Comprehensive genome analysis and comparisons of the swine pathogen, Chlamydia suis reveals unique ORFs and candidate host-specificity factors

ABSTRACT

Chlamydia suis, a ubiquitous swine pathogen, has the potential for zoonotic transmission to humans and often encodes for resistance to the primary treatment antibiotic, tetracycline. Because of this emerging threat, comparative genomics for swine isolate R19 with inter- and intra-species genomes was performed. A 1.094 Mb genome was determined through de novo assembly of Illumina high throughput sequencing reads. Annotation and subsystem analyses were conducted, revealing 986 putative genes (Chls_###) that are predominantly orthologs to other known Chlamydia genes. Subsequent comparative genomics revealed a high level of genomic synteny and overall sequence identity with other Chlamydia while 92 unique C. suis open reading frames were annotated. Direct comparison of Chlamydia-specific gene families that included the plasticity zone, inclusion membrane proteins, polymorphic membrane proteins and the major outer membrane protein, demonstrated high gene content identity with C. trachomatis and C. muridarum. These comparisons also identified diverse components that potentially could contribute to host-specificity. This study constitutes the first genome-wide comparative analysis for C. suis, generating a fully annotated reference genome. These studies will enable focused efforts on factors that provide key species specificity and adaptation to cognate hosts that are attributed to chlamydial infections, including humans.

Pathogenomic comparison of fully sequenced and annotated Chlamydia suis revealed unique genetic elements that could encode virulence factors and proteins that contribute to host-specificity.

BACKGROUND

Members of the genus, Chlamydia, are biologically distinctive, obligate intracellular bacterial pathogens that cause disease in both humans and agriculturally important livestock. Chlamydia suis is a near-ubiquitous swine pathogen causing reproductive disorders, conjunctivitis, enteritis, rhinitis and pneumonia (Rogers, Andersen and Hunsaker 1996; Schiller et al. 1997; Rogers and Andersen 1999; Sachse et al. 2004). Prevalence of C. suis disease within regions throughout the world has been investigated and those reports each found high incidence in farmed pigs. Many C. suis strains have acquired a tetracycline-resistance cassette with clinical resistance confirmed in over seven countries (Kaltenboeck, Kousoulas and Storz 1992; Lenart, Andersen and Rockey 2001; DiFrancesco et al. 2008; Borel et al. 2012; Schautteet et al. 2013). In vitro evidence has also demonstrated the ability for tetracycline resistance to be conveyed to C. trachomatis through horizontal gene transfer under conditions of co-infection (Suchland et al. 2009; Jeffrey et al. 2013). Though traditionally restricted to its swine host, there is some evidence that C. suis may be zoonotically transmitted, although active human infection and clinical symptoms have not been observed (Dean et al. 2013; De Puysseleyr et al. 2014, 2017).

The emerging potential threat of C. suis and ongoing concerns to the agricultural industry highlight a need for a comparison of the C. suis genome to prominent human pathogen, Chlamydia trachomatis. C. trachomatis is the most commonly reported bacterial infection worldwide and is the causative agent behind several important diseases including blinding trachoma and a set of different sexually transmitted conditions. Importantly, genome sequencing and comparative studies between Chlamydia species have enabled the identification and analysis of gene products that are diverse and may participate in host specific adaptations (Girjes, Carrick and Lavin 1994; Kalman et al. 1999; Read et al. 2000; Xie, Bonner and Jensen 2002). While there have been many intraspecies analyses of C. suis with a focus on the tetracycline-resistance genes (Donati et al. 2014; Joseph et al. 2016; Marti et al. 2017; Seth-Smith et al. 2017), interspecies comparisons with C. trachomatis and C. muridarum, the species most closely related to C. suis, have been limited. One key observation within Chlamydia is that despite the genetic similarity between species, there appear to be many host-restricting factors responsible for the variation in disease between species. Studying the C. suis genome in comparison to other Chlamydia sp. is expected to identify candidate host-specificity factors.

The C. suis R19 strain is a tetracycline resistant strain isolated from a Nebraska farm (Andersen and Rogers 1998) and has been widely studied for diagnostic and antibiotic resistance transfer capabilities (Suchland et al. 2009; De Puysseleyr et al. 2017; Marti et al. 2017). Previously, this strain was sequenced by Joseph et al with 14x coverage, was solved to the contig level and contains many ambiguous bases (Joseph et al. 2016) Additionally, there have been recent advances in gene function annotation methods that are expected to enhance the understanding of genetic content within bacteria. The PathoSystems Resource Integration Center (PATRIC), an NIAID funded bioinformatic toolbox, allows for streamlined workflows for assembly, annotation and protein comparisons (Wattam et al. 2014; Davis et al. 2020). While PATRIC contains a database of chlamydial genomes and proteins, this resource has not been utilized within the field, although, it has been demonstrated to be useful in the annotation of several other bacterial genomes (Al Dahouk et al. 2017; Viana et al. 2017; Banerjee et al. 2018; Viana et al. 2018). In this study, these public databases were used to annotate the C. suis genome, along with reannotation for C. trachomatis and C. muridarum, allowing for the identification of several new open reading frames and possible coded protein functions. These analyses have confirmed previous findings that the C. suis genome is highly similar in content and organization to both C. trachomatis and C. muridarum. These analyses also demonstrated that C. suis encodes for many genes similar to other Chlamydia. Finally, comparative analyses show specific genes which may play a role in the host-adaptation of Chlamydia.

