The role of Alg13 N-acetylglucosaminyl transferase in the expression of pathogenic features of Candida albicans

Highlights

• ALG13 gene encoding N-acetylglucosaminyl transferase in C. albicans

• Expression of ALG13 gene influences expression of all members of Alg7/13/14 complex.

• Defective N-glycosylation alters pathogenic attributes of C. albicans.

• Expression of genes encoding highly glycosylated adhesins Als1 and Hwp1 is changed.

• Cell wall of alg13 mutant contains much less mannose.

Abstract

Background

The pathogenic potential of Candida albicans depends on adhesion to the host cells mediated by highly glycosylated adhesins, hyphae formation and growth of biofilm. These factors require effective N-glycosylation of proteins.

Here, we present consequences of up- and down-regulation of the newly identified ALG13 gene encoding N-acetylglucosaminyl transferase, a potential member of the Alg7p/Alg13p/Alg14p complex catalyzing the first two initial reactions in the N-glycosylation process.

Methods

We constructed C. albicans strain alg13 ∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. Gene expression and enzyme activity were measured using RT-qPCR and radioactive substrate. Cell wall composition was estimated by HPLC DIONEX. Protein glycosylation status was analyzed by electrophoresis of HexNAcase, a model N-glycosylated protein in C. albicans.

Results

Both decreased and elevated expression of ALG13 changed expression of all members of the complex and resulted in a decreased activity of Alg7p and Alg13p and under-glycosylation of HexNAcase. The alg13 strain was also defective in hyphae formation and growth of biofilm. These defects could result from altered expression of genes encoding adhesins and from changes in the carbohydrate content of the cell wall of the mutant.

General significance

This work confirms the important role of protein N-glycosylation in the pathogenic potential of C. albicans.

Introduction

Candida albicans is a common component of human microflora and the most common cause of opportunistic fungal infections of immunocompromised patients, with a mortality rate around 30–50% [1], [2]. The pathogenic potential of C. albicans is attributed to several factors including expression of adhesins, the yeast-to-hyphae transition and biofilm formation. All these factors can be affected by changes in glycosylation of proteins which therefore plays an important role in Candida virulence [3], [4], [5], [6].

N-glycosylation is an essential protein modification highly conserved in evolution. In all eukaryotes, N-glycosylation is obligatory for viability since glycans have a common role in promoting protein folding, quality control, and certain sorting events and, finally, determination of protein activity [7], [8]. N-glycosylation can be divided into two phases: the first is the assembling of the core polysaccharide containing 14 monosaccharide residues which is then transferred en bloc to a growing polypeptide chain. The second step of N-glycosylation is further processing of the polycarbohydrate N-linked to the protein to the mature structures characteristic for the host [2], [9], [10], [11], [12], [13]. Glycosylation requires a phosphorylated isoprenoid lipid, dolichyl phosphate (Dol-P), as a carrier of the carbohydrates. During N-glycosylation the whole core polysaccharide is assembled on Dol-P [10]. Biosynthesis of the lipid-linked oligosaccharide (LLO) begins at the cytosolic side of the endoplasmic reticulum (ER) with a sequential addition of two N-acetylglucosamine (GlcNAc) residues and five mannoses to Dol-P with nucleotide diphosphate sugars, UDP-GlcNAc and GDP-mannose, as donors [14]. The resulting oligosaccharide, Dol-PP-GlcNAc₂ Man₅, is then flipped to the lumen of the ER and four mannosyl and three glucosyl residues from Dol-P-mannose and Dol-P-glucose are added to form Dol-PP-GlcNAc₂ Man₉ Glc₃. The assembled core oligosaccharide is transferred to the γ-amido group of asparagine residues located in a highly conserved motif Asn-X-Ser/Thr (X can be any amino acid except proline) of the modified protein.

The addition of the second GlcNAc residue to Dol-PP-GlcNAc is catalyzed by a hetero-oligomeric GlcNAc transferase that in most eukaryotes comprises the Alg13p and Alg14p subunits. Alg13p is the catalytic subunit recruited to the ER by the membrane protein Alg14p [15], [16], [17]. Bickel et al. [15] have demonstrated that yeast membranes depleted of Alg13p or Alg14p lack GlcNAc transferase activity in vitro and accumulate Dol-PP-GlcNAc in vivo. This activity is also present in bacteria, however, in E. coli the GlcNAc transferase (MurG) catalyzing peptidoglycan biosynthesis is a single protein with Alg13/Alg14 homologous domains. A structural comparison of Alg13p, Alg14p and MurGp based on the crystal structure of the latter has revealed that Alg13p corresponds to the C-terminal part of MurG thought to bind the UDP-GlcNAc donor, and Alg14p to the N-terminal part containing a glycine-rich motif postulated to be a membrane association site involved in Dol-P recognition [18]. The interaction between the two Alg13p/Alg14p subunits of UDP-GlcNAc transferase in yeast is limited to a C-terminal α-helix (comprised of fifteen amino acids) of Alg13p and three amino acids of Alg14p [16], [17], [19]. Furthermore, the N-terminal region of Alg14p interacts directly with one more protein, Alg7p. This enzymatic protein catalyzes the formation of Dol-PP-GlcNAc, the acceptor of the second GlcNAc added by Alg13p [17], [20]. Thus in S. cerevisiae Alg7p is, in fact, the third member of the Alg7p/Alg13p/Alg14p (Alg7/13/14) complex indispensable for the initial reaction in LLO biosynthesis. The genes coding for the proteins forming this complex are essential.

Here, we cloned and analyzed a previously uncharacterized orf19.6025 from C. albicans. This ORF was found by searching a C. albicans genomic data base with the Alg13p sequence from S. cerevisiae as probe.

To analyze the function of this hypothetical Alg13 protein we constructed a C. albicans strain alg13 ∆::hisG/TRp-ALG13 with one allele of ALG13 disrupted and the other under the control of a regulatable promoter, TRp. TRp is a strong promoter which can be repressed by doxycycline. Thus, depending on the composition of medium the constructed strain expressed ALG13 at a level exceeding that of the wild type (without doxycycline added) or markedly lower (with doxycycline).

The changes in expression of ALG13 influenced the expression of ALG7 and ALG14 and altered Alg7p and Alg13p activities causing under-N-glycosylation of the model N-glycosylated protein HexNAcase.

The impaired glycosylation resulted in defects in hyphae formation and biofilm growth — the two invasive forms of Candida. The background of these changes included altered expression of HWP1 (hyphae wall protein) and ALS1 (agglutinin-like sequence protein) genes encoding factors contributing to biofilm formation. Changes in the cell wall composition and in the composition of the extracellular matrix of biofilm were also observed.