Preliminary screening of putative homologs of CteG and of other C. trachomatis effectors within Chlamydiaceae

To reanalyse the distribution of putative CteG homologs across Chlamydiaceae, we started by performing a preliminary tBLASTx analysis against a local genome database. The top hits were subsequently used in a reciprocal tBLASTx against C. trachomatis, aiming to further verify putative homology to cteG, as illustrated in Fig. 1A. This revealed the presence of 36 putative homologs of CteG (including C. trachomatis CteG) encoded within Chlamydiaceae genomes with some species presenting several copies (four in C. caviae, C. felis, and C. poikilothermis; three in C. pecorum; and two in C. buteonis, C. crocodili, C. ibidis, C. pneumoniae, C. psittaci, C. serpentis, and C. suis) and only two species (C. avium and C. gallinacea) showing no putative CteG homologs (Table S4). Using this strategy, putative homologs of CteG were not found in species from a sister lineage of the Chlamydiaceae (CC-IV; [24]) or in closely related species belonging to the Chlamydiales/Parachlamydiales orders (E. lausannensis, S. negevensis, Ca. P. naegleriophila), suggesting that the ancestral cteG gene appeared after the divergence of Chlamydiaceae from other Chlamydiae families and from sister lineage CC-IV.

Fig. 1

CteG is the C. trachomatis effector with the higher relative number of putative homologs within Chlamydiaceae. (A) Graphical summary of the reciprocal tBLASTx approach to identify putative homologs of CteG within Chlamydiaceae. A similar procedure was used to identify putative homologs of non-Inc effectors and Incs. (B) The number of putative homologs found for each protein and in each Chlamydia or Chlamydiifrater species is coloured as indicated in the legend above the graph and depicted relative to the number of paralogs in C. trachomatis. The length of the coloured rectangles is proportional to the number of putative homologs in each species (e.g., only one homolog in CT222). See Table S4 for details

As previously described [17], amino acid similarity along the full-length of C. trachomatis CteG and its putative homologs was only observed in C. muridarum and C. suis (Fig. S1). All the other putative homologs of CteG only show sequence similarity to specific regions of CteG, ranging from amino acid residue 143 to the C-termini of CteG (Fig. S1). Furthermore, although C. buteonis HBN95_03995, C. caviae CCA_00297 and CCA_00298, C. crocodili H9Q19_03970, C. felis CF_0705 and CF_0706, C. poikilothermis C834K_0321 and C834K_0322, C. serpentis C10C_1043 were identified in the reciprocal BLAST analysis as putative homologs of CteG, they show a limited range of amino acid similarity (20 to 151 residues) and mostly display low sequence similarity (20–27% of identity; e-values from 0.013 to 3e− 6) to CteG (Fig. S1).

A procedure similar to what is illustrated in Fig. 1A was used to search for putative homologs within Chlamydiaceae of 54 additional C. trachomatis effectors, including 35 Incs experimentally shown to localize at the inclusion membrane (reviewed in [8] and references therein). Among the chlamydial effectors, most of the DUF582 proteins (CT619, CT620, CT621, CT711, CT712) [15] and the deubiquitinases Cdu1 and Cdu2 [42] led to the detection of different numbers of putative paralogs within C. trachomatis (Table S4). Therefore, for a visualization of how putative homologs of CteG and other C. trachomatis effectors are spread across Chlamydiaceae, the total number of putative homologs found in each case were plotted relative to the number of corresponding putative paralogs identified in C. trachomatis (Fig. 1B). Among the C. trachomatis effectors analysed, CteG displayed the higher relative number of putative homologs within Chlamydiaceae (Fig. 1B). By comparison to C. trachomatis, this is because of the existence of multiple CteG putative homologs in several Chlamydiaceae species, which is not seen to such an extent for any other C. trachomatis effector (Fig. 1B). This was intriguing and prompted us to analyse in further detail the evolutionary history of CteG, and if some of the putative CteG homologs are also T3S substrates delivered into host cells during infection.

