Gaspard Gervasi

Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris : evidence of hyper O-glycosylation

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris . The truncated protein lacked the C‐terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N‐glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C‐terminal of the construction to aid purification by immobilized metal‐chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O‐linked oligosaccharides containing α1‐2‐, α1‐3‐ or α1‐6‐linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human‐serum‐derived antibodies, specific for the native SAG1, which suggested that the O‐glycosylations had no major effect on the protein conformation. However, ELISA and Western‐blot analysis with human sera showed that the O‐carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Nonstructural protein NS1 immunodominant epitope detected specifically in dengue virus infected material by a SELDI-TOF/MS based assay

Dengue virus (DV) infection is the most common mosquito‐born viral disease of public health significance. Though most patients only suffer from flu‐like symptoms, a small group of patients experiences more severe forms of the disease. The viral nonstructural protein 1 (NS1), a secreted protein correlating with viremia, is a key element used for dengue diagnosis with potential implications in severe dengue prognosis. Capture‐ELISAs for the early detection of the NS1 protein in the sera during the acute febrile stage are commonly used in routine by diagnostic laboratories. In this study, the detection of NS1 protein in DV‐infected material was assessed by an alternative method combining a single NS1‐directed monoclonal antibody and the SELDI‐TOF/MS technology. According to the epitope mapping, the antibodies used are mainly directed against an immuno‐dominant peptide located on the C‐terminal part of the protein. The NS1 SELDI‐TOF assay is specific, has a sensitivity level close to capture‐ELISAs and is potentially useful for a coupled serotyping/detection assay or for the detection of subtle post‐translational modifications on the protein. J. Med. Virol. 84:490–499, 2012.

Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexahistidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni(2+)-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellmans reagent allowed the measurement respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with respectively, a diameter of 34 and 28-nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect anti-HBc antibodies in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

Erratum to “Characterization and diagnostic potential of hepatitis B virus nucleocapsid expressed in E. coli and P. pastoris”: [J. Virol. Methods 99 (2002) 99–114]

The hepatitis B core antigen (HBcAg) was expressed in Escherichia coli and in Pichia pastoris. A hexa-histidine tag was introduced at the C terminus of the E. coli expressed protein allowing its purification by Ni2+-chelate affinity chromatography. The P. pastoris expressed HBcAg was isolated following heat treatment. The two recombinant HBcAgs were purified further on a sucrose gradient. Mass spectrometry analysis suggested that HBcAg was N-acetylated only in P. pastoris and reaction with Ellmans reagent allowed the measurement, respectively, of 0.37 and 0.23 mole of free sulfydryl groups per mole of HBcAg monomer expressed in E. coli or P. pastoris. Electron microscopy indicated that the E. coli and the P. pastoris proteins formed capsid-like particles with, respectively, a diameter of 34 and 28 nm. Nucleic acid components were found entrapped in both particles but protected from enzymatic treatment only in the P. pastoris derived particles suggesting structural discrepancies between the two recombinant molecules. The high purity of these recombinant antigens allowed the development of a sandwich immunoassay to detect antibodies to HBcAg (anti-HBc) in human serum. The preliminary results indicate that the P. pastoris HBcAg produced intracellularly is more suitable than the renatured E. coli HBcAg for detection of anti-HBc in this diagnostic assay.

An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS)

Sepsis is the leading cause of death among patients in intensive care units (ICUs) requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID) of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek®MS slides directly from positive blood culture broth based on an “all-in-one” extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT® 3D (SA/SN blood culture bottles) or the BacT/ALERT VirtuoTM system (FA/FN Plus bottles), respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100%) when used with BacT/ALERT® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%), demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could potentially be improved by converting direct ID of positive blood cultures from a batch-based to real-time and “on-demand” process.