Characterization of Genetic Determinants That Modulate <i>Candida albicans</i> Filamentation in the Presence of Bacteria

<div><p>In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. <i>C. albicans</i> is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that <i>C. albicans</i> interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of <i>C. albicans</i> with bacteria and the impact on morphology we utilized a haploinsufficiency based <i>C. albicans</i> mutant screen to test for the ability of <i>C. albicans</i> to produce hyphae in the presence of three bacterial species (<i>Escherichia coli</i>, <i>Pseudomonas aeruginosa</i>, and <i>Staphylococcus aureus</i>). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, <i>CDR4</i>, displayed increased expression when co-cultured with <i>S. aureus</i>, but not <i>E. coli</i> or <i>P. aeruginosa</i>. Our data demonstrates the ability to use a large scale library screen to identify genes involved in <i>Candida</i>-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of <i>C. albicans</i> morphogenesis. </p> </div

Representative colony morphologies.

<p>(<b>A</b>) SC5314 wild-type non-filamentous growth on YPD agar (30° C); (<b>B</b>) SC5314 wild-type filamentation on M199 agar (37° C); (<b>C</b>) SC5314 wild-type filamentation inhibition in the presence of <i>P. aeruginosa</i> on M199 agar (37° C); (<b>D</b>) 4X magnification of A; (<b>E</b>) 4X magnification of B; (<b>F</b>) 4X magnification of C; (<b>G</b>), (<b>H</b>), (<b>I</b>) Representative examples of library mutant filamentation in the presence of bacteria due to haploinsufficiency on M199 agar (37° C) (4X magnification).</p

Representative photos of observed phenotypes of SC5314, library transposon candidates, and heterozygous and homozygous deletion strains.

<p>YPD growth control (30° C), 40x magnification; M199 filamentation control (37° C), 100x magnification; <i>E. coli</i> interactions with <i>C. albicans</i> strains, 100x magnification; <i>P. aeruginosa</i> interactions with <i>C. albicans</i> strains, 100x magnification; <i>S. aureus</i> interactions with <i>C. albicans</i> strains, 100x magnification.</p

Phenotypes observed when SC5314, library transposon candidates, heterozygous and homozygous deletion strains grown in liquid culture (37° C) with bacteria or spent media.

<p>A) <i>C. albicans</i> strains with M199 filamentation control and coculture with <i>E. coli</i>, <i>P. aeruginosa</i>, and <i>S. aureus</i>. Magnification 400x; B) <i>C. albicans</i> strains with M199 filamentation control and culture in spent media <i>E. coli</i>, <i>P. aeruginosa</i>, and <i>S. aureus</i>. Magnification 400x.</p

Distribution of mutants that filament in the presence of the three bacterial species tested.

<p>Overall 836 mutants were identified that filamented in the presence of the three bacterial species tested. Ec:<i>E</i>. <i>coli</i>; Pa: <i>P. aeruginosa</i>; Sa: <i>S. aureus</i>.</p

<i>C. albicans CDR4</i> transcript levels grown in the presence of bacteria.

<p><i>C. albicans</i> SC5314, <i>E. coli</i>, <i>P. aeruginosa</i>, and <i>S. aureus</i> were grown to mid log phase in liquid culture, mixed together in equal amounts, co-incubated at 37° C, and aliquots were taken at 0, 10, 20, 30, and 60 minutes post addition. RNA was isolated and expression of <i>CDR4</i> was measured by reverse transcription. <i>ENO1</i> was used as a loading control and reference gene for expression comparisons. Graphical representation of <i>CDR4</i> expression over time for SC5314 co-incubated with <i>E. coli</i>, <i>P. aeruginosa</i>, or <i>S. aureus</i>. Data is representative of three independent experiments with mean value and standard deviation bars shown. The asterisks indicate a statistically significant difference (P<0.05) in mean intensity of test conditions over the control.</p

Molecular modeling suggests one possible conformation that (1→6)-β-linked glucan side chains may exhibit, that is, a hook-like, bent structure.

<p>Structure A (top) is a linear polymer containing ten (1→3)-β-linked repeat units in the polymer chain. The linear (1→3)-β-linked glucan backbone structure assumes an open helical conformation. Structure B (bottom) is the same linear structure except a side chain branches from the third repeat unit. The side chain contains five (1→6)-β-linked repeat units. The curvature and hook-like structure of the side chain is evident in this model where the structure has been rotated slightly to optimize visualization of the curved side chain. The structures are rendered using JMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027614#pone.0027614-anonymous1" target="_blank">[39]</a>.</p

Initial identification of ¹³C and ¹H chemical shifts for the branchpoint repeat unit of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>HSQC-TOCSY 2D NMR spectrum shows correlations between C3 and C3Br carbons and methylene protons in the same spin system as C3Br.</p

2D NMR spectra of the glycosidic linkages and non-reducing termini of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>(a) The three different (1→6)-β-linked glycosidic bonds from the side chain are detailed in the NOESY 2D NMR spectrum for SC1, SC Internal, and SC NRT glucosyl groups associated with H1 SC1, SC H1 and SC NRT H1. A: H6Br,H6′Br/H1SC1; B: H6SCn,H6′SCn/H1SC(n+1); C: H6SC(n-1),H6′SC(n-1)/H1SCNRT. (b) Similarity of the structures of the glycosyl group associated with SC NRT H1 and NRT H1 is indicated in the TOCSY 2D NMR spectrum.</p

Comparison of average side chain lengths, SCn, and branching frequencies, Br Freq, for several strains of <i>C. glabrata</i> isolated by 2 different methods.

<p>Comparison of average side chain lengths, SCn, and branching frequencies, Br Freq, for several strains of <i>C. glabrata</i> isolated by 2 different methods.</p