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The penicillin-resistance phenotype was reconstructed by transforming the penicillin-susceptible R6 with PCR fragments amplified from the penicillin-resistant R6M1 or R6M2 mutants (Additional file Cheap Manchester For Cheap Valentino Garavani Rockstud cardholder Metallic Valentino Sale Cheapest Wide Range Of For Sale GvpOHzHO
). Selection was done on plates containing appropriate concentrations of penicillin. Competent cells were obtained by the dilution of an overnight culture 1:100 in C+Y medium, pH 6.8 [ 47 ]. The diluted cultures were grown up to the onset of exponential phase before being concentrated ten times and frozen in C+Y, pH 6.8, 15% glycerol. For transformation, competent cells were thawed on ice, diluted ten times with C+Y medium, pH 7.8, and complemented with 2 μg/ml of competence stimulating peptide 1 (csp-1) before being incubated for 15 minutes at 35°C under a 5% CO atmosphere. DNA was added to a final concentration of 2 μg/ml and the cultures were incubated for 1 hour at 30°C. Finally, the cultures were switched to 35°C under a 5% CO atmosphere for 1 hour before being plated on CAT medium containing the appropriate concentration of antibiotic. The plates were incubated for 48 hours at 35°C under a 5% CO atmosphere and the resistant colonies were picked for further studies.

The intracellular ROS accumulation was measured using the DCF-DA dye (Invitrogen, Grand Island, NY, USA) whose fluorescence is proportional to the level of ROS []. In a typical experiment, cells were grown to the onset of exponential phase (OD 0.12) before penicillin, ciprofloxacin, kanamycin, chloramphenicol or tetracycline were added at a final concentration of 0.1, 4.0, 400, 6.0 and 0.25 μg/ml, respectively. One milliliter aliquots were collected at baseline (prior to the addition of antibiotic) and at 1, 2 and 3 hours following the addition of antibiotic. The aliquots were washed once and resuspended in 500 μl of 1× PBS (pH 7.2) containing 5 μM DCF-DA and incubated at 37°C in the dark for 30 minutes. The labeled cells were washed once and resuspended in 500 μl of 1× PBS. The fluorescence signal of a 200 μl aliquot was analyzed using a Victor fluorometer (Perkin-Elmer, Waltham, MA, USA) at 485 nm excitation and 535 nm emission wavelengths. Results are expressed as relative fluorescence units (RFU) and were normalized according to the number of live cells at each time point. A minimum of three independent experiments have been performed for each antibiotic.

dichlorofluorescein diacetate

minimum inhibitory concentration

penicillin-binding protein

phosphate-buffered saline

polymerase chain reaction

penicillin G

reactive oxygen species

single-nucleotide polymorphism

whole genome sequencing

wild-type.

Acknowledgements

We thank Dr D Morrison (Chicago) for the Janus cassette. This work was funded by a CIHR grant to MO. FF received a studentship from the Fondation de l'Université Laval and MO holds the Canada Research Chair in Antimicrobial Resistance. We thank the McGill University Genome Quebec Innovation Centre for performing the sequencing.

13059_2011_2740_MOESM1_ESM.DOC Additional file 1: Circular maps of the genome of R6M1 and R6M2. (DOC 953 KB)
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Additional file 2: Chronological appearance of PBP mutations according to the levels of penicillin resistance in R6M2. (DOC 52 KB)
13059_2011_2740_MOESM3_ESM.PDF Additional file 3: PBP1a-targeting Janus cassette. (PDF 140 KB)
13059_2011_2740_MOESM4_ESM.DOC Additional file 4: Oligonucleotides used in this study. (DOC 58 KB)
13059_2011_2740_MOESM5_ESM.DOC Additional file 5: Plasmids used in this study. (DOC 42 KB)
13059_2011_2740_MOESM6_ESM.pdf Authors’ original file for figure 1
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Authors’ original file for figure 2

The authors declare that they have no competing interests.

Table 4. antigens selected by the ANTIGENome technology.

https://doi.org/10.1371/journal.pone.0064422.t004

Of the 23 candidates selected by the ANTIGENome technology, we evaluated 8 well conserved (see Table 5 ) and readily recombinant expressed antigens that had shown some promise in a preliminary mouse study in more detail for their potential to elicit protective immune response in vivo ( Figure 1 ). The rate of M. catarrhalis clearance from mouse lungs in response to immunization with recombinant antigens was assessed using a mouse pulmonary clearance model ( Figure 2 ). Mice were immunized intranasally 3 times at 3 week intervals and challenged intranasally with 40 µL of approximately 5×10 6 live M. catarrhalis RH4 (actual CFU varied between 3.8×10 6 to 5.9×10 6 ) 3 weeks after the last boost. Bacterial CFU were determined in lungs 6 hours post infection and systemic antibody titers after vaccination of mice were determined by ELISA ( Figure 2 ).

