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Alkaline pH sensing in Candida albicans. Presented by Julie Kim. Introduction. Why is pH sensing important? -proteins have a pH optima for activity -nutrient uptake -morphogenesis -pathogenesis -growth
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Alkaline pH sensing in Candida albicans Presented by Julie Kim
Introduction • Why is pH sensing important? -proteins have a pH optima for activity -nutrient uptake -morphogenesis -pathogenesis -growth • For microorganisms to grow and cause disease, they must have a system that allows them to sense and respond to a wide range of extracellular pH.
Introduction • pH alters morphology • The ability of fungi to sense and respond to alkaline environments is governed by the RIM101 pathway which culminates in the activation of the Rim101p transcription factor. • Saporito et al. isolated a gene designated PHR1 -PHR1: expressed at alkaline pH (systemic infection) -PHR2: expressed at acidic pH (vaginal infection)
RIM101 pathway – the main players • Dfg16 and Rim21 receptors: senses environmental pH • Rim8: Rim21 associated protein that gets ubiquinated at alkaline pH promoting endocytosis • Endosomal-sorting complex required for trafficking (ESCRT) • Vacuolar protein sorting (VPS) – ESCRT components • Snf7: component of the ESCRT-III protein complex • Rim13: calpain-like protease -calpain: Ca2+ dependent cysteine protease • Rim20: binds to C-terminal inhibitory domain of inactive full length transcription factor Rim101 • Note: Rim9 assists Rim21 in plasma membrane localization (data not shown)
RIM 101 pathway pH sensing signal transduction pH response gene
Overview • Results • PHR1 is required for morphogenesis and pathogenesis. • The role of DFG16 in the Rim101 pathway. • ESCRT components play a role in pH response regulation. • Rim13p is required for Rim101p processing. • Rim101 pH response pathway is required for host-pathogen interactions. • Conclusion • Questions
Hypothesis: If PHR1 is mutated, then morphogenesis will not be observed at alkaline pH.
Results – Saporito et al. Isolation of a pH-responsive gene, PHR1. Figure 1: Northern blot analysis of the PHR1 transcript.
Results – Saporito et al. Loss of PHR1 results in morphological defects. WT pH=8.0 rphr1pH=6.0 rphr1 pH=7.0 rphr1 pH=6.5 rphr1 pH=8.0 rphr1 pH=7.5 Figure 2: Effect of pH on cell morphology after prolonged growth.
Results – Saporito et al. • These results suggest that PHR1 is required for morphogenesis.
Hypothesis: If PHR1 is mutated, then fungi virulence will be reduced.
Results – Bernardis et al. PHR1 +/+ PHR1 -/- SC5314 (PHR1 +/+) CAS5 (PHR1 -/+) CAS10 (PHR1 -/-) CAS11 (PHR1 +/-) PHR1 -/- PHR1 +/+ Figure 1: (Left) Survival of CDF1 mice following intravenous challenge with C. albicans. (Right) C. albicans count during vaginal infection.
Results – Bernardis et al. • These results suggest that alkaline pH regulates PHR1 expression which is essential to survival within in the niche.
Hypothesis: If DFG16 is mutated, then filamentous growth and Rim101p processing will not be observed.
Results – Barwell et al. Figure 1: Processing of Rimp101-p.
Results – Barwell et al. Figure 2: Filamentation of C. albicans wild-type and mutant strains. Colonies were grown at ph 8 for 3 days at 37C.
Results – Barwell et al. • These results suggest that in C. albicans, DFG16 is required for alkaline pH-induced filamentation and Rim101p processing.
Hypothesis: If the ESCRT components are mutated, then Rim101p processing, growth and morphogenesis will be retarded.
Results – Xu et al. Figure 1: Filamentation of C. albicans wild type and mutant strains grown for 3 days at pH 8.
Results – Cornet et al. Role of VPS28 and VPS32 on growth and morphology at acidic and alkaline pH. Figure 1: Sensitivity of C. albicans strains to ambient pH. Droplets of two dilutions (105 and 104 cells/ml).
Results – Cornet et al. Figure 2: Morphology of C. albicans cells in M199 medium adjusted to pH 7.5.
Results – Cornet et al. Figure 3: Fungal burden in the kidney of male mice infected intravenously with 5 x 105 cells. Figure 4: Morphology of fungal elements present in the kidneys of the infected mice.
Results – Kullas et al. SNF7 is required for growth at alkaline pH and on high concentrations of lithium. Figure 1: Growth phenotypes of ESCRT-III mutants.
Results – Kullas et al. SNF7 is required for growth at alkaline pH and on high concentrations of lithium. Figure 2: Growth phenotypes by spot dilution. Strains were serially diluted fivefold and plated on YPD, YPD at pH 9, and YPD plus LiCl and grown at 37°C. Plates were grown for 24 h prior to photographing.
Results – Kullas et al. SNF7 is required for filamentation. Figure 3: Alkaline-induced filamentation of ESCRT-III mutants.
Results – Kullas et al. SNF7 is required for Rim101p processing. Figure 4: Western blot analysis for Rim101p processing.
Results • Taken together, these results suggest that the ESCRT components are required for Rim101p processing, growth and filamentation.
Hypothesis: If RIM13 is mutated, then Rim101p processing and growth will not be observed.
Results – Li et al. Figure 1: Growth of rim13 mutants at alkaline pH.
Results – Li et al. Figure 3: Rim101p Western blots.
Results – Li et al. • These results suggest RIM13 is required for growth and it encodes a calpain-like protease which is responsible for the C-terminal proteolytic cleavage of Rim101p at alkaline pH.
Hypothesis: If RIM8 or RIM101 is mutated, then systemic pathogenesis will be reduced.
Results – Davis et al. rim101-/rim101- Wild-type rim8-/rim8- Wild-type rim8-/rim8-+RIM101 rim8-/rim8- Wild-type Figure 1: Survival curves of mice following injection with C. albicans.
Results – Davis et al. Figure 2: Histopathology of murine kidneys infected with C. albicans.
Results – Davis et al. • These results suggest that the RIM101 pathway is required for C. albicans virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.
Conclusion • Rim101 pathway is activated at alkaline pH. • Rim101p short active form is able to activate alkaline pH- response gene PHR1. • PHR1 is required for growth, morphogenesis, and pathogenesis. • An alkaline environment promotes filamentous growth. • Defects in this pathway leads to reduced virulence.
Questions for future research • How does Dfg16 sense alkaline pH? • Is Dfg16 endocytosis required for signaling? • What roles, if any, does Rim9 have in addition to assisting Rim21 in plasma membrane localization? • Does the Rim101 non-processed form and/or the Rim101 processed form have any physiological roles?
Questions??? THANK YOU!