RESULTS

The C. suis genome architecture and composition

The C. suis R19 strain genome was sequenced using Illumina HiSeq with over 1000X coverage, enabling an unbiased, de novo assembly of the genome (Figure S1, Supporting Information). The completed genome includes a chromosome 1 094 719 bp in length and a plasmid of 7496 bp. These data agree with previously performed studies on C. suis strains. Read density analysis of the plasmid relative to the chromosome supported approximately two copies of the plasmid per chromosome. The genome was annotated using both direct ORF prediction and BLAST homology-based annotation through Geneious and the PATRIC annotation platform. This analysis revealed 986 coding regions (Chlamydia suis; Chls_###), with six rRNAs and 37 tRNAs, and an overall 41.7% G + C content (Fig. 1A, Table 1 and Table S1, Supporting Information). The origin of replication for this genome was assigned according to the reference genome C. trachomatis L2 434/Bu (NC_010 287; Thomson et al. 2008). Of the 986 coding sequences identified in C. suis, 686 were assigned through comparisons to other Chlamydia and through inferred functional assignment of known motifs with a 75% identity threshold. The assigned genes were divided into nine broad categories or defined as ‘other’ using the subsystems approach and the RAST toolkit (Overbeek et al. 2005, 2014). Overall, the largest division, at 165 genes, were predicted to be involved with protein processing followed closely by metabolism-related genes (163 ORFs; Fig. 1B). Additional subsystems include those involved with stress response, defense or virulence (50 genes), DNA processing (51 genes), energy (62 genes), RNA processing (31 genes), cellular processes (28 genes), cell envelope (28 genes) or membrane transport (3 genes). The remaining 282 genes had no homologues or identified motifs in Genbank and were annotated as hypothetical. The C. suis R19 tetracycline resistance cassette, likely integrated by the IS605 transposase and horizontal gene transfer, is thought to be spreading among C. suis strains (Dugan et al. 2004; Dugan, Andersen and Rockey 2007). Many analyses of the tet island demonstrate that the island is plastic, but that the key tet (C) gene was always intact, as it is for R19, and that the cassette is transferable between C. suis strains by homologous recombination without the need for the transposase (Kalman et al. 1999; Read et al. 2000; Marti et al. 2017; Seth-Smith et al. 2017). As in the other species in the genus, there is one plasmid with 8 genes encoding replicative machinery and putative virulence factors (Rockey 2011).

Figure 1.

(A) Annotated genome for C. suis R19. rings, from outside, show: chromosome (navy), all annotated coding regions in the forward direction (green), annotated coding regions in the reverse direction (violet), non-coding annotations including ribosomal RNAs (red), known virulence factors including type 3 secretion components, as automatically annotated through PATRIC annotation platforms (gold), functionally annotated transporters (blue), relative GC content (lavender ring) and GC skew (yellow ring), annotated membrane-associated proteins assigned through structural prediction and relatedness to reference Chlamydia and inclusion membrane proteins determined through comparative annotation and canonical hydrophobicity plots (cyan). B) Subsystems of functionally assigned genes excluding the 282 hypothetical genes. A total of 122 assigned genes were also not categorized into one of these subsystems and were left out of the chart. The remaining genes were divided into nine subsystems including protein processing, metabolism, stress response/defense/virulence, DNA processing, energy, RNA processing, cellular processes, cell envelope and membrane transport.

Table 1.

Characteristics of the C. suis R19 genome.

	Chlamydia suis R19	Chlamydia trachomatis 434/Bu	Chlamydia muridarum Nigg
Chromosome length (bp)	1 094 719	1 038 842	1 072 950
Chromosomal genes (PATRIC)	986	962	983
NCBI reported chromosomal genes	N/A	937	952
Plasmid length (bp)	7496	7499	7501
Plasmid coding regions	8	8	8
GC content (%)	41.7	41.3	40.3

Comparative analysis of the C. suis coding regions to C. trachomatis and C. muridarum representative strains

Chlamydia trachomatis L2/434/Bu contains a predicted 962 genes while the C. muridarum Nigg genome consists of 983 genes, as re-annotated under the PATRIC annotation platform (Table 1). There is approximately 90% synteny in the genome organization for these three genomes, as well as to other members of the phylum (Fig. 2A). Over 80% of proteins encoded by both C. trachomatis and C. muridarum have greater than 75% amino acid similarity with C. suis orthologs, further highlighting the evolutionary relationship of these species (Fig. 3A, Table S2, Supporting Information). Both this genetic comparison, and genome-wide sequence phylogenies (Fig. 2B) corroborate previous findings using 16S sequences that C. suis is in a phylogenetic clade with C. trachomatis and C. muridarum (Pudjiatmoko et al. 1997). Upon closer inspection of the genetic make-up of the C. suis genome, 20 proteins considered to be key for phylogenetic classification of Chlamydia are present, intact and highly similar to the other Chlamydiales (Pillonel et al. 2015).

Figure 2.

