Rebecca Lindsey

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non‐pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D‐threo‐1‐phenyl‐2‐palmitoyl‐3‐pyrrolidinopropanol (P4) and D‐threo‐3′,4′‐ethylenedioxy‐P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans.

Septins AspA and AspC are important for normal development and limit the emergence of new growth foci in the multicellular fungus Aspergillus nidulans.

ABSTRACT Septins are cytoskeletal proteins found in fungi, animals, and microsporidia, where they form multiseptin complexes that act as scaffolds recruiting and organizing other proteins to ensure normal cell division and development. Here we characterize the septins AspA and AspC in the multicellular, filamentous fungus Aspergillus nidulans. Mutants with deletions of aspA, aspC, or both aspA and aspC show early and increased germ tube and branch emergence, abnormal septation, and disorganized conidiophores. Strains in which the native aspA has been replaced with a single copy of aspA-GFP driven by the native septin promoter or in which aspC has been replaced with a single copy of aspC-GFP driven by the native promoter show wild-type phenotypes. AspA-GFP and AspC-GFP show identical localization patterns as discrete spots or bars in dormant and expanding conidia, as rings at forming septa and at the bases of emerging germ tubes and branches, and as punctate spots and filaments in the cytoplasm and at the cell cortex. In conidiophores, AspA-GFP and AspC-GFP localize as diffuse bands or rings at the bases of emerging layers and conidial chains and as discrete spots or bars in newly formed conidia. AspA-GFP forms abnormal structures in ΔaspC strains while AspC-GFP does not localize in ΔaspA strains. Our results suggest that AspA and AspC interact with each other and are important for normal development, especially for preventing the inappropriate emergence of germ tubes and branches. This is the first report of a septin limiting the emergence of new growth foci in any organism.

Analysis of glycosylinositol phosphorylceramides expressed by the opportunistic mycopathogen Aspergillus fumigatus.

Acidic glycosphingolipid components were extracted from the opportunistic mycopathogen Aspergillus fumigatus and identified as inositol phosphorylceramide and glycosylinositol phosphorylceramides (GIPCs). Using nuclear magnetic resonance sppectroscopy, mass spectrometry, and other techniques, the structures of six major components were elucidated as Ins-P-Cer (Af-0), Manp(α1→3)Manp(α1→2)Ins-P-Cer (Af-2), Manp(α1→2)Manp(α1→3)Manp(α1→2)Ins-P-Cer (Af-3a), Manp(α1→3)[Galf(β1→6)]Manp(α1→2)-Ins-P-Cer (Af-3b), Manp(α1→2)-Manp(α1→3)[Galf(β1→6)]Manp(α1→2)Ins-P-Cer (Af-4), and Manp(α1→3)Manp(α1→6)GlcpN(α1→2)Ins-P-Cer (Af-3c) (where Ins = myo-inositol and P = phosphodiester). A minor A. fumigatus GIPC was also identified as the N-acetylated version of Af-3c (Af-3c*), which suggests that formation of the GlcNα1→2Ins linkage may proceed by a two-step process, similar to the GlcNα1→6Ins linkage in glycosylphosphatidylinositol (GPI) anchors (transfer of GlcNAc, followed by enzymatic de-N-acetylation). The glycosylinositol of Af-3b, which bears a distinctive branching Galf(β1→6) residue, is identical to that of a GIPC isolated previously from the dimorphic mycopathogen Paracoccidioides brasiliensis (designated Pb-3), but components Af-3a and Af-4 have novel structures. Overlay immunostaining of A. fumigatus GIPCs separated on thin-layer chromatograms was used to assess their reactivity against sera from a patient with aspergillosis and against a murine monoclonal antibody (MEST-1) shown previously to react with the Galf(β1→6) residue in Pb-3. These results are discussed in relation to pathogenicity and potential approaches to the immunodiagnosis of A. fumigatus.

