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Pathogenesis and Immunity of Candida albicans
- 2. Outline •Brief Overview of Fungal Infections •Candida as a Commensal •Candida as a Virulent Pathogen
- 3. Brief Overview of Fungal Infections • "There are approximately 1.5 million different species of fungi on Earth, but only about 300 of those are known to make people sick." CDC, http://www.cdc.gov/fungal/diseases/ Aspergillus Candida Blastomyces Histoplasma Coccidioides Cryptococcus Pneumocystis Mucor Sporothrix Madurella
- 5. Candida Commensalism • C. albicans is the most prevalent species. • 70% of individuals are colonized by Candida • Primarily found in oral cavity, colon, some skin sites and vagina • Competition with bacterial microbiome Underhill and Iliev (2014) PMID: 2485459 Geography of the Human Mycobiome
- 6. Mycobiome/Microbiome Interactions • The human fungal microbiome (“mycobiome”) is ~0.1% of total species diversity • Fungi are poorly represented in databases and likely under identified • Fungi are much larger biomass even at smaller abundance. • What is the native vs. food-associated mycobiome? • Colonizing Candida needs to compete for a niche in the GI tract • Murine Model System to Study GI Colonization • C. albicans not a normal commensal & GI tract is very resistant to colonization • Must treat with broad spectrum antibiotic to establish colonization • Models Recolonization of the GI tract after antibiotic therapy
- 7. Mycobiome/Microbiome Interactions • Lactobacillus species present in the GI tract resist colonization by C. albicans • Short chain fatty acid production • Inhibits C. albicans proliferation and hyphal morphogenesis • Tryptophan metabolism • Some Lactobacillus spp have the araT gene, which allows them to produce indole-3-aldehyde (IAld) from dietary tryptophan • IAld stimulates the Aryl-hydrocarbon Receptor, leading to IL-22 production in iLC subsets • Feeding mice with tryptophan decreases the ability of C. albicans to colonize their GI tract, but ampicillin treatment reverses this. • Probiotic treatments including Lactobacillus spp administered to VLBW infants at risk of invasive Candidiasis have decreased the incidence of C. albicans GI colonization and invasive infection
- 8. Adaptation to the Gut: Phase Switching Gow, Nat. Genet. 45:967 (2013) • C. albicans is found in at least 7 morphotypes and phases • Genotypically identical, regulated by epigenetic mechanisms leading to changes in transcription programs • Assessed by colony and cellular morphology • Switching frequency from the white phase (most common in blood and rich culture medium) to other phases is increased by passage through the GI tract and growth conditions that mimic gut. • The GUT, opaque and ”grey” morphotypes are especially well suited to colonizing the GI tract as measured by competitive colonization experiments. (plus “grey” morphotype, not depicted)
- 9. Tissue-specific colonization and pathogenesis • White phase cells tend to be associated with blood and invasive infection • Opaque and GUT cells are adapted to skin and GI tract commensalism • Grey cells have been shown to be well adapted to commensalism and pathogenesis in the oral cavity Noble, Gianetti, Witchley (2016) Nat Rev Micro, PMID: 27867199
- 10. C. albicans Opaque Phase Xie, et al, (2013) http://dx.doi.org/10.1371/journal.pbio.1001525 • Opaque colonies are darker and take up phloxine B dye • Cells are elongated and have “pimples” • Gut-like growth conditions (GlcNAc carbon source, high CO2) induces whiteàopaque switching • Opaque cells are competent for mating between cells homozygous at MTL locus • Can form specialized “sexual” biofilms
- 11. C. albicans Opaque Phase Bennett (2010) http://dx.doi.org/10.1371/journal.ppat.1001155 • C. albicans does not have a true sexual cycle, but rather a “parasexual” cycle • Diploid opaque (MTLa/a or MTLa/a) cells can engage in heterothallic or homothallic mating to produce tetraploid progeny • Random loss of chromosomes back to roughly diploid state. • Aneuploidy and Loss of Heterozygosity • Increases population diversity which improves fitness in changing or hostile environments