(A) Circular alignment of C. suis R19 coding regions (outer ring) with canonical Chlamydia. Protein sequence identity is indicated by colorimetric scale where purple/blue represents higher identity than orange/red. White or blank areas represent an absence of coding regions annotated at that site. There are four key regions with a notable absence of coding regions. The first of these (around 140–160 kb) correlates with the ribosomal RNA operons. The second region (around 310 kb) has diversity which can be attributed to a polymorphic membrane protein (pmp) operon where not all genes are conserved between Chlamydia. The third region (around 530 kb), represents the plasticity zone which is where the greatest diversity is expected between species. The final region (around 850 kb) is an additional pmp operon. This alignment was performed using the PATRIC proteome comparison tool. (B) Phylogenetic analysis of Chlamydia using whole-genome sequences aligned through progressiveMauve demonstrates the positioning of representative C. suis genomes compared with selected C. trachomatis and C. muridarum reference genomes.

Figure 3.

(A) Amino acid similarity percentages for conserved protein coding sequences. C. trachomatis D/UW-3 was used in this comparison. (B) Protein families for C. muridarum and C. trachomatis that fall below 75% amino acid similarity with their C. suis orthologs.

C. suis encodes for 92 genes that are not present in either C. trachomatis or C. muridarum (Table 2). In each instance, C. trachomatis D/UW-3 and C. muridarum Nigg were investigated for syntenous orthologs and these appear to be unique to C. suis. Most of these are hypothetical proteins although several are known members of the tetracycline resistance island, an inclusion membrane protein and genes within the plasticity zone. Along with these unique C. suis genes appear to be a subset of orthologous that are shared between either C. suis and C. trachomatis or C. suis and C. muridarum (Table 3).

Table 2.

Unique C. suis open reading frames.

Gene	AA length	Annotated function	Gene	AA length	Annotated function
Chls 011	90	Hypothetical protein	Chls 331	51	Hypothetical protein
Chls 021	40	Hypothetical protein	Chls 334	41	Hypothetical protein
Chls 028	54	Hypothetical protein	Chls 339	41	Hypothetical protein
Chls 035	43	Hpothetical protein	Chls 363	62	Hypothetical protein
Chls 037	71	Hypothetical protein	Chls 378	42	Hypothetical protein
Chls 056	41	Hypothetical protein	Chls 396	55	Hypothetical protein
Chls 058	45	Hypothetical protein	Chls 404	285	Hypothetical protein
Chls 061	54	Hypothetical protein	Chls 405	105	Hypothetical protein
Chls 072	41	Hypothetical protein	Chls 406	91	Hypothetical protein
Chls 080	49	Hypothetical protein	Chls 407	44	hypothetical protein
Chls 100	62	Hypothetical protein	Chls 421	166	Hypothetical protein
Chls 105	37	Hypothetical protein	Chls 438	45	Hypothetical protein
Chls 113	44	Hypothetical protein	Chls 452	38	Hypothetical protein
Chls 123	459	Metal-dependent hydrolase	Chls 454	47	Hypothetical protein
Chls 124	292	Replication protein RepA	Chls 460	53	Hypothetical protein
Chls 125	108	Hypothetical protein	Chls 461	40	Hypothetical protein
Chls 126	107	Growth inhibitor, PemK family	Chls 462	67	Hypothetical protein
Chls 127	417	Relaxase	Chls 464	54	Hypothetical protein
Chls 128	167	Mobilization protein, MobB	Chls 468	92	Hypothetical protein
Chls 129	118	Mobilization protein, MobC	Chls 474	38	Hypothetical protein
Chls 130	227	Mobilization protein, MobD	Chls 475	261	Gamma-glutamylcyclotransferase
Chls 131	212	Mobilization protein, MobE	Chls 478	649	Hypothetical protein
Chls 132	250	OfxX fusion product	Chls 480	37	Hypothetical protein
Chls 133	101	Resolvase	Chls 503	29	Hypothetical protein
Chls 134	396	Tetracycline resistance, MFS efflux pump, Tet(C)	Chls 536	51	Hypothetical protein
Chls 135	211	Tetracycline resistance regulatory protein, TetR	Chls 540	50	Hypothetical protein
Chls 136	151	Transposase	Chls 542	37	Hypothetical protein
Chls 137	459	Mobile element protein	Chls 578	44	Hypothetical protein
Chls 138	868	Hemagglutinin	Chls 602	38	Hypothetical protein
Chls 143	44	Hypothetical protein	Chls 634	52	Hypothetical protein
Chls 150	71	Hypothetical protein	Chls 642	41	Hypothetical protein
Chls 151	37	Hypothetical protein	Chls 647	94	Hypothetical protein
Chls 175	52	Hypothetical protein	Chls 648	196	Hypothetical protein
Chls 188	51	Hypothetical protein	Chls 649	46	Hypothetical protein
Chls 213	44	Hypothetical protein	Chls 679	38	Hypothetical protein
Chls 218	39	Hypothetical protein	Chls 682	39	Hypothetical protein
Chls 234	47	Hypothetical protein	Chls 692	40	Hypothetical protein
Chls 235	41	Hypothetical protein	Chls 727	44	Hypothetical protein
Chls 236	58	Hypothetical protein	Chls 729	37	Hypothetical protein
Chls 237	48	Hypothetical protein	Chls 753	47	Hypothetical protein
Chls 248	30	Hypothetical protein	Chls 785	66	Hypothetical protein
Chls 269	50	Hypothetical protein	Chls 788	78	Hypothetical protein
Chls 285	41	Hypothetical protein	Chls 790	51	Hypothetical protein
Chls 289	342	Ribose-phosphate pyrophosphokinase	Chls 801	52	Hypothetical protein
Chls 290	44	Hypothetical protein	Chls 823	38	Hypothetical protein
Chls 300	39	Hypothetical protein	Chls 874	44	hypothetical protein

Light grey shading corresponds to the phage-origin tetracycline resistance island.

