Reversed Immunoglycomics Identifies α-Galactosyl-Bearing Glycotopes Specific for Leishmania major Infection

All healthy humans have high levels of natural anti-α-galactosyl (α-Gal) antibodies (elicited by yet uncharacterized glycotopes), which may play important roles in immunoglycomics: (a) potential protection against certain parasitic and viral zoonotic infections; (b) targeting of α-Gal-engineered cancer cells; (c) aiding in tissue repair; and (d) serving as adjuvants in α-Gal-based vaccines. Patients with certain protozoan infections have specific anti-α-Gal antibodies, elicited against parasite-derived α-Gal-bearing glycotopes. These glycotopes, however, remain elusive except for the well-characterized glycotope Galα1,3Galβ1,4GlcNAcα, expressed by Trypanosoma cruzi. The discovery of new parasitic glycotopes is greatly hindered by the enormous structural diversity of cell-surface glycans and the technical challenges of classical immunoglycomics, a top-down approach from cultivated parasites to isolated glycans. Here, we demonstrate that reversed immunoglycomics, a bottom-up approach, can identify parasite species-specific α-Gal-bearing glycotopes by probing synthetic oligosaccharides on neoglycoproteins. This method was tested here seeking to identify as-yet unknown glycotopes specific for Leishmania major, the causative agent of Old-World cutaneous leishmaniasis (OWCL). Neoglycoproteins decorated with synthetic α-Gal-containing oligosaccharides derived from L. major glycoinositolphospholipids served as antigens in a chemiluminescent enzyme-linked immunosorbent assay using sera from OWCL patients and noninfected individuals. Receiver-operating characteristic analysis identified Galpα1,3Galfβ and Galpα1,3Galfβ1,3Manpα glycotopes as diagnostic biomarkers for L. major-caused OWCL, which can distinguish with 100% specificity from heterologous diseases and L. tropica-caused OWCL. These glycotopes could prove useful in the development of rapid α-Gal-based diagnostics and vaccines for OWCL. Furthermore, this method could help unravel cryptic α-Gal-glycotopes of other protozoan parasites and enterobacteria that elicit the natural human anti-α-Gal antibodies

Biological Roles of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81-176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity

Influence of MeOPN on colonization.

<p>(<b>A</b>) Colonization levels of <i>C. jejuni</i> 81-176 wild-type and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant) in a chicken colonization model, 6 days post-infection. (<b>B</b>) Relative colonization levels of <i>C. jejuni</i> 81-176 wild-type and the MeOPN transferase mutant in a competitive piglet infection model. Note that no significant difference was observed between strains in the chicken colonization model. In the competitive piglet infection model, the MeOPN transferase mutant displays a 5-fold reduction in colonization relative to wild-type comparing the measured wild-type-to-mutant ratio of the inoculum to the ratio recovered from the piglet intestine following infection (p = 0.0005). Statistical significance was determined using a Mann−Whitney test. Horizontal bars represent median values.</p

A Branched and Double Alpha-Gal-Bearing Synthetic Neoglycoprotein as a Biomarker for Chagas Disease

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6–7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients

Loss of MeOPN results in a decrease in serum resistance.

<p>Survival of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant), and <i>kpsM</i>::<i>kan^r</i> (capsule mutant) in 10% natural human complement serum relative to survival in heat inactivated serum. Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type, the <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), and the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant at both 30 and 60 min (p<0.0001). Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type and the <i>kpsM</i>::Km^R mutant at both 30 and 60 min (p = 0.005 and p = 0.014, respectively) using an unpaired t-test. Results represent the mean (± SEM) of three independent experiments.</p

1D ¹H–³¹P HSQC spectra of select <i>Campylobacter</i> species containing orthologues of the <i>cj1416</i>-<i>cj1418</i> genes.

<p>Spectra contain the following resonances corresponding to MeOPN: <i>C. insulaenigrae</i> RM5435, peak at 3.74 ppm; <i>C. lari</i> UPTC NCTC 11845, peak at 3.70 ppm; <i>C. lari</i> subsp. <i>concheus</i> LMG 11760, peak at 3.70 ppm; <i>C. subantarcticus</i> RM8523, peak at 3.75 ppm; <i>C. cuniculorum</i> LMG 24588, peak at 3.76 ppm; <i>C. upsaliensis</i> RM3195, peak at 3.70 ppm; <i>C. upsaliensis</i> RM3940, peaks at 3.75 and 3.71 ppm; <i>C. helveticus</i> CCUG 30566, peak at 3.68 and 3.61 ppm; <i>C. jejuni</i> NCTC 11168, peaks at 3.71 and 3.68 ppm.</p

1D ¹H–³¹P HSQC analyses of <i>C. jejuni</i> 81-176 wild-type and the MeOPN biosynthesis and transferase mutants.

