Plasmids and Transitional Group (TRG) rickettsiae: a reply to Raoult et al.

Gillespie, Joseph J1,2, Beier, Magda S2, Rahman, M Sayeedur2, Ammerman, Nicole C2, Shallom, Joshua M1, Purkayastha, Anjan1, Sobral, Bruno S1, and Azad, Abdu F2*


1 Virginia Bioinformatics Institute at Virginia Tech, Blacksburg, VA 24061, USA
2 Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA



*Correspondence: Abdu F. Azad
Department of Microbiology and Immunology
660 West Redwood St.
HH Room 324,
University of Maryland (Baltimore)
Baltimore, MD 21201
e-mail: aazad@umaryland.edu






We were disappointed by the nature of the response posted by Raoult et al. to our paper (Gillespie et al., 2007). We felt that it was inappropriate for Raoult et al. to discuss topics not related directly to Gillespie et al. (2007). However, to respond to the criticisms of Raoult et al. our response addresses two separate issues: first, those related to Gillespie et al. (2007), and second, those dealing with the long-standing debate over Rickettsia felis.

1. Discussion directly related to Gillespie et al. (2007)

1a. The pRF-delta issues:
After the publication of R. felis genome (Ogata et al., 2005) and the revelation about the presence of two plasmids pRF and pRF-delta, interest was raised to harvest these plasmids, modify them and use them as shuttle vectors for genetic manipulation of rickettsial organisms (Kill et al., 2005; Holden et al., 2005; Gillespie et al., 2007). As reported earlier by Pornwiroon et al (2006), the PCR primers from Ogata et al. (2005) were unable to detect pRF-delta in the LSU isolate of R. felis. Of interest was that Jiang et al. (2006) reported that they were also unable to detect this plasmid, not only in the reference isolate (FleaData), but also in the LSU isolate and wild caught R. felis-infected fleas from several geographic regions. We also could not confirm the presence of pRF-delta in wild caught fleas (unpublished data). Thus, it is incumbent upon the Raoult group to definitively prove that two plasmids are present in his R. felis strain that was originated from the cat fleas obtained from FleaData. They could start by actually presenting data demonstrating the use of negative controls and DNA ladders (currently not in provided figures).
In our publication, Gillespie et al. (2007), we raised five points that we feel negate the presence of the smaller pRF-delta plasmid. Interestingly, the Raoult et al. reply to our manuscript failed to address any of these salient points, and instead chose to lump them together as "in silico" data inferior to results reported in Ogata et al. (2005), including poorly demonstrated pulse-field gel electrophoreses and unsubstantiated PCR. To reiterate our argument, we further discuss these five points in more detail.
First, in the larger plasmid, pRF, we identified through sequence comparison with other plasmid-containing bacteria several important genes likely involved in plasmid maintenance and replication. Most of these genes are absent in pRF-delta, including genes coding for two putative Dna-like chromosomal replication initiator proteins (pRF19 and pRF20), the putative cytokinesis regulatory protein ParA (pRF23), a putative structural maintenance of chromosomes protein (ABC_SMC_euk) (pRF27), and ParB, a protein implicated in the cleavage of ssDNA and supercoiled plasmid DNA (pRF35). Thus it is only natural for us (or any research group) to question the presence of this smaller plasmid if the essential genes likely involved in its maintenance and replication are deleted relative to the larger plasmid.
Second, and in a similar manner, we identified six more pRF proteins that have homology to proteins from other plasmid-containing bacteria, but are absent in pRF-delta. These include the ORF similar to P. syringae plasmid Ppsr1 ORF12 (pRF22), rickettsial hypothetical protein pRF29, the plasmid-encoded site specific recombinase TnpR (pRF32), a DNA polymerase III epsilon subunit-like protein with WGR domain (pRF34), and the putative conjugative transfer proteins TraD Ti (pRF37) and TraA Ti (pRF38). BLAST results imply that these proteins have counterparts in other plasmid-containing bacteria, suggesting that they too are likely important for the function of the plasmid. To us their absence in pRFdelta relative to pRF is suspicious.
Third, as discussed above, our recent attempts (unpublished data) and those of Pornwiroon et al. (2006) and Jiang, J. et al. (2006) to PCR-amplify pRF-delta in various strains of R. felis, including the samples from FleaData where the reference isolate came from, were unsuccessful. All of these attempts at amplifying pRF-delta were made using the prescribed primers from Ogata et al. (2005); thus, if no other independent research group can amplify this plasmid, we feel it is not unreasonable to doubt its presence, including when the fleas from FleaData were used by Jiang, J. et al. (2006).
Fourth, and perhaps our most convincing in silico evidence, is the lack of the predicted origin of replication (oriV) in the smaller plasmid. Our predicted oriV of pRF that is substantiated by gene composition, sharp change in coding strand and nucleotide compositional skew, is deleted in pRF-delta (Gillespie et al., 2007; Figure 4A). This suggests that another means of plasmid replication would be responsible for its continual inheritance. Using GenSkew (http://mips.gsf.de/servic...), an application for computing and plotting nucleotide skew data, and information from previous studies that demonstrate a sharp change in coding strand and nucleotide compositional skew around a plasmid partitioning gene (par) define plasmid oriV regions (e.g., Picardeau et al., 2000), we failed to identify an alternate replication region for the smaller plasmid. Thus, we challenge Raoult et al. to provide a mechanism for how the smaller plasmid replicates. As of now there is no evidence that pRF-delta is an autonomous replicon independent of chromosomal replication.
Finally, as stated in our manuscript,