Dark grey shading corresponds to the plasticity zone.

Table 3.

C. suis ORFs shared with only C. trachomatis or C. muridarum.

Gene	AA length	Annotated function	Orthologa	AA similarity (%)	ORF coverage (%)
Chls 472	370	Hypothetical protein	CT161b	56.4	94.3
Chls 473	540	Hypothetical protein	CT163	67.3	99.8
Chls 479	96	Transcriptional repressor protein TrpR	CT169	81.9	98.9
Chls 481	392	Tryptophan synthase beta chain (EC 4.2.1.20)	CT170	90.8	99.7
Chls 482	250	Tryptophan synthase alpha chain (EC 4.2.1.20)	CT171	68.8	99.6
Chls 484	580	Hypothetical protein	CT161b	45.2	88.6
Chls 534	181	Hypothetical protein	CT222	52.9	93.2
Chls 535	355	Inclusion membrane protein	CT223	41.1	65.6
Chls 538	102	Inclusion membrane protein	CT224	44.8	70.7
Chls 539	93	Hypothetical protein	CT225	59.3	73.8
Chls 547	111	Inclusion membrane protein B	CT232	84.2	92.6
Chls 476	3362	Hypothetical protein	TC0438	74.1	99.9
Chls 477	3224	Hypothetical protein	TC0439	75.2	99.9
Chls 529	829	DNA helicase IV (EC 3.6.4.12)	TC0490	80.5	96.0
Chls 537	323	Inclusion membrane protein	TC0496	36.1	66.0
Chls 585	105	Hypothetical protein	TC0541	59.8	99.1
Chls 617	184	Inclusion membrane protein	TC0574	56.9	57.1

^aCT—C. trachomatis and TC—C. muridarum.

^bChls 472 and Chls 484 show similarity to CT161 supporting a potential gene duplication in C. suis R19.

C. suis protein-coding sequences with lower similarity (<75%) to orthologs in C. trachomatis and C. muridarum are largely proteins of unknown function and also include potential host-interacting components such as inclusion membrane proteins, polymorphic membrane proteins and the phosopholipase D-like genes. Albeit a minority of those with low sequence similarity, proteins in other families such as those related to metabolism, DNA repair and other families, also are included (Fig. 3B). In Fig. 3B, genes within the ‘other’ category include; the MAC/perforin, a putative eukaryotic transcriptional activator, competence protein ComEC (LaBrie et al. 2019), late transcription unit B and Tol-Pal cell envelope complex TolA protein.

Individual proteins encoded by C. suis were compared to orthologs in six other Chlamydia species (Table S2, Supporting Information). As expected, those proteins with the highest primary sequence similarity include components critical for replication, transcription, translation and metabolism. Included in these highly conserved sequences are many hypothetical proteins that may contribute to those shared Chlamydia- and bacteria-specific processes. In contrast, for the other six chlamydial species represented here, this ratio flips and the majority of conserved proteins fall below the 75% similarity range. These results, particularly the subset of lowly conserved orthologs, highlight possible host-specificity candidates within C. suis: the hypothetical proteins, plasticity zone components and membrane proteins.

The C. suis plasticity zone

The chlamydial plasticity zone (PZ) is a variable region present in all species of the genus that is hypothesized to contain genes responsible for host-specific virulence factors. The PZ of C. muridarum contains a series of phospholipases (PLDs), three genes often annotated as adherence factors that have cytotoxic domains similar to those in Clostridium spp, as well as a purine interconversion operon (Read et al. 2000). In contrast, the significantly reduced PZ of C. trachomatis contains several of the phospholipases, but only contains a truncated version of one of the cytotoxin-resembling genes in specific serovars and has replaced the purine interconversion operon with a tryptophan biosynthesis pathway (Fig. 4A; Stephens et al. 1998). Other Chlamydia contain permutations of these plasticity zones where some contain no phospholipases, variable cytotoxin numbers with two present in C. pecorum and one ortholog in C. caviae, C. felis and C. psittaci. The presence or absence of the tryptophan operon and close investigation of the PZ has provided insight into the immunological interactions between host and bacteria (Sait et al. 2014; Jelocnik et al. 2015; Rajaram et al. 2015).

Figure 4.

(A) Gene organization for the plasticity zones of composite C. suis, C. muridarum and composite C. trachomatis strains. Mac/perforin annotated genes (orange) were used as the left margin for the PZ while the oppA2 genes (pink) was used as a right margin. Grey arrows represent hypothetical proteins which have no known function. Genes with dashed outlines are variable within the species. Key differences between these PZs include the presence and number of the cytotoxins or adherence factors (blue), the tryptophan biosynthesis operon (green) and phospholipase D-like genes (dark or light red depending on clade in B). (B) Phylogenetic cladogram comparing amino acid sequences for phospholipase D-like genes from eleven species of Chlamydia. Total alignment was 1000 aa in length. Marked in gray are the genes considered to be the ancestral chromosomal phospholipases. The PZ phospholipases from C. suis (red), C. muridarum (green) and C. trachomatis (blue) are indicated as well.

