Genomics of Rickettsiaceae: An Update

Abstract

Recent advancements in genomes sequencing of members of Rickettsiaceae family have led to set a new landmark in the study of these microorganisms. Genomic analyses of Rickettsia and Orientia reveal a history of genome reduction because of the interaction with intermediate and final hosts; the evidence shows that this is an ongoing process. The gene loss, the gain, and loss of plasmids in such an easy way, among other significant processes are the evidence of the evolutionary history of this bacterial group involving reductive processes. In particular, the integrative conjugative element called REIS, was necessary in the process of adaption to an intracellular lifestyle in eukaryotes. We present a genomic focusing on Rickettsia and Orientia species, due to the animal and human importance. In this analysis, the genomic evidence shows that genomes have been extensively shuffled; however, the existence of core genes has also been conserved.

1. Introduction

The Rickettsiales are an order within α-proteobacteria that comprises obligate intracellular endosymbionts of arthropods and mammals. Some authors have proposed three pathogenic genera of Rickettsiales: (1) Rickettsiaceae; (2) Bartonellaceae; and (3) Anaplasmataceae[1].

More recently, taxonomy of Rickettsiales has changed based on molecular systematics, phylogenomics, and bioinformatics studies. Today, four taxonomic families are recognized: Anaplasmataceae, Rickettsiaceae, Ca. Midichloriaceae, and Holosporaceae, with Rickettsiaceaebeing the most well-known group for they are human and animal pathogens [1, 2].

Rickettsiaceaefamily comprises a large and extremely diverse group of strictly intracellular Gram-negative rod-shaped, non-sporulating, coccoid, and small bacteria. Many of them are obligate intracellular parasites that can infect eukaryotic organisms, including animals and man, through arthropod bites and can cause from mild to severe and even fatal diseases such as epidemic typhus and Rocky Mountain Spotted Fever (RMSF) [1, 3]. This family comprises exclusively two genera: Rickettsiaand Orientia. Both genera contain many known and potential pathogens considered as causative agents of emerging and re-emerging human and animals diseases [1]. Genome size of bacteria of Rickettsiaceaeare typically small (0.8–2.3 Mbp) mainly due to reductive evolution [4].

These genomes contain split genes, gene remnants, and pseudogenes because of different steps of the genome degradation process. In Rickettsia, genomics has revealed extreme genome reduction and massive gene loss compared to less virulent or endosymbiotic species [5].

The Rickettsiaceaefamily has 42 species, 2 belonging to the genus Orientiathat have been sequenced, and 40 species of the genus Rickettsiaof which 37 genomes has been sequenced.

This wealth of information reveals a large field of study in comparative genomics to understand the evolution from a free-living to an intracellular or endosymbiotic lifestyle.

Adaptation to intracellular or endosymbiont lifestyle of the family Rickettsiaceaeis based on the genome degradation process as reducing genes. Additionally, to about 2135 [6] gene remnants and pseudogenes (1622), split genes, and horizontal transfer to other bacterial groups have also been observed. In fact, at least three events in Orientiareceived external genes and Rickettsiaspp. in six occasions [6].

The generation of de novogenes in 17 cases of Rickettsiaspecies has been reported, and at least two of them are functional [6]. Rickettsiaspp. contain gene families, selfish DNA, repeat palindromic elements, genes encoding eukaryotic-like motifs, large fraction of high conserved non-coding DNA, and large fraction of mobile genetic elements (MGEs), including plasmids [5].

The study of the dynamics of genomes evolution of the family Rickettsiaceaewith regard to new “omics” sciences are driven to understand this extraordinary bacterial group with importance for human and veterinary medicine.

2. Rickettsia

2.1. Rickettsiaevolution

Apparently, the clade Rickettsiadiverged from Claudobacter1650-2, 390 million years ago [7]. Then, the primarily lineages infecting arthropods emerged approximately 525–425 million years ago [5, 8]. The emerging of this group has been suggested approximately 150 million years ago after several transitions from a likely free-living ancestor of Rickettsiales to an intracellular life. Nowadays, Rickettsiahave been discovered in a different hosts as whiteflies, bruchid beetles, ladybird beetles, aphids, among others, which suggest that they are more common than expected [9–12].

