Development and Validation of a Novel Diagnostic Test for Human Brucellosis Using a Glyco-engineered Antigen Coupled to Magnetic Beads.

Brucellosis is a highly contagious zoonosis and still a major human health problem in endemic areas of the world. Although several diagnostic tools are available, most of them are difficult to implement especially in developing countries where complex health facilities are limited. Taking advantage of the identical structure and composition of the Brucella spp. and Yersinia enterocolitica O:9 O-polysaccharide, we explored the application of a recombinant Y. enterocolitica O:9-polysaccharide-protein conjugate (OAg-AcrA) as a novel antigen for diagnosis of human brucellosis. We have developed and validated an indirect immunoassay using OAg-AcrA coupled to magnetic beads. OAg-AcrA was produced and purified with high yields in Y. enterocolitica O:9 cells co-expressing the oligosaccharyltransferase PglB and the protein acceptor AcrA of Campylobacter jejuni without the need for culturing Brucella. Expression of PglB and AcrA in Y. enterocolitica resulted in the transfer of the host O-polysaccharide from its lipid carrier to AcrA. To validate the assay and determine the cutoff values a receiver-operating characteristic analysis was performed using a panel of characterized serum samples obtained from healthy individuals and patients of different clinical groups. Our results indicate that, using this assay, it is possible to detect infection caused by the three main human brucellosis agents (B. abortus, B. melitensis and B. suis) and select different cutoff points to adjust sensitivity and specificity levels as needed. A cutoff value of 13.20% gave a sensitivity of 100% and a specificity of 98.57%, and a cutoff value of 16.15% resulted in a test sensitivity and specificity of 93.48% and 100%, respectively. The high diagnostic accuracy, low cost, reduced assay time and simplicity of this new glycoconjugate-magnetic beads assay makes it an attractive diagnostic tool for using not only in clinics and brucellosis reference laboratories but also in locations with limited laboratory infrastructure and/or minimally trained community health workers.Fil: Ciocchini, Andres Eduardo. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Rey Serantes, Diego A.. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Melli, Luciano Jorge. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Iwashkiw, Jeremy A.. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Deodato, Bettina. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Wallach, Jorge. Hospital Múñiz. Unidad de Enfermedades Infecciosas; Argentina;Fil: Feldman, Mario F. University of Alberta . Department of Biological Sciences . Alberta Glycomics Centre; Estados Unidos de América;Fil: Ugalde, Juan E. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina;Fil: Comerci, Diego J. Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico Chascomús (San Martin); Argentina

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

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

Exploiting the <it>Campylobacter jejuni </it>protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

<p>Abstract</p> <p>Background</p> <p>Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods.</p> <p>Results</p> <p>In this work we expressed the <it>C. jejuni </it>oligosaccharyltansferase (OTase) PglB, responsible for <it>N</it>-linked protein glycosylation together with a suitable acceptor protein (AcrA) in <it>Yersinia enterocolitica </it>O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA <it>in vivo</it>. Because <it>Y. enterocolitica </it>O9 and <it>Brucella abortus </it>share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of <it>Brucella</it>, although this response was not protective against a challenge with a virulent <it>B. abortus </it>strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera.</p> <p>Conclusion</p> <p>Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.</p

Genomic and Functional Analysis of the Type VI Secretion System in <em>Acinetobacter</em>

<div><p>The genus <em>Acinetobacter</em> is comprised of a diverse group of species, several of which have raised interest due to potential applications in bioremediation and agricultural purposes. In this work, we show that many species within the genus <em>Acinetobacter</em> possess the genetic requirements to assemble a functional type VI secretion system (T6SS). This secretion system is widespread among Gram negative bacteria, and can be used for toxicity against other bacteria and eukaryotic cells. The most studied species within this genus is <em>A. baumannii,</em> an emerging nosocomial pathogen that has become a significant threat to healthcare systems worldwide. The ability of <em>A. baumannii</em> to develop multidrug resistance has severely reduced treatment options, and strains resistant to most clinically useful antibiotics are frequently being isolated. Despite the widespread dissemination of <em>A. baumannii</em>, little is known about the virulence factors this bacterium utilizes to cause infection. We determined that the T6SS is conserved and syntenic among <em>A. baumannii</em> strains, although expression and secretion of the hallmark protein Hcp varies between strains, and is dependent on TssM, a known structural protein required for T6SS function. Unlike other bacteria, <em>A. baumannii</em> ATCC 17978 does not appear to use its T6SS to kill <em>Escherichia coli</em> or other <em>Acinetobacter</em> species. Deletion of <em>tssM</em> does not affect virulence in several infection models, including mice, and did not alter biofilm formation. These results suggest that the T6SS fulfils an important but as-yet-unidentified role in the various lifestyles of the <em>Acinetobacter</em> spp.</p> </div

Genetic organization of T6SS loci.

<p>Selected genomes of sequenced <i>Acinetobacter</i> strains were probed for the presence of T6SS genes, with those genes predicted to be involved in T6SS colored and identified below the figure. Gene accession numbers are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055142#pone-0055142-t001" target="_blank">Table 1</a>.</p

<i>A. baumannii</i> ATCC 17978 requires the conserved TssM protein for T6SS activity.

<p>A) Whole cell and supernatant samples prepared from cultures of wild type 17978, the T6SS mutant 17978 Δ<i>hcp,</i> and its complemented (pHcp) or vector control (pWH1266) derivatives, were separated by SDS-PAGE and probed by Western blot with anti-Hcp (upper panel) or an anti-RNA polymerase (RNAP; lower panel) antibodies. B) Western blot of whole cell and supernatant samples prepared from cultures of wild type 17978, the T6SS mutant 17978 Δ<i>tssM,</i> and its complemented (pTssM) or vector control (pWH1266) derivatives probed for Hcp (upper panel) and RNAP (lower panel).</p

The T6SS is not required for virulence towards <i>G. mellonella</i> or in a mouse model of pneumonia.

<p>A) Groups of 10 larvae were injected with approximately 10⁶ or 10⁷ CFU of wild type 17978 or the <i>tssM</i> mutant, incubated at 37°C, and monitored for survival. No significant difference (p>0.05) in survival was observed (log-rank test). B) Bacterial burden of lung and liver tissue from mice infected intranasally with either wild type 17978 or Δ<i>tssM</i> 36h post infection. No significant difference (p>0.05; two-tailed, unpaired Student’s <i>t</i> test) in bacterial burden of the two strains was observed in either tissue.</p