Maureen J. Donlin

Chitosan, the Deacetylated Form of Chitin, Is Necessary for Cell Wall Integrity in Cryptococcus neoformans

ABSTRACT Cryptococcus neoformans is an opportunistic fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. The fungal cell wall is an excellent target for antifungal therapies as it is an essential organelle that provides cell structure and integrity, it is needed for the localization or attachment of known virulence factors, including the polysaccharide capsule, melanin, and phospholipase, and it is critical for host-pathogen interactions. In C. neoformans, chitosan produced by the enzymatic removal of acetyl groups from nascent chitin polymers has been implicated as an important component of the vegetative cell wall. In this study, we identify four putative chitin/polysaccharide deacetylases in C. neoformans. We have demonstrated that three of these deacetylases, Cda1, Cda2, and Cda3, can account for all of the chitosan produced during vegetative growth in culture, but the function for one, Fpd1, remains undetermined. The data suggest a model for chitosan production in vegetatively growing C. neoformans where the three chitin deacetylases convert chitin generated by the chitin synthase Chs3 into chitosan. Utilizing a collection of chitin/polysaccharide deacetylase deletion strains, we determined that during vegetative growth, chitosan helps to maintain cell integrity and aids in bud separation. Additionally, chitosan is necessary for maintaining normal capsule width and the lack of chitosan results in a “leaky melanin” phenotype. Our analysis indicates that chitin deacetylases and the chitosan made by them may prove to be excellent antifungal targets.

Using the Generic Genome Browser (GBrowse)

A genome browser is software that allows users to visualize DNA, protein, or other sequence features within the context of a reference sequence, such as a chromosome or contig. The Generic Genome Browser (GBrowse) is an open‐source browser developed as part of the Generic Model Organism Database project (Stein et al., 2002). GBrowse can be configured to display genomic sequence features for any organism and is the browser used for the model organisms Drosophila melanogaster (Grumbling and Strelets, 2006) and Caenorhabditis elegans (Schwarz et al., 2006), among others. The software package can be downloaded from the Web and run on a Windows, Mac OS X, or Unix‐type system. Version 1.64, as described in the original protocol, was released in November 2005, but the software is under active development and new versions are released about every six months. This update includes instructions on updating existing data sources with new files from NCBI. Curr. Protoc. Bioinform. 28:9.9.1‐9.9.25.

A Chitin Synthase and Its Regulator Protein Are Critical for Chitosan Production and Growth of the Fungal Pathogen Cryptococcus neoformans

ABSTRACT Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30°C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37°C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Δ and the csr2Δ mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.

Pretreatment Sequence Diversity Differences in the Full-Length Hepatitis C Virus Open Reading Frame Correlate with Early Response to Therapy

ABSTRACT Pegylated alpha interferon and ribavirin therapy for hepatitis C virus (HCV) genotype 1 infection fails for half of Caucasian American patients (CA) and more often for African Americans (AA). The reasons for these low response rates are unknown. HCV is highly genetically variable, but it is unknown how this variability affects response to therapy. To assess effects of viral diversity on response to therapy, the complete pretreatment genotype 1 HCV open reading frame was sequenced using samples from 94 participants in the Virahep-C study. Sequences from patients with >3.5 log declines in viral RNA levels by day 28 (marked responders) were more variable than those from patients with declines of <1.4 log (poor responders) in NS3 and NS5A for genotype 1a and in core and NS3 for genotype 1b. These correlations remained when all T-cell epitopes were excluded, indicating that these differences were not due to differential immune selection. When the sequences were compared by race of the patients, higher diversity in CA patients was found in E2 and NS2 but only for genotype 1b. Core, NS3, and NS5A can block the action of alpha interferon in vitro; hence, these genetic patterns are consistent with multiple amino acid variations independently impairing the function of HCV proteins that counteract interferon responses in humans, resulting in HCV strains with variable sensitivity to therapy. No evidence was found for novel HCV strains in the AA population, implying that AA patients may be infected with a higher proportion of the same resistant strains that are found in CA patients.

Cell wall integrity is dependent on the PKC1 signal transduction pathway in Cryptococcus neoformans

Cell wall biogenesis and integrity are crucial for fungal growth, pathogenesis and survival, and are attractive targets for antifungal therapy. In this study, we identify, delete and analyse mutant strains for 10 genes involved in the PKC1 signal transduction pathway and its regulation in Cryptococcus neoformans. The kinases Bck1 and Mkk2 are critical for maintaining integrity, and deletion of each of these causes severe phenotypes different from each other. In stark contrast to results seen in Saccharomyces cerevisiae, a deletion in LRG1 has severe repercussions for the cell, and one in ROM2 has little effect. Also surprisingly, the phosphatase Ppg1 is crucial for cell integrity. These data indicate that the mechanisms of maintaining cell integrity differ between the two fungi. Deletions in SSD1 and PUF4, potential alternative regulators of cell integrity, also exhibit phenotypes. This is the first comprehensive analysis examining genes involved the maintenance of cell integrity in C. neoformans and sets the foundation for future biochemical and virulence studies.

