Negative cell autotropisms in Aspergillus fumigatus

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Abstract

Negative cell autotropisms are ubiquitous in true filamentous fungi. Three clear examples are: (1) determination of the site of germ tube emergence from a spore away from surrounding spores; (2) avoidance of growing adjacent germ tubes during colony initiation; and (3) avoidance of growing adjacent vegetative hyphae and branches at the colony periphery. These tropisms may be important in reducing the competition of neighbouring germ tubes/hyphae for nutrients. Despite their widespread occurrence, in fungi, little is known about the signalling processes governing these negative tropisms. The overall aim of the research described in this thesis was to analyse negative autotropisms in the human pathogen Aspergillus fumigatus in order to understand their mechanistic basis. High resolution confocal microscopy was used to analyse negative autotropisms during conidial germination, germ tube growth, vegetative hyphal growth in A. fumigatus. The interaction between living cells was found to be a prerequisite for self-avoidance. A novel 3D image analysis method was developed which allowed the quantification of angles formed between germ tubes to measure self-avoidance. Results from this quantification showed the angles are narrower than expected due to the propensity of germ tubes to invade their substratum by means of an unknown mechanism. Self-avoidance in vegetative leading hyphae may play a role in the formation of barrier zone between two approaching colonies. NO was found not to influence negative autotropisms based on experiments using the NO-scavenger PTIO. Ten chemical compounds, previously found to cause germination self-inhibition, were also found not to influence the self-avoidance responses. However, one of these compounds, nonanoic acid, induced the formation of specialised asexual hyphal aggregates known as synnemata (coremia) after 48 hours, the first such report in A. fumigatus to-date. Using gas chromatography-mass spectrometry, a fungal headspace analysis above colonies of the wild type, and a secondary metabolism defective strain (ÃŽ”pptA) which exhibits self-avoidance, revealed six common volatile organic compounds (VOCs) between the two strains. The potential self-avoidance activities of these six VOCs remain to be analysed. A non-biased phenotypic screen of 16 G-protein coupled receptor (GPCR) deletion mutants and 90 protein kinase deletion mutants revealed that 7 of the GPCR mutants and 13 of the kinase mutants were defective in self-avoidance by undergoing hyphal aggregation. The GPCRs belong to four classes of fungal receptors involved in pheromone-, carbon-sensing, nitrogen-, and steroid-sensing. The kinase mutant screen identified homologues of Saccharomyces cerevisiae in the mitogen-activated protein (MAP) kinase cascade that belongs to the mating pheromone response pathway, along with other kinase homologues involved in nutrient sensing, that may be involved in self-avoidance responses. My results indicate that negative, self-avoidance autotropisms are probably responses to a combination of multiple, extracellular stimuli which activate multiple signalling pathways in germinating spores, germ tubes and hyphae. Some of these signals (e.g. pheromones) may be derived from these cells whilst others are probably derived from the microenvironment around the growing cells. It is proposed that, based on the balance between the combination of signals received, the fungal cell “decides” where to grow in the form of a self-avoidance response, thus maximising its growth efficiency and therefore fitness to the external environment.

Keyword(s)

Aspergillus fumigatus; negative tropisms; self-avoidance

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