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Lateral Gene Transfer Between Protozoa-Related Giant Viruses of Family Mimiviridae and Chlamydiae

Obligate intracellular chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. While pathogenic chlamydiae have adapted to a wide range of vertebrates, environmental chlamydiae inhabit unicellular amoebae, the free-living Acanthamoeba. However, how and why this divergence occurred remains unclear. Meanwhile, giant viruses consisting of protozoa-related and protozoa-unrelated viruses have been discovered, with the former group being suggested to have more influenced environmental chlamydiae during their evolution while cohabiting host amoebae. Against this background, we attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were significantly shared in both the chlamydia genomes depending on the giant virus type. In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high. Meanwhile, the prevalence of protozoa-related giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae; the actual number of sequences similar to giant virus was also significantly predominant compared with those in the environmental chlamydial genomes. Among them, the most prevalent of giant virus was in the case of chlamydiae with Megavirus chiliensis; total of 1338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) showed the presence of orthologs between these with several clustered. In addition, Pearson’s single regression analysis revealed that almost the prevalence of the genes from the giant viruses in chlamydial genomes was negatively and specifically correlated with the number of chlamydial open reading frames (ORFs). Thus, these results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of a putative linkage between chlamydiae and the giant viruses, providing us with a hint to understand chlamydial evolution.

viruses include the families Mimiviridae (eg, Mimivirus), Marseilleviridae, Pandoraviridae, Pithovirus, and Mollivirus, all of which can infect amoebae; they are ubiquitous in the environment, including in soil and the water supply. 16 The protozoan-unrelated giant viruses include the families Ascovirus, Irdovirus, and Poxvirus, all of which were isolated from vertebrates and invertebrates. 16 Meanwhile, some giant viruses have also been isolated from patients suffering from pneumonia, indicating their potential pathogenicity to humans, although this remains to be confirmed. 15,17,18 As mentioned above, similar to environmental chlamydiae, protozoa-related giant viruses also need to infect amoebae in order to replicate, indicating that the giant viruses could encounter environmental chlamydiae during the course of their life span; this would also have been the case for ancestral chlamydiae.
In the present study, we thus attempted to visualize genes of giant viruses in chlamydial genomes by bioinformatic analysis mainly with comparative genome and phylogenic analysis, seeking genes present in chlamydiae that are specifically shared with protozoa-related giant viruses. For the first time, we show a linkage between chlamydiae and protozoan-related giant viruses.

Analysis flow
The genome (or contig) information was obtained from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/genome/browse/), and the obtained sequence information was reconstructed as data sets with functional annotations into Rapid Annotation using Subsystem Technology (RAST) (http://rast.nmpdr.org/), which is an open-access genomic analysis tool that acts as a fully automated service for genomic annotation with Basic Local Alignment Search Tool (BLAST) analysis. 19 These reconstructed RAST data sets with annotated amino acid sequences are shown into Tables S1 to S4 (Table S1, protozoan-related  giant viruses; Table S2, protozoan-unrelated giant viruses; Table  S3, pathogenic chlamydiae; Table S4a and b, environmental chlamydiae). Then, BLAST analysis was performed using the RAST data sets with the default settings (cut-off 10 −10 identity >10%), and these sequences were furthermore selected with bidirectional hits and length cut-off (>30 amino acid residues). Numbers of orthologs were normalized with genome sizes of both chlamydiae and viruses. Specifically, the normalized numbers were obtained from raw numbers divided with each of the chlamydia and virus genome sizes; it is shown as ortholog numbers of giant virus assumed with 1 Mbp of genome size per 1 Mbp of chlamydial genome. Also, the cut-off value (>1.48%) as a background was defined by the prevalence of genes from Mimiviridae (Cafeteria roenbergensis virus, Megavirus (lba and chiliensis), Moumouvirus, Mimivirus) in the genome of Escherichia coli K12, which has never adapted to protozoa (mean ± 2SD: 1.28 ± 0.2%) ( Table S5). The identity of the extracted genes was finally determined by Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/), which is a domain research tool. 20 Functional annotation was also performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) 21 or the Universal Protein Resource (UniProt) (http://www.uniprot. org/). 22 Annotated functions were classified into "Metabolic process" (associated with the metabolism of proteins, lipid, DNA, RNA, and so on), "Regulation/modification" (associated with DNA/RNA repair, homologous recombination, chaperones and folding catalysts, protein-protein interaction, and so on), "Structure" (flagella, outer membrane protein, and so on), and "Others" (associated with structure and including those with an unknown function). Phylogenic analysis was performed with a maximum parsimony method by using MAFFT version 7 (https://mafft.cbrc.jp/alignment/software/). 23