The genus Rickettsiacomprises pathogenic bacteria causing RMSF, Mediterranean spotted fever, epidemic typhus, and murine typhus [13]. Traditionally, Rickettsiawas divided into spotted fever and typhus as major groups; however, based on molecular phylogenetic analyses now, it is classified into four groups: (1) ancestral, (2) typhus, (3) transitional, and (4) spotted fever (Figure 1). Based on whole genome sequence analysis transitional group was suggested, however, this grouping has generated some controversy based on genetic and genomic criteria and is not widely accepted [5, 14]. The controversy generated by these bacteria classification is because traditional classification methods used in bacteriology are hard to apply to Rickettsiaspp.

Figure 1.

Phylogenetic approximation obtained from amino acid sequences with the online program PATRIC, with the default pipeline (www.patricbrc.org/).

2.2. Genomic of Rickettsia

With the use of genome sequence techniques and the characterization of genomic sequences of microorganisms without the need of cultivation, the Rickettsiaceaediversity has been explored. Today, 79 genomic sequences of Rickettsiaand 11 Orientiastrains are known (Table 1).

Table 1.

Rickettsiaceaefamily genomes data reported on img.jgi.doe.gov.

The availability of complete genome sequences of different Rickettsiaspecies led to perform comparative genomic approaches in order to understand bacterial evolution and pathogenesis [5]. Genome reduction is a trait observed in Rickettsiaspecies, the gene loss has been an important and ongoing process in evolution of these bacteria. Some intragenus variations in size genome and gene content observed in Rickettsiaare the consequences of the large diversity of host and infection strategies that these bacteria have developed [5].

The Rickettsiagenomes exhibit a high degree of synteny punctuated by distinctive chromosome inversions, which goes diminishing as the phylogenetic relationship it is narrower (Figure 2).

Figure 2.

Comparison of pairwise syntenic dot plots of the nucleotide sequences: (A)Rickettsia rickettsiiArizona vs.R. akari; (B)R. rickettsiivs.R. canadensisCA410; (C)R. rickettsiivs.R. typhiWilmington; and (D)Orientia chutoFuller vs.O. tsutsugamushiSido.

In general, the genus Rickettsiamaintains GC content, rRNA, tRNA, and pseudogenes, with only some exceptions (Table 1). The aggregate characteristics (number, length, composition, and repeat identity) of tandem repeat sequences of Rickettsiawhich often exhibit recent and rapid divergence between closely related strains and species, are very conserved [15].

2.2.1. Plasmids in Rickettsia

The gene acquisition and gene loss are the major mechanism of adaptation interactions between bacteria and their host, in either the pathogenic or endosymbiotic lifestyle of Rickettsiales and other bacteria. To accomplish this process the preferential vehicle are the plasmids, that encompass very large genetic regions, even more than 100 kilobases (kb) including several set of genes. Their frequent integration at or near tRNA loci suggests that many of them were introduced into bacterial genomes via phage-mediated transfer events. In pathogenicity, they are called “pathogenic islands” and in endosymbionts “symbiotic islands.” Recently, the dogma that plasmids are not present in Rickettsiaceaewas refuted, with the pulsed-field gel electrophoresis (PFGE) and Southern blot analyses of DNAs in different species that suggests that they may be widespread in the genus. Plasmid existence in spite of pressure exerted by reductive genome evolution suggests an important role in rickettsial biology [18].

In the most relevant study of plasmids in Rickettsiawith 26 species, the authors found that 11 species had 1 to 4 plasmid(s) with a size ranging from 12 to 83 kb, and contained 15 to 85 genes. They elucidated that pRICO, the last common ancestor of the current rickettsial plasmids, was vertically inherited mainly from Rickettsia/Orientiachromosomes and diverged vertically into a single or multiple plasmid(s) in the species [3].

Out of 747 protein-coding genes, 65% were full-length genes and 35% were partially degraded. Degradation levels varied among plasmids, ranging from 16 to 40% in larger plasmids (size >47 kbp) and 44 to 59% in smaller plasmids [3].

It has been observed that plasmids are lost during long-term serial passage in cultured cells, which complicate studies of ancestry to elucidate a single or multiple ancestors. Nevertheless plasmids clustered into four putative groups (I–IV) (Figure 3): group I included four large and three small plasmids of five species: pRra2 in R. raoultii, pRhe in R. helvetica, pRfe, pRfeI1, and pdRfe in R. felis, pRam32 in Candidatus R. amblyomii, and pRau in R. rhipicephalii; group II clustered two large and four small plasmids belonging to five species: pReis1 and pReis2 in R.endosymbiont of Ixodes, pRaf in R. africae, pRam23 in C. R. amblyomii, pRmo in R. monacensis, and pRpe in R. peacokii; group III contain five small plasmids of four species: pRam18 in C. R. amblyomii, pRrh in R. rhipicephalii, pRra1 in R. raoultiiand pRma and pRmaB in R. massiliae; and group IV gathered one large and one small plasmids from two species: pReis3 in R.endosymbiont of Ixodes and pRra3 in R. raoultii. At inter-species level, plasmids of the same group showed variable sequence conservations [6].