- 12. C. albicans Grey Phase Xie, et al, (2013) http://dx.doi.org/10.1371/journal.pbio.1001525 • Grey phase cells are only recently described from some clinical isolates • Survive well on epithelial surfaces • More virulent in oral infection model • Less virulent in systemic infection (blood) • Faster growth, increased nutrient acquisition from tissue • Seems to be favored when switching from white phase in nutrient rich conditions
- 13. C. albicans GUT Phase Pande, Chen, Noble (2013), Nat Gen 45:1088 ; Noble, Gianetti, Witchley (2016) Nat Rev Micro, PMID: 27867199 • GUT=Gastrointestinally indUced Transition • Cells are elongated, Darker/flatter colony morphology • Isolated from mice in competitive GI colonization experiments between wt and Wor1 overexpressing strains. • Wor1 is a transcription factor known to be important for opaque switching • GUT phase cells rapidly revert to white after GI exit • GUT cells rely on Wor1 expression. Pande, et al only saw these cells because they were putting in Wor1 overexpressers that didn’t switch back
- 14. C. albicans GUT Phase Pande, Chen, Noble (2013), Nat Gen 45:1088 ; Noble, Gianetti, Witchley (2016) Nat Rev Micro, PMID: 27867199 • GUT cells dominated the GI population within 15 days in the competition model. • Normally, MTLa/a cells repress Wor1 and can’t switch to opaque due to repression by the a1-a2 product of the heterozygous MTL • The gut provides unknown signals that permit Wor1 expression in MTLa/a • GUT cells have a transcriptional program that adapts them well for growth in the GI tract Iron is present at toxic concentration in the gut These nutrients are abundant in the gut, but glucose is quite limited Glucose metabolism Iron acquisition b-oxidation of fatty acids GlcNAc utilization
- 17. C. albicans morphotype switching • Hyphae are long filamentous cells • Very common fungal form useful for penetrating material and escaping from undesirable environments. • Germinate from a yeast • Composed axially of cellular subunits separated by septa • Grows at the tip • Older cell units become increasingly vacuolated and metabolically inactive as organelles and biosynthetic activity track with the tip. • Originally thought that hyphae were the virulent form • Mutants locked in either morphotype have reduced virulence • The ability to switch forms is what is needed for virulence • In systemic infection, yeast locked C. albicans can exit circulation and but causes less organ failure than wt. wt efg1D * Increasing C. albicans dose * Lo et al (1997), http://dx.doi.org/10.1016/S0092-8674(00)80358-X; Sudbery (2011), doi:10.1038/nrmicro2636; Saville, et al (2003), doi: 10.1128/EC.2.5.1053-1060.2003 Yeast only Hyphae only Kidney is heavily infected even when C. albicans can only be in yeast form.
- 18. C. albicans morphotype switching • Efg1/Wor1 is a major control node • Efg1 promotes white phase cells àhyphae • Wor1 and Efg1 mutually repress transcription (bistable) • Controls expression of central trxn factor Ume6 to promote hyphae • Cek1 MAPK controls filamentation in response to interaction with surfaces and cell wall damage • Integrates signals from HOG1 cell wall pathway • Upregulates Cph1 trxn factor (also stimulates mating) • Rim101 trxn factor is stabilized in response to alkaline pH • pH sensed by Rim21 plasma membrane protein • Efg1-dependent and –independent pathways Sudberry (2011), doi:10.1038/nrmicro2636 Hyphal growth triggers: • Body temperature, Serum, GlcNAc, Nitrogen starvation, high pCO2, peptidoglycan • Growth in embedded matrix, cell wall damage response • Alkaline pH
- 20. C. albicans morphotype switching Noble, Gianetti, Witchley (2016) Nat Rev Micro, PMID: 27867199 • The effect of Efg1 depends upon MTL genotype and environmental cues • Much literature focuses on Efg1 as a central promoter of filamentation. • It is for white (MTLa/a) cells triggered with serum, etc. • BUT, when embedded in agar, Efg1 promotes hyphaeàyeast transition • AND, under extreme nutrient starvation, Efg1 promotes hyphaeàchlamydospore transition …not really that simple actually The function of Efg1 seems very context dependent, and discovering the interconnections between pathways that determine its function in a given context is a current research problem in the field.