Evolution of CteG in Chlamydiaceae

To deepen the study of the evolution of cteG among Chlamydiaceae species and closest relatives, and recover the highest number of homologs possible, we next inferred a species tree of the Chlamydiaceae (Fig. 2) and searched for all putative CteG homologs in a local proteome database, lowering the stringency of the BLASTp search (e-value < 0.1). The species tree was constructed using a phylogenomic approach, consisting of an alignment of 214 concatenated proteins obtained from Orthofinder (Fig. 2). The phylogenetic relationships among the Chlamydiaceae members is either consistent [43] or slightly differs from that described in previous studies [3, 24, 44]. Such variations could be attributed to the inclusion of more chlamydial species or to the use of different strategies for phylogenetic inference.

Fig. 2

Phylogenomic tree of the Chlamydiaceae and closest related species of the Chlamydiae Clade IV (CC-IV) clade. The phylogeny was constructed with 214 single copy orthologs and rooted with S. negevensis, E. lausannensis and Ca. P. naegleriophila. Only bootstrap values below 100 are shown next to the respective nodes. Presence and absence of CteG homologs is illustrated as indicated next to each species name according to the local BLASTp analyses using the respective whole proteomes (see Fig. 3). Blue represents CteG belonging to clade I, red CteG belonging to clade II, and white means that CteG was absent, as also shown in Fig. 3. Original tree files and alignments can be found in Figshare: https://figshare.com/s/b5ee1bbacc3d9ec347fb

We next inferred the phylogenetic tree of CteG using all putative homologs recovered by the local BLASTp search (total of 63 sequences, including C. trachomatis CteG; Fig. 3 and Table S5). The phylogeny separated the CteG homologs in two main clades: CteG I (C. trachomatis CteG and 25 other proteins) and CteG II (37 proteins). Clade CteG I includes CteG from C. trachomatis and putative orthologs from closely related species, grouping essentially according to the species phylogeny (Figs. 2 and 3). However, and in line with our initial screen for CteG homologs (Fig. 1), several paralogs could be identified. These include inparalogs (e.g., in C. pecorum) and outparalogs (e.g., in C. cavie, C. crocodili or C. poikilothermis). This suggests that several cteG duplication events occurred at different time points during the evolution of Chlamydiaceae. Clade CteG II includes more distantly related homologs from Chlamydiaceae species, but not from C. trachomatis and closest relatives C. suis and C. muridarum. Interestingly, this clade also includes CteG homologs from distantly related Chlamydiifrater species, which are seemingly absent from CteG I clade. As in CteG I, in CteG II, the global relationships between species/clades follow those from the species tree (Figs. 2 and 3). However, inparalogs (C. volucris) and several outparalogs (e.g., in C. abortus, C. caviae, or C. poikilothermis) could also be inferred (Fig. 3).

Fig. 3

Evolutionary history of CteG across Chlamydiaceae and closest relatives. CteG phylogeny comprising all top CteG BLASTp hits (e-value < 0.1). The two sequences used as queries for the BLASTp searches are highlighted in bold. Proteins selected for further analyses of protein secretion and delivery into host cells are highlighted by black filled squares. Phylogeny was midpoint rooted using iTOL v5 [71]. Only bootstrap values higher than 95 are shown as black dots next to the respective nodes. Phylogenies were visualized and rooted using iTOL [71]. Original tree files and alignments can be found in Figshare: https://figshare.com/s/b5ee1bbacc3d9ec347fb