<p>Intact whole cells of <i>C. jejuni</i> were analysed by HR-MAS NMR after 48 h growth on MH agar. Depicted are the 1D ¹H–³¹P HSQC spectra, which specifically show the MeOPN resonance at 3.8 ppm in 81-176 wt (A), but not in the <i>cjj81176_1415</i>::<i>kan^r</i> MeOPN biosynthesis mutant (B), and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> MeOPN transferase mutant (C).</p

MeOPN modifications provide protection against serum mediated killing by interfering with the classical complement activation pathway.

<p>Survival of <i>C. jejuni</i> 81-176 wild-type and <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant) in 10% natural human complement serum. The samples were tested in the presence or absence of 50 mM EGTA after 60 min incubation, relative to survival in heat inactivated serum. Serum resistance was significantly different between <i>C. jejuni</i> 81-176 wild-type in the presence versus absence of EGTA (p = 0.0030), the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant in the presence versus absence of EGTA (p = 0.0014), and the 81-176 wild-type versus the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> mutant in the absence of EGTA (p = 0.0047) using an unpaired t-test. Results represent the mean (± SEM) of three independent experiments. Note that both the wild-type and MeOPN transferase mutant approach 100% survival in the presence of EGTA.</p

MeOPN does not have insecticidal activity against <i>Galleria mellonella</i>.

<p>(A) Insecticidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> (MeOPN transferase mutant), <i>C. jejuni</i> 81-176 <i>kpsM</i>::<i>kan^r</i> (capsule mutant), <i>C. jejuni</i> 11168H wild-type, and <i>C. jejuni</i> 11168H <i>cj1421-1422</i>::<i>kan^r</i> (MeOPN transferase mutant). (B) Insecticidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>cjj81176_1415</i>::<i>kan^r</i> (MeOPN biosynthesis mutant), <i>C. jejuni</i> 11168H wild-type, and <i>C. jejuni</i> 11168H <i>cj</i>1416::<i>kan^r</i> (MeOPN biosynthesis mutant). (C) Insectidal activity of <i>C. jejuni</i> 81-176 wild-type, <i>C. jejuni</i> 81-176 purified CPS, or MeOPN-containing compounds in a <i>G. mellonella</i> larval model. (D) Chemical structures of MeOPN-containing compounds. Glc – methyl α-D-glucopyranoside; 2-MeOPN-Glc - methyl 2-<i>O</i>-(methylphosphoramidyl)-α-D-glucopyranoside; 6-MeOPN-Glc - methyl 6-<i>O</i>-(methylphosphoramidyl)-α-D-glucopyranoside; Gal – methyl α-D-galactopyranoside; 2-MeOPN-Gal - methyl 2-<i>O</i>-(methylphosphoramidyl)-α-D-galactopyranoside; 6-MeOPN-Glc - methyl 6-<i>O</i>-(methylphosphoramidyl)-α-D-galactopyranoside; CPS – purified capsular polysaccharide from 81-176 wild-type. Survival percentages were significantly different between wild-type 81-176 and the <i>kpsM</i>::<i>kan^r</i> mutant (p = 0.0031), the <i>cjj81176_1420</i>::<i>kan^r/cjj81176_1435</i>::<i>cam^r</i> and <i>kpsM</i>::<i>kan^r</i> mutants (p = 0.0026), and between wild-type 81-176 and all tested MeOPN-containing compounds (p<0.005 in all cases). Note that no statistically significant difference was observed between the wild-type and MeOPN transferase mutant in either the 81-176 (p = 0.264) or 11168H (p = 0.063) strains. Results represent the mean (±SEM) of five independent experiments in (A) and four independent experiments in (C) except for 81-176 CPS, which was the result of a single experiment.</p

Prevalence of MeOPN biosynthesis genes within the <i>Campylobacter</i> genus.

<p>Dendrogram of AtpA amino acid sequences of representative <i>Campylobacter</i> species, with strains containing orthologues of the MeOPN biosynthesis genes (<i>cj1416-1418</i>) indicated with an asterisk. The scale bar represents substitutions per site. Bootstrap values of >75%, generated from 1000 replicates using the Neighbor-Joining algorithm, are shown in the nodes. The Cj1416-Cj1418 orthologues for <i>C. jejuni, C. lari</i> and <i>C. fetus</i> are present in the NCBI database: the accession number for <i>C. jejuni subsp. jejuni</i> strain NCTC 11168 is AL111168; the accession number for <i>C. coli</i> strain 76339 (genes BN865_14280-14300) is HG326877; the accession number for <i>C. lari subsp. lari</i> strain RM2100 (genes Cla_0314-316) is CP000932; the accession number for <i>C. fetus subsp. fetus</i> strain 82–40 (genes CFF8240_1630-1632) is CP000487; and the accession number for <i>C. fetus subsp. venerealis</i> strain NCTC 10354 (genes CFV354_1762-1764) is CM001228. The accession numbers for the <i>atp</i> sequences and new Cj1416-Cj1418 orthologues from the other <i>Campylobacter</i> species are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087051#pone.0087051.s002" target="_blank">Table S1</a>.</p