"the fifth reservation we have with the existence of pRF-delta in R. felis deals with plasmid incompatibility. Plasmid incompatibility is the failure of two co-resident plasmids to be stably inherited without external selection (Novick et al., 1976). Incompatibility arises either by conflict in common replication or maintenance elements found in each unique plasmid, or by interference with the ability to correct stochastic fluctuations in copy number of the co-resident plasmids (Novick, 1987). Even though pRF-delta is lacking several of the important genes suspected in plasmid replication and maintenance, the presence of other genes identical to pRF would likely result in plasmid incompatibility, either by conflict in maintenance, replication or regulation of copy number".

Put simply, if pRF-delta is real, how would it be stably inherited, given that it is identical to pRF, when incompatibility arises through conflict in common replication or maintenance elements found in each unique plasmid? Thus, it is apparent that characterized plasmid system in R. felis (Ogata et al., 2005) defies the theoretical basis for all other known plasmid systems (Novick, 1976, 1987).
In summation, two comparative genomic approaches (BLAST analyses), laboratory evidence from several independent research groups (PCR using Raoult's primers), in silico methods for determining oriV, and long-standing genetic theory for plasmid maintenance and inheritance, collectively warrant the questioning of the presence of a second smaller plasmid in the R. felis genome. Additionally, we were confused by the most recent paper from this group (Blanc et al., 2007) as it provides genome statistics for several rickettsial genomes and contains a footnote explaining the size of the R. felis genome (their Table 1). It reads:

" aFor R. felis, the size for the chromosome (1,485,148 bp) plus the pRF plasmid (62,829 bp) is shown"

We wonder why the smaller plasmid was not included in this estimation, as pRF-delta adds an additional 39,263 bp to the R. felis genome). This only adds confusion to the literature regarding not only R. felis but also rickettsial and bacterial genomics and will certainly present confusion to genomic databases that attempt to list these important genome statistics.
Finally, It should be noted that we use the words "likely" and "possibly" often in this section of our manuscript, as Raoult et al. are correct in that we have no direct biological evidence to negate the existence of pRF-delta. However, neither the reviewers nor the editor had trouble with our use of in silico, empirical (PCR) or theoretical approaches for questioning the presence of pRF-delta. In fact, one reviewer suggested that we make an entire section based on our findings and include this finding in the Abstract (which we did, as it was initially a much more minimal part of the original manuscript). We also add that we are in an age of science where the advancement of bioinformatic tools has begun (and will continue) to provide powerful resources for in silico predictions as well as indicators for in vitro or in vivo experimental errors. Thus, we fail to see the argument by Raoult et al. that the pRF-delta plasmid is real when it is only known from their lab and an overwhelming amount of other evidence suggests it is not likely to occur in cells that harbor the larger pRF plasmid.

