Genome sequencing and comparative analysis of three Chlamydia pecorum strains associated with different pathogenic outcomes
© Sait et al.; licensee BioMed Central Ltd. 2014
Received: 24 September 2013
Accepted: 6 January 2014
Published: 14 January 2014
Chlamydia pecorum is the causative agent of a number of acute diseases, but most often causes persistent, subclinical infection in ruminants, swine and birds. In this study, the genome sequences of three C. pecorum strains isolated from the faeces of a sheep with inapparent enteric infection (strain W73), from the synovial fluid of a sheep with polyarthritis (strain P787) and from a cervical swab taken from a cow with metritis (strain PV3056/3) were determined using Illumina/Solexa and Roche 454 genome sequencing.
Gene order and synteny was almost identical between C. pecorum strains and C. psittaci. Differences between C. pecorum and other chlamydiae occurred at a number of loci, including the plasticity zone, which contained a MAC/perforin domain protein, two copies of a >3400 amino acid putative cytotoxin gene and four (PV3056/3) or five (P787 and W73) genes encoding phospholipase D. Chlamydia pecorum contains an almost intact tryptophan biosynthesis operon encoding trpABCDFR and has the ability to sequester kynurenine from its host, however it lacks the genes folA, folKP and folB required for folate metabolism found in other chlamydiae. A total of 15 polymorphic membrane proteins were identified, belonging to six pmp families. Strains possess an intact type III secretion system composed of 18 structural genes and accessory proteins, however a number of putative inc effector proteins widely distributed in chlamydiae are absent from C. pecorum. Two genes encoding the hypothetical protein ORF663 and IncA contain variable numbers of repeat sequences that could be associated with persistence of infection.
Genome sequencing of three C. pecorum strains, originating from animals with different disease manifestations, has identified differences in ORF663 and pseudogene content between strains and has identified genes and metabolic traits that may influence intracellular survival, pathogenicity and evasion of the host immune system.
Members of the genus Chlamydia are Gram-negative, obligate intracellular pathogens that share a biphasic developmental cycle. Chlamydia pecorum infects a broad host range, including small and large ruminants, swine, birds and marsupials. Seroprevalence and PCR-based studies suggest that infection or exposure to C. pecorum and/or C. abortus is almost ubiquitous in cattle and sheep [1–5]. In the majority of these cases, infection is subclinical, with C. pecorum being routinely detected in the intestine and genital tract. The incidence and severity of disease caused by C. pecorum appears to be heightened in koalas and is associated with clinical disease such as conjunctivitis, urinary- and reproductive tract disease, and infertility . Many chlamydial species, including C. pecorum can enter persistent states, characterised in vitro by enlarged, morphologically aberrant, non-fusogenic reticulate bodies (RBs). Persistence can be induced in vitro by antibiotic exposure , amino acid-  or iron-  deficiencies and exposure to IFN-γ  and it is likely that C. pecorum causes a persistent, subclinical infection in the host. Subclinical infections can have detrimental effects on the animal’s health. Animals with inapparent chlamydiae infections have higher body temperatures, lower body weights, reduced growth rates, reduced iron, haemoglobin, haematocrit and leukocyte levels and a higher incidence of follicular bronchiolitis [11–13]. C. pecorum can also cause clinical disease including encephalomyelitis, vaginitis, endometritis, mastitis, conjunctivitis, polyarthritis, pneumonia, enteritis, orchitis, pleuritis, infertility or pericarditis .
Genetic variation has been reported to occur between C. pecorum strains in ompA, the rrn-nqr F intergenic region, incA, rRNAs, a number of housekeeping genes and the hypothetical protein ORF663 [14–22]. These and other unidentified genomic differences may enable differentiation between strains isolated from asymptomatic or diseased animals. However, to date, only the genome sequence of a single C. pecorum strain (E58) has been published . The genetic factors responsible for the diverse host range, tissue tropism, disease outcomes and associated sequelae of C. pecorum infections are thus still poorly understood. In this study, we present the complete genome sequences of three C. pecorum strains isolated from animals exhibiting different disease manifestations and use comparative genomics to provide insights into the biology of C. pecorum and to identify both genus- and species-specific virulence factors.
