a bacterium’s G-C content. While the relationship between bacteria and human has been studied from many angles, their proteomic similarity still needs to be examined in more detail. This paper sheds further light on this relationship, particularly with respect to immunity and pathogenicity

Synthesis and evaluation of troponoids as a new class of antibiotics

Novel antibiotics are urgently needed. The troponoids [tropones, tropolones, and α-hydroxytropolones (α-HT)] can have anti-bacterial activity. We synthesized or purchased 92 troponoids and evaluated their antibacterial activities against Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa. Preliminary hits were assessed for minimum inhibitory concentrations (MIC80) and cytotoxicity (CC50) against human hepatoma cells. Sixteen troponoids inhibited S. aureus/E. coli/A. baumannii growth by ≥80% growth at 50 values >50 μM. Two selected tropolones (63 and 285) inhibited 18 methicillin-resistant S. aureus (MRSA) strains with similar MIC80 values as against a reference strain. Two selected thiotropolones (284 and 363) inhibited multidrug-resistant (MDR) E. coli with MIC80 ≤30 μM. One α-HT (261) inhibited MDR-A. baumannii with MIC80 ≤30 μM. This study opens new avenues for development of novel troponoid antibiotics to address the critical need to combat MDR bacterial infections

Global transcriptome profile of Cryptococcus neoformans during exposure to hydrogen peroxide induced oxidative stress

The ability of the opportunistic fungal pathogen Cryptococcus neoformans to resist oxidative stress is one of its most important virulence related traits. To cope with the deleterious effect of cellular damage caused by the oxidative burst inside the macrophages, C. neoformans has developed multilayered redundant molecular responses to neutralize the stress, to repair the damage and to eventually grow inside the hostile environment of the phagosome. We used microarray analysis of cells treated with hydrogen peroxide (H(2)O(2)) at multiple time points in a nutrient defined medium to identify a transcriptional signature associated with oxidative stress. We discovered that the composition of the medium in which fungal cells were grown and treated had a profound effect on their capacity to degrade exogenous H(2)O(2). We determined the kinetics of H(2)O(2) breakdown by growing yeast cells under different conditions and accordingly selected an appropriate media composition and range of time points for isolating RNA for hybridization. Microarray analysis revealed a robust transient transcriptional response and the intensity of the global response was consistent with the kinetics of H(2)O(2) breakdown by treated cells. Gene ontology analysis of differentially expressed genes related to oxidation-reduction, metabolic process and protein catabolic processes identified potential roles of mitochondrial function and protein ubiquitination in oxidative stress resistance. Interestingly, the metabolic pathway adaptation of C. neoformans to H(2)O(2) treatment was remarkably distinct from the response of other fungal organisms to oxidative stress. We also identified the induction of an antifungal drug resistance response upon the treatment of C. neoformans with H(2)O(2). These results highlight the complexity of the oxidative stress response and offer possible new avenues for improving our understanding of mechanisms of oxidative stress resistance in C. neoformans

Prospects for personalizing antiviral therapy for hepatitis C virus with pharmacogenetics

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide. HCV infection is currently treated with IFNα plus ribavirin for 24 to 48 weeks. This demanding therapy fails in up to 50% of patients, so the use of pharmacogenetic biomarkers to predict the outcome of treatment would reduce futile treatment of non-responders and help identify patients in whom therapy would be justified. Both IFNα and ribavirin primarily act by modulating the immune system of the patient, and HCV uses multiple mechanisms to counteract the antiviral effects stimulated by therapy. Therefore, response to therapy is influenced by variations in human genes governing the immune system and by differences in HCV genes that blunt antiviral immune responses. This article summarizes recent advances in understanding how host and viral genetic variation affect outcome of therapy. The most notable human associations are polymorphisms within the IL28B gene, but variations in human leukocyte antigen and cytokine genes have also been associated with treatment outcome. The most prominent viral genetic association with outcome of therapy is that HCV genotype 1 is much less sensitive to treatment than genotypes 2 and 3, but genetic differences below the genotype level also influence outcome of therapy, presumably by modulating the ability of viral genes to blunt antiviral immune responses. Pharmacogenetic prediction of the outcome of IFN-based therapy for HCV will require integrating the efficacies of the immunosuppressive mechanisms of a viral isolate, and then interpreting the viral resistance potential in context of the genetic profile of the patient at loci associated with outcome of therapy. Direct-acting inhibitors of HCV that will be used in combination with IFNα are nearing approval, so genetic prediction for anti-HCV therapy will soon need to incorporate viral genetic markers of viral resistance to the new drugs