The C. suis PZ contains two putative cytotoxins, both of which are similar to two of the C. muridarum cytotoxin-like genes as also determined by Manuela et al. (2014) for C. suis MD56 (Fig. 4A). To investigate the presence of these cytotoxins in other strains of C. suis, full or partial genomes were analyzed for 29 deposited SRA samples using the C. suis R19 genome as a reference (Seth-Smith et al. 2017). Interestingly, when these strains of C. suis were examined, seven were discovered to contain only one of the adherence factors, indicating that both copies are likely non-essential for the in vivo success of the organism, as also noted by Seth-Smith et al. (2017). It is unclear as of yet, whether the two cytotoxins have distinct biological functions in vivo or whether they play a role in host or biovar selectivity. Also found in the C. suis PZ is an intact tryptophan biosynthesis pathway, homologous to the one in C. trachomatis genital strains.

As is seen in C. muridarum and C. trachomatis, the PZ of C. suis contains a putative operon encoding phospholipases (PLDs). Importantly, these proteins have some of the most lowly conserved amino acid similarities. The operon in C. suis, however, seems to contain an increased number of PLDs relative to both C. trachomatis and C. muridarum (Fig. 4A). Phylogenetic analysis of the repertoire of PLD enzymes, as identified through their HxKx₄Dx₆G domains, revealed a distinct clade of non-PZ PLD which mirror classic phylogenetic relationships for the Chlamydia (Fig. 4B). This indicates that this non-PZ PLD is likely truly ancestral and has evolved with the speciation of Chlamydia. The pzPLDs, however, are not so simply categorized. There is a great diversity in these sequences, with a subset of proteins more closely related the chromosomal PLD, perhaps representing duplication events. The clade more distantly related the chromosomal PLDs include the greatest number of pzPLDs, potentially pointing to a unique role for this subset of PLDs. The division between clades could suggest differential selection for these PLDs which would support a distinct biological function.

C. suis membrane proteins

One key protein differentiating members of Chlamydiaceae is the major outer membrane protein (Momp, ompA). This porin-like structure is integrated into the outer membrane of all Chlamydia species and has been shown to make up approximately 60% of the chlamydial outer membrane (Caldwell, Kromhout and Schachter 1981). While structural analyses of this protein suggest it to be a general porin, its conserved and highly present nature indicates that this gene is crucial to bacterial survival in the host. In 2016, a study was conducted which looked at variation in ompA among strains of C. muridarum collected from wild mice and found a 99% identity between ompA genes (Ramsey et al. 2009). Similarly, several strains of Chlamydia pecorum, Chlamydia abortus, Chlamydia pneumoniae and Chlamydia psittaci (Schofl et al. 2011), have been deposited and close examination of these strains shows little ompA diversity. In contrast, C. trachomatis ompA has increased variability between strains when compared with C. muridarum and has allowed classification into unique serovars or clades based solely on ompA-typing.

While it has been shown that genotyping by ompA does not indicate genomic relatedness (Suchland et al. 2017), these divisions do appear to correlate with specific pathobiovars. In C. trachomatis, ompA appears to be evolving at a faster rate than the rest of the genome, likely due to unique selective pressures, (Nunes et al. 2009) and is characterized by four variable domains (VD) which are used to distinguish serovars. These variable domains are generally regions of lower hydrophobicity and potentially indicate outward facing domains, leading to the hypothesis for the biovar-specificity attributed to this gene. The variability in ompA could confer an evolutionary advantage specific to the host organism and the specific tissues involved in infection.

A recent analysis by Chahota et al. (2017)investigated the potential for serotyping by Momp in C. suis using PCR analysis of VD2 and VD3. When the deposited contigs were investigated for completed genes for ompA, and compared to R19, similar results were found. C. suis Momp also contains four variable domains which directly correspond to those seen in C. trachomatis, though are divergent (Fig. 5). Phylogenetic analysis of the C. suis ompA divides the strains into distinct clades, corroborating the observation by Chahota et al. that C. suis strains could be serotyped and a larger study could be performed to investigate the potential for distinctions between the clades.

Figure 5.

(A) Alignment of C. suis Momp primary sequences with differences are marked in black. Regions of the greatest variation appear are labelled as variable domains (VD). Mean hydrophobicity is plotted in red and blue along the top. (B) Phylogenetic tree from (A), with the addition of select C. trachomatis Momp sequences (blue). R19 is indicated in red. Scale bar is in substitutions per site.

There are several other important gene families that show some variability between the Chlamydiales, including a family of inclusion membrane proteins (Incs), and polymorphic membrane proteins (Pmps). Inclusion membrane proteins, in particular, may be targets for host specificity, as the conserved Incs share disproportionately lower amino acid sequence similarity between species. Both families have been shown to be correlated with tissue tropism and therefore, may play a role in speciation (Almeida et al. 2012). The chlamydial inclusion is a modified vacuole in which chlamydia are able to grow and divide. Relatively little is known about the modifications made to this vacuole that allow for the bacteria's survival, but the Inc family of proteins are secreted into the inclusion membrane and are known to interact with the host cytosol. Inc proteins make up approximately 7–10% of the chlamydial proteome and have a high degree of diversity (Dehoux et al. 2011). Despite this diversity, most Incs are defined by a characteristic bilobed hydrophobicity motif. This domain is conserved among predicted Inc proteins and seems to be a Chlamydia-specific motif.