Figure 3.

Evolutionary events that shaped rickettsial plasmids. Plasmid supertree obtained from 10 genes ofRickettsiaandOrientiaspecies. The genes used were hsp, dnaA, sca12, transposase 1 and 2, phospholipase, traD, leucine repeat, helix-turn-helix, and thymidylate kinase. Figure based on [3].

Rickettsiaplasmids are a mirror of the evolutionary history of this bacterial group involving reductive processes, duplication events, and horizontal acquisition of genes necessary to adapt to an intracellular lifestyle in eukaryotes. It is now necessary to determine their distribution, evolution, and their role in host adaptation and virulence [16].

2.2.2. Gene loss and evolution of Rickettsia

The mechanism of gene loss it has been a fairly widespread strategy in the evolution of the Rickettsiales genomes, and was discovered in the Rickettsiaendosymbiont Ixodes scapularis(REIS). It was found that proliferation of mobile genetic elements, in particular, an integrative conjugative element RAGE (for Rickettsiales Amplified Genetic Element) is present in chromosome and plasmids [6].

REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to other in Rickettsiagenomes. These comprise 35% of the total genome, making REIS one of the most plastic and repetitive bacterial mobile elements. The presence of REIS provides the most convincing evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE. This, probably through a continuum of genomic invasions, provides insights about the origin of mechanisms of rickettsial pathogenicity [17].

The RAGEs are the fusion of tra-like family genes that encoding the conjugal transfer protein. Inserted genes can be found between traAand traDgenes. We present a phylogeny with 60 sequences of traDgenes of 16 genomes species (Figure 4), including ancestral, transitional, and spotted fever group.

Figure 4.

The evolutionary history of gen TraD was inferred by using MEGA7 using ML method and GTR model, with the highest log likelihood (−237.1216). The analysis involved 60 nucleotide sequences from genomes of the IMG (img.jgi.doe.gov) of 16 species and tree outgroup from NCBI.

Rickettsiaspp. share 1027 genes that probably were vertically transferred from “proto-Rickettsia” R. belliimaintained all these genes and other species lost a large part of them, like R. prowazekiiand R. typhi(128 lost genes). It is well supported that differential gene loss contributes to creation of new rickettsial species [6].

In conclusion, the loss of regulatory genes causes an increase of virulence in rickettsial species in ticks and mammals, and the traoperon is presumably involved [18].

2.2.3. Phylogeny and taxonomy of Rickettsia

The taxonomy of Rickettsiawas historically based on the phenotypic criteria, the phylogenetic approaches with gene 16S rRNA defined three groups typhus group (TG) which includes: R. prowazekiiand R. typhi; classic spotted fever group (SFG), which includes a large collection of mostly tick-borne rickettsials; and an ancestral group (AG), which included R. belliiand R. canadensis[19], even thus they remain unresolved clades at species level.

Other evolutionary gen reconstructions are inconsistent when using different portions of the genome [20]. An analysis based on the whole genome sequence analysis (WGSA) allows emerging of transitional group (TRG) consisting of Rickettsia feliswhich was primarily associated with Ctenocephalides felisand the sister group to the neighboring: R. akari[19, 20].

In different studies of Rickettsiausing WGSA, we can observe resolved trees with single topology, which is supported by multiple sources of phylogenetic signal, which describes the evolutionary history of the core genome [20].

Unfortunately, we cannot have always the powerful tool of WGSA, so the search of new molecular markers is necessary to provide a well-supported phylogenetic approach, at least at species level. As we can see in Figure 5, a reliable phylogeny can be obtained using several sequences of all Rickettsiaspecies and the conserved gen rpoB, offering high-resolution clades at species level.

Figure 5.

rpoBPhylogeny ofRickettsiaandOrientiawas inferred by using MEGA7 using ML method and GTR model, with the highest log likelihood (−14425.5668). The analysis involved 87 nucleotide sequences obtained from genomes of the IMG siteimg.jgi.doe.gov. There were a total of 2721 positions in the final dataset.