There are differences in the number and identity of putative CteG homologs retrieved from the preliminary reciprocal tBLASTx approach (Fig. 1A and Table S4) and the homologs identified by the BLASTp and subsequent CteG phylogeny (Fig. 3 and Table S5). However, the expansion of cteG-related genes among Chlamydiaceae suggested by the initial screen revealed to be more significative with the more robust analysis of CteG phylogeny: 63 CteG homologs (including C. trachomatis CteG) instead of the 35 putative homologs initially found (Figs. 1 and 3). Only C. trachomatis and C. muridarum do not possess CteG paralogs encoded in their genomes. All other Chlamydiaceae species encode 2 (C. suis, C. corallus, C. avium, C. gallinacea, C. phoenicopteri), 3 (C. serpentis, C. sanzinia, C. abortus, C. ibidis, C. volucris), 4 (C. pneumoniae, C. pecorum, C. buteonis, C. psittaci), or 5 (C. crocodili, C. poikilotermis, C. caviae, C. felis) proteins evolutionarily related to C. trachomatis CteG. As cteG homologs show significant divergence among each other it is plausible that other putative cteG homologs were overlooked using our approach. However, we constructed an HMM profile for CteG using the 62 homologs (recovered by BLASTp), used a phmmer-based search [45] and did not find additional homologs. Overall, when considering the distribution (Fig. 2) and phylogenetic relationships (Fig. 3) of CteG proteins, it seems plausible that an ancient duplication in the most recent common ancestor (MRCA) of the Chlamydiaceae took place originating two distinct cteG ‘alleles’, followed by multiple gene duplication and differential loss events.

Synteny of cteG homologs

We next analysed gene organization in the vicinity of the cteG homologs. While the analysis was performed based on the genomic sequence of the C. trachomatis L2/434 strain, we used the nomenclature of the C. trachomatis serovar D UW3 strain (D/UW3). The genomic nucleotide sequences of the two strains are syntenic and 99.55% identical [46], and as strain D/UW3 corresponds to the first sequenced chlamydial genome [11] its gene nomenclature is often used as reference. The analysis revealed the existence of homologs that are syntenic to cteG (ct105 in C. trachomatis D/UW3) and that almost all localize in a locus encompassing C. trachomatis ct102 to ct107 homologous genes (Fig. 4). They all correspond to the CteG I clade in the phylogenetic tree of CteG, suggesting they are all cteG orthologs (Fig. 3). In addition, the genes encoding the CteG homologs corresponding to the CteG II clade in the phylogenetic tree of CteG (Fig. 3) are non-syntenic to C. trachomatis cteG (Fig. S2). However, they are all syntenic with each other occupying a locus flanked by homologs of C. trachomatis ct009-ct008-ct007 on one side (except C. corallus, C. pneumoniae, and C. serpentis where only homologs of ct009-ct008 were found), and tRNA-Ser and ct356-ct355-ct354 (most Chlamydia species) or tRNA-Ser and ct003-ct002-ct001 (C. serpentis, C. corallus, and C. pneumoniae) on the other side (Fig. S2). In the Chlamydiifrater species, the locus containing the CteG homologs also includes several pmp (polymorphic membrane protein) genes [47] and homologs of ct009, ct008, and ct007 with altered localization relative to C. trachomatis (Fig. S2). This locus is flanked by homologs of ct356-ct355-ct354 on one side and of ct004-ct003-ct002-ct001 on the other side (Fig. S2), thus showing further rearrangements relative to the syntenic locus in most Chlamydia species.

Fig. 4

Genomic region of cteG and of each of its syntenic homologs in other Chlamydia and Chlamydiifrater species. cteG homologs identified by the analysis of CteG phylogeny depicted in Fig. 2 are coloured in red. Our preliminary search for putative CteG homologs (Fig. 1 and Table S4) retrieved a C. ibidis protein (H359_0450) that was not retrieved by the CteG phylogeny. The corresponding gene is indicated coloured in red and white stripes. Other genes are coloured as indicated within the figure. Genes for which no putative homologs were found are coloured in white. C. abortus cab377 was not identified as a putative homolog of cteG and is annotated in NCBI databases as a fragmented pseudogene. Genomic regions are depicted according to the species tree in Fig. 2A. The nomenclature in the legend for each group of putative homologs is from C. trachomatis D/UW3 strain [11]

In summary, the synteny of cteG homologs (from the CteG I and CteG II clades) within Chlamydiaceae further supports that they are evolutionarily related.

