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

Heather Taff, Kora Bongen, Garry Kennebrew, Jr., Amy Peterson, and John Stark

Four members of the class of 2004 speak to their experiences at IMSA in an interview recorded at their 10-year reunion. Heather Taff recalls that she heard about IMSA when her parents came home with information about it. Kora Bongen says her parents wanted to send her to boarding school and she said no, thinking they meant in England, only to later realize they actually meant IMSA. Garry Kennebrew, Jr. says he and his parents were always on the lookout for other educational opportunities and they had neighbors who had sent children to IMSA, which is how he found out about it. Amy Peterson was a \u27shmen - she had done some of the summer programs in middle school and then she applied in eighth grade. In terms of residential life and extracurriculars, Taff recalls being the only person on the women\u27s junior varsity tennis team who had ever played any tennis before, a novel experience compared to her home high school where you had to actually be good at sports to play! Bongen joined the fine arts club, because she wanted to try something different. Kennebrew had played football before coming to IMSA, but instead joined the soccer team and ran track. He also joined the acapella group, Mod 21, as the beatboxer. Peterson didn\u27t play sports, but managed the boys soccer and basketball teams. She also participated in the yearbook club. They all participated in work service during their time at IMSA as well - in the music department, the college and academic counseling office, and in the library. Kennebrew has particularly fond memories of the library staff. Kennebrew first came to IMSA for the Excel program and his IMSA memories start there with his roommate. Bongen also recalls meeting her first roommate sophomore year, and while they didn\u27t stay roommates, they ended up being good friends. Taff and her roommate lived together all three years, though by senior year it got rather messy. They also speak about pranks students would play on each other. On the academic side of things, they recall the teachers all having a lot of character and not being afraid to challenge the students or experiment with their teaching. Bongen recalls how science teacher Don Dosch walked out when a class was messing around and it forced them to be more serious about their work. Taff recalls setting things on fire with lenses in Branson Lawrence\u27s physics class. Peterson remembers Micah Fogel\u27s creative Halloween costumes. Classes could also be very challenging and intimidating, as well as memorable in other ways. They all reflect on how difficult the classes were, even compared to their college experiences. Asked how the IMSA experience changed them, Taff says it gave her a lot of motivation to go out and do things. Bongen says she probably wouldn\u27t have ended up in her field, computer science, without the extra math classes she was able to take at IMSA. Kennebrew likewise says he wouldn\u27t be a successful without IMSA because struggling with the academics made him a much better student and that helped him to well in college. Peterson ended up getting a PhD from the same lab where she did an IMSA Mentorship. Though they all ended up in STEM fields, they also speak very highly of the humanities and language classes. While some of them had intended to participate in trips abroad as part of language programs, in their sophomore year all trips were cancelled after 9/11 and in their junior year an intersession trip was cancelled due to airfares. Kennebrew had the opportunity to go to Mexico with a Spanish class. To conclude, Peterson reflects on how unique and significant her IMSA experience was. Kennebrew also mentions how his RC, Quintin Backstrom, was an important mentor to him. At the time of interview: Taff held a PhD in microbiology and was finishing her third year in medical school; Bongen studied computer science and worked at Cisco; Kennebrew had completed medical school and worked as a physician and captain in the US Army; and Peterson got a PhD in chemical engineering and was an assistant professor at Worcester Polytechnic. Duration: 33:34https://digitalcommons.imsa.edu/oral_histories/1012/thumbnail.jp

Comparative Phenotypic Analysis of the Major Fungal Pathogens Candida parapsilosis and Candida albicans.

Candida parapsilosis and Candida albicans are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to C. albicans, relatively little is known about the virulence properties of C. parapsilosis, a pathogen particularly associated with infections of premature neonates. We describe here the construction of C. parapsilosis strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to C. albicans strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of CAP1 in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in C. parapsilosis but not in C. albicans. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in C. parapsilosis. Only three (Efg1, Bcr1 and Ace2) have similar effects on C. albicans biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in C. parapsilosis only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in C. albicans do not have the same function in C. parapsilosis. We also compared the transcription profile of C. parapsilosis and C. albicans biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in C. parapsilosis

Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was compared to conventional biochemical testing methods and nucleic acid analyses (16S rDNA sequencing, hippurate hydrolysis gene testing, whole genome sequencing [WGS]) for species identification of Campylobacter isolates obtained from chickens ( Gallus gallus domesticus, n = 8), American crows ( Corvus brachyrhynchos, n = 17), a mallard duck ( Anas platyrhynchos, n = 1), and a western scrub-jay ( Aphelocoma californica, n = 1). The test results for all 27 isolates were in 100% agreement between MALDI-TOF MS, the combined results of 16S rDNA sequencing, and the hippurate hydrolysis gene PCR ( p = 0.0027, kappa = 1). Likewise, the identifications derived from WGS from a subset of 14 isolates were in 100% agreement with the MALDI-TOF MS identification. In contrast, biochemical testing misclassified 5 isolates of C. jejuni as C. coli, and 16S rDNA sequencing alone was not able to differentiate between C. coli and C. jejuni for 11 sequences ( p = 0.1573, kappa = 0.0857) when compared to MALDI-TOF MS and WGS. No agreement was observed between MALDI-TOF MS dendrograms and the phylogenetic relationships revealed by rDNA sequencing or WGS. Our results confirm that MALDI-TOF MS is a fast and reliable method for identifying Campylobacter isolates to the species level from wild birds and chickens, but not for elucidating phylogenetic relationships among Campylobacter isolates

A <em>Candida</em> Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance

<div><p>Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of <em>Candida albicans</em> biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by <em>BGL2</em>, <em>PHR1</em>, and <em>XOG1</em>, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by <em>FKS1</em>. Furthermore, our observations suggest that this matrix delivery pathway works independently from the <em>C. albicans ZAP1</em> matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics.</p> </div

Comparative Phenotypic Analysis of the Major Fungal Pathogens <i>Candida parapsilosis</i> and <i>Candida albicans</i>

<div><p><i>Candida parapsilosis</i> and <i>Candida albicans</i> are human fungal pathogens that belong to the CTG clade in the Saccharomycotina. In contrast to <i>C. albicans</i>, relatively little is known about the virulence properties of <i>C. parapsilosis</i>, a pathogen particularly associated with infections of premature neonates. We describe here the construction of <i>C. parapsilosis</i> strains carrying double allele deletions of 100 transcription factors, protein kinases and species-specific genes. Two independent deletions were constructed for each target gene. Growth in >40 conditions was tested, including carbon source, temperature, and the presence of antifungal drugs. The phenotypes were compared to <i>C. albicans</i> strains with deletions of orthologous transcription factors. We found that many phenotypes are shared between the two species, such as the role of Upc2 as a regulator of azole resistance, and of <i>CAP1</i> in the oxidative stress response. Others are unique to one species. For example, Cph2 plays a role in the hypoxic response in <i>C. parapsilosis</i> but not in <i>C. albicans</i>. We found extensive divergence between the biofilm regulators of the two species. We identified seven transcription factors and one protein kinase that are required for biofilm development in <i>C. parapsilosis</i>. Only three (Efg1, Bcr1 and Ace2) have similar effects on <i>C. albicans</i> biofilms, whereas Cph2, Czf1, Gzf3 and Ume6 have major roles in <i>C. parapsilosis</i> only. Two transcription factors (Brg1 and Tec1) with well-characterized roles in biofilm formation in <i>C. albicans</i> do not have the same function in <i>C. parapsilosis</i>. We also compared the transcription profile of <i>C. parapsilosis</i> and <i>C. albicans</i> biofilms. Our analysis suggests the processes shared between the two species are predominantly metabolic, and that Cph2 and Bcr1 are major biofilm regulators in <i>C. parapsilosis</i>.</p></div

Relationship between β-1,3 glucan synthase and modification enzymes during biofilm growth.

<p>(A) RNA was isolated from reference and <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutant biofilms. Real-time RT-PCR assays were used to measure transcript levels in triplicate. Data are shown as a normalized ratio of transcript in the mutant strain to that in the reference strain. (B) The glucan modifier genes, <i>BGL2</i>, <i>XOG1</i>, and <i>PHR1</i> were placed under the control of an inserted <i>TDH3</i> promoter for overexpression of these genes in the homozygous <i>FKS1</i>−/+ mutant. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown), and drug impact was determined using an XTT reduction assay. The * symbol indicates biofilm susceptibilities were significantly different (p value<0.008) based upon ANOVA with pairwise comparison. (C) The <i>FKS1</i> gene was placed under the control of an inserted <i>TDH3</i> promoter for overexpression of this genes in the <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutants. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown) and drug impact was determined using an XTT reduction assay. (D) The glucan modifier genes, <i>BGL2</i>, <i>XOG1</i>, and <i>PHR1</i> were placed under the control of an inserted <i>TDH3</i> promoter for overexpression of these genes in each of the homozygous <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutants. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown) and drug impact was determined using an XTT reduction assay. Data for all XTT assays above are expressed as percent reduction compared to untreated controls. Standard errors are shown. Student's <i>t</i> test was used for (C) and (D) to compare the mutant strains at each drug concentration <b>*</b>, p<0.05.</p

Analysis of glucan modifier double knockout strains.

<p>(A) The glucan modifier double knockout strains and the reference strain were assayed for filamentation in YPD. Representative images from light microscopy are shown (top row). The strains were also assayed for adherence to coverslips following 2 h incubation. Representative light micrographs are shown (bottom row). (B) The double knockouts were examined for overall biofilm growth in both YPD and RPMI by comparing the ODs of the untreated control with those of the reference strain in an XTT assay. Strains were also examined for relative planktonic growth in YPD using a turbidity endpoint. (C) The glucan modifier double knockout strains (<i>bgl2</i>−/− <i>phr1</i>−/− and <i>bgl2</i>−/− <i>xog1</i>−/−), the modifier single knockouts, and the reference strain were assayed for biofilm susceptibility to fluconazole (125 µg/ml shown) using the XTT assay as described above. Data for all XTT assays above are expressed as percent reduction compared to untreated controls. Standard errors are shown.</p