Results and discussion
Genome features and comparative analysis
General features of C. pecorum PV3056/3, P787 and W73 compared with the type strain E58 [CP002608]
C. pecorum(E58) []
Date of isolation
Cow, cervical swab
Sheep, synovial fluid
Genome size (bp)
% GC of genome
Predicted no. of pseudogenes
No. of CDS with functional assignment
No. of pmp proteins
No. of tRNA genes
No. of rRNA operons
No. of sRNA molecules
Location of OriC region
1104229–147 nt (471 nt)
1106263–147 nt (296 nt)
1106389–147 nt (295 nt)
305941–306155 nt (215 nt)
Comparative genomics of chlamydial species has identified a number of genes coding for metabolic functions, such as tryptophan metabolism, biotin biosynthesis and folate biosynthesis, where subtle variations in gene content may contribute to growth of the organism in vivo and the ability to evade the host immune system [23, 25–29].
The 3 sequenced C. pecorum strains and E58 contain the biotin biosynthesis operon encoding bioBFDA. (CPE1_0687-CPE1_0690; CPE2_0688-CPE2_0691; CPE3_0688-CPE3_0691). This region shows significant variability between chlamydial species, being absent in C. caviae, C. trachomatis and C. muridarum but present in C. abortus, C. psittaci, C. felis and C. pneumoniae. The ability to synthesise biotin is hypothesised to assist in the colonization of biotin-limited niches and contribute to the tissue tropism differences observed in the chlamydiae . Upstream of bioBFDA, located between dapB and bioB, a series of genes encoding hypothetical proteins with unknown function and limited distribution across chlamydial species are present. Chlamydia abortus, C. psittaci and C. felis genomes contain four genes (in C. abortus, CAB681, CAB682, CAB683 and CAB684), C. pneumoniae contains 2 genes in this region that are homologues of CAB681 and CAB682, while C. pecorum contains one gene in this region that is homologous to CAB684 (Additional file 2: Figure S1).
Bacterial secretion systems
The Type III secretion system (T3SS) consists of 18 genes encoding the major structural components of the secretion apparatus, accessory proteins and chaperones and is arranged in 4 genetic loci (Additional file 1: Table S5). In sequenced chlamydial genomes, a number of putative T3SS effector proteins belonging to the Inc or transmembrane head (TMH) protein family are located in the region extending between pmpD and lpxB (Additional file 2: Figure S3). The distance between the 3′ ends of these genes in C. pecorum is ~2.8 kb (2 genes) compared to 18.1 kb C. abortus (11 genes), 17.7 kb in C. psittaci (11 genes), 16.4 kb in C. caviae (13 genes), 15.9 kb in C. felis (11 genes) and 1.7 kb in C. pneumoniae (1 gene). The two genes present in this region in C. pecorum (CPE1_0764 (pseudogene), CPE1_0765, CPE2_0765, CPE2_0766, CPE3_0765, CPE3_0766) possess an N-terminal signal sequence, a single N-terminal transmembrane domain and two domains of unknown function (DUF1539 and DUF1548). Members of this protein family are present in C. abortus, C. psittaci, C. caviae and C. felis (3 CDSs each) and C. pneumoniae (1 CDS).
Simple sequence repeats
A region of variability between C. pecorum and other chlamydial species is located immediately upstream of the 5S rRNA gene. This region, between the 3′ ends of the 5S rRNA and nqrF genes range in size from 261–269 bp in C. pecorum to 4464 bp in C. caviae. In C. caviae this region encodes a 1291aa residue pseudogene identified as a member of the virulence-associated invasion/intimin family of outer membrane proteins of Gram-negative bacteria. The genome of C. abortus contains two CDSs in place of the intimin family gene in this region, encoding a conserved membrane protein and a unique hypothetical protein. In C. psittaci, C. felis and C. muridarum these two proteins are fused to encode a single hypothetical protein. In C. pecorum there are no predicted CDSs in this region and the intergenic region between the 5S rRNA and nqrF genes comprises an 8 bp simple sequence repeat sequence AAAGCACT repeated 12 (W73, PV3056/3 and E58) or 13 times (P787) (Additional file 2: Figure S4).
Clustered tandem repeat (CTR) sequences observed in orthologs of hypothetical protein ORF663
CTR motif sequence
Clustered tandem repeat sequences (CTR) observed in IncA
CTR motif sequence
In chlamydial species, the plasticity zone is defined as the region between inosine-5′-monophosphate dehydrogenase (guaB) and acetyl-CoA carboxylase (accB) and is the region of the genome that is most variable in gene content and sequence. In C. pecorum, this region is 40.3-42.1 kb in size and contains 16 (PV3056/3) or 17 (W73 and P787) genes encoding GMP synthase, an adenosine deaminase superfamily-protein, a MAC/perforin domain-containing protein, 3 (PV3056/3) or 4 (W73 and P787) phospholipase D family proteins, 2 cytotoxins and 4 hypothetical proteins (Additional file 1: Table S6).