2.2.4. Comparative genomics of Rickettsia

In the existing 82 sequenced genomes of Rickettsiaspecies, 77 belong to 37 species and 5 in Candidatusstatus. In general, the genomes size is constant, between 0.8 and 2.3 Gb; and the average size is 1.3 Gb.

The genomes shows 865 genes as minimum and a maximum of 2634 genes, the average is 1360 genes; the GC content is very constant among genomes with 33%. Only four genomes have pseudogenes and the average of horizontal gene transfer is 2.87% (Table 1). The presence of conjugative elements in some of these genomes correlates with an increased number of transposons, breakpoints, and a general breakdown in genome synteny, which is very conserved in nearby groups, with some inversions. However, as they move away phylogenetically, more inversions are observed and still synteny is conserved (Figure 2).

The genomic and metabolic impairment of Rickettsiagenomes is mainly due to population bottlenecks in free live style and genome size reduction is related to the gene loss, split genes, and pseudogene formation during endosymbiosis. The presence of plasmids and their sporadically integration into the chromosome leading to emergence of pathogenicity and loss of regulation are also factors that influence in Rickettsiagenomes variability [5].

When comparing synteny between Rickettsia rickettsii, member of spotted fever group, and R. felis, member of transitional group, (Figure 6) a panoramic view of the genome dynamics at large scale can be observed. A point cut of 100 pb events shows a significant difference respect to 1000 pb. This difference is also observed in the gen itself and the alignment with the Vista tool in the IMG site (img.jgi.doe.gov/cgi-bin/w/main.cgi?section=Vista&page=vista).

Figure 6.

Genomic rearrangements with different cutoff: (A) 100 bp; (B) 1000 pb.

The comparative genomic studies reveal the relation between small size and more virulent species strains, this fact supports Rickettsiavirulence and is the result of a reduction genome ongoing process. The reconstructions of inactive genes revealed that deletions strongly predominate over insertions with an excess of GC-to-AT substitutions, which explain the low GC content (32% in genome average) [21].

3. Orientia

This genus comprises Orientia tsutsugamushi, the causative agent of scrub typhus or Tsutsugamushi disease, and the novel species Orientia chutoidentified in Dubai, in the United Arab Emirates [22, 23]. Humans are the final host of the bacteria and the symptoms include a simple febrile illness to a life threatening fatal infection (meningitis, eschar, disseminated intravascular coagulation) and complicated with dysfunction in several organs [24].

O. tsutsugamushiis widely spread in the Asia-Pacific region comprising Siberia, Japan, Korea, Papua New Guinea, Thailand, Philippines, the Kamchatka Peninsula in the east, Pakistan in the west, and down to Australia in the south [22, 25].

These bacteria are an obligate intracellular Gram-negative rod-shaped and its vector is Leptotrombidiumspp. species mite populations, where vertically is maintained. Transmission to humans occurs by the bite of infected larval-stages mites called chiggers [26]. Although, some other vectors have been reported, including ticks of rodents from different geographic origins [27, 28]. Vertical or transovarial transmission of Orientiaspp. would be essential to the maintenance of the infection due to mites have a role as vectors and reservoir [25].

In the recent years, a dramatic variation in phenotypes and genotypes of O. tsutsugamushihas been observed in humans, animal host, and vector mites using immunological and molecular methods [25].

3.1. Genotyping of Orientia

Strain classification of Orientiaand serotyping were performed based on the immunodominant 56 kDa type-specific antigen (TSA) located on the surface of the bacteria [29].

With this method, three antigenic prototypes were primarily described: Karp, Kato, and Gilliam; and then many more variations of different serotypes were described in several countries [30, 31]. As primary attempts, genotyping was made by sing RFLP (Restriction Fragment Length Polymorphism) to identify unique isolates or directly by sequence analysis of the TSA gene by PCR. A comparison between nested polymerase chain reaction (nPCR) of 56-kDa antigen gene, the most used molecular technique for confirmation of scrub typhus and genotyping of O. tsutsugamushi,and single-step conventional PCR (cPCR) revealed that nPCR products have more variation among strains than cPCR, which emphasizes cPCR advantages [32, 33].

The antigenic variation of the strains Karp, Kato, and Gilliam, subsequent strains, and recently isolates discovered depends on the diversity of the TSA located on the surface of O. tsutsugamushi [34].

