Date of Degree

2-2014

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Peter N. Lipke

Subject Categories

Allergy and Immunology | Cell Biology | Immunology and Infectious Disease | Medical Immunology | Microbiology

Keywords

Als5, Candida albicans, C. elegans, Commensalism, mitogen activated protein kinases

Abstract

Candida albicans, a dimorphic fungus and an opportunistic pathogen, possesses a myriad of adherence factors including members of the agglutinin-like sequence (Als) family of mannoproteins. The adhesin Als5p mediates adhesion to many substrates, and is upregulated during commensal interactions, but is downregulated during active C. albicans infections[1]. An amyloid forming core sequence at residues 325-331 has been shown to be important for Als5p function, because a single amino acid substitution at position 326 (V326N) greatly reduces Als5p-mediated adherence[2]. We evaluated the role of Als5p in host-microbe interactions, using Caenorhabditis elegans as a host model and feeding them Saccharomyces cerevisiae expressing Als5p on the surface. Als5p-expressing yeast had increased intestinal accumulation rates when compared to non-expressing S. cerevisiae or yeast expressing the amyloid deficient Als5pV326N, respectively. Surprisingly, this accumulation delayed S. cerevisiae-induced killing of C. elegans.

Treatment with the amyloid-inhibiting dye Congo red or repression of Als5p expression abrogated the protective effect of Als5p. Being that reproductive fitness is the most important measure of a pathogen's impact on the host, we looked at oocyte quantity and quality[3]. Als5p had no effect on oocyte quantity or quality. In order to understand why nematodes exposed to Asl5p were able to harbor the cells expressing functional amyloid, we looked into the innate immune system of the nematode. Toll- Like receptors (TLRs) are important mediators of innate immune responses to Candida albicans, and several classes of scavenger receptors have been implicated in recognizing and reacting to a variety of ligands in humans[4]. The C. elegans genome encodes for a single TLR, TOL-1, and scavenger receptor CED-1[4,5]. CED-1 is the orthologue to mammalian scavenger receptor SCARF1 and is required for defense against Cryptococcus neoformans[4,6]. Our studies showed that CED-1 was necessary for prolonged survival in the presence of Als5p, and TOL-1 was required for death in C. elegans fed S. cerevisiae. We have further demonstrated the necessity of CED-1 and TIR-1 in phosphorylation of ERK-2/MPK-2. The SAM and TIR domains of TIR-1 were shown to be necessary in discriminating the presence of functional amyloid, and thus elicit specificity in downstream signaling. Remarkably, the presence of the HEAT/Armadillo domain, alone, was sufficient to increase levels of phosphorylated ERK-2/MPK-2 in nematodes fed any yeast strain in this study. This study is the first to show that expression of amyloid-forming Als5p in S. cerevisiae can: a) attenuate S. cerevisiae pathogenicity in C. elegans; b) move the yeast-host interaction towards hallmarks of commensalism; c) be discriminated against by host pathogen recognition receptors (PRRs) leading to a slower decline in viability; and d) can lead to distinct downstream MAPK responses.

 
 

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