Bacterial strains and culture conditions
E. coli strain JM109 [20] was used as a host strain for propagating all plasmids used in this study. Functional expression of plasmid constructs was tested using S. flexneri serotype Y strain (SFL 124) which is an attenuated vaccine candidate strain [21]. Details of the strains created are described in Additional File 1: Table AFT2 and Additional file 1: Table AFT3. All cultures were grown at 37°C aerobically in Luria-Bertani media with chloramphenicol (Cm-25 μ g/ml final concentration).
DNA techniques
Plasmid vectors were derived from pBCSK + (Stratagene). Plasmids were isolated using the alkaline lysis method either using the QIAprep spin miniprep kit (Qiagen) or by the method described by Sambrook et al.
[20]. Details of the plasmid constructs made in this study are described in Additional File 1: Table AFT2. Standard procedures as outlined in Sambrook et al.
[22] were used for cloning and transformation by electroporation.
Sequencing was performed at the Biomolecular Resource Facility, John Curtin School of Medical Research, Australian National University, using the Big Dye Terminator v3.1 Cycle Sequencing Kit.
Creation of oac-pho A-lac Zα fusion construct pNV1644
The construction of the plasmid pNV1644 (in B2012) and SFL1899 has previously been described in [3]. When B2012 (carrying pNV1644) was plated on DI media, a red coloured fusion product was observed indicating that the point of fusion, that is the C-terminal end, was localized in the cytoplasm. Furthermore, as described in [3], SFL1899 containing pNV1644 was confirmed by slide agglutination and LPS Western immunoblotting using MASF 6 antibodies to carry a functional oac gene.
pNV1644 was used as a parent plasmid to make constructs containing oac point mutations. All subsequent constructs generated could be assessed against the parent strain for Oac functionality and assembly in the membrane. For instance, the observation of colonies with red colouration similar to that seen for pNV1644 (B2012) would indicate that the mutated protein was fully and correctly assembled similar to that of the wild-type Oac. Moreover, with the use of anti-PhoA antibodies in Western immunoblotting, the level of expression of the modified Oac-PhoA-LacZα fusion proteins could be assessed and compared with the unmodified Oac-PhoA-LacZα fusion protein.
Oac functionality testing
O-acetylation establishes the group 6 specificity of the O-antigen. This was tested by transforming a serotype Y strain, SFL124 with the appropriate construct encoding Oac and observing whether the serotype Y strain (group specificity 3, 4) is converted to serotype 3b, having group specificities of 6 and 3, 4. All tests were done in parallel with SFL1899 (having functional oac in pNV1644) serving as a positive control and SFL124 serving as negative control for Oac functionality.
Slide agglutination was performed using antisera (Denka-Seiken, Tokyo) or monoclonal antibodies such as MASF6 (Reagensia AB, Sweden). Absence of autoagglutination was always confirmed using saline instead of the antibodies. All samples were subjected to LPS Western immunoblotting as a confirmatory test after the preliminary result obtained from the slide agglutination. All functional results are thus shown for both the slide agglutination and the LPS Western blotting.
Western blots for LPS samples were prepared and analysed using 12% resolving gels and silver-staining as described by Hitchcock and Brown [23]. Silver staining helped ensure equal loading of samples. The same volume of sample was then used to run another gel for the Western blotting using the MASF 6 as the primary antibody (diluted 1 in 200) and anti-mouse IgG HRP-conjugated as the secondary antibody (diluted 1 in 8000). Detection was performed using the Super Sugnal West Pico Chemiluminescent Substrate according to the manufacturer’s directions (Pierce).
Assessment of the role of the residues in protein assembly in the membrane - qualitative analysis
Membrane fractions of all mutant proteins were prepared as described by Morona et al.
[24] and used in Western immunoblotting. Prior to loading samples on SDS-PAGE, the total amount of protein present in the samples was estimated by the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific). Approximately 10 μg of protein was loaded in a 10% SDS-PAGE gel. Western blots were carried out using anti-PhoA antisera (Chemicon) in a 1 in 1000 dilution as the primary antibody and anti-mouse IgG-HRP conjugated secondary antibody (Sigma) in a 1 in 8000 dilution as the secondary antibody. Detection was performed as described above.
Quantitative assessment of the extent of protein assembly in the membrane- BG assay
pNV1644 carries the pho A-lac Zα reporter gene and hence codes for functional LacZα (otherwise BG). All mutant constructs were assessed against pNV1644 to determine whether the mutation affected the assembly of the Oac-PhoA-LacZα protein and hence altered the BG activity. A quantitative assay was used to measure BG activities based on the cleavage of the substrate o- nitrophenyl- β-D-galactoside as described by Miller et al.
[25]. Before the BG assay was performed, strain B2280 (containing pNV1870) was generated from pNV1644. pNV1870 was constructed to have the dual reporter fused immediately after the oac promoter with the rest of oac deleted. In short, the dual reporter would be transcribed under the influence of the oac promoter in the same background vector pNV1644 used to make all the mutant constructs. The rationale behind constructing this vector was that it was necessary to see what levels of BG activity were obtained for the LacZα fragment alone, which was fully assembled and active in the cytoplasm without being dependent on the assembly and folding of the Oac membrane protein. Thus pNV1870 was constructed using pNV1644 as the template DNA using a reverse PCR technique (as described for pNV1644 in [3]). Primers Pholac(BamHI)_For and Oac_K3(BamHI)_Rev (Additional File 1: Table AFT4) which bound downstream and upstream, respectively, of the oac sequence, amplified the entire vector excluding the oac coding sequence.
Negative controls were also included in the assay to monitor leaky expression of BG. Baseline BG activities recorded were then subtracted from sample values. Values obtained are an average of two independent experimental repeats with internal replicates of the samples.
Site directed mutagenesis
Mutagenesis was performed using the Stratagene’s QuickChange Site-Directed Mutagenesis protocol. Details of the primers used for site-directed mutagenesis are presented in Additional File 1: Table AFT4. All constructs were sequenced entirely using appropriate primers to ensure that the intended mutation was in place and that no other extraneous mutations had been introduced during the PCR process.