Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Abstract

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

In Vitro Activity of Neisseria meningitidis PglL O-Oligosaccharyltransferase with Diverse Synthetic Lipid Donors and a UDP-activated Sugar

“…The protein targets of O-linked glycosylation systems in bacteria are implicated in activities as varied as protein folding, adhesion, disulfide bond formation, and solute uptake, as well as both aerobic and anaerobic respiration (16). Lack of glycosylation may affect biofilm formation and virulence (12,17,18).…”

“…In this context, understanding of the molecular mechanisms of O-OTases is relevant to improve their biotechnological potential. Furthermore, the presence of O-OTases in many important bacterial pathogens and the observation that in some of them O-glycosylation appears to play a role during infection, suggests that they could be targets for inhibitors with antimicrobial properties (12).…”

Diversity, Abundance and Distribution of O-linked Glycosylation Pathway Enzymes in Prokaryotes-A Comparative Genomics Study

“…All are mesophiles and live in aerobic environment. Recently, the antibiotic-resistant Acinetobacter baumannii ATCC 17978 has been reported to have the O-glycosylation pathway [9]. Even the present study shows that this organism has homologs for all five enzyme categories and hence can potentially glycosylate the proteins.…”

“…These are the O-glycosylation pathways of Neisseria [1][2][3][4][5], Helicobacter pylori [6], Pseudomonas aeruginosa [7], Bacteroides fragilis [8] and Acinetobacter baumannii [9], and the N-glycosylation pathways of Campylobacter jejuni [10][11][12], Haloferax volcanii [13] and Methanococcus voltae [14]. In the genus Neisseria, the O-glycosylation pathway ( Figure S1) has been delineated in the species gonorrhoeae [1,5], lactamica [15] and meningitidis [2,3].…”

“…These also help in understanding the role of glycans in processes such as virulence and pathogenesis. GTs are potential drug targets (see, for example, [9]). In addition, their promiscuous substrate specificity in response to variations in the assay conditions is advantageous for in vitro glycan synthesis [23,24].…”