Drug resistance and virulence of the human fungal pathogen Candida glabrata

Candida glabrata ist ein opportunistischer humanpathogener Pilz. Nach Candida albicans stellt er die zweithäufigste Ursache für Pilzerkrankungen durch Candida spp. dar. Die Infektion mit C. glabrata kann zu Erkrankungen der Haut oder der Schleimhäute bis hin zur lebensbedrohlichen systemischen Infektion bei immunsupprimierten Patienten führen. Hauptvirulenzfaktoren von C. glabrata sind sowohl eine erhöhte natürliche Resistenz gegen Azolverbindungen, als auch eine große Anzahl verschiedener Adhesine. Welche molekularen Mechanismen diesen Virulenzfaktoren zu Grunde liegen, ist größtenteils noch unbekannt. Genomik und phänotypische Analyse von Deletionsmutanten stellen einen Ansatz dar, um neue Virulenzfaktoren pathogener Pilzen zu identifizieren. Für diese Doktorarbeit wurden C. glabrata Sequenzdaten verwendet, um nicht essentielle, zu Saccharomyces cerevisiae homologe Gene auszuwählen. Mit Hilfe eines revers-genetischen Ansatzes wurde dann eine Kollektion von Deletionsmutanten erstellt. Diese Stammbibliothek diente als Ausgangsbasis für die phänotypische Analyse der Mutanten zur Identifizierung neuer Gene, die die Pathogenität und Azolresistenz von C. glabrata beeinflussen. Insgesamt umfasst die Kollektion 476 einzelne, mit einem molekularen Barcode versehene Stämme, bei denen Signaltransduktionsgene, Transkriptionsfaktoren, Zellwandbiosynthesegene, Resistenzgene und C. glabrata spezifische Gene deletiert wurden. Es wurden 103 Mutanten identifiziert, die Wachstumsdefekte unter Zellwandstress, in Gegenwart von Antimykotika, bei Temperaturstress, Osmolaritätsänderungen, Kontakt mit Detergenzien oder beim Wachstum auf Minimalmedium aufzeigen. Die Interaktion zwischen Makrophagen und C. glabrata wurde in vitro durch die Detektion von reaktiven Sauerstoffmolekülen (ROS) getestet. Dadurch wurden mehrere Zellwandmutanten entdeckt, die eine erhöhte ROS-Produktion durch die Makrophagen verursachen. Mehrere Deletionsstämme zeigten Phänotypen, die sich von den bekannten S. cerevisiae Mutanten unterscheiden. Es konnten Gene identifiziert werden, die noch nicht mit Caspofunginsensitivität assoziiert worden sind. Darunter befanden sich auch Gene, die keine Orthologe in S. cerevisiae haben. CgCBK1 wurde aufgrund des schweren Zellteilungsdefekts näher charakterisiert. Die Transkriptionsanalyse des Cgcbk1Δ Stammes zeigte, dass bestimmte Zellwandgene unterschiedlich exprimiert werden. Diese Sammlung von Deletionsstämmen ist eine der größten weltweit und ist somit von sehr großem Nutzen für das Studium der Pathogenität von C. glabrata. Zukünftige in vivo Experimente werden unter Ausnützung der integrierten „molekularen Barcodes“ die Identifizierung neuer Virulenzfaktoren ermöglichen.Candida glabrata is an opportunistic human fungal pathogen. It is the second most frequent cause of Candida-derived infections after Candida albicans. Infection with either one of the two pathogenic fungi can result in diseases ranging from superficial cutaneous or mucosal to life-threatening systemic infections in immunocompromised individuals. Welldocumented virulence attributes of C. glabrata are the inherent reduced azole susceptibility and a large repertoire of adhesin genes, which are regulated by transcriptional silencing. However, the molecular basis of C. glabrata antifungal drug resistance and additional virulence factors is not well understood. The combination of fungal genomics and large-scale phenotypic profiling of deletion mutants represents a powerful approach to identify new factors contributing to fungal virulence. For this doctoral thesis, the C. glabrata genome sequence data were used to select genes with non-essential functional orthologues of the non-pathogenic yeast Saccharomyces cerevisiae. Based on this selection, a large-scale reverse genetics approach was initiated to identify novel genes implicated in C. glabrata pathogenicity and drug resistance. A bar-coded C. glabrata deletion strain collection was engineered comprising some 500 single gene deletion mutants affected in signaling functions, regulation of gene expression, cell wall biogenesis, transport processes, drug resistance, stress response and metabolism. Phenotypic profiling identified a total of 103 C. glabrata genes involved in resistance to cell wall-perturbing compounds, antifungal drugs, heat stress, osmosensitivity, metal ion or detergent tolerance, growth on minimal medium and phenotypic switching. Host-pathogen interaction related phenotypes were analyzed using an in vitro assay detecting reactive oxygen species (ROS). Screening for ROS elicited by primary mouse bone-marrow-derived macrophages co-incubated with C. glabrata mutants resulted in the identification of mutants lacking cell wall-related genes. Taken together, numerous deletion strains showed growth phenotypes different from known phenotypes of S. cerevisiae. For example, genes were identified, which have previously not been associated with sensitivity to the glucan synthase inhibitor Caspofungin. This also included C. glabrata genes, which do not have orthologues in baker’s yeast. The function of CgCBK1 gene has been studied in more detail, because it exhibits severe cell separation defects. Transcriptional profiling of this mutant showed differential expression of a distinct set of genes with cell wall-associated functions. In summary, the generated C. glabrata gene deletion strain collection is one of the largest collections of fungal deletion strains in the world and represents a powerful tool to study the virulence of a human fungal pathogen. Future studies based on the in vitro results will exploit the signature-tag strategy to identify novel virulence-associated factors in vivo