Statistical analysis
Comparison of the prevalence of giant virus genes between pathogenic and environmental chlamydiae was performed by Mann-Whitney's U test. The presence of a correlation between the prevalence of genes from giant viruses within chlamydial genomes and annotated chlamydial ORF numbers was determined by Pearson's single regression analysis. A correlation coefficient value of >0.5 or <−0.05 with a P-value of less than .05 was considered significant. Calculations were performed in Excel for Mac (2011) with Statcel3C.

Results and Discussion
Several genes of protozoa-related giant viruses of the family Mimiviridae are significantly conserved in the genomes of both chlamydiae To explore the traces of an encounter with giant viruses in chlamydiae, the prevalence of genes from giant viruses in chlamydiae was assessed by BLAST analysis using RAST with genomic information from multiple species from each group (environmental chlamydiae n = 14, pathogenic chlamydiae n = 12, protozoan-related giant viruses n = 15, protozoan-unrelated giant viruses n = 11)(see Table 1). To ensure a uniform annotation of all the genes, pre-existing annotations in the database were re-annotated by RAST (http://rast.nmpdr.org/), which is an open-access genomic analysis tool that acts as a fully automated service for genomic annotation with BLAST analysis. Also, the cut-off value (>1.48%) as a background was defined by the prevalence of genes from Mimiviridae in the genome of Escherichia coli K12, which has never adapted to protozoa (see Table S5). As a result, in contrast to protozoa-unrelated giant viruses, the genes of protozoa-related giant viruses were Evolutionary Bioinformatics significantly shared in both the chlamydia genomes depending on the giant virus type (Figure 1). In particular, the prevalence of Mimiviridae genes among the protozoa-related giant virus genes in chlamydial genomes was significantly high, exceeded the cut-off value ( Figure 1A, green circles into Mimiviridae). Meanwhile, the prevalence of the genes of giant viruses within chlamydial genomes varied from 0.2% to 3.5% depending on not only the giant virus type but also the chlamydial strain   Table S5).

7
( Figures S1 and S2). Furthermore, the prevalence of protozoarelated giant virus genes in pathogenic chlamydia genomes was consistently higher than those of environmental chlamydiae ( Figure 1A and B), corresponding to number of sequences normalized with genome sizes of both chlamydiae and viruses similar to giant virus that was significantly predominant as compared with those in the environmental chlamydial genomes ( Figure S3). In addition, phylogenic analysis with most prevalent sets (Megavirus chiliensis and Protochlamydia EI2 or Chlamydia trachomatis L2 434Bu) clearly showed that there was several clusters, indicating the presence of orthologs between the chlamydiae and the giant viruses ( Figure 2). As expected, we found that, in contrast to the protozoaunrelated viruses, several genes of protozoa-related giant viruses, the family Mimiviridae (Megavirus chiliensis was most prevalent) were significantly conserved in the genomes of both the chlamydiae. Meanwhile, as compared with those of pathogenic chlamydiae, the prevalence of Mimiviridae genes was found to more differ among the various genera of environmental chlamydiae, being particularly high in Neochlamydia (S13, TUM1, EPS4) and Protochlamydia (UWE25, R18) and contrastingly low in Parachlamydia (UV-7, KNic, OEW1, Bn 9 , Hall's coccus) (see Figure S1). It is possible that the selection and maintenance of the giant virus genes occurred preferentially in some environmental chlamydiae through the ongoing interaction, presumably into cohabiting amoebae. Thus, these findings indicated that, in contrast to the protozoa-unrelated viruses, several orthologs of protozoa-related giant viruses, in particular Mimiviridae, were more conserved in the genomes of either environmental or pathogenic chlamydiae, suggesting that chlamydiae and Mimiviridae did interact in the host cells that both cohabited.

