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We recently described a protein O-glycosylation pathway conserved in all species of the Burkholderia genus that results in the synthesis and incorporation of a trisaccharide glycan to membrane-exported proteins. Here, we exploited this system to construct and evaluate a diagnostic tool for glanders. Burkholderia mallei, the causative agent of glanders, is a highly infectious and fatal zoonotic pathogen that infects horses, mules, donkeys, and occasionally humans. A highly sensitive and specific diagnostic tool is crucial for the control, elimination, and eradication of B. mallei infections. We constructed plasmids carrying synthetic genes encoding a modified, previously unannotated Burkholderia glycoprotein containing three glycosylation sequons fused to the cholera toxin B-subunit. The resulting proteins were glycosylated in the B. cenocepacia K56-2 parental strain, but not in glycosylation-deficient mutants, as determined by SDS-PAGE and fluorescent lectin blots. One of these glycoproteins was used as an antigen in ELISA and western blots to screen a panel of serum samples collected from glanders-infected and healthy horses, which were previously investigated by complement fixation test and indirect ELISA based on a semi-purified fraction of B. mallei. We show that ELISA and western blot assays based on our glycoprotein antigen provide 100% specificity, with a sensitivity greater than 88%. The glycoprotein antigen was recognized by serum samples collected from patients infected with B. pseudomallei, B. mallei, B. multivorans, and B. cenocepacia. Our results indicate that protein O-glycosylation in Burkholderia can be exploited as a biomarker for diagnosis of Burkholderia-associated infections.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS–based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei. Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan–specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

The Burkholderia genus encompasses many Gram-negative bacteria living in the rhizosphere. Some Burkholderia species can cause life-threatening human infections, highlighting the need for clinical interventions targeting specific lipopolysaccharide proteins. Burkholderia cenocepacia O-linked protein glycosylation has been reported, but the chemical structure of the O-glycan and the machinery required for its biosynthesis are unknown and could reveal potential therapeutic targets. Here, using bioinformatics approaches, gene-knockout mutants, purified recombinant proteins, LC-MS–based analyses of O-glycans, and NMR-based structural analyses, we identified a B. cenocepacia O-glycosylation (ogc) gene cluster necessary for synthesis, assembly, and membrane translocation of a lipid-linked O-glycan, as well as its structure, which consists of a β-Gal-(1,3)–α-GalNAc-(1,3)–β-GalNAc trisaccharide. We demonstrate that the ogc cluster is conserved in the Burkholderia genus, and we confirm the production of glycoproteins with similar glycans in the Burkholderia species: B. thailandensis, B. gladioli, and B. pseudomallei. Furthermore, we show that absence of protein O-glycosylation severely affects bacterial fitness and accelerates bacterial clearance in a Galleria mellonella larva infection model. Finally, our experiments revealed that patients infected with B. cenocepacia, Burkholderia multivorans, B. pseudomallei, or Burkholderia mallei develop O-glycan–specific antibodies. Together, these results highlight the importance of general protein O-glycosylation in the biology of the Burkholderia genus and its potential as a target for inhibition or immunotherapy approaches to control Burkholderia infections.

Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that “virulence” depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-l-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection., IMPORTANCE Virulence and pathogenicity are properties ascribed to microbes, which actually require careful consideration of the host. Using the term “pathogen” to define a microbe without considering its host has recently been debated, since the microbe’s capacity to establish a niche in a given host is a critical feature associated with infection. Opportunistic bacteria are a perfect example of microbes whose ability to cause disease is intimately related to the host’s ability to recognize and respond to the infection. Here, we use the opportunistic bacterium Burkholderia cenocepacia and the host plant Arabidopsis thaliana to investigate the role of bacterial surface molecules, namely, lipopolysaccharide and flagellin, in contributing to infection and also in eliciting a host response. We reveal that both molecules can be modified by glycosylation, and although the modifications are critical for the bacteria to establish an infection, they do not impact the host’s ability to recognize the pathogen.

ArnT is a glycosyltransferase that catalyses the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyse lipid A modification with L-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.