There are varying predictions for the numbers of Inc proteins across the genus with 23 conserved incs encoded in five examined species (Lutter, Martens and Hackstadt 2012). The C. suis R19 genome encodes each of these conserved incs as well as 34 non-conserved predicted Incs for a total of 57 predicted Incs. According to Lutter, Martens and Hackstadt (2012), C. trachomatis also encodes for 55 incs with 6 that differ from C. muridarum, which has 53 Incs. Analysis of the 57 putative inclusion membrane proteins in C. suis that were identified and aligned with C. trachomatis and C. muridarum revealed an additional 2 putative Incs for C. muridarum. A Venn diagram and phylogeny were constructed to display Incs shared between or specific to the three species and their evolutionary relationship (Fig. 6). Briefly, 51 Incs are conserved within the three species of the clade. One Inc is unique to C. suis (Chls_474) and one to C. muridarum (TC0011; Fig. 6C, panels 1 and 8). Several Incs are shared between species. Three Incs are found in C. trachomatis and C. suis only; CT222 (Chls_534), CT224 (Chls_538) and CT225 (Chls_539). These are in an operon of inclusion membrane protein genes and possibly function together as a complex. While CT222 and Chls534 are syntenous, phylogenetic analysis suggests that these may not be direct orthologs (Fig. 6C, panels 2 and 6). Additionally, two Incs are unique to C. muridarum and C. suis, TC0496 (Chls_537) and TC0573 (Chls_617). Interestingly, and unexpectedly, due to the placement of C. suis as an intermediate between C. trachomatis and C. muridarum, there appears to be one Inc that is absent in C. suis but found in the other two species (CT227/TC0498; Fig. 6C, panel 1). This Inc is closely related to the neighboring Inc CT226/TC0497 which is also present in C. suis. In general, the conservation of Incs between C. suis, C. muridarum and C. trachomatis suggests that the protein functions may be retained in all three species and that they may share cognate host interactions. Non-conserved Incs may indicate key differences in host interactions or the host environment in general. Functional analysis of host-binding or -interacting partners for each of these Incs, could reveal their specific roles in their host species.

Figure 6.

(A) Venn diagram displaying the shared or specific inclusion membrane proteins (Incs) between C. trachomatis D/UW-3 (blue), C. suis R19 (red) and C. muridarum Nigg (green). (B) Phylogenetic analysis of putative Inc protein sequences. (C) Highlighted in boxes 1–8 are the Incs that are not conserved between C. trachomatis (blue), C. muridarum (green) and C. suis (red). Tree is transformed with ordered branching.

Like Incs, polymorphic membrane proteins (Pmps) share in varying numbers across species, though the specific roles for each Pmp are unknown. Pmps are outer membrane proteins and are immunogenic for humans. These genes make up approximately 1–2% of the chlamydial gene content and each contain a C-terminal phenylalanine and as well as multiple GGAI motifs which are associated with host cell adhesion (Gomes et al. 2006). Nine putative pmps were annotated in C. suis R19 through a genome wide search for multiple GGAI motifs. Each of these correspond directly to a Pmp in C. trachomatis and C. muridarum. These Pmp primary sequences range from high identity between the three species to low identity: PmpA (79.8%), PmpI (78.8%), PmpH (77.0%), PmpB (74.9%), PmpG (74.0%), PmpE (73.9%), PmpD (72.9%), PmpF (65.2%) and PmpC (46.3%).A total of seven out of nine C. suis R19 Pmps are more similar to their C. muridarum orthologs while two (PmpH and PmpF) are more similar to C. trachomatis. Interestingly, Pmps have been shown to be involved in cellular tropism as six of the nine Pmps in C. trachomatis analyzed from different serovars were able to be phylogenetically clustered based on disease properties, suggesting a role for these membrane proteins in adhesion or differential biovar tropism (Gomes et al. 2006).

DISCUSSION

The C. suis R19 genome was assembled through whole genome sequencing and fully annotated using PATRIC. One key aspect of this study was the use of NIAID annotation service, PATRIC, for independent annotation. As indicated in Table 1, the PATRIC platform has annotated several genes not previously annotated by other methods in both C. trachomatis and C. muridarum. It is likely that these new annotations correspond with known open reading frames that were not originally included in the first depositions of these genomes. These could correspond with pseudogenes or untranscribed genes, as many are small < 300 bp open reading frames. However, in recent years, it has been shown that small open reading frames (smORFs), previously discarded in many genome annotation pipelines, do produce transcribed and translated proteins in both bacteria and eukaryotes (Couso and Patraquim 2017; Sharma et al. 2018). These smORFs are predicted to have functional roles in the stress response and nutrient sensing (Khitun, Ness and Slavoff 2019). While it would be important to investigate the transcription of these genes in C. suis, these studies reveal their presence and potential importance, emphasizing the need for continual evaluation of genomic content and locus tag numbering system.

Interestingly, individual Chlamydia can display diverse but limited ranges of mammalian hosts. Livestock pathogens like C. pecorum, which cause chlamydiosis in a variety of animals particularly ruminants and swine, and is notably the leading cause of infectious disease in koalas and C. abortus, which also infects ruminants and has been associated with spontaneous abortions in swine and sheep, are among the few able to infect and cause disease in a multiple ruminants species, while the majority of Chlamydia, including C. suis, C. trachomatis and C. muridarum appear to be largely restricted to a single host (Jelocnik et al. 2015; Borel, Polkinghorne and Pospichil 2018). Comparative genomic studies, like this report, will continue to identify molecular candidates potentially involved in restricting host range and enable direct genetic analyses to investigate the role of these host-specific factors.

