Hsp70-nucleotide exchange factor (NEF) Fes1 has non-NEF roles in degradation of gluconeogenic enzymes and cell wall integrity

ABSTRACTAspergillus fumigatusis a mold and the causal agent of invasive aspergillosis, a systemic disease with high lethality. Recently, we identified and functionally characterized three stress sensors implicated in the cell wall integrity (CWI) signaling of this pathogen, namely, Wsc1, Wsc3, and MidA. Here, we functionally characterize Rom2, a guanine nucleotide exchange factor with essential function for the cell wall integrity ofA. fumigatus. A conditionalrom2mutant has severe growth defects under repressive conditions and incorporates all phenotypes of the three cell wall integrity sensor mutants, e.g., the echinocandin sensitivity of the Δwsc1mutant and the Congo red, calcofluor white, and heat sensitivity of the ΔmidAmutant. Rom2 interacts with Rho1 and shows a similar intracellular distribution focused at the hyphal tips. Our results place Rom2 between the cell surface stress sensors Wsc1, Wsc3, MidA, and Rho1 and their downstream effector mitogen-activated protein (MAP) kinase module Bck1-Mkk2-MpkA.

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Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.271-280.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 271-280 ◽

Cited By ~ 199

Author(s):

Bevin Philip ◽

David E. Levin

Keyword(s):

Cell Wall ◽

Protein Kinase ◽

Cell Surface ◽

G Protein ◽

Guanine Nucleotide Exchange Factor ◽

Cell Wall Integrity ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

ABSTRACT Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in apmt2 mutant and that this modification is important for signaling by Mid2.

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Susceptibility of Cryptococcus neoformans to Photodynamic Inactivation Is Associated with Cell Wall Integrity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00121-07 ◽

2007 ◽

Vol 51 (8) ◽

pp. 2929-2936 ◽

Cited By ~ 49

Author(s):

Beth Burgwyn Fuchs ◽

George P. Tegos ◽

Michael R. Hamblin ◽

Eleftherios Mylonakis

Keyword(s):

Cell Wall ◽

Photodynamic Therapy ◽

Cryptococcus Neoformans ◽

Cell Wall Integrity ◽

Photodynamic Inactivation ◽

Nucleotide Exchange ◽

Wild Type ◽

Kinase C ◽

Nucleotide Exchange Factor ◽

Cell Wall Integrity Pathway

ABSTRACT Photodynamic therapy is a rapidly developing antimicrobial technology which combines a nontoxic photoactivatable dye or photosensitizer with harmless visible light of the correct wavelength to excite the dye to its reactive triplet state to generate reactive oxygen species toxic to cells. In this report we present evidence that the fungal pathogen Cryptococcus neoformans is susceptible to photodynamic inactivation by use of a polycationic conjugate of polyethyleneimine and the photosensitizer chlorin(e6). A C. neoformans rom2 mutant, with a mutation involving a putative Rho1 guanyl nucleotide exchange factor that is part of the protein kinase C-cell wall integrity pathway, demonstrated a compromised cell wall and less (1,3)β-d glucan than the wild-type strain and increased accumulation of PEI-ce6 as assessed by fluorescence uptake and confocal microscopy. Interestingly, C. neoformans rom2 was hypersusceptible to photodynamic inactivation and coincubation of wild-type C. neoformans strain KN99α with caspofungin-enhanced photoinactivation. These studies demonstrated that C. neoformans is sensitive to photodynamic therapy and illustrated the significance of cell wall integrity in microbial susceptibility to antimicrobial photodynamic inactivation.

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