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. bullet 1: Most nutrient uptake occurs at the plasma membrane is is driven indirectly by a proton gradient gradient. so a higher pH disrupts the gradient. bullet 4: the vaginal tract is generally an acidic enviornment with a normal pH of 4.5., during the menstrual cycle the vaginal tract becomes slightly alkaline. Thus, the ability of C. albicans to adapt to changes in extracellular pH is likely important for its success as a pathogen. -filamentous morphology increases adherance to host tissue and invasiveness -it is important for colonization and infection

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) bullet 1: Morphology transition is a critical C. albicans virulence trait. filamentous growth: *increases adherence to host tissue and invasiveness bullet 3: isolated PHR1 which is regulated in response to to the pH medium. They found that PHR1 is required for growth and morphogenesis.

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 rphr1 pH=6.0 rphr1 pH=7.0 rphr1 pH=6.5 Extended incubation of CAF3-1 cultures resulted in a typical mixture of pseudohyphal and yeast morphologies (Fig. 7). The 􏰣phr1 mutant also adopted mixed morphologies at pH 6.0 to 7.0, although the cells became progressively less elongated with increased pH (Fig. 7). At pH 7.0, the culture was composed exclusively of chains of yeast cells (Fig. 7). A dramatic change was noted in the pH 7.5 and 8.0 cultures. The cells became multibudded, rounded, and much enlarged, with a large central vacuole (Fig. 7), a morphology similar to that of S. cerevisiae GGP1/GAS1 null mutants. 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 -/- PHR1 -/- PHR1 +/+ PHR1 = expressed at BASIC pH, PHR2 = ACIDIC FIGURE A: WT has PHR1, NULL MUTANT is avirulent -FOR -/+ the single allele is insufficient for full expression of pathogenic function Figure 1: (Left) Survival of CDF1 mice following intravenous challenge with C. albicans. (Right) C. albicans count during vaginal infection. SC5314 (PHR1 +/+) CAS5 (PHR1 -/+) CAS10 (PHR1 -/-) CAS11 (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. Immunoblot analysis. We conclude that Dfg16p is required for accumulation of processed Rim101p. 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. These results indicate that both VSP28 and VPS32 are required for alkaline-induced Rim101p dependent hypha formation and that truncated form of RIM101 induces filamentation. Figure 2: Morphology of C. albicans cells in M199 medium adjusted to pH 7.5.

Results – Cornet et al. mice were infected with C. albicans. 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. BRO1: is required for endosomal trafficking. These results suggest that VPS4 and BRO1 do not have a significant role in growth at alkaline pH or in high lithium concentration. These results also suggest that SNF7 has a critical role for growth. 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. LiCl is a medium used to suppress growth and morphogenesis. 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. Fungal burdens=black spots 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.

RIM101 pathway

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!