Most CteG homologs within Chlamydiaceae are T3S substrates

To analyse if the CteG homologs identified within Chlamydiaceae are also T3S substrates, we selected both CteG I and CteG II proteins and tested if they can be secreted as full-length proteins by Yersinia enterocolitica. Bacteria such as Shigella flexneri or Yersinia spp. with well-characterized T3S systems were used as heterologous systems to show that Chlamydia encodes T3S substrates [10, 12], and have been used to screen for putative chlamydial effectors [35, 48, 49] and to test if specific chlamydial proteins are T3S substrates [41, 50,51,52,53].

The CteG homologs analysed were from C. muridarum (AAF39239.1/TC_0381), C. suis (ESN89684.1/Q499_0113, ESN89662.1/Q499_0114), C. pneumoniae (AAD18548.1/Cpn_0404, AAD18549.1/Cpn_0405), C. abortus (CAH63829.1/CAB376), C. pecorum (AEB41680.1/G5S_0729, AEB41682.1/G5S_0731, AEB41684.1/G5S_0733) and C. caviae (AAP05046.1/CCA_00297, AAP05047.1/CCA_00298, AAP05136.1/CCA_00389, AAP05137.1/CCA_00390). This includes CteG I proteins (TC_0381, Q499_0113, which are closely related to CteG; and Q499_0114, Cpn_0404, Cpn0405, CAB376, G5S_0729, G5S_0731, G5S_0733, CCA_00389, CCA_00390, which by comparison to TC_0381 and Q499_0113 show less similarity to CteG) and CteG II proteins (CCA_00297, CCA_00298) (Fig. 3 and Fig. S1).

We first generated plasmids enabling gene expression from the Yersinia yopE T3S effector gene promoter of CteG homologs with a C-terminal haemagglutinin (HA) epitope tag. While generating the plasmids encoding C. suis Q499_0114-HA and C. pecorum G5S_0729-HA, we noted by DNA sequencing that the genes were truncated relative to what is annotated in the NCBI database. For C. suis Q499_0114 we generated two plasmids encoding two putative proteins that we named Q499_0114A (residues 1 to 283 of the annotated Q499_0114 sequence) and Q499_0114B (residues 314 to 529 of the annotated Q499_0114 sequence) while for C. pecorum G5S_0729 we only analysed the truncated protein (residues 1 to 120 and 274 to 337 of the annotated sequence).

The plasmids encoding HA-tagged CteG homologs were then separately introduced into T3S-proficient Y. enterocolitica (ΔHOPEMT), which lacks most Yersinia effectors [32, 33, 35]. As positive and negative controls in the secretion assays, we used Y. enterocolitica strains encoding CteG-HA and a HA-tagged chlamydial ribosomal protein (RplJ-HA), respectively. In general, the proteins migrated according to their predicted molecular mass (Fig. 5A and B). Many CteG homologs revealed multiple bands besides the band corresponding to the predicted molecular mass (Fig. 5A and B), a feature of unknown relevance that we consistently found in our studies involving CteG [17, 22, 35]. This revealed that 11 out of the 14 (∼ 80%) CteG homologs tested were secreted except for C. suis Q499_0114B and C. pecorum G5S_0731, which were not secreted, and for C. pecorum G5S_0733, where the results were unclear (Fig. 5A and B; summarized in Table 1). Then, the plasmids encoding the HA-tagged CteG homologs that were secreted (including the plasmid encoding G5S_0733) were individually introduced into T3S-deficient Y. enterocolitica ΔHOPEMT ΔYscU. Assays with the resulting strains confirmed that secretion of these CteG homologs was T3S-dependent (Fig. S3). In summary, these data suggest that, overall, the CteG homologs within Chlamydiaceae are also T3S substrates, even those more distantly related such as C. caviae CCA_00297 and CCA_00298.