Flanking the cytotoxin genes in C. pecorum are 4 (PV3056/3) or 5 (P787, W73 and E58) phospholipase D (PLD) genes each containing the conserved HxKx4Dx6GSxN (HKD) motif essential for the initiation of phosphodiesterase activity and amino acid motifs that are responsible for catalytic activity. PLD genes identified in the plasticity zone of P787, W73 and E58 share 95-99% amino acid sequence identity (CPE2_0554, CPE3_0554, G5S_0938; CPE2_0553, CPE3_0553, G5S_0935; CPE2_0551, CPE3_0551, G5S_0931; CPE2_0550, CPE3_0550, G5S_0930) whereas orthologous PLD genes in PV3056/3 are more divergent (58-71% sequence identity) (CPE1_0553, CPE1_0551, CPE1_0550). The remaining PLD gene is almost identical in E58 and W73 (98% identity, CPE2_0556, G5S_0945) but divergent in the remaining strains (55-79% identity, CPE1_0555, CPE3_0556). The presence of poly(G) and poly(C) homopolymeric tracts ranging in size from 5–19 nucleotides within the PLD genes and the presence of intact variants in the sequence reads of pseudogenes could indicate that these proteins are subject to phase variation by slip-strand pairing . Whilst the function of PLD in C. pecorum is currently unknown, PLD can perform numerous functions ranging from DNA hydrolysis, to protein-protein interactions with host signalling pathways, to the more classic lipase function. In C. trachomatis, PLD genes located in the PZ have been associated with inclusion formation , whereas in other bacteria PLD has been identified as an important virulence determinant involved in dissemination, serum resistance and invasion of epithelial cells [48, 49].
The complete genome sequence of C. pecorum P787, W73 and PV3056/3 was determined by Illumina/Solexa and Roche 454 genome sequencing. Despite the differences in the clinical manifestations of infections caused by the strains, comparative analysis revealed a high level of sequence conservation, gene content and order between the genomes. Additional genomic analyses of strains originating from other non-ruminant host species, such as pig and koala, will determine if the high level of sequence similarity is common to all, or just ruminant strains of C. pecorum. In agreement with previous studies , differences in the number of clustered tandem repeat sequences in ORF663 were observed between strains isolated from diseased (PV3056/3 and P787) or asymptomatic (W73) animals however, no other genetic differences were observed that may account for the different disease manifestations. A number of metabolic traits were identified in C. pecorum that may contribute to its ability to evade the host immune system and enable persistent infections to be established in the host. Specifically, this study has particularly highlighted the absence of genes involved in folate biosynthesis and the presence of tryptophan and biotin biosynthesis pathways. The presence of clustered tandem repeats in surface expressed proteins, 15 polymorphic membrane proteins, two cytotoxin genes and multiple phospholipase D genes that are likely to be subject to phase variable expression may play a role in the invasion of host cells and trigger the switching between persistent and acute disease in the host.
C. pecorum strain information, propagation and preparation of gDNA
Three C. pecorum strains originating from different geographical regions and disease manifestations were selected for genome sequencing. Strain P787 was isolated in Scotland, in 1977, from the affected synovial fluid of a sheep with polyarthritis. Strain PV3056/3 was isolated in Italy, in 1991, from a cervical swab of a cow with purulent metritis and has subsequently been shown to induce a purulent metritis following inoculation into the uterine body and cervix of cattle . Strain W73 was isolated in Northern Ireland, in 1989, from the faeces of a sheep with an inapparent enteric infection and has subsequently been found to be non-invasive in a mouse model of infection .
Strains were propagated in Caco-2 cells grown in RPMI medium supplemented with 5% FBS and 1 μg/ml cyclohexamide. Genomic DNA from PV3056/3 and P787 was derived from the 7th tissue culture passage of original strains propagated in fertile hens’ eggs. W73 was derived from the 6th tissue culture passage of a strain propagated in fertile hens’ eggs, however the passage history prior to this is unknown. Flasks of infected cells were harvested using glass beads followed by centrifugation at 22,000 × g for 40 mins. Pellets were washed in ice-cold PBS and re-centrifuged as before. Pellets were resuspended in 20 mM Tris–HCl (pH 7.5)/150 mM KCl/1% sarkosyl and lightly homogenised using a ground glass homogeniser. Homogenised cells were layered onto cushions of 15% sucrose in 20 mM Tris–HCl (pH 7.5)/150 mM KCl/1% sarkosyl and centrifuged at 70,000 × g for 45 min at 4°C. Genomic DNA was extracted from pellets using the Wizard DNA extraction kit (Promega).
