Flagellin

Strains of P. aeruginosa were genotyped using the ArrayTube (AT) system (CLONDIAG, Alere Technologies, Köln, Germany) as described previously (Wiehlmann et al. 2007 (link)). The AT microarray chip enables strains to be classified according to 13 core genome single-nucleotide polymorphisms (SNPs), and also screens for 38 variable genetic markers of the P. aeruginosa accessory genome. These include several previously reported genomic islands (Arora et al. 2001 (link); Liang et al. 2001 (link); Larbig et al. 2002 (link); de Chial et al. 2003 (link); Spencer et al. 2003 (link); He et al. 2004 (link); Klockgether et al. 2004 (link); Lee et al. 2006 (link)). Data from the 13 SNPs are combined with flagellin type (a/b) and the presence of the genes encoding mutually exclusive type III secretion exotoxins (S or U), to generate a strain-specific “hexadecimal code” represented by four digits (Wiehlmann et al. 2007 (link)). This code can be used to search a large database of P. aeruginosa strains (Cramer et al. 2012 (link)). Subsequently, eBURST (version 3.0) (Feil et al. 2004 (link); Spratt et al. 2004 (link)) analysis of data generated using the AT from the 13 SNPs, flagellin type (a or b) and presence of the mutually exclusive type III secretion exotoxins (S or U), was used to visualize the position of panel strains within the wider P. aeruginosa population structure using a database of 955 genotyped strains (Cramer et al. 2012 (link)).

LP02 is a streptomycin-resistant thymidine auxotroph derived from L. pneumophila LP01. An unmarked deletion of flaA (corresponding to nucleotides 1478105–1479574 of the LP01 genome [49 (link)]) was generated in LP02 by use of the allelic exchange vector pSR47S [22 (link)]. The ΔflaA strain was also complemented by inserting the flaA gene and its own promoter (corresponding to nucleotides 1478136–1479915 of the LP01 genome) on the chromosome just after the ahpC gene (at position 3354877 of the LP01 genome). The ahpC locus is highly expressed but is not essential for Legionella virulence [50 (link)]. The fliI null strain LP02 fliI::Cm [22 (link)] was the kind gift of R. Isberg (Tufts University School of Medicine). The broad-host-range plasmid pBBR1-MCS2 was used to express flagellin from Legionella, E. coli MG1655 (fliC, b1923), S. flexneri 2457T (fliC, S2062), and Salmonella typhimurium LT2 (fliC, STM1959). The various flagellin open reading frames were first cloned into pET28a (NcoI, XhoI) and then transferred to pBBR1-MCS2 (XbaI, PvuI), such that all flagellins were expressed from the lac promoter of pBBR and the ribosome-binding site of pET28a. Expression was induced with 1mM IPTG. The pBBR-flagellin constructs were also expressed in E. coli CM735ΔfliC [51 (link)], along with LLO (pACYC184-LLOc) [41 (link)]. Salmonella strains were on the LT2 background and were the kind gift of the Starnbach laboratory.

STs were deduced from the genome assemblies using mlst (v2.16.1; https://github.com/tseemann/mlst) and assigned to pre-defined CCs on PubMLST [40 (link)] (https://pubmlst.org/campylobacter). The same database was used to calculate core-genome MLST (cgMLST) (Oxford scheme) on PubMLST [40 (link)]. The relationship between STs, phenotypic resistance, seasonality and the presence of pTet plasmid was assessed using Fisher’s exact test (GraphPad Prism 9.1.2). Source attribution patterns were assigned based on ST–ecotype associations described in recent publications [41–43 (link)]. Gene annotation was carried out from the draft assemblies using Prokka v.1.13 [44 (link)]. The presence of plasmids or prophages was inferred from the assemblies using MOB-suite v.2.0.1 [45 (link)]. The predicted plasmid sequences were used as queries in blastn with threshold values set to >80 % coverage and >95 % identity. The sequences of accession numbers CP017866 and CP014746 were used to define the predicted sequences as pTet and pVir, respectively. Pan-genome analyses were carried out using Roary v.3.12.0 [46 (link)] with an amino acid identity cut-off of 95 % and splitting homologous groups containing paralogues into groups of true orthologues. A summary of the pan-genome composition and visualization of gene diversity is provided in Fig. S1(a–c), available with the online version of this article.
In parallel, whole-genome SNP (wgSNP)-based alignments were built from trimmed reads using the Snippy v.4.3.6 pipeline (https://github.com/tseemann/snippy). The closed genome of strain C. jejuni subsp. jejuni NCTC 11168 (GenBank assembly accession no. GCA_000009085.1) was used as a reference in read mapping. Areas of putative recombination were removed from the resulting alignment using Gubbins v.2.2.0 [47 (link)] and default settings (five iterations and >3 base substitutions to identify a recombination event). Maximum-likelihood phylogenies were obtained from the recombination-removed alignments using the tree building option FastTree v2.1.4 [48 (link)]. The core-genome phylogeny was visualized using iTOL [49 (link)] and the pan-genome genes calculated in Roary were displayed alongside the recombination-removed phylogenetic tree using Phandango [50 (link)] (https://jameshadfield.github.io/phandango). Virulence gene detection was carried out using ABRicate (version 0.8.10; https://github.com/tseemann/abricate) equipped with VFDB (Virulence Factor Database) [51 (link)]. Hits with less than 80 % identity or coverage were filtered out of the analysis.
The PubMLST C. jejuni database was screened for the major flagellin protein, FlaA (encoded by the flaA gene). All 2058 deposited C. jejuni sequences classified as CC-257 (as of November 4th 2022) were searched for the presence of the flaA sequence by blastn [NCBI, National Institutes of Health (NIH)] analysis using the DNA sequence from C. jejuni strain NCTC 11168 as reference. Presence of the gene was determined by >90 % alignment and identity with the query sequence (E value=0). Finally, the presence of type VI secretion system (T6SS) genes, encoding a total of 13 core components (TagH, TssA–TssG, TssI–TssM), was assessed using previously published reference sequences [52 (link)] and the blastn tool. The presence of genes was defined as DNA identity and coverage of ≥90 %.

Surface plasmon resonance studies were performed as described previously [43 (link)]. Amine coupling was used to ligate either HD6 or flagellin to individual flow cells on a CM5 sensor chip. The running buffer (pH 7.4) contained: 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and surfactant P20 (0.005%). The multi-cycle experiments were performed with an analyte flow rate of 20 μl/min for 12.5 min in each cycle. This was followed by a 2.5 min interval during which the flow cells were perfused by analyte-free buffer until the sample loop had acquired the next 250 μl aliquot of analyte solution. The final analyte injection was in "kinetic mode", with a 10 min dissociation period.