Insights into Onchocerca volvulus population biology through multilocus immunophenotyping

We have developed a serologically based immunophenotyping approach to study Onchocerca volvulus (Ov) population diversity. Using genomic sequence data and polymerase chain reaction-based genotyping, we identified nonsynonymous single-nucleotide polymorphisms (SNPs) in the genes of 16 major immunogenic Ov proteins: Ov-CHI-1/Ov-CHI-2, Ov16, Ov-FAR-1, Ov-CPI-1, Ov-B20, Ov-ASP-1, Ov-TMY-1, OvSOD1, OvGST1, Ov-CAL-1, M3/M4, Ov-RAL-1, Ov-RAL-2, Ov-ALT-1, Ov-FBA-1, and Ov-B8. We assessed the immunoreactivity of onchocerciasis patient sera (n = 152) from the Americas, West Africa, Central Africa, and East Africa against peptides derived from 10 of these proteins containing SNPs. Statistically significant variation in immunoreactivity among the regions was seen in SNP-containing peptides derived from 8 of 10 proteins tested: OVOC1192(1-15), OVOC9988(28-42), OVOC9225(320-334), OVOC7453(22-36), OVOC11517(14-28), OVOC3177(283-297), OVOC7911(594-608), and OVOC12628(174-188). Our data show that differences in immunoreactivity to variant antigenic peptides may be used to characterize Ov populations, thereby elucidating features of Ov population biology previously inaccessible because of the limited availability of parasite material

Portrait of Candida albicans Adherence Regulators

Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans

Requirement for Candida albicans Sun41 in Biofilm Formation and Virulence▿

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41, Orf19.5412, Orf19.1277, MSB2, Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41Δ/sun41Δ deletion mutants and sun41Δ/sun41Δ+pSUN41-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41Δ/sun41Δ mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41Δ/sun41Δ cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target

Portrait of Candida albicans adherence regulators.

<p>Cell-substrate adherence is a fundamental property of microorganisms that enables them to exist in biofilms. Our study focuses on adherence of the fungal pathogen Candida albicans to one substrate, silicone, that is relevant to device-associated infection. We conducted a mutant screen with a quantitative flow-cell assay to identify thirty transcription factors that are required for adherence. We then combined nanoString gene expression profiling with functional analysis to elucidate relationships among these transcription factors, with two major goals: to extend our understanding of transcription factors previously known to govern adherence or biofilm formation, and to gain insight into the many transcription factors we identified that were relatively uncharacterized, particularly in the context of adherence or cell surface biogenesis. With regard to the first goal, we have discovered a role for biofilm regulator Bcr1 in adherence, and found that biofilm regulator Ace2 is a major functional target of chromatin remodeling factor Snf5. In addition, Bcr1 and Ace2 share several target genes, pointing to a new connection between them. With regard to the second goal, our findings reveal existence of a large regulatory network that connects eleven adherence regulators, the zinc-response regulator Zap1, and approximately one quarter of the predicted cell surface protein genes in this organism. This limited yet sensitive glimpse of mutant gene expression changes had thus defined one of the broadest cell surface regulatory networks in C. albicans.</p

Gene expression profiles of adherence mutants.

<p>Panel A. Hierarchical clustering of gene expression data. NanoString expression data (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s004" target="_blank">Table S2</a>) were analyzed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#s4" target="_blank">Methods</a>. Briefly, averages of three independent determinations for each mutant strain were divided by averages of six independent determinations of the reference wild-type strain DAY185 to obtain the fold change values for each of 293 genes. All mutant strains were insertion homozygotes except for <i>ace2, arg81, crz2, zap1,</i> and <i>zfu2</i>, which were deletion homozygotes. Transcription factor mutants with adherence defects are indicated with underlined gene names; the remaining mutants were controls included for comparison. Color scale limits were set at (−2.0, 0.0, 2.0), so that the brightest yellow represents 4 fold up-regulation compared to wild-type, and the brightest blue represents 4 fold down-regulation. We define the clusters by representative genes. HYVIR: over 50% of the genes in this cluster are known to play roles in <u>hy</u>phal growth or <u>vir</u>ulence. RAM: top targets of Ace2 (<u>R</u>egulation of <u>A</u>ce2 and polarized <u>m</u>orphogenesis), which are also regulated by Cbk1, Snf5, Cas5, Bcr1, and Met4. ZAPT: known Zap1 targets. CSTAR: <u>C</u>ell <u>s</u>urface <u>t</u>argets of <u>a</u>dherence <u>r</u>egulators. Additional small clusters of co-regulated genes did not have unifying functional or structural features. Panel B. Summary of regulatory relationships among the 30 adherence regulators, Zap1, and the four clusters of target genes defined in panel 2A. Black circles: target gene clusters. Yellow circles: transcription factors. Yellow circles with black border: adherence regulators whose defects in adherence can be rescued by <i>ZAP1</i> overexpression. Blue lines: negative regulation for at least 2/3 of the target genes in the cluster. Orange lines: positive regulation for at least 2/3 of the target genes in the cluster.</p

Adherence of wild-type and mutant strains.