1b. The phylogenetic position of R. felis:
Raoult et al. state:

"In a previous paper, we left little doubt as to the phylogenetic position of R. felis (Blanc et al., 2007). The phylogenetic tree that we inferred from the comparison of 704 concatenated core genes of Rickettsia placed R. felis within the SFG (Figure 1). However, we do not understand the desire to create a new tree in this paper based only on 15 genes! There is also no obvious reason to create a new subgroup as the distance to other SFG is not greater than for R. helvetica or R. akari for example [25]".

There are several problems with this statement that will be discussed in detail. First, the statement

"In a previous paper, we left little doubt as to the phylogenetic position of R. felis"

cannot possibly be made in all seriousness as we hope that Raoult et al. do not think that sampling such a small portion of the known diversity of rickettsiae (see Perlman et al., 2006) has resulted in a definitive placement of R. felis in the rickettsial tree. On the contrary, it is very important for all students of evolutionary biology to realize that phylogenies are hypotheses. They are estimates of historical divergence based on the available data (taxa and characters) that we have. For these bacteria, we will likely never have access to extinct DNA sequences, thus we are left entirely to estimating ancestry, as recently demonstrated by Blanc et al. (2007). However, it is important to note that phylogenetic structure will likely change with the addition of new taxa to the analyses. And herein lies the fundamental flaw of the Raoult et al. argument. This group, while having access to ten available rickettsial genomes for well over a year, did not include any of the genome sequences generated from the CDC (R. akari, R. rickettsii, R. canadensis, and R. bellii str. OSU85-389) nor the genome sequence of R. sibirica (Malek et al., 2004) in their latest phylogeny estimation (Blanc et al. 2007). Thus, the statement

"the phylogenetic tree that we inferred from the comparison of 704 concatenated core genes of Rickettsia placed R. felis within the SFG (Figure 1)"

is only a reflection of the taxa that were included in their analysis. We wonder, why were the five genomes discussed above not included in their analysis? We find it important to include all of these genomes in phylogenomic analyses as assumptions based on limited taxon sampling may result from poor representation of particular lineages (e.g., the exclusion of R. akari in Blanc et al. (2007) of course did not result in a monophyletic TRG rickettsiae). When we include them (attached Figure 1a) and analyze 731 core rickettsial proteins across ten genomes we recover the same tree topology that we presented in Gillespie et al. (2007) from 716 fewer genes! We stated in our paper

"Thus, the failure for this phylogenetic position of R. felis to be recovered in many previous studies is likely due to the fewer number of genes analyzed. For instance, when we analyzed nine genes, we recovered the same tree topology but with weaker bootstrap support (data not shown). Furthermore, analyses of fewer than nine genes did not consistently recover the R. akari/ R. felis clade"

Thus, the statement

"However, we do not understand the desire to create a new tree in this paper based only on 15 genes!"

is unwarranted. Thus, the Blanc et al (2007) analysis is not comparable to ours because the same taxa were not compared. We now show in our unpublished tree (attached Figure 1a) that when all available rickettsial genomes are included with the core genes analyzed, our rickettsiae phylogeny is still recovered and that the position of R. felis is well supported in a clade with R. akari. Thus we believe that this strongly supported monophyletic clade is worthy of reclassification, given the biological features that define its members.
Finally, it is apparent that the Raoult group is unhappy with the naming of a new group of rickettsiae based on its clade having strong support in our estimated phylogenies (and the future phylogenies of Raoult et al. once they analyze all of the available genomes...for instance their Figure 1 accompanying their new post now includes R. akari within the TRG rickettsiae clade). We therefore disagree with their statement

