Whole genome de novo sequencing and comparative genomic analyses suggests that Chlamydia psittaci strain 84/2334 should be reclassified as Chlamydia abortus species

Abstract

Background: Chlamydia abortus and Chlamydia psittaci are important pathogens of livestock and avian species, respectively. While C. abortus is recognized as descended from C. psittaci species, there is emerging evidence of strains that are intermediary between the two species, suggesting they are recent evolutionary ancestors of C. abortus. Such strains include C. psittaci strain 84/2334 that was isolated from a parrot. Our aim was to classify this strain by sequencing its genome and explore its evolutionary relationship to both C. abortus and C. psittaci.

Results: In this study, methods based on multi-locus sequence typing (MLST) of seven housekeeping genes and on typing of five species discriminant proteins showed that strain 84/2334 clustered with C. abortus species. Furthermore, whole genome de novo sequencing of the strain revealed greater similarity to C. abortus in terms of GC content, while 16S rRNA and whole genome phylogenetic analysis, as well as network and recombination analysis showed that the strain clusters more closely with C. abortus strains. The analysis also suggested a closer evolutionary relationship between this strain and the major C. abortus clade, than to two other intermediary avian C. abortus strains or C. psittaci strains. Molecular analyses of genes (polymorphic membrane protein and transmembrane head protein genes) and loci (plasticity zone), found in key virulence-associated regions that exhibit greatest diversity within and between chlamydial species, reveal greater diversity than present in sequenced C. abortus genomes as well as similar features to both C. abortus and C. psittaci species. The strain also possesses an extrachromosomal plasmid, as found in most C. psittaci species but absent from all sequenced classical C. abortus strains.

Conclusion: Overall, the results show that C. psittaci strain 84/2334 clusters very closely with C. abortus strains, and are consistent with the strain being a recent C. abortus ancestral species. This suggests that the strain should be reclassified as C. abortus. Furthermore, the identification of a C. abortus strain bearing an extra-chromosomal plasmid has implications for plasmid-based transformation studies to investigate gene function as well as providing a potential route for the development of a next generation vaccine to protect livestock from C. abortus infection.

Keywords: Chlamydia abortus; Chlamydia psittaci; Comparative genomic analysis; Genome sequence; MLST; Phylogenomics; Plasticity zone; Polymorphic membrane proteins.

Fig. 1

MLST phylogenetic tree for C. psittaci strain 84/2334 and representative strains of Chlamydiaceae species. The consensus PhyML phylogenetic tree of a 3147 bp alignment of concatenated sequences of seven MLST housekeeping gene fragments (enoA, fumC, gatA, gidA, hemN, hlfX and oppA) was estimated in TOPALi by Maximum Likelihood using a GTR + G + I substitution and rate heterogeneity model, according to BIC model selection, and 100 non-parametric bootstrap replicates. The tree is rooted on the C. trachomatis/muridarum/suis branch and bootstrap support is indicated by the number at the node. The scale bar indicates the expected substitutions per site. Genotypes are given in square brackets. The tree was prepared for publication in Dendroscope. Strain 84/2334 is in bold and red font. Classical and avian C. abortus strains are in blue and green fonts, respectively

Fig. 2

Circular representation of the genome of C. psittaci strain 84/2334. Circles from the outside in show: the positions of protein-coding genes (blue), tRNA genes (orange) and rRNA genes (Pink) on the positive (circle 1) and negative (circle 2) strands, respectively. Circles 3–5 shows positions of BLAST hits determined through blastn comparisons of C. abortus S26/3 (circle 3), C. psittaci 6BC (circle 4) and C. psittaci GR9 (circle 5) with the following settings: query split size = 50,000 bp, query split overlap size = 0, expect value cut off = 0.00001. Low complexity sequences were eliminated from the analysis. The height of the shading in the BLAST results rings is proportional to the percent identity of the hit. Overlapping hits appear as darker shading. Circles 6 and 7 show plots of the GC content and GC skew plotted as the deviation from the average for the entire sequence. The origin of replication is indicated by the vertical dashed line. The four Pmp loci, PZ region and TMH loci are highlighted using rectangular boxes. The figure was generated using the program CGView