Figure 1. Structural features of 8 potential vaccine candidates.

MCR_0076, TonB-dependent receptor; MCR_0196, MltB; lytic murein transglycosylase; MCR_0686, peptide methionine sulfoxide reductase MsrA/MsrB; MCR_0996, hypothetical protein; MCR_1003, LysM domain protein; MCR_1010, D-alanyl-D-alanine carboxypeptidase; MCR_1303, oligopeptide ABC transport system substrate binding protein; MCR_1416, cytochrome c class II, Msp22. SP, signal peptide; LP, signal peptide for lipidation; Plug, an independent folding subunit blocking the pore until the channel is bound by a ligand; PGBD1, peptidoglycan binding-like; MsrA, methionine sulfoxide reductase A; SelR, seleno protein R; LysM, lysine motif; SBP bac 5, bacterial extracellular solute-binding protein family 5. Light grey bars represent the recombinant protein (fragments). Thin black bars delineate epitope containing regions covered by clones selected by the ANTIGENome technology with human IgGs.

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Figure 2. Pulmonary clearance of RH4 after intranasal challenge following intranasal immunization with 8 selected antigens.

Pulmonary clearance 6 hours after intranasal challenge with ∼5×10 6 CFU M. catarrhalis , in mice immunized with purified, IC31 ® adjuvanted recombinant proteins, IC31 ® adjuvant without proteins in PBS, or PBS without adjuvant. The mean values of the combined, normalized results from 2 to 6 independent experiments are shown. Error bars represent the standard error of the mean. (A) Bacterial CFU recovered from all experiments; (B) bacterial CFU recovered from experiments after exclusion of sterile lung cultures. Black bars: negative and positive controls (data from 6 experiments), grey bars: data from 2 to 3 independent experiments in which different antigens were tested. (C) ELISA measuring IgG levels to the respective recombinant proteins in serum from mice immunized intranasally with purified recombinant proteins as noted below the x-axis. For the controls (IC31 ® alone or PBS), IgG levels were determined using a mix of all recombinant proteins. Endpoint titers were expressed as the last dilution that gave an absorbance of at least 0.1 at 405 nm. Median values with the interquartile range from 2 to 6 independent experiments using 10 sera (10 mice per group) per experiment are shown. **, statistically highly significant (P<0.01), *, statistically significant (P<0.05).

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Chemical Science

A modular synthetic route to size-defined immunogenic b antigens is key to the identification of an octasaccharide lead vaccine candidate

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D. Meierhofer , C. L. Pereira * and P. H. Seeberger *

* Corresponding authors

Max Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany Peter.Seeberger@mpikg.mpg.de , claney.pereira@vaxxilon.com

Freie Universität Berlin, Department of Chemistry and Biochemistry, 14195 Berlin, Germany

Max-Planck Institute for Molecular Genetics (MPIMG), 14195 Berlin, Germany

The first glycoconjugate vaccine using isolated glycans was licensed to protect children from serotype b (Hib) infections. Subsequently, the first semisynthetic glycoconjugate vaccine using a mixture of antigens derived by polymerization targeted the same pathogen. Still, a detailed understanding concerning the correlation between oligosaccharide chain length and the immune response towards the polyribosyl-ribitol-phosphate (PRP) capsular polysaccharide that surrounds Hib remains elusive. The design of semisynthetic and synthetic Hib vaccines critically depends on the identification of the minimally protective epitope. Here, we demonstrate that an octasaccharide antigen containing four repeating disaccharide units resembles PRP polysaccharide in terms of immunogenicity and recognition by anti-Hib antibodies. Key to this discovery was the development of a modular synthesis that enabled access to oligosaccharides up to decamers. Glycan arrays containing the synthetic oligosaccharides were used to analyze anti-PRP sera for antibodies. Conjugates of the synthetic antigens and the carrier protein CRM197, which is used in licensed vaccines, were employed in immunization studies in rabbits.

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Publication details

The article was received on 18 Oct 2017, accepted on 05 Dec 2017 and first published on 11 Dec 2017

Article type: Edge Article
DOI: 10.1039/C7SC04521B
Citation: , 2018, 9 , 1279-1288
Chem. Sci.

BRIAN DEER

Brian Deer is a British investigative journalist, best known for inquiries into medicine, the drug industry, and social issues for The Sunday Times of London
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