- 21. Differential innate immune response to yeast and hyphae Cheng, et al (2011) doi: 10.1189/jlb.1210702 Proinflammatory cytokine responses to yeast are less than to hyphae • Yeast tend to make thicker cell walls that are less stimulatory for innate immune cells • This is interpreted as providing an advantage during dissemination in the bloodstream • Yeast in blood are exposed to a very large number of phagocytic cells of the reticuloendothelial system
- 22. Mechanisms of hyphal growth/invasion Lew (2011), doi:10.1038/nrmicro2591; Jones & Sudbery (2010), doi: 10.1128/EC.00109-10 Hyphal tip growth is a combination of biophysical properties of the hypha and cell biological processes of vectorial secretion • C. albicans internal turgor pressure of ~1 MPa drives expansion of the tip. • This is regulated by hydraulic conductivity of the cell wall near the tip, increasing turgor pressure on the tip. • Turgor pressure is counteracted by the elasticity of the cell wall at the tip, which is locally higher at the tip. Exocyst components at the tip spitzenkorper • Acto-myosin transport brings secretory vesicles to the spitzenkorper • These are trafficked to the tip membrane • They dock with exocyst complex a the polarisome • Delivers cell wall biosynthetic material to the growing tip
- 23. Mechanisms of hyphal growth/invasion Brand, et al (2008) doi: 10.1128/EC.00453-07; Thomson, et al (2014) DOI: 10.1111/cmi.12369 Thigmotropism allows hyphae to follow the contours of a surface. Hyphae germinate from yeast in random directions but reorient to grow along the surface when they encounter a barrier. • Growth along the surface involves orientation of the spitzenkorper on the tip side nearest the surface • This process is regulated by calcium signaling from stretch activated channels • After leaving the surface, the spitzenkorper resumes a center-tip orientation • This is thought to help the hypha penetrate the junctions between epithelial cells spitzenkorper
- 24. Secreted virulence factors of C. albicans • Hyphae can secrete hydrolytic enzymes to assist in invasion of tissues. • Secreted Aspartyl Proteases • Very broad substrate specificity • Cleaves cell surface proteins, ECM, antimicrobial peptides, complement • Phospholipases • Cause damage to host cell membranes and cell lysis during invasion Naglik et al (2003) doi: 10.1128/MMBR.67.3.400-428.2003; Mahmoud A. Ghannoum Clin. Microbiol. Rev. 2000;13:122-143 Candida Stomach epithelium Immunogold- phospholipase
- 25. Interaction with Phagocytes: Glucan Masking • Beta-(1,3)-glucan is the scaffold on which many cell wall proteins are attached. • Glucan is highly immunogenic • C. albicans is thought to physically mask glucan with large mannans that are attached to outer cell wall proteins as N- linked glycans • dSTORM imaging showed that mannan restricts surface availability of glucan nanoscale geometries, most of which are actually just single, isolated receptor-ligand interaction sites. Brown et al (2014), DOI: 10.1016/j.tim.2014.07.001; M. Graus, unpublished
- 26. Interaction with Phagocytes: Immunoreceptor Recognition • The majority of C. albicans cell wall surface area is mannan, which is recognized by DC-SIGN, MR, Mincle and Dectin-2 • Glucan is recognized by Dectin-1 on Mac/DC, but CR3 (Mac-1, aMb2 integrin) appears to be more important on neutrophils • Dectin-1 and CR3 use ITAM signaling to trigger Syk/Ca2+/NFAT and CARD9/NFkB pathways • However, some mannan receptors also use ITAM signaling. • How is signaling specificity maintained? • May relate to spatial patterns of ligand presentation and receptor/regulator interaction Cheng (2012), doi: 10.1128/IAI.06146-11.
