Diversity within the O-linked protein glycosylation systems of acinetobacter species

Abstract

The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii.

Fig. 1.

Major O-glycan structure identified using ZIC-HILIC enrichment. ITMS-CID fragmentation results in near exclusive glycan fragmentation of A. baumannii glycopeptides leading to the identification of A, the previously pentamer glycan (14) within strain ATCC 19606 (¹¹⁰RPQPPVNAPAPVASQAK¹²⁶ of D0CDA9_ACIBA), B, a 4-mer glycan containing a 316 Da residue in strain SDF (³⁰²AKPASTPAVK³¹¹ of B0VKN6_ACIBS), C, a novel pentamer glycan within strain Arg1 containing a 258 residue (³⁰²AKPASTPAVK³¹¹ of A3M265_ACIBT), D, a novel 4-mer glycan within strain Arg2 (¹¹⁰RPQPLVNAPAPVASQAK¹²⁶ of J5IPS4_ACIBA) containing a similar 316 Da residue to SDF, and E, the novel trisaccaride identified in strains 1441 C1 and C3 (²³KEEATQAGQDAASTAVADK⁴¹ of A7FB63_ACIBT) containing the 316 Da residue of SDF and Arg 1.

Fig. 2.

Alterative O-glycan structure identified using ZIC-HILIC enrichment with A. baumannii strains. ITMS-CID fragmentation enabled the identification of A. baumannii glycopeptides containing alterative glycoforms within all strains examined including: A, A tetramer glycan composed of HexNAc₂-Hex-NulOAc within A. baumannii SDF (⁴⁸SAGDQAASDIATATDNASAKIDAATDHAADATAK⁸¹ of B0VLI0_ACIBS), B, A trisaccharide composed of HexNAc-dHexNAc-NulOCH3 within A. baumannii 1441 C1 (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of A7FB63_ACIBT), C, A disaccharide composed of HexNAc₂ within A. baumannii SDF (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of B0VLI0_ACIBS), D, A pentamer glycan within A. baumannii 19606 composed of HexNAc-Hex₂-HexNAc-GlcNAc3NAcA (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of D0CEI7_ACIBA), E, A pentamer glycan within A. baumannii 19606 and 17978 composed of HexNAc-Hex₂-HexNAc-GlcNAc3NAcA4OAcCH3 (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of A7FB63_ACIBT), F, The known pentamer glycan of A. baumannii ATCC 17978 where a Hex has been exchanged for a HexNAc residue compared with the previously reported glycan (14) (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of A7FB63_ACIBT), G, The known pentamer glycan of A. baumannii ATCC 17978 where the linking HexNAc residue has been exchanged for the unique 386.11 Da residues compared with the previously reported glycan (14) (⁴⁸SAGDQAASDIATATDNASAK⁶⁷ of A7FB63_ACIBT), I, A dimer of the trisaccharide O-glycan of A. baumannii 1441 C1 (¹⁹NDGMHEASDPATSHDMNK³⁶ of A7FB95_ACIBT), and J, A trimer of the trisaccharide O-glycan of A. baumannii 1441 C1 (³⁰²AKPASTPAVK³¹¹ of A3M265_ACIBT).

Fig. 3.

A. baumannii prominently glycosylates Serine residues with Alanine residues in the −1 position. Analysis of glycoproteins and glycosylation sites identified of A. baumannii strains. A, Comparison of glycoproteins identified within each strain. Showing the high level of overlap between strains. B, Analysis of the overlap between strains glycoproteins identified in all seven A. baumannii strains. C, Comparison of the sequence identity of proteins between strains of A. baumannii demonstrating the high level of sequence identity between strains of the identified glycoproteins. D, Motif analysis of identified glycosylation sites showing a strong preference for the sequence AS. E, Comparison of the region of disorder around the identified sites of glycosylation, a disordered prediction >0.5 is consisted to be disordered according to PreDisorder, http://casp.rnet.missouri.edu/predisorder.html.

Fig. 4.

Western blot analysis used to resolve the mass difference between glycosylated and unglycosylated A1S_1193. A, Anti-Histidine Western blot analysis of Acinetobacter strains recombinantly expressing Acinetobacter glycoprotein A1S_1193 with a C-terminal Histidine tag. The slight increase in molecular weight indicates the protein has been post-translationally modified. B–J, ESI-QTOF MS/MS Analysis of the fished A. baumannii glycoprotein A1S_1193 to elucidate the glycan structure. ESI-QTOF-MS and MS/MS was carried out on purified A1S_1193, expressed in various Acinetobacter strains, to characterize the posttranslational modification. B–C, ESI-QTOF-MS/MS analysis of tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ expressed in A. baylyi ADP1 revealed either the pentasaccharide 285-217–245₂-HexNAc or 285-217–245-HexNAc₂ attached to the glycopeptide. D, MS/MS fragmentation of ²⁰³AASGVEAAAAPATLTLSTDDK²²³ expressed in A. calcoaceticus 1217 displays modification with the pentasaccharide HexNAc-176-HexNAc₂-Hex. E, A. calcoaceticus 1218 glycosylates the tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with the tetrasaccharide HexNAc-217-HexNAc₂. F, Fragmentation of tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ from A1S_1193 expressed in A. pittii 1219 reveals glycosylation with a pentasaccharide identical to 1217, HexNAc-176-HexNAc₂-Hex. G, A. nosocomialis 1222 modifies glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with the trisaccharide repeat unit HexNAc-Hex₂. H, A. baumannii 1224 uses the hexasaccharide HexNAc-Hex-HexNAc₂-158-Hex to glycosylate glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³. I–J, A. baumannii 1225 modifies glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with one of two tetrasaccharides, 272–258-HexNAc-258 or 228–258-HexNAc-258.

Fig. 4.

Western blot analysis used to resolve the mass difference between glycosylated and unglycosylated A1S_1193. A, Anti-Histidine Western blot analysis of Acinetobacter strains recombinantly expressing Acinetobacter glycoprotein A1S_1193 with a C-terminal Histidine tag. The slight increase in molecular weight indicates the protein has been post-translationally modified. B–J, ESI-QTOF MS/MS Analysis of the fished A. baumannii glycoprotein A1S_1193 to elucidate the glycan structure. ESI-QTOF-MS and MS/MS was carried out on purified A1S_1193, expressed in various Acinetobacter strains, to characterize the posttranslational modification. B–C, ESI-QTOF-MS/MS analysis of tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ expressed in A. baylyi ADP1 revealed either the pentasaccharide 285-217–245₂-HexNAc or 285-217–245-HexNAc₂ attached to the glycopeptide. D, MS/MS fragmentation of ²⁰³AASGVEAAAAPATLTLSTDDK²²³ expressed in A. calcoaceticus 1217 displays modification with the pentasaccharide HexNAc-176-HexNAc₂-Hex. E, A. calcoaceticus 1218 glycosylates the tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with the tetrasaccharide HexNAc-217-HexNAc₂. F, Fragmentation of tryptic peptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ from A1S_1193 expressed in A. pittii 1219 reveals glycosylation with a pentasaccharide identical to 1217, HexNAc-176-HexNAc₂-Hex. G, A. nosocomialis 1222 modifies glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with the trisaccharide repeat unit HexNAc-Hex₂. H, A. baumannii 1224 uses the hexasaccharide HexNAc-Hex-HexNAc₂-158-Hex to glycosylate glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³. I–J, A. baumannii 1225 modifies glycopeptide ²⁰³AASGVEAAAAPATLTLSTDDK²²³ with one of two tetrasaccharides, 272–258-HexNAc-258 or 228–258-HexNAc-258.