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, 7 (6), e1000133

Biofilm Matrix Regulation by Candida Albicans Zap1

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Biofilm Matrix Regulation by Candida Albicans Zap1

Clarissa J Nobile et al. PLoS Biol.

Abstract

A biofilm is a surface-associated population of microorganisms embedded in a matrix of extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix of Candida albicans, the major fungal pathogen of humans. We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator of a major matrix component, soluble beta-1,3 glucan, in both in vitro and in vivo biofilm models. To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target genes through expression profiling and full genome chromatin immunoprecipitation. On the basis of these results, we designed additional experiments showing that two glucoamylases, Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of insoluble beta-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5, Csh1, and Ifd6 have roles in matrix production: Adh5 acts positively, and Csh1 and Ifd6, negatively. We propose that these alcohol dehydrogenases generate quorum-sensing aryl and acyl alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel regulatory circuit and its mechanism of control of a process central to infection.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Analysis of biofilm and matrix production.
The mutant strain CJN1201 (zap1Δ/zap1Δ), complemented strain CJN1193 (zap1Δ/zap1Δ+pZAP1), and reference wild-type strain DAY185 (ZAP1/ZAP1) were assayed for (A) in vitro-grown biofilm biomass, (B) in vitro-grown biofilm soluble β-1,3 glucan production, and (C) in vitro planktonic culture soluble β-1,3 glucan production. In addition, (D) soluble β-1,3 glucan production was assayed in a rat catheter biofilm infection model. The symbol “*” indicates that glucan measurements were significantly different (p<0.0005) from the zap1Δ/zap1Δ strain.
Figure 2
Figure 2. CSLM analysis of in vitro biofilm structure.
In vitro-grown biofilms of the mutant strain CJN1201 (zap1Δ/zap1Δ, [A–C]) and complemented strain CJN1193 (zap1Δ/zap1Δ+pZAP1, [D–F]) were visualized by CSLM. (A,D) Depth views show the x-y plane. (B, E) Magnified depth views with pseudocolor scale. (C, F) Side views show the y-z plane.
Figure 3
Figure 3. Scanning electron microscopy of in vivo biofilms.
The mutant strain CJN1201 (zap1Δ/zap1Δ, [A,B]), complemented strain CJN1193 (zap1Δ/zap1Δ+pZAP1, [C,D]), and reference wild-type strain DAY185 (ZAP1/ZAP1, [E,F]) were inoculated into rat intravenous catheters, and resulting biofilms were visualized after 24 h of growth. Images show catheter luminal surfaces at (A,C,E) 1,000× and (B,D,F) 50× magnification.
Figure 4
Figure 4. ChIP mapping of genomic Zap1 binding sites.
Zap1 myc-tagged strain CJN1688 versus untagged wild-type strain DAY185 immunoprecipitation binding data were performed under biofilm conditions. The x-axis represents ORF chromosomal locations (See Dataset S6, sheet 1 for exact location values). The y-axis is the Agilent normalized enrichment value (log2) for binding of Zap1 (See Dataset S6, sheet 1 for exact enrichment values). Zap1-myc strain (blue line) and untagged wild-type (red line) ChIP–chip array binding data were mapped and plotted onto the chromosomes containing ZRT1 and PRA1 located on Chromosome 4 (A), ZRT2 located on Chromosome 2 (B), ZRT3 located on Chromosome 2 (C), CSH1 located on Chromosome 1 (D), IFD6 located on Chromosome 1 (E), and itself ZAP1 located on Chromosome 4 (F) using ChipView v0.954. The promoters of these genes show significant peak enrichment (determined using Agilent Chip Analytics software v1.2) for the binding of Zap1. The blue track under the peak indicates that the Agilent segment p-value (−log10) for the binding of Zap1 is significant (See Dataset S6, sheet 1 for actual segment p-values). Genes plotted above the bold line read in the sense direction; genes plotted below the bold line read in the antisense direction. Identical binding sites with similar peak enrichment values were observed for the independently isolated Zap1 myc-tagged strain CJN1694 versus untagged wild-type strain DAY185 (unpublished data).