Genome of O. tsutsugamushistrain Boryong has 2,217,051 bp, with 2179 potential protein-coding sequences and 963 sequences of fragmented genes, which represent a coding capacity of only 49.6%. A core genome is composed of 512 genes that share with seven Rickettsiaspecies (Figure 7). The fragmented regions have a significant interest since they correspond to repeated DNA regions distributed throughout the whole genome. The absent of collinearity with other Rickettsiagenomes and the no systematic pattern in the GC plot suggest that the genome has been extensively shuffled [35].

Figure 7.

The mapping of different regions in the circular genome ofO. tsutsugamushi. Figure reproduced from [35].

3.2. Comparative genomics

Nakayama et al. [36] compared two genomes of O. tsutsugamushi: Boryong and Ikeda strains. Both genomes recently reported and isolated in Korea and Japan [35, 37]. In this comparative analysis, a phylogenetic relationship of O. tsutsugamushi strainswas reconstructed using 11 conserved genes in O. tsutsugamushiand closely related Rickettsiaspecies. The multilocus sequencing analysis of 10 O. tsutsugamushistrains representing each TSA subtype revealed the distribution of strain-specific sequences identified in Boryong or Ikeda among the O. tsutsugamushistrains.

The analysis revealed an extensive reductive genome evolution and a significant amplification of repetitive sequences. In fact, the repetitive sequences identified in Ikeda strain were classified in three types: (1) Integrative and conjugative element (ICE) named OT amplified genetic element (OtAGE); (2) Transposable elements (TE); and (3) Short repetitive sequences of unknown origin (short repeat). Both genomes of Orientiacontain the same set of repetitive sequences, which have been amplified in both strains and caused an extensive genome shuffling. Additional to this, the existence of core genes set of family Rickettsiaceae is also highly conserved. It seems that the extensive genome rearrangements generated by repetitive sequences have occurred between the two strains, although the high complex and repeat-rich feature of the Orientiagenomes and some genomics differences still have to be clarified [36].

3.3. Gene loss, gene gain, and evolution

Reductive evolution can be studied in members of Rickettsiales, because genome degradation is a process that occurs in members of this order. Gene loss has shaped the content of some Rickettsialesgenomes, and horizontal gene transfer (HGT) has played an important role in the genome evolution of these bacteria [38].

An evolution study based on gene loss and HGT events in Rickettsiaspp., Anaplasmaspp. and Orientiaspp. showed that three possible HGT event occurred from various organisms to Orientiaand six events to Rickettsiaspp., and three possible HGT event from Rickettsiaand Orientiato other bacteria (Figure 8) [38].

Figure 8.

Probable gene gain events occurred inRickettsiaandOrientia. Orientiawould have been gained genes from viruses, other bacteria and even archaea. Figure taken from [38].

Gene gain is a known event that has occurred throughout rickettsial evolution. In O. tsutsugamushiIkeda one HGT event was identified and none in O. tsutsugamushiBoyrong. Many of the genes transferred by HGT were gained ancestrally, and include transposases and ankyrin repeat-containing proteins that appear to have been transferred from viruses and protist to Orientiaspecies; the genes donated by Orientiawere gained by Firmicutesspp., Bacteroidetesspp., and Gamma-proteobacteriaspp. [38].

The whole genome analysis of O. tsutsugamushiBoryong has revealed the presence of type IV secretion system histidine kinases, SpoT, Tra, and ankyrin repeat- and tetratricopeptide repeat (TPR) containing proteins. Histidine kinases are proteins that act as sensor and signal transduction in response to changes in the environment; SpoT family proteins have a role in the response to energy starvation; Tra family proteins participate in gene transfer between rickettsia and other bacteria [39].

In a shotgun proteomics analysis using SDS-PAGE and LC-MSMS, many expressed proteins and the protein profiles were identified. 584 out of 1152 proteins of O. tsutsugamushiwere identified by trypsin and Lys-C digestion and LC-MSMS, which corresponds to 49.4% proteins, annotated on the genome of the bacteria. It seems that during evolution the obligate intracellular bacteria lacked some proteins of important function (i.e., metabolism) and conserved proteins that allow them to survive in the host cells [39].

4. Conclusions

Rickettsiaceae family comprises widely distributed and genomically diverse microorganisms. Genome analysis of the members of the family has revealed an extraordinary evolution process throughout the time driven by the constant interactions with host cells and other bacteria. Recently, genomics analyses have revealed the presence of core genes in this family, as well as genes encoding proteins with significant function in Orientiaspp.