Fig. 5

Analysis of Type III secretion (T3S) of CteG homologs within Chlamydiaceae using Y. enterocolitica as heterologous host. (A) Type III secretion (T3S)-proficient Y. enterocolitica ΔHOPEMT was used to analyse secretion of CteG homologs within Chlamydiaceae with a C-terminal HA epitope tag. Immunoblots show the result of T3S assays in which proteins in culture supernatants (S, secreted proteins) and in bacterial pellets (P, non-secreted proteins) from ∼ 5 × 108 and ∼ 5 × 107 bacteria, respectively, were loaded per lane. CteG, a known C. trachomatis T3S substrate [35], was used as positive control, and the C. trachomatis ribosomal protein RplJ was used as a negative control [35]. The bands corresponding to the predicted molecular mass of the proteins analysed are indicated with a white asterisk (RplJ-HA, ∼ 19 kDa; CteG-HA, ∼ 68 kDa; Cpn_0404-HA, ∼ 37 kDa; Cpn_0405-HA, ∼ 26 kDa; TC_0381-HA, ∼ 71 kDa; CAB376-HA, ∼ 80 kDa; CCA_00389-HA, ∼ 79 kDa; CCA_00390-HA, ∼ 94 kDa; CCA_00297-HA, ∼ 49 kDa; CCA_00298-HA, ∼ 60 kDa; G5S_0729-HA, ∼ 19 kDa; G5S_0733-HA, ∼ 63 kDa; G5S_0731-HA, ∼ 38 kDa; Q499_0113-HA, ∼ 68 kDa; Q499_0114A-HA, ∼ 29 kDa; Q499_0114A-HA, ∼ 32 kDa). SycO is a strictly cytosolic Yersinia T3S chaperone [72] and its immunodetection ensured that the presence of HA-tagged proteins in the culture supernatants was not a result of bacterial lysis or contamination. (B) The percentage (%) of secretion of each protein by Y. enterocolitica ΔHOPEMT was calculated by densitometry of the bands in the immunoblots, as the ratio between the amount of secreted and total protein. Data are the mean ± standard error of the mean from at least 3 independent experiments

Table 1 Summary of the data on type III secretion and delivery into host cells of CteG homologs within Chlamydiaceae analysed in this studyMany CteG homologs can be delivered into host cells by C. trachomatis

Next, we sought to test if the CteG homologs that are type III secreted by Y. enterocolitica, including G5S_0733, could be delivered by C. trachomatis into infected host cells. To avoid possible toxic effects in C. trachomatis, we initially generated plasmids where the expression of the genes encoding the CteG homologs was under control of the tetracycline-inducible tetA promoter. In all cases, the constructs were designed for the CteG homologs to have a C-terminal double (2HA) epitope tag. After individual introduction of the plasmids into CteG-deficient C. trachomatis (cteG::aadA), we successfully obtained transformants in eleven of the twelve cases. The exception was for the strain that should encode CCA_00390-2HA, and this protein was not further analysed.

To confirm that the generated C. trachomatis strains were producing the desired proteins, HeLa cells were individually infected for 24 and 46 h with each of the eleven strains, and with a derivative of the cteG::aadA mutant harbouring a plasmid encoding CteG-2HA, also expressed from the tetA promoter [CteG-2HA(PtetA)]. Immunoblotting of whole cell extracts confirmed the production of Q499_0113-2HA, Cpn0404-2HA, Cpn0405-2HA, CAB376-2HA, G5S0729-2HA, G5S0733-2HA, CCA_00297-2HA, CCA_00298-2HA, and CCA_00389-2HA (Fig. S4). However, we could not detect production of TC_0381-2HA and Q499_0114A-2HA. Alternatively, we generated strains derived from the cteG::aadA mutant harbouring plasmids encoding these two proteins expressed from the cteG promoter. Immunoblotting from whole cell extracts of HeLa cells infected for 24 and 46 h with these two strains, and with a derivative of the cteG::aadA mutant harbouring a plasmid encoding CteG-2HA expressed from its own promoter [CteG-2HA(PcteG)], confirmed the production of both TC_0381-2HA and Q499_0114A-2HA (Fig. S5). As previously observed in Fig. 5A and B and in previous studies of C. trachomatis CteG [17, 22, 35], the detection by immunoblotting of the production of many of its homologs in Chlamydiaceae revealed multiple bands besides the band corresponding to the predicted molecular mass (Figs. S4 and S5).