<p>Adherence to silicone was measured in a Fluxion flow cell as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#s4" target="_blank">Methods</a>, and is expressed relative to the wild-type reference strain. Panel A. Transcription factor insertion mutants. The mutants presented had statistically significant decreases (p value ≤0.05) in adherence when compared to reference strain DAY286. The <i>zap1Δ/Δ</i> mutant is included for reference. Measurements indicate mean and standard deviation for 1–3 isolates, as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s003" target="_blank">Table S1</a> worksheet 1A. Panel B. Analysis of Bcr1 and its target genes. The wild-type strain DAY185 was used as a standard for comparison to mutants <i>bcr1Δ/Δ</i> (CJN702), <i>bcr1Δ/Δ+pBCR1</i> (CJN698), <i>hwp1Δ/Δ</i> (CAH7-1A1E2), <i>als3Δ/Δ</i> (CAYF178U), <i>als1Δ/Δ</i> (CAYC2YF1U), <i>bcr1Δ/Δ+ALS1-OE</i> (CJN1144), and <i>als1Δ/Δ+pALS1</i> (CAYC1). Asterisks indicate statistically significant decreases in adherence compared to the wild-type strain.</p

Summary of adherence mutant properties.

<p>Footnotes:</p>a<p>These columns list each mutant according to the mutated gene (orf19 numbers and gene names).</p>b<p><i>S. cerevisiae</i> orthologs or best hits, or transcription factor classes, are indicated as listed in the Candida Genome Database.</p>c<p>Column that indicates whether a deletion transcription factor mutant was available for adherence testing. All deletion mutants were created in the SN152 parent strain as described in Homann et al. 2009.</p>d<p>Column that indicates whether a deletion transcription factor mutant was created and test for adherence. Strains were created in the BWP17 background and genotypes are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s006" target="_blank">Table S4</a>.</p>e<p>These columns list the relative adherence for each mutant strain, and for each mutant strain derivative that carries the <i>ZAP1-OE</i> allele. The complete dataset for adherence measurements is in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s003" target="_blank">Table S1</a>. All of the mutants and <i>ZAP1-OE</i> strains were insertion homozygotes except for <i>ace2, arg81, bcr1, crz2, snf5,</i> and <i>zfu2</i>, which were deletion homozygotes.</p>f<p>This column lists the number of genes that were differentially expressed in each mutant compared to the wild-type control strain DAY185, as indicated by nanoString profiling. A cutoff p value of 0.05 was applied. Complete data and p values are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s004" target="_blank">Table S2</a>.</p

Functional relationship between Snf5 and Ace2.

<p>Strains indicated at the top of each column include <i>SNF5/SNF5</i> (DAY185), <i>snf5Δ/Δ</i> (DHY02), <i>snf5Δ/Δ+pSNF5</i> (DHY8), and <i>snf5Δ/Δ+ACE2-OE</i> (DHY20). Panel A. Adherence and biofilm formation assays. Each strain was assayed for adherence, biofilm formation in vitro (48 hr biomass measurements and 24 hr confocal imaging assays), and 24 hr biofilm formation in vivo (catheter lumen surfaces imaged via scanning electron microscopy at 50× or 1000× magnification as indicated). Panel B. Pleiotropic phenotypic assays. Yeast cells were visualized to assess aggregation after 8 hr growth (mid-exponential phase) in YPD at 30°C. Hypha formation visualized after 4 hr of growth in Spider medium at 37°C. Cell wall inhibitor sensitivity was measured by spot dilution assays: overnight cultures were serially diluted five-fold from left to right and assayed for growth on YPD, YPD+200 µg/ml Congo Red and YPD+62.5 µg/ml caspofungin after 48 hours at 30°C.</p

Portrait of <i>C. albicans</i> adherence regulators.

<p>Our main findings are summarized with transcription factors (blue boxes) connected to cell surface genes (green boxes) and the target process of adherence. Bcr1 promotes adherence through stimulation of <i>ALS1</i> expression. Snf5 promotes adherence through stimulation of <i>ACE2</i> expression. Try2, Try3, Try4, Try5, Suc1, Fgr27, Zcf28, and Uga33 are required for adherence and required for the expression of CSTAR genes. CSTAR gene products include numerous predicted cell wall proteins; we hypothesize that many CSTAR gene products mediate adherence. Zap1 is also a positive regulator of CSTAR genes, but it is not required for adherence in our assays. Ada2, Met4, and Try6 are negative regulators of many CSTAR genes. Finally, many transcription factors are required for adherence (Arg81, Cas5, Czf1, Crz2, Dal81, Fcr3, Leu3, Not3, Taf14, War1, Znc1, Zfu2, Zcf8, Zcf31, Zcf34, Zcf39), and govern expression of one or several classes of genes (summarized in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat-1002525-g002" target="_blank">Figure 2B</a>), but cannot be connected to specific functional targets.</p

Expression of <i>ZAP1</i> and novel Zap1 dependent genes.

<p>Strains were grown in Spider medium for 8 hr at 37°C and QRTPCR assays were used to determine RNA levels for of <i>ZAP1, ORF19.4652, PGA39</i> and <i>QDR1.</i> RNA levels were normalized to control <i>TDH3</i> RNA and then expressed as relative units compared to each RNA in the wild-type strain. Strains included wild type (DAY185), <i>zap1Δ/Δ</i> (CJN1201), <i>zcf28Δ/Δ</i> (JF144), <i>zcf28Δ/Δ+ZAP1-OE</i> (JFY261), <i>try2−/−</i> (EHY97), <i>try2Δ/Δ+ZAP1-OE</i> (JFY337), <i>try3−/−</i> (EHY30), and <i>try3−/−+ZAP1-OE</i> (JFY251).</p