As with a full dN/dS analysis, comparing of amino acid similarity in primary sequences, allows for a direct analysis of evolutionary relationships between species. In this study, approximately 10–20% of the C. suis genome appears to have increased evolutionary pressure, likely stemming from the differences in host species and ability to interact with cognate host factors. Further study of this subset of genes could lead to insights in chlamydia–host interactions and chlamydial tissue tropisms. Even in the more distantly related species, like C. pecorum which has the fewest highly conserved genes (over 90%) with C. suis, 88% of genes appear to be conserved to some degree.

Several distinct aspects of C. suis emerge, however, likely due to adaptations to the porcine host. These include the those in the plasticity zone: a tryptophan biosynthesis operon absent in C. muridarum, cytotoxins lost in C. trachomatis that show variable representation in C. suis and a unique subset of phospholipase D-like genes. Complete tryptophan biosynthesis operons are also found inC. pecorumandC. felis. Tryptophan levels are known to play a key role in inhibiting the pathogenesis of intracellular organisms, including Chlamydia. Human (Roshick et al. 2006) and pig (Meurens et al. 2012) innate immune responses are able to limit the available tryptophan through the indoleamine 2,3-dioxygenase (Bailey, Christoforidou and Lewis 2013) response pathway mediated by interferon gamma (IFN-γ). Briefly, IFN-γ, activated by chlamydial infection, will induce IDO to catalyze the breakdown of L-tryptophan into N-formylkynurenine effectively depleting available tryptophan available for the bacteria. Indole producers, like many members of the human vaginal microbiome, provide the necessary input for salvage by the trp operon. Importantly, ocular C. trachomatis do not encode the trp operon, potentially due to the absence of indole-producing microbes. Sherchand and Aiyar (2019) demonstrate that the trp operon can be deleterious to Chlamydia in the absence of indole-producers. It would follow that perhaps a strong negative selection on the trp operon due to a lack of indole-producers in C. muridarum-infected tissues would explain the absence of the trp operon in C. muridarum. There is no current evidence to support an IFN-γ-mediated IDO response in mice which further supports the negative selection of the trp operon in mouse-infecting Chlamydia. Overall, this pathway could provide a hypothesis as to why there are no incidences of C. muridarum infection in humans, but there have been reports of C. suis present in human samples while detection of active human infection or symptomology has not been reported.

Eukaryotic phospholipase D has roles in lipid metabolism and vesicle regulation and this family of proteins has been exploited by pathogens and used to increase virulence (Selvy et al. 2011). While the in vivo role of these genes in Chlamydia remains relatively unknown, a study done with C. trachomatis in HeLa cells provides evidence that the PZ phospholipases (pzPLDs) may be important for inclusion formation and lipid acquisition (Nelson et al. 2006). Other bacterial phospholipase D genes have been characterized, however, and could provide insight into their role in Chlamydia. A study in Neisseria gonorrhea showed that a phospholipase D homolog acts to increase adherence and invasion to cervical cells by stimulating complement receptor type 3-mediated endocytosis (Edwards, Entz and Apicella 2003). This effect was species specific, as other bacterial phospholipase D proteins were not able to rescue a knock-out mutant. In Yersinia pestis, a plasmid-encoded phospholipase D allows the pathogen to survive in its arthropod host further suggesting that these proteins may play a role in host-specificity (Hinnebusch et al. 2002). As noted, C. suis contains more PLD genes within its PZ than almost any other chlamydial species. In fact, several species including C. caviae, C. pneumoniae and many parachlamydiae are missing any PLD family genes in the plasticity zone but retain ancestral chromosomal PLD outside of the PZ (Nelson et al. 2006). The variability in number and sequence of this operon, and the putative roles in virulence and host specificity from other bacterial species suggests that these enzymes could be providing an essential function in the manipulation of the unique host cell allowing the species to infect and survive within its specific host.

Outside of the plasticity zone, the presence of variable regions in ompA suggest a similar role to that in C. trachomatis, given that C. suis is known to inhabit several distinct tissues within pigs including eyes, gastrointestinal tract, respiratory tract and reproductive tracts. Other membrane proteins, incs and pmps, have a similar repertoire as found in C. trachomatis and C. muridarum. Additional studies on the roles of the non-conserved genes may provide insight into the biological role they may play as well as to provide insight into the host environment. One limitation of this study is in the use of few or single representative genomes from each species. To fully investigate the roles for these key families of proteins, a global analysis of C. suis strains, as well as other clinical Chlamydia isolates, collected from individual tissues or geographic locations, may be necessary. As with the small-scale studies performed by Chahota et al and Seth-Smith et al on ompA and the tet island, respectively, a larger-scale study could reveal the dynamics between host and gene content (Chahota et al. 2017; Seth-Smith et al. 2017).

Based on the evidence for C. suis evolution with C. trachomatis and C. muridarum and the presence of several genes present in C. trachomatis and absent in C. muridarum, C. suis may provide a better model for human chlamydial infection than C. muridarum. In order to evaluate this, several questions would need to be answered. Primarily, does C. suis infect, ascend and cause pathology in a mouse? One study has provided some evidence that C. suis may result in a more robust infection in mice than C. trachomatis (Donati et al. 2015), but the absence of direct infection analyses and comparison with C. muridarum leaves some uncertainty related to this hypothesis. Comparative analysis of the C. suis genome opens the door for further studies into the complexities of the chlamydial genetic repertoire, proteome and host specificity adaptations.