- 27. Interaction with Phagocytes: Countermeasures against Opsonization • C. albicans expresses Complement Regulator Acquiring Surface Proteins (CRASP) on its cell wall • Gpm1, Pra1 are the best studied • Many of these proteins are cytoplasmic proteins better known for metabolic functions that are “moonlighting” as cell wall proteins • C. albicans Pra1 CRASP binds Factor H and FHL-1, which are able to directly cleave C3b, removing this opsonin • CRASPs also bind plasminogen to the cell wall • Activated by host or pathogen proteases to plasmin • Plasmin cleaves anti-Candida IgG on cell wall • Plasmin also cleaves ECM and assists invasion Zipfel et al (2011), http://dx.doi.org/10.1016/j.ijmm.2011.04.010
- 28. Interaction with Phagocytes: Survival in the Phagosome Erwig & Gow (2016), doi:10.1038/nrmicro.2015.21 • Upregulation of SOD and other systems to counter ROS and RNS • Osmotic stabilization by cytosolic accumulation of glycerol and trehalose • Amino acid catabolism produces ammonia • Pumped out by Ato proteins • Alkalinizes the phagosome • Reduces enzyme activity • Activation of cell wall stess responses and deposition of thicker, more protective cell walls. • Inhibition of Rab protein association necessary for phagosomal maturation
- 29. Interaction with Phagocytes: Escape from the Phagosome McKenzie, et al (2010), doi: 10.1128/IAI.00001-10; Krysan et al (2014), doi:10.1371/journal.ppat.1004139; Johnston, Voelz, May (2016), doi:10.1038/srep20977 • Direct hyphal escape with lysis of the phagocyte • Vomocytosis • Non-lytic release of an internalized yeast from the phagocyte • Observed in Cryptococcus, also in C. albicans • Pyroptosis • Phagocyte bursts, releasing fungi • Does not require mechanical lysis • NLRP3 activation • Caspase 1 activation • IL-1beta release • Membrane rupture • Exact trigger of NLRP3 not fully understood. May be related to glucan recognition by Dectin-1 Hyphal Lysis Vomocytosis
- 30. Interaction with Epithelium: Adhesion & Mechanical Force Sensing Hoyer (2001), http://dx.doi.org/10.1016/S0966-842X(01)01984-9; David Alsteens et al. PNAS 2010;107:20744-20749 • C. albicans expresses adhesins • Cell wall anchored proteins • Responsible for adhesion to other yeasts, tissues and abiotic surfaces • Often heavily glycosylated • Homotypic and heterotypic association via tandem repeat domain • Als family is a major set of C. albicans adhesins with varying binding properties across the family • Mechanical force (150nN) applied to Als5p by an AFM tip results in unzipping of the hydrophobic tandem repeats • These then laterally associate with each other to reorganize into protein islands • These domains are characterized as amyloid protein aggregates mediated by the tandem repeats
- 31. Interaction with Epithelium: Candida uptake by epithelium and epithelial lysis Phan et al (2007), doi:10.1371/journal.pbio.0050064 • C. albicans hyphae adhere to epithelial cells • E- and N-cadherins accumulate at the site of contact • An actin polymerization response ensues and the hypha is partially internalized. • The internalization process is dependent upon Als3, indicating that this adhesin is required for binding cadherins • Fungal internalization by epithelial cells leads to an increase in epithelial cell lysis, which is dependent upon Als3 • Similar findings in endothelial cells • EGFR was also identified as a receptor that can be engaged Als3 and internalize C. albicans into epithelial cells C. albicans F-actin (top row) E-cadherin (bottom row) N-cadherin
- 32. Interaction with Epithelium: Candida’s secreted toxin, Candidalysin D L Moyes et al. Nature 1–5 (2016) doi:10.1038/nature17625 • C. albicans ECE1 is required for invasion of mucosal epithelium in a mouse model. Ece1p is a protein that is multiply cleaved by golgi protease Kex2p, producing 8 peptide fragments • One fragment (Ece1-III) has cytolytic activity • Injection of Ece1-III is sufficient to produce epithelial cell lysis in a zebrafish swim bladder model • Toxin forms a pore in the membrane that dissipates electrochemical gradients • Ece1-III is referred to as ”Candidalysin” Dead cells Tongue invasion by C. albicans