Figure 5
Figure 5. Effect of altered Zap1 target gene expression.
Soluble β-1,3 glucan levels were determined after biofilm growth (A) in vitro or (B) in the rat catheter model. Determinations were carried out with zap1Δ/zap1Δ strains carrying either no promoter fusion or TDH3 promoter fusions to genes ZRT2, ZRT1, PRA1, CSH1, or IFD6, as indicated in the figure. Determinations were also carried out with ZAP1/ZAP1 strains carrying either no promoter fusion, or TDH3 promoter fusions to genes YWP1, orf19.3499, HXT5, GCA1, GCA2, HGT2, or ADH5, as indicated in the figure. A single asterisk indicates that glucan measurements were significantly different (p<0.05) from the zap1Δ/zap1Δ strain carrying no promoter fusion; a double asterisk indicates that glucan measurements were significantly different (p<0.05) from the ZAP1/ZAP1 strain carrying no promoter fusion; both assessments are based upon Student's t-tests. In (B), the pound symbol (#) indicates that the respective strain was not assayed in the in vivo biofilm model.
Figure 6
Figure 6. Comparison of C. albicans and S. cerevisiae Zap1 regulons.
Expression of Zap1-responsive genes in C. albicans (complemented strain versus zap1Δ/zap1Δ mutant, x-axis) was compared with their S. cerevisiae orthologs and best hits (wild-type strain versus zap1 mutant, y-axis). Definitions of orthologous genes and best hits were provided by the Candida Genome Database (see Dataset S4; worksheet 3; (http://www.candidagenome.org/download/homology/orthologs/Calb_Scer_by_inparanoid/Assem21orthologs/CA_SC_orthologs.txt and http://www.candidagenome.org/download/homology/best_hits/Calb_Scer_best_hits_Assem21.txt). Expression data for S. cerevisiae were for growth in 61 nM zinc from Lyons et al. . This graph presents the 40 most downregulated genes (purple triangles) and 40 most upregulated genes (blue triangles) in the zap1Δ/zal1Δ mutant compared to S. cerevisiae orthologs, and the 40 most downregulated genes (purple squares) and 40 most upregulated genes (blue squares) in the zap1Δ/zal1Δ mutant compared to S. cerevisiae best hits. In addition, all C. albicans ERG genes are graphed against their orthologs or best hits (green squares). Finally, the five genes shown to be functionally relevant for biofilm matrix are graphed against their orthologs or best hits (red circles).
Figure 7
Figure 7. Integration of Zap1 function into biofilm formation.
Zap1 functions as a negative regulator of biofilm matrix accumulation. It does so through activation of expression of CSH1 and IFD6, which inhibit matrix accumulation, and through repression of expression of GCA1, GCA2, and ADH5, which promote matrix accumulation. Zap1 binds to the CSH1 and IFD6 promoter regions and thus is likely to activate their expression directly. Zap1 is a negative regulator of two gene classes—ERG genes and HXT genes—that that are upregulated during biofilm development . We suggest that Zap1 functions as a negative regulator of several aspects of biofilm maturation.

Comment in

  • Zap1 sticks it to Candida biofilms.
    Heller K. Heller K. PLoS Biol. 2009 Jun 16;7(6):e1000117. doi: 10.1371/journal.pbio.1000117. PLoS Biol. 2009. PMID: 20076744 Free PMC article. No abstract available.

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References

    1. Donlan RM. Biofilm formation: a clinically relevant microbiological process. Clin Infect Dis. 2001;33:1387–1392. - PubMed
    1. Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev. 2002;15:167–193. - PMC - PubMed
    1. Sutherland IW. The biofilm matrix–an immobilized but dynamic microbial environment. Trends Microbiol. 2001;9:222–227. - PubMed
    1. Blankenship JR, Mitchell AP. How to build a biofilm: a fungal perspective. Curr Opin Microbiol. 2006;9:588–594. - PubMed
    1. Nobile CJ, Mitchell AP. Microbial biofilms: e pluribus unum. Curr Biol. 2007;17:R349–R353. - PubMed

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