METHODS

Whole genome sequencing and de novo assembly

Using the DNeasy Blood and Tissue Kit (Qiagen, Hilden Germany), DNA was isolated and purified from C. suis strain R19, provided by Dr Daniel D Rockey (Lenart, Andersen and Rockey 2001). Library generation was performed at the University of Kansas Genome Sequencing Core using the NEBNext Ultra II DNA Library kit. Libraries were run on the Illumina Miseq PE100. Over 90 million 151 bp reads were obtained. Reads were trimmed and quality filtered using BBDuk. Subsequent de novo assembly was performed in the Geneious (version 9.1.8) software suite (https://www.geneious.com) using Velvet v1.2.10 with the Velvet optimizer to determine an optimal Kmer (Kearse et al. 2012). When needed, to resolve larger gaps and to verify final assembly, PCR and Sanger sequencing was used. Predicted coverage to the assembled R19 genome was over 1000x. The origin of replication and the first nucleotide was assigned using C. trachomatis L2 434/Bu (NC_010 287) as a reference and genomes were adjusted accordingly.

Annotation

Annotation was performed using the RAST tool kit (RASTtk) through PATRIC web resources (Wattam et al. 2014; Davis et al. 2020) and using open reading frame prediction through Geneious. Functional assignments of Geneious-identified open reading frames were verified through BLAST analysis against protein sequences in the NCBI database. Subsystems were determined through PATRIC automatic annotation. Amino acid similarities were generated through PATRIC proteome comparisons with a minimum % coverage of 30%, a minimum similarity of 10% and an E-value below 1e-5.

Inclusion membrane proteins were identified by manually searching translated open reading frames for two hydrophobic domains. Putative Inc proteins were analyzed for closest ortholog in C. trachomatis D/UW-3 and C. muridarum Nigg using the phylogenetic analyses methods for single gene phylogenies as described below. In each case, individual Incs were translated and compared against all known incs from all three species. Pmps were identified by manually searching the genome for the GGAI motif and C-terminal phenylalanine. All pmps were compared with those found in C. trachomatis D/UW-3 and C. muridarum Nigg using the same methods as for the incs (data not shown).

Phylogenetic analyses

Comparison of genetic content was performed using the following reference genomes: C. suis SWA-2 (NZ_LT821323), C. suis SWA-14 (NZLT860207), C. suis SWA-86 (NZ_LT860209), C. suis 2–26b (NZ_LT999997), C. suis 3–25b (NZ_LT999998), C. trachomatis D/UW-3/CX, C. trachomatis L2 434/Bu, C. trachomatis A/HAR-13 (NC_007 429.1), C. trachomatis B/TZ1A828/OT (NC_012 687.1), C. trachomatis E/Bour (NC_020 971.1), C. trachomatis F/SW4 (NC_017 951.1), C. trachomatis G/SotonG1 (NC_020 941.1), C. trachomatis Ia/SotonIA1 (NC_020 970.1), C. trachomatis K/SotonK1 (NC_020 965.1), C. muridarum Nigg (NC_002 620), C. muridarum MopnTet14 (NZ_ACUJ01000001.3), C. muridarum Nigg3 (NZ_CP009760.1) C. muridarum Weiss (ACOW01000004.1), C. pneumoniae strain Wien 1 (NZ_LN846980), C. pecorum strain strain E58 (NC_015 408), C. psittaci GR9 (NC_018 620), C. abortus strain GIMC 2006:CabB577 (NZ_CP024084), C. caviae GPIC (NC_003 361), C. felis Fe/C-56 (NC_007 899) and C. avium 10DC88 (NZ_CP006571). Genomic alignments were performed in Geneious using MAFFT (v7.309) alignment tools and coding sequence alignments were performed with the PATRIC proteome comparison web tool which uses bidirectional BLASTP with the following parameters: minimum coverage of 30%, minimum identity of 10%, BLAST E-value of 1e-5.

Phylogenetic trees comparing single genes were constructed using the Geneious Tree Builder for a global alignment with free end gaps and a cost matrix set to Blosum65. Distances were obtained from pairwise alignments of all sequence pairs. To assemble the trees, a Jukes–Cantor genetic distance model was selected and UPGMA tree-building was performed. Nucleotide-based alignments and analyses were also performed in all cases and resulting phylogenies showed similar relationships to protein sequences (Data not shown). Whole genome alignments and subsequent phylogenies to determine genomic evolutionary relationships were performed using the progressiveMauve algorithm with a match seed weight of 15 and a minimum LCB score of 30 000. Gaps were aligned using Muscle 3.6 and a phylogenetic tree was determined using the neighbor-joining method with bootstrapping.

DECLARATIONS

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

AVAILABILITY OF DATA AND MATERIALS

The datasets generated and/or analyzed during the current study are available in the NCBI repository, www.ncbi.nlm.nih.gov under the accession numbers CP034310 and CP034311.

AUTHOR'S CONTRIBUTIONS

ZED assembled and annotated the genome, performed all subsequent comparative analyses and wrote the manuscript. PSH was a major contributor of analytical direction and in revising the manuscript. All authors read and approved the final manuscript.

FUNDING

ZED was supported by NIH AI126785. PSH was supported by NIH AI126785 and P20GM113117. Genome sequencing was supported by NIH P20GM103638. NIH or associated administrative personnel had no direct influence on study design, analysis, interpretation of the data, or writing this manuscript.

Conflicts of Interest

None declared.