Enhanced immune recognition of acidic adapted cells is mediated via Dectin-1.

<p><b>a)</b> Fc-Dectin-1 binding to wild type <i>C</i>. <i>albicans</i> cells grown to mid-log phase in YPD buffered to the appropriate pH, as quantified by FACS counting 10,000 events per repeat. Fold increased is relative to unbuffered YPD. <b>b)</b> <i>C</i>. <i>albicans</i> was grown in YPD at the appropriate pH to mid-log phase, co-incubated with fibroblasts for 1 h, fixed and the association index (number of fungal cells either attached and phagocytosed/100 macrophages) determined. <b>c)</b> Association of acidic adapted <i>C</i>. <i>albicans</i> cells to fibroblasts expressing Dectin-1 in the presence of glucan phosphate. <b>d)</b> J774.1A macrophages were pre-incubated with glucan phosphate and infected with <i>C</i>. <i>albicans</i> at an MOI of 5 and the phagocytosis index (number of fungal cells phagocytosed/100 macrophages) determined after 1 h. <b>e)</b> J774.1A macrophages were pre-incubated with glucan phosphate and infected with <i>C</i>. <i>albicans</i> at an MOI of 5 and the association index determined after 1 h. Data represent the mean ± SEM from three independent repeats (* p > 0.05. *** p > 0.001).</p

Adaptation to acidic environments promotes surface exposure of chitin.

<p><b>a)</b> Wild type cells (NYG152) were grown in YPD buffered at the appropriate pH, stained with CFW and fluorescence quantified by FACS. The fold increase in CFW staining was determined from the background subtracted MFI values from FACS analysis, and normalised to YPD grown cells. <b>b)</b> Fold increase in FITC fluorescence of FITC-WGA stained wild type cells (NYG152) grown at different pH, relative to growth in YPD as quantified by FACS analysis. All data represent the mean ± SEM from three independent experiments. <b>c)</b> Microscopy of WGA stained cells. Arrowheads indicate chitin exposure. Scale bar = 10 μm.</p

Mkc1, Hog1 and Crz1 are not required for de-cloaking of chitin in response to acidic pH.

<p><b>a)</b> A <i>C</i>. <i>albicans</i> strain expressing Hog1-GFP was grown in YPD buffered at the appropriate pH for 4 h and the localisation (cytoplasmic vs. nuclear) of Hog1 scored in 200 cells per condition. As a positive control of Hog1 activation, cells grown in YPD were incubated with 1 M NaCl for 30 min prior to imaging. The data represent the mean ± SEM from three independent experiments. <b>b)</b> Increase in WGA and CFW staining in kinase mutants grown at pH4 relative to YPD as quantified by FACS analysis. Data represent the mean ± SEM from three independent experiments. DAY286 is the parental control strain of the two kinase mutants <b>c)</b> Wild type <i>C</i>. <i>albicans</i> (NGY152) was grown to mid-log phase in YPD buffered at the appropriate pH, total protein extracted and activation of Mkc1 and Cek1 assessed via western blot using the MAPK p42/p44 antibody which cross reacts with both kinases. Arrow indicates Mkc1, while arrowhead indicates Cek1. <b>d)</b> Increase in WGA and CFW staining in transcription factor mutants at pH4 relative to YPD as quantified by FACS analysis. SN152 is the parental control strain of the two transcription factor mutants. Data represent the mean ± SEM from three independent experiments.</p

<i>C</i>. <i>albicans</i> cells adapted to acidic environments recruit more innate immune cells <i>in vivo</i>.

<p><b>a)</b> Total number of CD45+, CD11b+ and Ly-6B.2+ (7/4 clone) cells (including monocytes and neutrophils) recruited to the peritoneal cavity following 4 h exposure to <i>C</i>. <i>albicans</i> incubated in pH6 or pH4 YPD (p = 0.007). <b>b)</b> Total number of neutrophils (further identified using F4/80) recruited to the peritoneal cavity (p = 0.010) <b>c)</b> Percentage of neutrophils in the total population of recruited innate immune cells (p = 0.540).</p

Exposure of β-glucan and chitin do not co-localise.

<p>Wild type <i>Candida albicans</i> cells (NGY152) were grown to mid-log phase in YPD buffered at pH2, pH4 and pH6, washed, fixed with 4% PFA and stained with CFW, TRITC-labeled WGA, and Fc-Dectin-1 (FITC). White arrowheads indicate patches of β-glucan exposure. Scale bar represents 10 μm.</p

Regulation of cell wall remodeling during adaptation to environmental pH.

