Rationale: This section introduces all the knockout and complement mutant strains used in this study in order to facilitate the readability of the thesis. Schematic representations of these strains are summarized in Figure 13. All constructs for strain generation were analyzed by PCR and confirmed by sequencing prior to transformation of C. jejuni. Strains were also verified by PCR analysis and sequencing.
Mutants used in this study
C. jejuni NCTC 11168 was chosen for this study since it was the first sequenced strain and our laboratory has already constructed protein glycosylation and capsular heptose mutants in this strain (Wong et al., 2015). It also encodes for Cj1319 which we had surmised could initiate the capsular heptose synthesis pathway. Cj1319 is not found in all strains of C. jejuni, but has been found in 82% of isolates (of 151 strains tested, 3 from chickens and the rest from patients) (Huang et al., 2013). In addition, this strain has one of the most complex protein glycosylation islands compared to other C. jejuni strains (Gundogdu et al., 2007; Parkhill et al., 2000). The cj1319 knockout mutant, cj1319::CAT, was constructed by disruption of the gene with a chloramphenicol resistance cassette (Zebian et al., 2015).
For analysis of capsule related phenotypes, we compared the cj1319::CAT mutant to mutants of the capsule heptose modification pathway, cj1427::CAT, cj1428::CAT, cj1429::CAT and cj1430::CAT, as well as the capsuless mutant KpsM (Wong et al., 2015). Cj1427 or WcaG, is a negative regulator of the pathway (McCallum et al., 2011). Cj1430 and Cj1428 are the MlghB epimerase and MlghC reductase of this pathway respectively (Figure 3B) (McCallum et al., 2013). Cj1429 is thought to be a regulator of capsule synthesis, but its function is unknown (Wong et al., 2015). We also compared the cj1319::CAT mutant to a protein N-glycosylation pathway mutant, cj1121c::CAT, whereby Cj1121c is the aminotransferase of this pathway (Vijayakumar et al., 2006), and a mutant of the protein O-glycosylation pathway, cj1294::CAT, whereby Cj1294 is the
aminotransferase of this pathway(Obhi and Creuzenet, 2005; Vijayakumar et al., 2006) in order to investigate if Cj1319 is related to either of these two pathways or if it could be part of an independent protein glycosylation pathway. These studies also complemented our laboratory’s previous studies on the role of protein glycosylation in virulence.
Figure 13. Schematic summary of strains used and created for this study.
For each strain, cj1319 and its flanking genes are shown as well as the three copies of 16S and 23S rRNA genomic regions used for complementation as the site for chromosomal integration. Drawing not to scale. ‘R’ indicates that the complementation construct integrated into a copy of 16S 23S rRNA genomic region. ‘O’ indicates the complementation construct replaced the genes in the original cj1319 locus. ‘SP’ denotes ‘strong promoter’ and corresponds to the use of the constitutive strong promoter of OmpE to drive expression of the genes in the complementation construct. ‘NP’ denotes ‘native promoter’ whereby the native promoter of cj1319 was cloned into the complementation construct. The CAT and KAN antibiotic resistance cassettes are in the same orientation as cj1319.
Generation and qRT-PCR characterization of complements
In this study, we originally complemented the cj1319 knockout mutant according to a published method for C. jejuni chromosomal integration-based complementation (Karlyshev and Wren, 2005). Due to the instability of shuttle vectors in C. jejuni, this method of chromosomal integration of the complement construct into one of the three copies of 16S-23S rRNA genomic regions was chosen. Briefly. we cloned the strong constitutive promoter of the outer membrane protein, OmpE, as described by (Karlyshev and Wren, 2005) upstream of cj1319 and inserted a kanamycin resistance cassette downstream of cj1319. This was done using gene splicing by overlap extension (SOEing). This construct was flanked by a region of 16S rRNA upstream and 23S rRNA downstream. This construct was transformed into both the cj1319 knockout mutant and the WT by natural transformation and homologous recombination into a 16S-23S rRNA genomic region, generated cj1319comp_RSP and WTcj1319comp_RSP respectively where ‘RSP’ indicates that the complement construct integrated into a redundant 16S 23S region and that cj1319 at this site is under the control of the strong constitutive promoter of OmpE. This complement was used in the majority of the work in this thesis. Similarly, complement constructs using cj1121c and cj1294 instead of cj1319 were made and transformed into either cj1121c::CAT or cj1294::CAT and WT, yielding cj1121ccomp_RSP and WTcj1121ccomp_RSP as well as cj1294comp_RSP and WTcj1294comp_RSP respectively.
As cj1319comp_RSP was able to complement some phenotypes, but not others, we hypothesized that this complement may be overexpressing Cj1319, and thus not restoring the phenotype to that of WT. We determined by qRT-PCR that the level of cj1319 gene expression in cj1319comp_RSP was ~7 fold higher compared to that of the WT (Figure 14). Therefore, at the latest stages of this work, we altered the complement construct to encode the native promoter of cj1319 rather than that of ompE. This construct was transformed into the cj1319 knockout mutant and WT as described above, yielding cj1319comp_RNP and WTcj1319comp_RNP respectively, where ‘RNP’ indicates that the complement construct integrated into a redundant 16S 23S rRNA region and that cj1319
at this site is under the control of its nature promoter (Figure 13). The cj1319comp_RNP expressed WT levels of Cj1319 (Figure 14).
To determine if secondary mutations occurred in the cj1319::CAT mutant that contribute to the phenotypes that we tested, we constructed a third complement, whereby we replaced the cj1319 gene disrupted with CAT in the knockout mutant with a complete cj1319 gene under its native promotor. We inserted a KAN cassette downstream of cj1319 in order to select for the new complement. This construct was introduced into the cj1319::CAT mutant and WT as described above resulting in strains cj1319comp_ONP and WTcj1319comp_ONP respectively, where ‘ONP’ indicates the complement construct replaced the knockout construct and cj1319 is under the control of its native promoter. As these two new strains are theoretically genetically identical, they should not differ in phenotypes.
Figure 14. Fold change of cj1319 locus in cj1319comp_RSP and cj1319comp_RNP compared to WT as determined through qRT-PCR.
Genetic organization of the genes targeted for the qRT-PCR analysis is depicted (not drawn to scale). The interstrain change in gene expression is reported in the table shown as a heat map of low to high gene expression compared to the WT. The fold change in expression was determined from two biological replicates, each tested in duplicate. The brackets indicate 2(ΔΔCT-SD) and 2(ΔΔCT+SD), where SD represents the standard error propagated through the fold calculations. No significant difference was determined by Student’s t test.