The prevalence of genes from protozoa-related giant viruses in chlamydiae is negatively and specifically correlated with chlamydial ORF numbers
If the prevalence of genes from giant viruses in chlamydial genomes specifically revealed that chlamydiae had encountered protozoa-related giant viruses presumably in ancestral amoebae, this would also suggest that this encounter resulted in specific modifications of the chlamydial genome, such as changes of the ORF numbers. To assess this hypothesis, the correlation between the prevalence of giant virus genes in chlamydial genomes and the chlamydial ORF numbers was assessed by Pearson's single regression analysis. The results showed significant correlation coefficients of <−0.5 with a P-value <.05 for almost combinations of chlamydiae with giant viruses, indicating the prevalence of the genes from giant virus in chlamydial genomes could be a factor predicting the number of chlamydial open reading frames (ORFs) ( Table S6).
The prevalence of the genes from almost giant viruses in each of the chlamydiae was negatively and specifically correlated with the number of chlamydial ORFs. These results suggest that these giant viruses changed the chlamydial genome and influenced chlamydial evolution. Interestingly, studies have shown that Protochlamydia (UWE25 or R18) can induce cell death such as apoptosis in insect cells or human immortal HEp-2 cells, [24][25][26] while Neochlamydia (S13) was found to exhibit complete loss of its ability to perform secondary infection of amoebae. 27 These findings also suggest the presence of a sympatric lifestyle between the viruses and chlamydiae and that such selection and maintenance of the giant virus genes were required for the successful specific adaptation of chlamydiae to the host niche.
In contrast to the pathogenic chlamydiae, the environmental chlamydiae specifically possess genes conserved among the Mimiviridae (Megavirus chiliensis) Since information on the specific genetic material that was shared would be critical for understanding the forces driving the evolution and divergence of chlamydiae, we explored the specific genes of chlamydiae commonly shared with protozoa-related giant viruses, the Mimiviridae. Meanwhile, because of most prevalent, Megavirus chiliensis as a representative virus was used for this analysis. As shown in Figure 3 and Table S7, a total of 1,338 genes of the chlamydiae were found to be shared with the virus (444 genes specific to environmental chlamydiae, 892 genes shared between both chlamydiae, only two genes in the pathogenic chlamydiae). Although these genes were classified into the categories of "Metabolic process," "Regulation/modification," "Structure", and "Others", almost genes (approximately 60%) were assigned to "Metabolic process" regardless of pathogenic or environmental chlamyidae (Figure 3, pie charts in the center). Meanwhile, as well as some genes of "Metabolic process", the genes assigned to "Structure" (surface protein Sur1, phage tail fiber protein, outer membrane lipoprotein Blc, flagellar hooklength control protein FliK) was specifically seen into environmental chlamydiae ( Figure 3 and Table S3). Furthermore, few genes were multiply conserved in almost all of the environmental chlamydiae used in this study (Figure 3 and Table S3).
Thus, these findings indicated that, in contrast to the pathogenic chlamydiae, the environmental chlamydiae specifically possessed functional genes conserved among the Mimiviridae responsible for "Metabolic process" or "structure" presumably as a platform for survival into harsh natural environments and as a trace of ongoing interaction of the chlamydiae with giant viruses. It is possible that because of the presence of genes from Mimiviridae presumably with adverse effects, the loss of such genes in chlamydiae may have been a critical event required for adaptation to mammalian cells. Furthermore, a large number of protozoa-related giant virus genes shared between both chlamydiae. It appeared that the some giant virus genes were passed down through the generations and became fixed evenly 8

Evolutionary Bioinformatics
in both environmental and pathogenic chlamydiae, implying before dividing two chlamydial lineages, ancestral chlamydiae had encountered giant viruses. Interestingly, only two genes specific to the pathogenic chlamydiae were detected into Megavirus chiliensis. The results revealed that in contrast to environmental chlamydiae, ongoing interaction of pathogenic  Table S1 to S4b). Black circles show these chlamydial genes. Phylogenic trees were constructed with a maximum parsimony method by using MAFFT version 7 (https://mafft.cbrc.jp/alignment/ software/). 23  23 or the Universal Protein Resource (UniProt). 24 Upper panel: specific to environmental chlamydiae; Middle panel: shared between both chlamydiae; Lower panel: specific to pathogenic chlamydiae. Colors show distinct gene functions annotated by KEGG or UniProt. Pie charts in the center show the prevalence of genes classified into the categories of "Metabolic process," "Regulation/ modification," "Structure," and "Others." chlamydiae with giant viruses may be minimal, presumably prompting pathogenic chlamydial genome reduction. 28

Conclusions
Altogether, our study showed a putative linkage between chlamydiae and protozoa-related giant viruses, in particular Mimiviridae. These results indicated the trace of lateral gene transfer between protozoa-related giant viruses of family Mimiviridae and chlamydiae. This is the first demonstration of the linkage, providing us with a hint to understand chlamydial evolution via encounters with giant viruses in host niche.