The Putative α-1,2-Mannosyltransferase AfMnt1 of the Opportunistic Fungal Pathogen Aspergillus fumigatus Is Required for Cell Wall Stability and Full Virulence

Abstract

Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative ␣-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A ⌬afmnt1 mutant is viable and grows normally at 37°C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48°C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48°C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the ⌬afmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that ␣-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.

A Single UDP-galactofuranose Transporter Is Required for Galactofuranosylation in Aspergillus fumigatus

“…The A. fumigatus genome contains a clear GDP-Man transporter homolog (AFUA_ 5G05740) whose importance is difficult to predict. It has recently been shown that protein O-mannosylation is dispensable in this fungus but is required for cell wall stability and full virulence (66,67). Yet the importance of N-glycan branching and elongation, and mannosylation of GIPCs, is currently undetermined.…”

Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

“…pJW121 was constructed by cloning the tetracycline-dependent transactivator, the crgA terminator, and the tetO7::Pmin (a blunt-ended fragment derived from pVG2.2 after digestion with EcoRI and PmeI [22]) into the PmeI site of pSK379. pJW103 and pSK379 were described previously (7,23). Strains for the doxycycline-inducible expression of the C-terminally 6ϫHis-tagged full-length Rom2 and N-terminally hemagglutinin (HA)-tagged Rom2 with amino acids 733 to 1199 [HA-Rom2(733-1199)] were generated by PCR amplifying full-length Rom2 with primers Rom2-5g08550-fwd and His-Rom2-5g08550-rev and Rom2(733-1199) with the primers HA-Rom2GEF-5g08550-f and Rom2-5g08550-rev and cloning the respective PCR products in pSS005, yielding pSS006 and pSS009.…”

Aspergillus fumigatus and Related Species

“…In addition, conidia are in contact with many different fungicidal factors present in the upper layer of the mucous epithelium. Among them, (Mellado et al 1996;Langfelder et al 1998;Tsai et al 1998;Mouyna et al 2005;Maubon et al 2006;Romano et al 2006;Li et al 2007;Schmalhorst et al 2008), signaling and metabolism regulation (Panepinto et al 2003;Krappmann et al 2004;Liebmann et al 2004;Fortwendel et al 2005Fortwendel et al , 2012Steinbach et al 2006;Zhao et al 2006;da Silva Ferreira et al 2007;Cramer et al 2008;Ibrahim-Granet et al 2008;Soriani et al 2008;Ejzykowicz et al 2009Ejzykowicz et al , 2010Gravelat et al 2010), thermotolerance (Bhabhra et al 2004;Wagener et al 2008), nutrition (Hensel et al 1998;Schrettl et al 2004Schrettl et al , 2007Hissen et al 2005;Moreno et al 2007;Liu et al 2010), toxin genes Sugui et al 2007;Spikes et al 2008), and response to stress (Paris et al 2003;Tsitsigiannis et al 2005;Reeves et al 2006;Willger et al 2008;Feng et al 2011;Dinamarco et al 2012;Grahl et al 2012). …”