Membrane integrity contributes to resistance of Cryptococcus neoformans to the cell wall inhibitor caspofungin

The fungal pathogen Cryptococcus neoformans causes up to 278 000 infections each year globally, resulting in up to 180,000 deaths annually, mostly impacting immunocompromised people. Therapeutic options for C. neoformans infections are very limited. Caspofungin, a member of the echinocandin class of antifungals, is generally well tolerated but clinically ineffective against C. neoformans. We sought to identify biological processes that can be targeted to render the cell more susceptible to echinocandins by screening the available libraries of gene deletion mutants made in the KN99α background for caspofungin sensitivity. We adapted a Candida albicans fungal biofilm assay for the growth characteristics of C. neoformans and systematically screened 4,030 individual gene deletion mutants in triplicate plate assays. We identified 25 strains that showed caspofungin sensitivity. We followed up with a dose dependence assay, and 17 of the 25 were confirmed sensitive, 5 of which were also sensitive in an agar plate assay. We made new deletion mutant strains for four of these genes

The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformans IMPORTANCE Cryptococcus neoformans is a fungal pathogen that kills approximately 200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents

Cross talk between the Cell Wall Integrity and Cyclic AMP/Protein Kinase A pathways in Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis

Contribution of Genome-Wide HCV Genetic Differences to Outcome of Interferon-Based Therapy in Caucasian American and African American Patients

Background: Hepatitis C virus (HCV) has six major genotypes, and patients infected with genotype 1 respond less well to interferon-based therapy than other genotypes. African American patients respond to interferon α-based therapy at about half the rate of Caucasian Americans. The effect of HCV's genetic variation on treatment outcome in both racial groups is poorly understood. Methodology:We determined the near full-length pre-therapy consensus sequences from 94 patients infected with HCV genotype 1a or 1b undergoing treatment with peginterferon α-2a and ribavirin through the Virahep-C study. The sequences were stratified by genotype, race and treatment outcome to identify HCV genetic differences associated with treatment efficacy. Principal Findings:HCV sequences from patients who achieved sustained viral response were more diverse than sequences from non-responders. These inter-patient diversity differences were found primarily in the NS5A gene in genotype 1a and in core and NS2 in genotype 1b. These differences could not be explained by host selection pressures. Genotype 1b but not 1a African American patients had viral genetic differences that correlated with treatment outcome. Conclusions & Significance: Higher inter-patient viral genetic diversity correlated with successful treatment, implying that there are HCV genotype 1 strains with intrinsic differences in sensitivity to therapy. Core, NS3 and NS5A have interferonsuppressive activities detectable through in vitro assays, and hence these activities also appear to function in human patients. Both preferential infection with relatively resistant HCV variants and host-specific factors appear to contribute to the unusually poor response to therapy in African American patients. © 2010 Donlin et al

ConDeTri - A Content Dependent Read Trimmer for Illumina Data

During the last few years, DNA and RNA sequencing have started to play an increasingly important role in biological and medical applications, especially due to the greater amount of sequencing data yielded from the new sequencing machines and the enormous decrease in sequencing costs. Particularly, Illumina/Solexa sequencing has had an increasing impact on gathering data from model and non-model organisms. However, accurate and easy to use tools for quality filtering have not yet been established. We present ConDeTri, a method for content dependent read trimming for next generation sequencing data using quality scores of each individual base. The main focus of the method is to remove sequencing errors from reads so that sequencing reads can be standardized. Another aspect of the method is to incorporate read trimming in next-generation sequencing data processing and analysis pipelines. It can process single-end and paired-end sequence data of arbitrary length and it is independent from sequencing coverage and user interaction. ConDeTri is able to trim and remove reads with low quality scores to save computational time and memory usage during de novo assemblies. Low coverage or large genome sequencing projects will especially gain from trimming reads. The method can easily be incorporated into preprocessing and analysis pipelines for Illumina data