Fig. 3

Circular representation of the C. psittaci 84/2334 plasmid sequence and comparative phylogenetic analysis. a Circular representation of the plasmid sequence of strain 84/2334; Circles from the outside in show: the positions of the eight coding sequences on the forward (circle 1) and reverse (circle 2) strands of p84/2334. Circles 3–5 show positions of BLAST hits determined through blastn comparisons of C. psittaci 6BC (circle 3), VS225 (circle 4) and M56 (circle 5) with the following settings: query split size = 50,000 bp, query split overlap size = 0, expect value cut off = 0.0001. The height of the shading in the BLAST results rings is proportional to the percent identity of the hit. Overlapping hits appear as darker shading. Circles 6 and 7 show plots of the GC content and GC skew plotted as the deviation from the average for the entire sequence. The figure was generated using the program CGView. b Phylogenetic tree of an alignment of the plasmid sequences of C. psittaci (Cps), avian C. abortus (Cab), C. caviae (Cca), C. felis (Cfe) and C. pneumoniae (Cpn) strains. The consensus tree was estimated in TOPALi by Maximum Likelihood (PhyML) using a TIM + G substitution and rate heterogeneity model, according to BIC model selection, and 100 non-parametric bootstrap replicates. The tree is midpoint rooted and bootstrap support is indicated by the number at the node. The scale bar indicates the expected substitutions per site. Genotypes are given in square brackets. The tree was prepared in Dendroscope. Strain 84/2334 plasmid is in bold and red font. Avian C. abortus strain plasmids are in green font

Fig. 4

Whole genome phylogenetic analysis informed by recombination. Phylogenetic tree of a whole genome sequence MAFFT alignment of the C. abortus (Cab) and C. psittaci (Cps) strains shown in Table 2 as derived by Gubbins after removing genomic regions affected by recombination. Genotypes are given in square brackets. FastTree was used as tree builder, and a maximum of 100 iterations was specified in order to guarantee convergence. The tree was midpoint rooted and prepared for publication in Dendroscope. Strain 84/2334 is in bold and red font. Classical and avian C. abortus strains are in blue and green fonts, respectively

Fig. 5

Summary of Gubbins recombination analysis excluding avian C. abortus strains 15-59d/3 and 15-70d/24. The red blocks represent recombination events occurring on an internal branch of the phylogenetic tree, which are shared by several strains by common descent. The blue blocks indicate recombination events occurring on terminal branches of the phylogenetic tree, which are unique to a specific strain. The parameters used for the run are those described in Fig. 4

Fig. 6

Comparative analysis of the genes present in the plasticity zone of C. psittaci strain 84/2334. Comparison of nucleotide matches (computed using blastn) between the genes accB (orange) and guaB (pink) in C. abortus and C. psittaci species and genotypes. Chlamydia psittaci genotypes B, E, F (not shown) are identical in gene content to genotypes A, D and WC. The presence of accC (dark blue), guaA (green), MACP (purple), cytotoxin (red) and hypothetical protein (brown) genes are as indicated. Vertical lines through the arrows indicate point or frame-shift mutations. The orientation of coding sequences in the forward and reverse frames are indicated by the direction of the block arrows. The level of BLAST identity between the sequences is indicated by the degree of grey shading in the vertical bars. The figure was generated using EasyFig

Fig. 7

Comparative analysis of the genes present in the transmembrane head of C. psittaci strain 84/2334. Comparison of nucleotide matches (computed using blastn) between the genes lpxB and pmpD (both orange) in C. abortus and C. psittaci species and genotypes. Chlamydia psittaci genotypes B, E, F (not shown) are identical in gene content to genotypes A, D and WC. The presence of TMH/Inc. (green) and hypothetical protein genes/gene remnants (brown) are as indicated. The 3 genes coding for proteins of unknown function are also indicated (blue). Vertical lines through the arrows indicate point or frame-shift mutations. The orientation of coding sequences in the forward and reverse frames are indicated by the direction of the block arrows. The level of BLAST identity between the sequences is indicated by the degree of grey shading in the vertical bars. The figure was generated using EasyFig

Fig. 8

Polymorphic membrane proteins in C. psittaci strain 84/2334. a Gene families and gene organisation in the four Pmp loci (as indicated) showing intact genes (orange) and pseudogenes (grey), identified following BLAST and phylogenetic comparison with other published Pmps. Pmp gene designations are indicated under each block arrow. b Schematic diagram showing the conserved Pmp features, comprising the predicted PmpM domains (blue rectangular block), autotransporter domains (red rectangular block) and pmp passenger domain repeat motifs GG[A/L/V/I][I/L/V/Y] (blue vertical lines) and FXXN (red vertical lines) (Only motifs in which FXXN follows GG[A/L/V/I][I/L/V/Y] are shown). The predicted number of amino acids (aa) is indicated to the right of each gene. Pmp gene identification numbers and gene identification tags (indicated as DZK34_0xxxx) are indicated to the left of each gene