Genome-wide hepatitis C virus amino acid covariance networks can predict response to antiviral therapy in humans

Hepatitis C virus (HCV) is a common RNA virus that causes hepatitis and liver cancer. Infection is treated with IFN-alpha and ribavirin, but this expensive and physically demanding therapy fails in half of patients. The genomic sequences of independent HCV isolates differ by approximately 10%, but the effects of this variation on the response to therapy are unknown. To address this question, we analyzed amino acid covariance within the full viral coding region of pretherapy HCV sequences from 94 participants in the Viral Resistance to Antiviral Therapy of Chronic Hepatitis C (Virahep-C) clinical study. Covarying positions were common and linked together into networks that differed by response to therapy. There were 3-fold more hydrophobic amino acid pairs in HCV from nonresponding patients, and these hydrophobic interactions were predicted to contribute to failure of therapy by stabilizing viral protein complexes. Using our analysis to detect patterns within the networks, we could predict the outcome of therapy with greater than 95% coverage and 100% accuracy, raising the possibility of a prognostic test to reduce therapeutic failures. Furthermore, the hub positions in the networks are attractive antiviral targets because of their genetic linkage with many other positions that we predict would suppress evolution of resistant variants. Finally, covariance network analysis could be applicable to any virus with sufficient genetic variation, including most human RNA viruses.

Baby, Be Safe: the effect of tailored communications for pediatric injury prevention provided in a primary care setting

Injuries are a major cause of morbidity and mortality to young children. The provision of individually tailored educational materials in primary care settings may be an effective and efficient way to promote adoption of injury prevention measures by parents. A randomized controlled study compared the effectiveness of tailored and generic persuasive communications delivered in a primary care setting on the adoption of home and car safety behaviors. During routine well-child visits, a primarily African-American sample of parents of children ages 6-20 months (n=213) was randomized to receive either tailored or generic information regarding the prevention of injuries to their child. At follow-up, participants who received tailored information reported greater adoption of home and car safety behaviors than those receiving generic information. In addition, within the tailored information group, those who discussed the information with their physician showed significantly greater change than those who did not. However, this difference was not observed among those receiving generic information. Findings support the use of office-based tailored injury prevention education as a component of routine well-child care.

Posttranslational, Translational, and Transcriptional Responses to Nitric Oxide Stress in Cryptococcus neoformans: Implications for Virulence

ABSTRACT The ability of the fungal pathogen Cryptococcus neoformans to evade the mammalian innate immune response and cause disease is partially due to its ability to respond to and survive nitrosative stress. In this study, we use proteomic and genomic approaches to elucidate the response of C. neoformans to nitric oxide stress. This nitrosative stress response involves both transcriptional, translational, and posttranslational regulation. Proteomic and genomic analyses reveal changes in expression of stress response genes. In addition, genes involved in cell wall organization, respiration, signal transduction, transport, transcriptional control, and metabolism show altered expression under nitrosative conditions. Posttranslational modifications of transaldolase (Tal1), aconitase (Aco1), and the thiol peroxidase, Tsa1, are regulated during nitrosative stress. One stress-related protein up-regulated in the presence of nitric oxide stress is glutathione reductase (Glr1). To further investigate its functional role during nitrosative stress, a deletion mutant was generated. We show that this glr1Δ mutant is sensitive to nitrosative stress and macrophage killing in addition to being avirulent in mice. These studies define the response to nitrosative stress in this important fungal pathogen.

Effectiveness of Individually Tailored Calendars in Promoting Childhood Immunization in Urban Public Health Centers

OBJECTIVESnWe examined the effectiveness of tailored calendars in increasing childhood immunization rates.nnnMETHODSnParents of babies aged birth to 1 year (n = 321) received individually tailored calendars promoting immunization from 2 urban public health centers. For each baby, an age- and sex-matched control was selected from the same center. Immunization status was tracked through age 24 months.nnnRESULTSnA higher proportion of intervention than of control babies were up to date at the end of a 9-month enrollment period (82% vs 65%, P <.001) and at age 24 months (66% vs 47%, P <.001). The younger the babys age at enrollment in the program, the greater was the intervention effect.nnnCONCLUSIONSnTailored immunization calendars can help increase child immunization rates.

Hepatitis C virus diversity and evolution in the full open-reading frame during antiviral therapy.

Background Pegylated interferon plus ribavirin therapy for hepatitis C virus (HCV) fails in approximately half of genotype 1 patients. Treatment failure occurs either by nonresponse (minimal declines in viral titer) or relapse (robust initial responses followed by rebounds of viral titers during or after therapy). HCV is highly variable genetically. To determine if viral genetic differences contribute to the difference between response and relapse, we examined the inter-patient genetic diversity and mutation pattern in the full open reading frame HCV genotype 1a consensus sequences. Methodology/Principal Findings Pre- and post-therapy sequences were analyzed for 10 nonresponders and 10 relapsers from the Virahep-C clinical study. Pre-therapy interpatient diversity among the relapsers was higher than in the nonresponders in the viral NS2 and NS3 genes, and post-therapy diversity was higher in the relapsers for most of HCVs ten genes. Pre-therapy diversity among the relapsers was intermediate between that of the non-responders and responders to therapy. The average mutation rate was just 0.9% at the amino acid level and similar numbers of mutations occurred in the nonresponder and relapser sequences, but the mutations in NS2 of relapsers were less conservative than in nonresponders. Finally, the number and distribution of regions under positive selection was similar between the two groups, although the nonresponders had more foci of positive selection in E2. Conclusions/Significance The HCV sequences were unexpectedly stable during failed antiviral therapy, both nonresponder and relapser sequences were under selective pressure during therapy, and variation in NS2 may have contributed to the difference in response between the nonresponder and relapser groups. These data support a role for viral genetic variability in determining the outcome of anti-HCV therapy, with those sequences that are more distant from an optimal sequence being less able to resist the pressures of interferon-based therapy. Trial registration ClinicalTrials.gov NCT00038974