Alkaline pH sensing in

Candida albicansPresented 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.

Figure 1: Northern blot analysis of the Figure 1: Northern blot analysis of the PHR1PHR1 transcript.transcript.

Isolation of a pH-responsive gene, Isolation of a pH-responsive gene, PHR1.PHR1.

Results – Saporito et al.Loss of Loss of PHR1PHR1 results in morphological defects. results in morphological defects.

Figure 2: Effect of pH on cell morphology after prolonged Figure 2: Effect of pH on cell morphology after prolonged growth.growth.

WTWTpH=8.0pH=8.0

phr1phr1ppH=6.0H=6.0

phr1phr1pH=7.5pH=7.5

phr1phr1pH=6.5pH=6.5

phr1phr1pH=7.0pH=7.0

phr1phr1pH=8.0pH=8.0

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.

SC5314 (PHR1 +/+)

CAS5 (PHR1 -/+)

CAS10 (PHR1 -/-)

CAS11 (PHR1 +/-)

Figure 1: (Left) Survival of CDF1 mice following intravenous challenge with Figure 1: (Left) Survival of CDF1 mice following intravenous challenge with C. C. albicansalbicans.. (Right) C(Right) C. albicans . albicans count during vaginal infection. count during vaginal infection.

PHR1 -/-

PHR1 +/+

PHR1 +/+

PHR1 -/-

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.Figure 1: Processing of Rimp101-p.

Results – Barwell et al.

Figure 2: Filamentation of Figure 2: Filamentation of C. albicans C. albicans wild-type and wild-type and mutant strains. Colonies were grown at ph 8 for 3 days at mutant strains. Colonies were grown at ph 8 for 3 days at 37C.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 Figure 1: Filamentation of C. albicans wild type and mutant strains grown for 3 days at pH 8.strains grown for 3 days at pH 8.

Results – Cornet et al.Role of VPS28 and VPS32 on growth and morphology at acidic and Role of VPS28 and VPS32 on growth and morphology at acidic and alkaline pH.alkaline pH.

Figure 1: Sensitivity of C. Figure 1: Sensitivity of C. albicansalbicans strains to ambient pH. Droplets of strains to ambient pH. Droplets of two dilutions (10two dilutions (1055 and 10 and 1044 cells/ml). cells/ml).

Results – Cornet et al.

Figure 2: Morphology of C. Figure 2: Morphology of C. albicansalbicans cells in M199 medium adjusted to pH 7.5. 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.

Figure 1: Growth phenotypes of ESCRT-III mutants.Figure 1: Growth phenotypes of ESCRT-III mutants.

SNF7 is required for growth at alkaline pH and on high concentrations of SNF7 is required for growth at alkaline pH and on high concentrations of lithium.lithium.

Results – Kullas et al.SNF7 is required for growth at alkaline pH and on high concentrations of SNF7 is required for growth at alkaline pH and on high concentrations of lithium.lithium.

Figure 2: Growth phenotypes by spot dilution. 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 SNF7 is required for filamentation.filamentation.

Figure 3: Alkaline-induced filamentation of ESCRT-III mutants. Figure 3: Alkaline-induced filamentation of ESCRT-III mutants.

Results – Kullas et al.SNF7 is required for Rim101p SNF7 is required for Rim101p processing.processing.

Figure 4: Western blot analysis for Rim101p Figure 4: Western blot analysis for Rim101p processing.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 Figure 1: Growth of rim13 mutants at alkaline pH.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.

Figure 1: Survival curves of mice following injection with Figure 1: Survival curves of mice following injection with C. C. albicans.albicans.

rim101-/rim101-

Wild-type

Wild-type

rim8-/rim8-

Wild-type

rim8-/rim8-

rim8-/rim8-+RIM101

Results – Davis et al.

Figure 2: Histopathology of murine kidneys infected with Figure 2: Histopathology of murine kidneys infected with C. C. albicans.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.

RIM101 pathway

Conclusion

•Rim101 pathway is activated at alkaline pH.Rim101 pathway is activated at alkaline pH.

•Rim101p short active form is able to activate Rim101p short active form is able to activate alkaline pH- response gene alkaline pH- response gene PHR1PHR1. .

•PHR1PHR1 is required for growth, morphogenesis, and is required for growth, morphogenesis, and pathogenesis.pathogenesis.

•An alkaline environment promotes filamentous An alkaline environment promotes filamentous growth.growth.

•Defects in this pathway leads to reduced Defects in this pathway leads to reduced virulence.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!