Genome sequencing was performed by The Gene Pool genomic facility in The University of Edinburgh using Roche 454 GS-FLX and Solexa/Illumina 35-bp paired-end sequencing on standard libraries constructed according to the manufacturers instructions. Reads were assembled using Newbler v2 (Roche) and Velvet v.0.7 , combined using minimus2 and mapped to the reference genome of C. pecorum E58  to generate 13, 10 and 9 contigs for P787, W73 and PV3056/3 respectively. In total, 12,926,259 (PV3056/3), 8,169,259 (W73) and 10,039,539 (P787) reads obtained from Solexa/Illumina sequencing and 95,683 (PV3056/3), 101,405 (W73) and 65,050 (P787) reads from Roche 454 GS-FLX sequencing were obtained. Following quality filtering, sequencing reads were mapped to the reference genome providing approximately 253× (PV3056/3), 136× (W73) and 59.4× (P787) sequencing coverage. Regions spanning the contig ends were PCR-amplified using Phusion High-fidelity DNA polymerase (NEB) and the sequence determined ensuring that each base was covered by sequence in each direction.
Sequence annotation and analysis
Protein-encoding genes were predicted using Prodigal  and open reading frames (ORFs) consisting of fewer than 30 codons or those overlapping larger open reading frames were eliminated. Frameshifts, point mutations and pseudogenes were corrected or confirmed by visual inspection of mapped reads using Tablet . The origin of replication was determined using Ori-finder  and the genomes were adjusted so that the first base was upstream of the hemB gene in the oriC region. Ribosomal RNA genes and tRNA genes were identified using RNAmmer and ARAGORN [56, 57]. Sequences of experimentally validated small non-coding RNAs (sRNA) from chlamydia were downloaded from BSRD  and identified in C. pecorum genomes using blastn. Functional assignments were made based on homology searches using blastp  against protein sequences present in the NCBI nr database and the identification of conserved domains using Pfam  and InterProScan protein databases . Signal sequences were predicted using the LipoP 1.0 . KEGG orthology assignments were performed using KAAS . Data collation and annotation was performed using Artemis .
Comparative analysis were performed using the following genomes: C. pecorum E58 [GenBank: CP002608] , C. abortus S26/3 [GenBank: CR848038] , C. caviae GPIC [GenBank: AE015925] , C. felis Fe/C-56 [GenBank: AP006861] , C. psittaci 6BC [GenBank: CP002586] , C trachomatis D/UW-3/CX [GenBank: AE001273] ), C. pneumoniae AR39 [GenBank: AE002161] and C. muridarum Nigg [GenBank: AE002160] . Global genomic comparisons were visualised using ACT  with input files generated by the tblastx function in DoubleAct http://www.hpa-bioinfotools.org.uk/pise/double_act.html# with a cutoff score of 0. Comparisons of regions flanking the PZ were performed using default blastn settings in EasyFig . Orthologous gene sets were identified by OrthoMCL-DB using reciprocal blastp with a cutoff of e-5 and 50% match . Genome maps were generated using the CGView Server .
Reference sequences were obtained from GenBank and aligned with relevant C. pecorum CDSs using MUSCLE . Phylogenetic alignments and tree files are available from the Dryad Digital repository http://doi.org/10.5061/dryad.np597. For ribosomal proteins, 48 individual alignments were concatenated into a single alignment for analysis. For the phylogenetic analysis of cytotoxin genes, GBlocks v 0.91  was used to eliminate regions that could not be unambiguously aligned resulting in 2845 positions being analysed (75% of the original 3766 positions). Phylogenetic analyses were performed using PhyML (for ribosomal proteins and polymorphic membrane proteins) or MrBayes (for cytotoxins) software  launched from the TOPALi v2.5package  generated using the JTT + G (ribosomal proteins), JTT + I + G (polymorphic membrane proteins) or WAG + I + G (cytotoxins) substitution model that was determined to be the model of best fit based on the BIC criterion. For MrBayes phylogeny, trees were generated using Markov Chain Monte Carlo (MCMC) settings of 2 runs of 625,000 generations with a burn-in of 125,000 generations with trees sampled every 100 runs. For PhyML phylogeny, bootstrap analysis was performed based on 100 replicate trees. Phylogenetic network analysis was performed using SplitsTree .
Nucleotide sequence accession number
Genome sequences of C. pecorum strains PV3056/3, W73 and P787 have been deposited in GenBank under the accession numbers CP004033, CP004034, and CP004035, respectively.
This work as well as MS, NW, LS and EMC was funded by grant no. BB/E018939/1 from the Biotechnology and Biological Sciences Research Council (BBSRC) and by the Scottish Government Rural and Environment Science and Analytical Services division (RESAS).
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