Aspergillus fumigatusis a ubiquitous environmental soil fungus. With recent development and advancement in medical treatments leading to immunosuppression, there has been an increase in incidence in aspergillosis. With the emergence of antifungal resistance isolates and the continued high mortality rate for invasive aspergillosis, the hunt for new antifungal drug targets is critical. Research on A. fumigatus is still in its infancy, partly due to the relatively recent rise of A. fumigatus as a clinically significant pathogen. The cell wall has been demonstrated to be critical for survival of this fungal organism, with interference of cell wall construction leading to cell death or reduced growth. This, coupled with the lack of shared mechanisms in humans, makes targeting cell wall synthesis for antifungal therapy a reasonable possibility. The cell wall of A. fumigatus shares a few similarities to S. cerevisiae. However, major differences exist, including the presence of β-1,3;1,4-glucan in the cell wall of A. fumigatus. In fact, the presence of β-1,3;1,4-glucan was never previously described in fungi before Latge's group reported it a number of years ago. It comprises about 10% of the glucans in the cell wall of A. fumigatus, but its role in the cell wall is unknown. In 2006 and 2009, two papers were published that demonstrated the role of CslF and CslH(Cellulose like synthases) in the production of β-1,3;1,4-glucan of the cell wall in rice and barley, respectively. Taking both protein sequences for these genes, we blasted it against the A. fumigatus database for any possible orthologues. A single orthologue, albeit with weak homology, was identified that named TFT1. We hypothesize that TFT1a plays a direct role in A. fumigatus β-1,3;1,4-glucan synthesis. Through Agrobacterium tumefaciens mediated transformation, an A. fumigatus strain lacking this enzyme (tft1Δ) was generated. From tft1Δ a revertant strain (revtft1) was created where the gene was reintroduced. Immunofluorescence staining with antibodies against β-1,3;1,4-glucan and biochemical quantification both demonstrated complete loss of β-1,3;1,4-glucan within the cell wall of the tft1Δ strain, with recovery detected in revtft1. This strongly suggests that this enzyme does indeed play a role in β-1,3;1,4-glucan synthesis in A. fumigatus. Growth experiments, spore size determination and an in vitro model of virulence also indicated that the loss of TFT1 leads to additional phenotypes. While the precise mechanism for β-1,3;1,4-glucan synthesis is unknown, the results shown herein indicate a pivotal role forTFT1 in its biosynthesis, and resulting phenotypes upon loss of mixed linkage glucan adds some clues to its role in the cell wall of A. fumigatus.

Free-living pathogenic amoebae Acanthamoeba spp. and Balamuthia mandrillaris are causative agents of important diseases of human: rarely occurring but highly fatal granulomatous amoebic encephalitis (both) and keratitis (Acanthamoeba). One of the reasons for the problematic therapy is differentiation into highly resistant cysts often found in affected tissues. In our study we have found that correct encystation in Acanthamoeba requires apart from others, the presence of functioning Golgi apparatus transporting the cyst wall material to the cell surface; glycogen phosphorylase degrading glycogen into glucose which seems to be further used for cellulose synthesis and two non-constitutive cellulose synthases. Acanthamoeba cellulose synthases seem not to be inhibited by known herbicides. In the cyst wall of acanthamoebae we detected cellulose, -mannan, and -1, 3-1, 4-linked glucan [lichenin or mixed-linkage glucan (MLG)]. Cellulose is present in the inner (endocyst) and the outer (exocyst) layers of the cyst wall, whereas-mannan and MLG are found in the endocyst. In a protozoan organism, MLG was detected for the first time. The MLG of Acanthamoeba has a similar composition to that found in barley with high amount of cellobiosyl and cellotriosyl followed by cellotetraosyl units. In contrast, with...