<p><b>a)</b> In environments above pH5.5, the Rim101 pathway is activated resulting in C-terminal processing of Rim101 and enhance expression of <i>CHT2</i>. The She3 complex then transports <i>CHT2</i> mRNA to the cell wall, where it is translated and attached via its GPI anchor. At pH4 Rim101 is not activated, resulting in reduced expression of Cht2. <b>b) (i)</b> When present in the cell wall (i.e. above pH5.5), Cht2 hydrolyses the growing chitin polymer into shorter fragments which may hydrogen bond and form short chitin microfibrils that are embedded deep within the cell wall. <b>(ii)</b> When there is reduced amounts of Cht2 in the cell wall (i.e. environments below pH4), the growing chitin polymer is cleaved less efficiently, resulting in the incorporation of longer chitin polymers that are more exposed on the outer surface of the cell wall.</p

Unmasking of β-glucan in response to environmental pH is specific to <i>C</i>. <i>albicans</i> and <i>C tropicalis</i>.

<p><b>a)</b> Exponentially growing cells in YPD, YPD buffered to pH4 and YPD buffered to pH8 were incubated with recombinant β1,3-glucanase. The decrease in OD₆₀₀ represents cell lysis as the β1,3-glucanase digests the cell wall and is expressed as a % of the starting OD₆₀₀. Data represent the mean ± SEM from four independent experiments. <b>b)</b> Initial rate of cell lysis was calculated from the first 100 min after the addition of β-glucanase. Data represent the mean ± SEM from four independent repeats (* p < 0.05, ** p < 0.01).</p

Acidic environments unmask β-glucan in <i>C</i>. <i>albicans</i>.

<p><b>a i)</b> Wild type <i>C</i>. <i>albicans</i> (NGY152) was grown to mid-log phase in YPD buffered at the appropriate pH, and incubated with recombinant β1,3-glucanase. The decrease in OD₆₀₀ represents cell lysis as the β1,3-glucanase digests the cell wall and is expressed as a percentage of the starting OD₆₀₀. <b>ii)</b> The initial rate of cell lysis as calculated from the first 100 min. Data represent the mean ± SEM from four independent repeats <b>b)</b> Immunofluorescent imaging of β-glucan exposure of exponentially growing NGY152 cells using a anti-β1,3-glucan monoclonal antibody. Scale bar = 10 μm. <b>c)</b> Quantification of β-glucan exposure by FACS counting 10,000 events per repeat. Fold increased is relative to unbuffered YPD. Data represent the mean ± SEM from six independent experiments. <b>d)</b> Quantification of Aniline Blue staining of exponentially growing NGY152 cells by FACS analysis counting 10,000 events per repeat. Fold increased is relative to unbuffered YPD. Data represent the mean ± SEM from three independent experiments. <b>e)</b> β-glucan exposure of clinical <i>C</i>. <i>albicans</i> isolates grown to mid-log phase in YPD buffered to pH4 relative to YPD. Data represent the mean ± SEM from three independent experiments (* p < 0.05).</p

Adaptation to environmental pH alters the ultrastructure of the fungal cell wall.

<p><b>a)</b> Electron micrographs showing the ultrastructure of the cell walls of wild type <i>C</i>. <i>albicans</i> (NGY152) grown to mid-log phase in YPD buffered to the appropriate pH. Scale bar represents 100 nm. <b>b)</b> Quantification of the thickness of the inner cell wall layer and mannoprotein fibril length. Data represent the mean ± SEM from 5 individual cells. Each cell was measured at 30 different points around the cell periphery (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Masking of chitin in the cell wall is regulated by Rim101 and Bcr1 and requires Cht2.

<p><b>a)</b> Respective strains were grown in YPD buffered at pH4 or pH6 to mid-log phase, cells were fixed, stained with FITC-WGA and fluorescence quantified by FACS. <b>b)</b> SC5314 was grown to mid-log phase in YPD buffered at pH4, pH6 and pH8, snap frozen and total RNA extracted. Expression of <i>CHT2</i> was determined by semi quantitative RT-PCR using 50 ng of total RNA. Expression levels were normalized to <i>ACT1</i>. <b>c)</b> Relative expression of <i>CHT2</i> in <i>rim101</i>Δ and <i>bcr1</i>Δ mutants exponentially growth in YPD buffered at pH4, pH6 and pH8 and determined by semi quantitative RT-PCR. Expression levels were normalized to <i>ACT1</i>. <b>d)</b> FACS analysis of WGA staining in <i>rim101</i>Δ and <i>bcr1</i>Δ mutants. <b>e)</b> FACS analysis of WGA staining in CARP1-1 which expresses constitutively active Rim101 <b>f)</b> FACS analysis of WGA staining in <i>she3</i>Δ mutants. Data represent the mean ± SEM from three independent experiments. (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05).</p