To analyse if the various proteins were delivered by C. trachomatis into host cells, HeLa cells were infected for 24 and 46 h with each of the newly generated strains encoding homologs of CteG and also with the strains producing CteG-2HA(PtetA) and CteG-2HA(PcteG). We then immunolabelled the infected cells with antibodies against HA and Cap1 [a C. trachomatis effector known to localize at the inclusion membrane; [54]], followed by fluorophore-conjugated secondary antibodies and DAPI (to stain the DNA in the host cell nucleus and within chlamydiae). Analysis by fluorescence microscopy revealed that among the 11 proteins tested, ∼ 65%/7 (C. muridarum TC_0381, C. suis Q499_0113, C. abortus CAB376, C. pecorum G5S_0733, and C. caviae CCA_00297, CCA_00298, and CCA_00389) were delivered by C. trachomatis into host cells, while for ∼ 35%/4 (C. suis Q499_0114A, C. pneumoniae Cpn_0404 and Cpn_0405, and C. pecorum G5S_0729) their delivery into host cells by C. trachomatis was not observed (Fig. 6 and Fig. S6; summarized in Table 1). In a few cases (C. abortus CAB376, C. pecorum G5S_0733, and C. caviae CCA_00389), the proteins were only found to be delivered into host cells at 46 h p.i., but not at 24 h p.i. (Fig. 6 and Fig. S6; summarized in Table 1). The C. trachomatis strain producing C. caviae CCA_00298 showed a clear growth defect and at 46 h p.i. almost only small inclusions were observed (Fig. 6).

Fig. 6

Delivery into host cells by C. trachomatis of CteG homologs in Chlamydiaceae. HeLa cells were infected for 46 h with C. trachomatis cteG::aadA harboring plasmids encoding CteG or CteG homologs within Chlamydiaceae (Q499_0113 and Q499_0114A, from C. suis; Cpn0404 and Cpn0405, from C. pneumoniae; CAB376 from C. abortus; CCA00389, CCA00297 and CCA00298, from C. caviae; G5S_0733 and G5S_0729, from C. pecorum; TC_0381, from C. muridarum) with a 2HA C-terminal epitope tag. The gene encoding CteG was expressed from its own promoter (PcteG) or from the tetA promoter (PtetA)) and the genes encoding its homologs within Chlamydiaceae were mostly expressed from PtetA, except for the genes encoding TC_0381 and Q499_0114A that were expressed from PcteG. Infected cells were fixed with 4% (w/v) paraformaldehyde and immunolabelled with antibodies against HA (red) and the inclusion membrane-localized protein Cap1 (green) and appropriate fluorophore-conjugated secondary antibodies. The host and chlamydial were also stained with DAPI (blue). The immunolabeled and stained cells were analysed by fluorescence microscopy. Scale bars, 5 μm

To compare the subcellular localization of the CteG homologs that were delivered into HeLa cells by C. trachomatis to the known localization of CteG at the Golgi (at about 24 h p.i.) and at the plasma membrane (at late infection times such as 46 h p.i.), HeLa cells infected for 24 and 46 h were immunolabelled with antibodies against HA, C. trachomatis MOMP, and the cis-Golgi protein GM130, followed by fluorophore-conjugated secondary antibodies. Analysis by fluorescence microscopy of cells infected for 24 h showed that CteG homologs that were delivered by C. trachomatis into host cells also localized at the Golgi region (C. muridarum TC_0381, C. suis Q499_0113, C. caviae CCA_00297, and C. caviae CCA_00298; Fig.