Glycosphingolipids of the model fungus Aspergillus nidulans characterization of GIPCs with oligo-α-mannose-type glycans

Aspergillus nidulans is a well-established nonpathogenic laboratory model for the opportunistic mycopathogen, A. fumigatus. Some recent studies have focused on possible functional roles of glycosphingolipids (GSLs) in these fungi. It has been demonstrated that biosynthesis of glycosylinositol phosphorylceramides (GIPCs) is required for normal cell cycle progression and polarized growth in A. nidulans (Cheng, J., T.-S. Park, A. S. Fischl, and X. S. Ye. 2001. Mol. Cell Biol. 21: 6198–6209); however, the structures of A. nidulans GIPCs were not addressed in that study, nor were the functional significance of individual structural variants and the downstream steps in their biosynthesis. To initiate such studies, acidic GSL components (designated An-2, -3, and -5) were isolated from A. nidulans and subjected to structural characterization by a combination of one-dimensional (1-D) and 2-D NMR spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), ESI-MS/collision-induced decomposition-MS (MS/CID-MS), ESI-pseudo-[CID-MS]2, and gas chromatography-MS methods. All three were determined to be GIPCs, with mannose as the only monosaccharide present in the headgroup glycans; An-2 and An-3 were identified as di- and trimannosyl inositol phosphorylceramides (IPCs) with the structures Manα1→3Manα1→2Ins1-P-1Cer and Manα1→3(Manα1→6)Manα1→2Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester, and Cer = ceramide). An-5 was partially characterized, and is proposed to be a pentamannosyl IPC, based on the trimannosyl core structure of An-3.

A septin from the filamentous fungus A. nidulans induces atypical pseudohyphae in the budding yeast S. cerevisiae.

Background Septins, novel cytoskeletal proteins, form rings at the bases of emerging round buds in yeasts and at the bases of emerging elongated hyphal initials in filamentous fungi. Methodology/Principal Findings When introduced into the yeast Saccharomyces cerevisiae, the septin AspC from the filamentous fungus Aspergillus nidulans induced highly elongated atypical pseudohyphae and spore-producing structures similar to those of hyphal fungi. AspC induced atypical pseudohyphae when S. cerevisiae pseudohyphal or haploid invasive genes were deleted, but not when the CDC10 septin gene was deleted. AspC also induced atypical pseudohyphae when S. cerevisiae genes encoding Cdc12-interacting proteins Bem4, Cla4, Gic1 and Gic2 were deleted, but not when BNI1, a Cdc12-interacting formin gene, was deleted. AspC localized to bud and pseudohypha necks, while its S. cerevisiae ortholog, Cdc12, localized only to bud necks. Conclusions/Significance Our results suggest that AspC competes with Cdc12 for incorporation into the yeast septin scaffold and once there alters cell shape by altering interactions with the formin Bni1. That introduction of the A. nidulans septin AspC into S. cerevisiae induces a shift from formation of buds to formation of atypical pseudohyphae suggests that septins play an important role in the morphological plasticity of fungi.

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in Salmonella enterica serotypes from food animals

While antimicrobial resistance in Salmonella enterica is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (KanR) in various S. enterica serotypes from diagnostic samples of human or animals were reported. In this study, over 200 KanR S. enterica isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three KanR ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that KanR ColE1-like plasmids are widely distributed among different S. enterica serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.

Corrigendum to: Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc: ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth (FEBS 26342)

aDepartment of Chemistry, University of New Hampshire, Durham, NH 03824, USA bDepartment of Botany, University of Georgia, Athens, GA 30602-7229, USA cDepartment of Biochemistry, Universidade Federal de Sa‹o Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023-900 Sa‹o Paulo SP, Brazil dDivision of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA eThe Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA

Erratum: Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc: Ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth (FEBS 26342): (FEBS Letters (2002) 525 (59-64) PII S001457930030673)

aDepartment of Chemistry, University of New Hampshire, Durham, NH 03824, USA bDepartment of Botany, University of Georgia, Athens, GA 30602-7229, USA cDepartment of Biochemistry, Universidade Federal de Sa‹o Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023-900 Sa‹o Paulo SP, Brazil dDivision of Nephrology, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 48109, USA eThe Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA