Home > Research Groups > cheng jin > Researsh Interests and Results

Research Areas

Microbial physiology and biochemistry

 

Major achievements in this group:
1. Characterization of glycosylation in Aspergillus fumigatus
The investigation of A. fumigatus in this lab was initiated in 1997. The basic premise is that investigating glycosylation pathways and identifying glycol-genes involved in growth and virulence of A. fumigatus, which helps to understand the functional roles of glycosylation during fungal development and thus be helpful for screening of antifungal drugs. At the beginning, we focused on the purification and characterization of chitinase, a scretory glycoprotein produced by A. fumigatus (Eur. J. Biochem., 268(14):4079, 2001). After then we resolved the crystal structure of this protein (Acta Crystallogr. D Biol. Crystallogr., 60(Pt 5):939, 2004). Further studies showed that the N-glycan on secreted glycoprotein consists of 6 residues of mannose and 2 residues of N-acetylglucosamine, which is similar to the core structure of mammalian N-glycan (Microbiol., 154:1960, 2008). The O-glycan attached to chitinase is Man-Ser/Thr (Eukaryotic Cell, 6(12):2260, 2007).
 
2. Identification of genes involved in glycosylation
We have identified over 50 genes involved in biosynthesis of N-glycosylation, O-mannosylation and GPI-anchoring in A. fumigatus. Based on the structural information of glycans, we were able to construct a draft of putative pathway of glycosylation. Using the genetic systems established for functional analysis, over 10 putative genes in glycosylation pathway have been investigated(Fig.1). The present data showes that glycosylation is involved in morphogenesis, thermal tolerance, and virulence in A. fumigatus.
 
3. Functional analyses of putative glycol-genes
3.1 Activation of mannose
As mannose is one of important components in glycans and cell wall in A. fumigatus, we evaluated the importance of mannose synthesis. The precursor of all mannose residues found in galactomannan, glycoprotein, and GPI anchor in A. fumigatus is GDP-mannose. The pmi1 and srb1 genes are responsible for synthesis of GDP-mannose and essential for survival. Repression of the srb1 expression leads to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance and branching site selectionMicrobiol, 154:2730, 2008. Deletion of the pmi1 gene leads to uncoupling of the link between energy production and glycosylation and accumulation of Man-6-P, which then results in defects in cell wall integrity, conidiation, and morphology. Although extracellular mannose can rescue the growth of PMI deficient mutants in A. fumigatus, both lower and higher concentrations of mannose lead to a reduction in the levels of a-glucan in the cell wall and an accumulation of Man-6-P. The phenotypes associated with the mutant under mannose-starvation are mainly due to an insufficient supply of GDP-Man required for cell wall synthesis. The abnormal morphology associated with the Dpmi1 mutant under mannose-replete conditions is mainly ascribed to an accumulation of Man-6-P,which cannot efficiently enter glycolysis, instead becoming trapped in a cycle of de-phosphorylation and re-phosphorylation resulting in depletion of intracellular ATP. Interestingly, the A. fumigatus PMI mainly catalyzes the conversion of Fru-6-P to Man-6-P and its binding affinity for Man-6-P is similar to that ofyeasts but different from the ones from bacteria or animals. This suggests that it may be possible to design a specific inhibitor for fungal PMIs.
These results show that glycosylation is essential for cell wall integrity, morphology, and viability in A. fumigatus.
 
3.2 Function of N-glycosylation
The N-glycans on mature secreted glycoprotein produced by A. fumigatus are Man6GlcNAc2, Man7GlcNAc2 and Man8GlcNAc2, in which Man6GlcNAc2 is the major glycoform.The formation of N-linked oligosaccharides is initiated by the assembly of a lipid-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol by a series of glycosyltransferases. Subsequently, the Dol-PP-linked Glc3Man9GlcNAc2 is transferred as a whole to an asparagine residue within an N-X-T/S consensus sequence of a nascent peptide, which is catalyzed by the oligosaccharyltransferase (OST). Once Glc3Man9GlcNAc2 is transferred to proteins, the N-glycan is processed sequentially in the ER and Golgi.
N-Glycan processing is initiated by the removal of the glucose residues catalyzed by ER glucosidase I and glucosidase II. The cwh41 has been identified to encode ER glucosidase I. Deletion of the cwh41 gene in A. fumigatus results in defective N-glycan processing of the proteins secreted by A. fumigatus. Although cwh41 is not essential for hyphal growth and virulence, a severe reduction in conidial formation, abnormalities of polar growth and septation and a temperature-sensitive deficiency of cell wall integrity were documented. In addition, we found that the genes encoding Rho-type GTPases (Rho-type GTPase/Cdc42) were upregulated, which suggests that the cell wall integrity (CWI) pathway was activated in the mutant (FEMS Microbiol Lett, 289:155, 2008). 2-D gel analysis reveals that deletion of the cwh41 gene in A. fumigatus leads to ER stress, which induces over-expression of HSP70 and calnexin chaperone and activates the ERAD. Meanwhile, the proteins required for actin re-arrangement are found to be under-expressed or missing, which is consistent with the observation of random localization of actin fibers in the mutant (Microbiol, 155: 2157, 2009). These observations, for the first time, clearly suggest that N-glycosylation contributes to proper folding and trafficking of proteins in A. fumigatus. It appears that proteins involved in cell wall biosynthesis in A. fumigatus are more dependent on the N-glycan dependent folding system.
MsdSp (XP_752825.1) has been identified as a Class I α1, 2-mannosidase and acts on Man8GlcNAc2 to produce Man6GlcNAc2 in Golgi. Deletion of the msdS gene leads to a defect in N-glycan processing, as well as a reduction of cell wall components (including a-glucan, b-glucan, mannopretein and chitin) and reduced conidiation. Morphological analysis reveals abnormal polarity and septation. However, deletion of the msdS has no effect on fungal growth and virulence (Microbiol, 154:1960, 2008).
Based on these findings, we proposed the molecular model of function of the N-glycosylation as (Fig.2): The proteins required for cell wall synthesis or cell wall stress sensing are substrates of A. fumigatus Cwh41p and require glucose-trimming for their proper localization and function. Misfolding of these proteins would cause cell wall defects, which then leads to activation of the ERAD and Rho-type GTPases-mediated CWI pathway. Moreover, activation of Cdc42 in the CWI pathway also activates SepA, an upstream organizer of actin ring formation at septation sites, and thus causes abnormal polarized growth associated with the Dafcwh41 mutant (FEMS Microbiol Lett, 289:155, 2008; Microbiol, 155: 2157, 2009). Although the phenotypes associated with different N-glycosylation mutants vary, the finding that all of these mutants exhibit phenotypes associated with cell wall defects, abnormal polarization and morphological changes, can all be explained by this proposed model.
In addition, we found that the A. fumigatus Ams1p is responsible for degradation of N-glycan. Deletion of the A. fumigatus ams1 leads to a severe defect in conidial formation, especially at a higher temperature. In addition, abnormalities of polarity and septation are associated with the DAfams1 mutant. These results show that the ams1 gene is required for morphogenesis and cellular function in A. fumigatus (Glycobiol, 19: 624, 2009). The involvement of the ams1 gene in polarized growth demonstrates that the processes involved in N-glycan degradation are important for A. fumigatus. It is likely that the Ams1p is involved in cell wall synthesis and thus polarity through the CWI pathway.
3.3 Funtional of O-glycosylation
O-mannose glycosylated proteins were first discovered in yeast and filamentous fungi, and recently this type of glycoproteins has also been described in mammals. Protein O-mannosylation is initiated by a family of protein O-mannosyltransferases (PMTs) that are evolutionarily conserved from yeast to human. Mutations in human POMT1 cause Walker-Warburg Syndrome (WWS), which is characterized by severe congenital muscular dystrophy, neuronal-migration defects, and structural abnormalities of the eye. Targeted deletion of Pomt1 in mice results in embryonic lethality due to defects in the formation of the Reicherts membrane, the first basement membrane to form in the embryo.
We have identified that the pmt1 and pmt2 are responsible for O-mannosylation in A. fumigatus. Deletion of pmt1 results in temperature-sensitive phenotypes (Eukaryotic Cell, 6:2260, 2007). When the A. fumigatus Dpmt1 mutant was grown on solid complete medium at 37ºC, no difference was found between the mutant and the wild-type. A strongly retarded growth, however, was observed when this mutant was grown at 42°C and 50°C.This temperature sensitive phenotype could be complemented by the addition of 1M sucrose in the media. Further analysis shows that the mannoprotein, a-glucan and chitin in the cell wall of the mutant grown at 37°C are increased, while b-glucan is reduced. When the A. fumigatus Dpmt1 mutant was cultured at 42°C, the a-glucan was increased, while the b-glucan was decreased, and the mannoprotein and chitin content remained unchanged. Moreover, deficient conidiation and reduced germination have been documented at 42°C.
On the other hand, the single pmt2 deletion is lethal. Reduced expression of pmt2 leads toretarded growth, cell wall defects, abnormal polarity and reduced conidiation, however, no temperature-sensitive growth was found. Interestingly, this is the first time that Pmt2p is revealed to be involved in polarized growth. These observations suggest that A. fumigatus Pmt2p is required for cell wall synthesis and morphogenesis and itsfunction is distinct from that of A. fumigatus Pmt1p (Glycobiol, 20:542, 2010). Our results not only increase our understanding of the function of O-mannosylation in A. fumigatus, but also may deepen our understanding of the molecular basis of the human Walker-Warburg Syndrome (WWS).
 
3.4 Function of GPI anchoring

GPI anchoring is also a conserved glycosylation process in eukaryotes, which enables many cell surface proteins such as cell surface enzymes, receptors and adhesion molecules to be covalently anchored to the cell membrane. The A. fumigatus pig-a gene has been investigated in this group. Deletion of Dpig-a results in a phenotype characterized by increased cell lysis. Also, an increased content of b-glucan and mannoprotein was observed in the mycelial cell wall of the Dafpig-a mutant. The Dafpig-a can survive at temperatures from 30°C to 50°C. Completely blocking GPI anchor synthesis in A. fumigatusDpig-a leads to cell wall defects, abnormal hyphal growth, rapid conidial germination, and aberrant conidiation. In vivo assays reveal that the mutant exhibits reduced virulence in immunocompromised mice. Therefore, the GPI anchor seems not essential for viability, but required for cell wall integrity, morphogenesis and virulence in A. fumigatus (Mol Microbiol, 64:1014, 2007). Indeed, this is the first report that a deficiency in GPI-anchor synthesis does not lead to a temperature-sensitive or conditional lethal phenotype in microbes, which provides an opportunity to identify the basic function of GPI-anchoring in fungi.