Identification of Candida glabrata genes involved in pH modulation and modification of the phagosomal environment in macrophages

notes: PMCID: PMC4006850types: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov'tCandida glabrata currently ranks as the second most frequent cause of invasive candidiasis. Our previous work has shown that C. glabrata is adapted to intracellular survival in macrophages and replicates within non-acidified late endosomal-stage phagosomes. In contrast, heat killed yeasts are found in acidified matured phagosomes. In the present study, we aimed at elucidating the processes leading to inhibition of phagosome acidification and maturation. We show that phagosomes containing viable C. glabrata cells do not fuse with pre-labeled lysosomes and possess low phagosomal hydrolase activity. Inhibition of acidification occurs independent of macrophage type (human/murine), differentiation (M1-/M2-type) or activation status (vitamin D3 stimulation). We observed no differential activation of macrophage MAPK or NFκB signaling cascades downstream of pattern recognition receptors after internalization of viable compared to heat killed yeasts, but Syk activation decayed faster in macrophages containing viable yeasts. Thus, delivery of viable yeasts to non-matured phagosomes is likely not triggered by initial recognition events via MAPK or NFκB signaling, but Syk activation may be involved. Although V-ATPase is abundant in C. glabrata phagosomes, the influence of this proton pump on intracellular survival is low since blocking V-ATPase activity with bafilomycin A1 has no influence on fungal viability. Active pH modulation is one possible fungal strategy to change phagosome pH. In fact, C. glabrata is able to alkalinize its extracellular environment, when growing on amino acids as the sole carbon source in vitro. By screening a C. glabrata mutant library we identified genes important for environmental alkalinization that were further tested for their impact on phagosome pH. We found that the lack of fungal mannosyltransferases resulted in severely reduced alkalinization in vitro and in the delivery of C. glabrata to acidified phagosomes. Therefore, protein mannosylation may play a key role in alterations of phagosomal properties caused by C. glabrata.Deutsche ForschungsgemeinschaftNational Institutes for HealthWellcome TrustBBSR

The high-osmolarity glycerol response pathway in the human fungal pathogen Candida glabrata strain ATCC 2001 lacks a signaling branch that operates in Baker's yeast

The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1? (Cgsho1?) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1? mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1? cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgsSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1? mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions. Copyright © 2007, American Society for Microbiology. All Rights Reserved

Alkalinization-defective <i>C. glabrata</i> mutants.

<p>Listed are mutants that showed reduced <i>in vitro</i> alkalinization of phenol red containing YNB medium with 1% casamino acids as sole carbon and nitrogen source in a screen of 647 mutants. Alkalinization defects were verified in independent assays and with two independent clones.</p>A<p>+reduced alkalinization (same phenotype as <i>bcy1</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>), ++ strongly reduced alkalinization (same phenotype as <i>put3</i>Δ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone-0096015-g005" target="_blank">Fig. 5A</a>) as compared to the wild type.</p>B, C<p>Growth was monitored in parallel in YPD and in YNB medium with 1% casamino acids without phenol red by measuring absorption at 600 nm. ++ strong growth defect, + weak growth defect, - unaltered growth as compared to the wild type.</p>D<p>Mutants were co-incubated with MDMs for 90 min and phagosome acidification was monitored by LysoTracker staining. At least three independent microscopic fields were scored per mutant. ++ strong increase in LysoTracker signal, + medium increase in LysoTracker signal, - no change in LysoTracker signal as compared to the wild type.</p

<i>C. glabrata</i> does not induce MAP-kinase or NFκB signaling cascades upon phagocytosis but activates Syk.

<p>RAW264.7 macrophages were stimulated with LPS (1 µg/ml) or infected with viable or heat killed (Hk) <i>C. glabrata</i> (MOI of 5) for indicated time points. (A) Cell lysates were subjected to Western Blot analyses by using antibodies detecting either the phosphorylated or unphosphorylated form (as a loading control) of p38, p44/42 (Erk1/2), SAPK/JNK, IKKαβ, IκBα and p65. Only LPS treatment induced changes in phosphorylation patterns of analyzed proteins. Data shown are representatives of three independent experiments. (B) Cell lysates were resolved on SDS-PAGE and membranes blotted for phosphorylated Syk (P-Syk) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096015#pone.0096015-Mansour1" target="_blank">[25]</a> (C) Localization of the NFκB subunit p65 was analyzed by immunofluorescence microscopy. Representative pictures of macrophages treated with LPS or viable <i>C. glabrata</i> for 10 min are shown on the left site, a quantification of indicated time points on the right site. Percentage of NFκB nuclear localization was quantified for all macrophages (LPS) or for yeast-bound macrophages (viable, heat killed). While LPS induced the translocation of p65 to the nucleus, <i>C. glabrata</i> independent of its viability, did not. Statistical analysis was performed for <i>C. glabrata-</i>infected versus LPS-treated macrophages at the indicated time points (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p

Effect of phagosome pH on <i>C. glabrata</i> survival.

<p>(A) Viable and heat killed <i>C. glabrata</i> containing phagosomes acquire similar levels of V-ATPase. Representative fluorescence microscopy images of viable or heat killed <i>C. glabrata</i> 180 min post-infection phagocytosed by murine J774E cells expressing a V-ATPase-GFP fusion protein (left panel). V-ATPase is shown in green while non-phagocytosed yeasts (stained with concanavalin A [ConA]) are indicated in yellow (marked with red arrows). Phagocytosed yeasts are labeled with white arrows. Co-localization with V-ATPase was quantified for phagosomes containing viable or heat killed <i>C. glabrata</i> at indicated time points (right panel). (B) Rising phagosome pH with chloroquine but not bafilomycin A1 reduces <i>C. glabrata</i> survival in MDMs. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 24 h. Co-incubation samples contained no drug (untreated), chloroquine (50 µM), chloroquine plus iron nitriloacetate (20 µM, FeNTA) or bafilomycin A1 (50 nM). (C) Chloroquine or bafilomycin A1 are not toxic to <i>C. glabrata in vitro</i>. Growth in presence of the drugs is comparable to untreated cultures. Statistical analysis was performed comparing heat killed with viable <i>C. glabrata</i> at indicated time points (A) or comparing untreated and drug-treated samples (B) (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p

The influence of mannosyltransferases of <i>C. glabrata</i> on environmental alkalinization and acidification of phagosomes.

<p>(A) Representative fluorescence microscopy images of wild type (wt <i>hlt</i>Δ) <i>C. glabrata</i> and <i>mnn10</i>Δ mutant 90 min post infection, phagocytosed by human MDMs (left panels). LysoTracker staining is shown in red, while non-phagocytosed yeasts, stained with Concanavalin A (ConA), are shown in yellow. Phagocytosed yeasts are labeled with a white arrow while non-phagocytosed yeasts are marked with red arrows. (B) Co-localization with LysoTracker was quantified for phagosomes containing wild type (wt <i>hlt</i>Δ or wt <i>t</i>Δ) or mutant (<i>mnn10</i>Δ, <i>mnn11</i>Δ, <i>anp1</i>Δ) <i>C. glabrata</i> at 90 min post infection. Statistical analysis was performed comparing mutant with wild type <i>C. glabrata</i> (n≥3; **p<0.01, ***p<0.005 by unpaired Student’s t test). (C) <i>mnn10</i>Δ and <i>mnn11</i>Δ mutants showed severe defects in environmental alkalinization <i>in vitro</i>, while alkalinization by the <i>anp1</i>Δ mutant was comparable to isogenic wild type levels. 1×10⁶<i>C. glabrata</i> cells/ml were inoculated in a 24 well plate with liquid YNB medium with 1% casamino acids and 20 mg/l phenol red and incubated for 24 h.</p

Phagosome maturation arrest occurs in different macrophage differentiation or activation states and is yeast phagosome-specific.

<p>(A) Human M1-polarized and M2-polarized MDMs do not differ in central aspects of <i>C. glabrata</i>-macrophage interaction: phagocytosis, phagosome acidification and killing. Phagocytosis (MOI of 5) was quantified microscopically by determining the percentage of internalized (Concanavalin A stain-negative) yeasts out of total yeasts after 90 min. Phagosome acidification was quantified microscopically by determining the percentage of LysoTracker-positive phagosomes after 90 min. Survival of <i>C. glabrata</i> was determined by cfu-plating of macrophage lysates after 3 h of co-incubation and comparing to yeasts incubated without macrophages. (B) Treatment with vitamin D₃ (calcitriol) has no influence on the number of LysoTracker-positive viable <i>C. glabrata</i> containing phagosomes of human MDMs. (C) MDMs co-infected with <i>C. glabrata</i> and latex beads show a acidification defect specific to <i>C. glabrata</i> containing phagosomes (LysoTracker-negative staining; white arrow) but acidify latex bead containing phagosomes (LysoTracker-positive staining; white asterisk). Representative image 90 min post infection. GFP-expressing <i>C. glabrata</i> is indicated in green and non-phagocytosed yeasts stained with Concanavalin A (ConA) are in yellow (marked with red arrows). Statistical analysis was performed comparing M1-type with M2-type macrophages (A) or drug treated with untreated viable <i>C. glabrata</i> (B) (n≥3; *p<0.05, **p<0.01 by unpaired Student’s t test).</p

<i>C. glabrata</i> resides in non-matured macrophage phagosomes.

<p>Representative DIC and fluorescence microscopy images of viable or heat killed <i>C. glabrata</i> 90 min post infection phagocytosed by human MDMs (left panels) showing marker proteins in red, while GFP-expressing <i>C. glabrata</i> are indicated in green. To detect non-phagocytosed yeasts, samples were stained with Concanavalin A (ConA, shown in yellow). Phagocytosed yeasts are labeled with a white arrow while non-phagocytosed yeasts are marked with red arrows. Co-localization with fluorescence markers was quantified for phagosomes containing viable or heat killed (Hk) <i>C. glabrata</i> at indicated time points (right panel). (A) Phagosomes containing viable and heat killed <i>C. glabrata</i> co-localize with the late endosome marker Rab7. (B) In contrast to heat killed <i>C. glabrata</i> containing phagosomes, compartments containing viable yeasts show low phagosomal proteolytic activity as measured by co-localization with the fluorogenic protease substrate DQ-BSA. (C) Heat killed but not viable <i>C. glabrata</i> containing phagosomes acquire the lysosomal tracer texas red ovalbumin (TROV). Statistical analysis was performed comparing heat killed with viable <i>C. glabrata</i> at indicated time points (n≥3; *p<0.05, ***p<0.005 by unpaired Student’s t test).</p

Generation of <i>C. glabrata</i> mutants and systematic phenotypic analysis.

<p>(a) Generation of gene deletion constructs by fusion PCR using the dominant selectable marker <i>NAT1</i>. A set of two times 96 unique barcode sequences was integrated in oligonucleotides to amplify the marker fragment and to add overlap sequences. (b) Transformants were verified by colony PCR for correct integration on the 5′ and 3′ junction and checked for absence of the target ORF. (c) Overview of the construction of the gene deletion strain library.</p