PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome.

BACKGROUND: Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. RESULTS: We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. CONCLUSIONS: We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level

Major colonic bacterial groups detected by FISH in the culture broths recovered from the vessel 1 (A), vessel 2 (B), and vessel 3 (C) of the colonic model before (SS1) and after (0, 4, 6, 24, 48, 72, and 96 h and SS2) inoculation of <i>S. aureus</i>.

<p>Results are reported as means (Log₁₀ CFU/mL) of the data of three colonic models ± standard error of mean. For each colonic model, measurements were performed in triplicate at SS1 (days 11, 12 and 13) and SS2 (days 21, 22 and 23). ^*<i>P</i><0.05.</p

Schematic diagram of the <i>in vitro</i> three-stage culture colonic model system (human colonic model).

<p>Each vessel was continually sparged with O₂ free N₂. The pH of each vessel was individually maintained via automated addition of 1M HCl or 1M NaOH, as required. The system was maintained at 37°C and stirred continuously.</p

Primers and PCR conditions for colony screening.

<p>Primers and PCR conditions for colony screening.</p

Oligonucleotide probes used in this study and hybridization conditions for FISH analysis.

†<p>R = G/A; ^‡Y = T/C.</p><p>*20% Formamide was incorporated in the hybridization buffer.</p><p>**These probes were used together in equimolar concentrations (50 ng/µL) and 35% formamide was incorporated in the hybridization buffer.</p

Short-chain fatty acids concentrations in the culture broths recovered from the vessel 1 (A), vessel 2 (B), and vessel 3 (C) of the colonic model before (SS1) and after (24, 48, 72, and 96 h and SS2) inoculation of <i>S. aureus</i>.

<p>Results are reported as means (mM) of the data of three colonic models ± standard error of mean. For each colonic model, measurements were performed in triplicate at SS1 (days 11, 12 and 13) and SS2 (days 21, 22 and 23). ^*<i>P</i><0.05; ^**<i>P</i><0.01.</p

The influence of Staphylococcus aureus on gut microbial ecology in an in vitro continuous culture human colonic model system

An anaerobic three-stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine, was used to study the effect of S. aureus infection of the gut on the resident faecal microbiota. Studies on the development of the microbiota in the three vessels were performed and bacteria identified by culture independent fluorescence in situ hybridization (FISH). Furtheremore, short chain fatty acids (SCFA), as principal end products of gut bacterial metabolism, were measured along with a quantitative assessment of the predominant microbiota. During steady state conditions, numbers of S. aureus cells stabilised until they were washed out, but populations of indigenous bacteria were transiently altered; thus S. aureus was able to compromise colonisation resistance by the colonic microbiota. Furthermore, the concentration of butyric acid in the vessel representing the proximal colon was significantly decreased by infection. Thus infection by S. aureus appears to be able to alter the overall structure of the human colonic microbiota and the microbial metabolic profiles. This work provides an initial in vitro model to analyse interactions with pathogens