"There is also no obvious reason to create a new subgroup as the distance to other SFG is not greater than for R. helvetica or R. akari for example"

because it is not only unsubstantiated, but also inaccurate and contradictory to the cladistic method. This statement is unsubstantiated because our inclusion of R. helvetica within a phylogeny of 16 genes places this taxon basal to the remaining SFG rickettsiae, all of which are monophyletic and diverged from a separate monophyletic TRG rickettsiae (attached Figure 1B). The statement is inaccurate because R. akari was included by us in the TRG rickettsiae and has a longer branch from the common ancestor of the R. felis/R. akari clade (attached Figure 1A); hence neither R. felis nor R. akari are as close to SFG as R. helvetica. Furthermore, the inclusion of R. australis in our analyses further supports this unique monophyletic TRG rickettsial clade, and further separates R. akari from the SFG rickettsiae, as it groups with R. australis and is nested within a basal R. felis with high bootstrap support (attached Figure 1B). Finally, the statement by Raoult et al. contradicts the cladistic method, which is based on the naming of monophyletic clades that are supported by shared-derived characters, or synapomorphies (theory by Willi Hennig). Our unpublished trees (attached Figure 1A & B) and trees estimated in Gillespie et al. (2007) are in agreement and reveal a strongly supported monophyletic TRG rickettsial clade that is distinct from SFG rickettsiae. Both TRG and SFG rickettsiae form a monophyletic clade that is unique from the remaining TG and AG rickettsiae; hence, in this regard, they are closer to one another in tree topology than to other rickettsiae. However, as we stated in our manuscript, TRG is unique in that it colonizes a range of arthropod hosts (not just ticks as in SFG rickettsiae) and it harbours plasmids and plasmid-associated genes that make it more similar to AG rickettsiae than SFG is. Unpublished data from our labs also further supports this as TRG and AG rickettsiae share more unique genes than do TRG and SFG rickettsiae. In this regard, the statement by Raoult et al.

"...the authors discussed on the surprising presence of a plasmid in R. felis and not in other Rickettsia. This is also highly speculative"

is very false because we not only highlight the presence of a plasmid in R. bellii str. OSU85-389 (as reported by Eremeeva et al., 2006) but we also predict that other members of the TRG rickettsiae will likely contain plasmids, including R. akari. In fact, the very premise of the paper, the evolution of plasmids within rickettsiae, touches on the likelihood of more plasmids in other rickettsial species, as we state:

"We stress that the discovery of a plasmid system in R. felis, as well as the presence of conjugative pili in R. bellii and close genomic similarities between the two species, should be considered the opening of Pandora's box, as subsequent completed rickettsial genomes will likely yield more plasmid systems and other means for non-vertical exchange of genetic material within rickettsiae and between rickettsiae and other organisms. For instance, it was recently presented at the annual meetings of The American Society of Rickettsiology (Eremeeva et al., 2006) that R. bellii str. OSU85-389 contains a putative conjugative plasmid (sequence as yet unpublished). Based on this finding, R. akari str. Hartford may also contain a conjugative plasmid, as it shares a unique clade (TRG rickettsiae) in the rickettsial tree with R. felis that is unique in that it colonizes mite and insect hosts respectively".

In addition, we clearly state:

"We predict that as more genomic sequences become available for other Rickettsia spp., the four clades defined herein using phylogenetic estimation (AG, TG, TRG, SFG) will remain strongly supported, and that R. australis and other rickettsiae with either recent host switches or the presence of plasmids, will likely fall within the AG and TRG rickettsiae. However, because the recently sequenced genome of another member of the SFG rickettsiae, R. massiliae, revealed a large genome size and the presence of a tra gene cluster similar to that found in R. bellii (Blanc et al., 2007), plasmids may be uncovered in other as yet unsequenced SFG rickettsiae".

Thus, the following statement by Raoult et al.:

"The lack of plasmid in other SFG rickettsia mainly results from a lack of investigation concerning rickettsial agents. We have already identified plasmids in 2 other SFG rickettsial species (unpublished)"

is unwarranted, because we allude to the likelihood that other plasmids will appear in newly sequenced rickettsiae (thus we didn't even need empirical data because our robust phylogeny and knowledge of these organisms already predicted what Raoult et al. have yet to publish). Thus these last two arguments by Raoult et al. do not appear to take into account the thesis of our manuscript discussed in the Conclusion.
In closing, we feel no different about the above issues in light of the Raoult et al. reply to our manuscript. Our current work supports our findings in Gillespie et al. (2007) and we doubt that the issues raised by Raoult et al. will contribute anything relevant to the science already published. Until now, our manuscript has been received very favorably by our peers and has been deemed a significant contribution to the genomics, systematics and classification of this wonderful group of organisms.

2. Discussion indirectly related to Gillespie et al. (2007)

NOTE: This section of the discussion is a direct response to the Raoult et al. discussion unrelated to Gillespie et al. (2007). Thus, with the exception of Azad, none of the authors listed in Gillespie et al. (2007) has anything to do with the issues raised regarding the disputed R. felis isolate.

2a. History and Koch's postulate:
Since 1989, our investigations of R. felis (formerly known as ELB agent) have focused on the epidemiological, ecological and molecular diagnosis of this organism. It all started because the cat flea, Ctenocephalides felis, had been suspected in the transmission of Neorickettsia rictici (Ehrlichia rictici), which was killing prize-winning thoroughbred horses in Maryland and elsewhere. We obtained cat fleas from a commercial laboratory in California and attempted to infect them with N. rictici but to no avail. Instead, and rather surprisingly, we found the fleas were loaded with Rickettsia-like organisms that reacted with rickettsial antisera. Subsequently, we showed, via electron microscopy, the presence of rickettsiae in the gut epithelial lining, malpighian tubules, muscles, and ovaries of fleas. We then studied the genetic characterization of the ELB agent and its maintenance via transovarial transmission. We found that cat fleas from eight commercial colonies from various regions of the USA were infected with this bacterium. These flea colonies were initiated either with fleas from one supplier (EL Laboratories), in which the ELB agent was first identified, or were started with fleas from stray cats and dogs and later replenished with infected EL fleas from time to time. Infection rates in these colonies ranged from 43% to 93%. In light of these findings, we obtained cat fleas from opossums, cats, dogs, and a bobcat and found that <10% were infected with this agent. This agent was named R. felis based on its molecular characteristics. Identification of R. felis in fleas within several foci of murine typhus in the U.S. prompted a retrospective investigation for this bacterium among human murine typhus patients. In 1994, R. felis was detected by PCR in a blood sample from a patient diagnosed with murine typhus. Our initial work was followed by other investigators and the search for this pathogen generated surprises when R. felis was identified in clinically masquerading dengue fever cases in patients from Yucatan, Mexico. Additionally, both serological and PCR-based evidence of R. felis infection is reported in patients from France, Brazil, Germany, Algier, Portugal, Thailand and Spain. Collectively, our published data and these recent reports not only support the pathogenic role of R. felis but also demonstrate its wide geographic distribution and its potential to cause human infections. Unfortunately, neither we nor others have any credible human isolates as of yet and although PCR positive patients demonstrate general symptoms of rickettsial diseases there is no specific and/or clear picture discriminating R. felis as a real human pathogen (hence our question mark in Figure 1 of Gillespie et al., 2007). This point is noteworthy since Raoult et al. have used serology or PCR evidence to define every rickettsial species as pathogenic thus ignoring the fact that microbial contents of bloodsucking arthropods could be acquired by the host that they feed upon (incidental occurrence outside of natural vector and host range). In this regard, their claim:

"...Gillespie’s paper gives a very archaic vision of Rickettsia, which is contradicted by recent findings. There is no test to prove that a particular rickettsia is non-pathogenic".

is invalid due to a lack of data to substantiate claims for this pathogenicity as well as laboratory studies demonstrating the true hosts and vectors (passing and fitness studies) versus those arising from limited incidental interactions in nature. Similarly, the statement:

"Moreover, by deliberately omitting SFG rickettsiae of unknown pathogenicity (such as R. montanensis) the authors propose in their figure 1 a false representation of what is currently known about rickettsial pathogenicity. Therefore, they speculated there was an increase in virulence after the divergence of R. canadensis".

is unnecessary because we certainly did not "deliberately omit" any taxa from our analyses, as we clearly state in the first sentence the Materials and Methods on page 14 of Gillespie et al. (2007) the following:

"We analyzed only those Rickettsia spp. for which a genome sequence was available: Rickettsia bellii str. RML369-C (NC_007940), R. bellii str. OSU85 389 (NZ_AARC00000000), R. canadensis str. McKiel (NZ_AAFF01000001), R. prowazekii str. Madrid E (NC_000963), R. typhi str. Wilmington (NC_006142), R. akari str. Hartford (NZ_AAFE01000001), R. felis str. URRWXCal2 (NC_007109), R. conorii str. Malish 7 (NC_003103), R. rickettsii (NZ_AADJ01000001), and R. sibirica str. 246 (NZ_AABW01000001)".

Thus, our methodology was objective and not contrived as Raoult et al. suggest. Furthermore, our mapping of the gain of virulence over our estimated phylogeny of the sampled rickettsial taxa (NOT ALL OF RICKETTSIAE) is accompanied by a question mark to denote that we do not really know for sure that some (e.g., R. felis) of these sampled taxa are really pathogenetic (see above).

2b. R. felis, the real issues:
Isolation of R. felis has been a difficult task in our hands and took several years because we were using mammalian cell lines to isolate this bacterium from naturally infected fleas. We finally obtained an isolate that grows very slowly and could not be maintained via repeated passages in Vero cells. It is not unexpected to maintain insect-adapted intracellular bacteria in mammalian cell lines and particularly at much higher temperatures. Nevertheless, several groups (e.g., CDC, LSU) had similar problems maintaining R. felis in mammalian cells, although long-term maintenance at higher temperature results in the loss of the organism. Upon request to provide ELB isolate to several laboratories, one of our postdoctoral fellows seeded a flask of Vero cells and hand delivered them to Dr. Raoult and two other laboratories. Almost a year later three laboratories told us that the sample of isolate given to them was R. typhi and not R. felis. We later found that the tubes were mislabeled and this was a source of the problem. We reported and corrected the mix-up where needed. Raoult in at least 10 of his papers also reported the incidence over and over. However, the mislabeled rickettsiae were not used in any of the published work, and molecular analysis clearly demonstrates the true identity of the R. felis original isolate.
In fact, we purchased the cat fleas from FleaData and paid for the shipment to Dr. Raoult’s laboratory where they isolated R. felis in a frog cell line. We would like to emphasize that the isolate came from New York fleas (mixed with wild caught) not California fleas as published. Raoult’s isolate is not available outside of his own laboratory and, although he has deposited this strain at the ATCC (ATCC VR-1525), the isolate is not available and currently on “administrative hold”. Attempts to acquire ATCC VR-1525 from the ATCC were met with the response “there is no plan to have VR1525 available to order any time soon”. This means the Raoult et al. isolate is either not pure or contaminated, or simply not growing. Furthermore, Dr. Raoult was not forthcoming in providing either the strain or his specific R. felis monoclonal antibodies to us and other labs that have inquired. In fact, upon our request for three MAbs (F11E9A2G1, F24A2D1F9, F19F8H3H4) they told us two crucial ones are no longer available and they never sent us the remaining one that they had at hand. Therefore, there is no way to verify the specificity of their published MAbs. What Dr. Raoult is not revealing is the issue of the “turf fight” since he could not get the initial credit for describing R. felis. Before his publication of the R. felis isolation paper he asked our lab to join the authorship in his description of the isolate as a new species. We politely refused. Thus, what is included in their discussion are totally out of line issues pertaining to an outdated subject.
In closing, it is important to realize that scientists make mistakes, and indeed, several published papers from Raoult’s group, regardless of the subject matter, contain misleading information [e.g., estimation of genome size for R. rickettsii as 2.1 Mbp (Roux et al., 1992), several new species of supposed pathogenic Rickettsia, phylogenies based on single genes and/or inadequate sequence analyses, and genome sequencing papers (to be debated shortly)]. Rather than choose to attack these publications inappropriately within an open access format pertaining to an unrelated study, we will deal with them properly in publications as they arise in pertinent studies.


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Figure 1. Estimated phylogenies of rickettsiae. (A) Analysis of 731 proteins from 10 rickettsial taxa and one outgroup (Wolbachia). Tree is from an exhaustive search using parsimony, with branch support from one million bootstrap replicates. (B) Analysis of 16 selected proteins across 21 members of the Rickettsiales and one outgroup (Pelagibacter). Tree is from a heuristic search using parsimony, with 1000 random sequence additions saving only the 100 best trees from each addition. Branch support is from one million bootstrap replicates.

Link to figure 1: https://patric.vbi.vt.edu...