Bacterium Rickettsia Research Articles

To Tree or Not to Tree? Genome-Wide Quantification of Recombination and Reticulate Evolution during the Diversification of Strict Intracellular Bacteria

It is well known that horizontal gene transfer (HGT) is a major force in the evolution of prokaryotes. During the adaptation of a bacterial population to a new ecological niche, and particularly for intracellular bacteria, selective pressures are shifted and ecological niches reduced, resulting in a lower rate of genetic connectivity. HGT and positive selection are therefore two important evolutionary forces in microbial pathogens that drive adaptation to new hosts. In this study, we use genomic distance analyses, phylogenomic networks, tree topology comparisons, and Bayesian inference methods to investigate to what extent HGT has occurred during the evolution of the genus Rickettsia, the effect of the use of different genomic regions in estimating reticulate evolution and HGT events, and the link of these to host range. We show that ecological specialization restricts recombination occurrence in Rickettsia, but other evolutionary processes and genome architecture are also important for the occurrence of HGT. We found that recombination, genomic rearrangements, and genome conservation all show evidence of network-like evolution at whole-genome scale. We show that reticulation occurred mainly, but not only, during the early Rickettsia radiation, and that core proteome genes of every major functional category have experienced reticulated evolution and possibly HGT. Overall, the evolution of Rickettsia bacteria has been tree-like, with evidence of HGT and reticulated evolution for around 10–25% of the core Rickettsia genome. We present evidence of extensive recombination/incomplete lineage sorting (ILS) during the radiation of the genus, probably linked with the emergence of intracellularity in a wide range of hosts.

Crystal structure of the N‐terminal domains of the surface cell antigen 4 of Rickettsia

The obligate intracellular, gram-negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the co-localization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N-terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all-helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved β-strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.

‘Candidatus Megaira polyxenophila’ gen. nov., sp. nov.: Considerations on Evolutionary History, Host Range and Shift of Early Divergent Rickettsiae

“Neglected Rickettsiaceae” (i.e. those harboured by non-hematophagous eukaryotic hosts) display greater phylogenetic variability and more widespread dispersal than pathogenic ones; yet, the knowledge about their actual host range and host shift mechanism is scarce. The present work reports the characterization following the full-cycle rRNA approach (SSU rRNA sequence, specific in situ hybridization, and ultrastructure) of a novel rickettsial bacterium, herewith proposed as 'Candidatus Megaira polyxenophila' gen. nov., sp. nov. We found it in association with four different free-living ciliates (Diophrys oligothrix, Euplotes octocarinatus, Paramecium caudatum, and Spirostomum sp., all belonging to Alveolata, Ciliophora); furthermore it was recently observed as intracellular occurring in Carteria cerasiformis and Pleodorina japonica (Chlorophyceae, Chlorophyta). Phylogenetic analyses demonstrated the belonging of the candidate new genus to the family Rickettsiaceae (Alphaproteobacteria, Rickettsiales) as a sister group of the genus Rickettsia. In situ observations revealed the ability of the candidate new species to colonize either nuclear or cytoplasmic compartments, depending on the host organism. The presence of the same bacterial species within different, evolutionary distant, hosts indicates that 'Candidatus Megaira polyxenophila' recently underwent several distinct host shifts, thus suggesting the existence of horizontal transmission pathways. We consider these findings as indicative of an unexpected spread of rickettsial infections in aquatic communities, possibly by means of trophic interactions, and hence propose a new interpretation of the origin and phylogenetic diversification of rickettsial bacteria.

Prevalence of Theileria equi, Babesia caballi, and Anaplasma phagocytophilum in horses from the north of Portugal

Piroplasmid protozoa Theileria equi and Babesia caballi and zoonotic rickettsial bacterium Anaplasma phagocytophilum are important agents of equine vector-borne diseases (EVBD). This study aimed at investigating the prevalence of infections with or exposure to these pathogens in horses from the north of Portugal. Blood was randomly collected from 162 horses, living in 72 different stables, to prepare Giemsa-stained slide smears. Additionally, plasma samples were tested for antibodies to T. equi and B. caballi by two competitive enzyme-linked immunosorbent assays and to A. phagocytophilum by an indirect fluorescence antibody test. Five horses were positive to T. equi by microscopy (3.1 %), three to B. caballi (1.9 %), and none to A. phagocytophilum with no horse simultaneously positive for the two piroplasms. Clinically suspect animals had a significantly higher positivity to T. equi by microscopy in comparison with the nonsuspect ones (21.4 vs. 1.4 %). Twenty-nine horses were seropositive to T. equi (17.9 %), 18 to B. caballi (11.1 %), and 21 to A. phagocytophilum (13.0 %). Combined serology and microscopy positive results to T. equi and B. caballi were 19.1 and 11.7 %, respectively, with 33.3 % of the horses found positive to at least one agent. Forty horses were positive to single agents and 14 to more than one agent. An outdoor or mixed outdoor/indoor type of housing was found to be a risk factor for the combined positivity to T. equi. Infections with T. equi, B. caballi, and A. phagocytophilum are endemic in the north of Portugal. In addition to the treatment of positive horses, preventive measures should be put in practice to reduce exposure to and infection with agents of EVBD.

Effects of Infection byArsenophonusandRickettsiaBacteria on the Locomotive Ability of the TicksAmblyomma americanum,Dermacentor variabilis, andIxodes scapularis

The goal of this study was to determine the effect of vertically transmitted Arsenophonus and Rickettsia bacteria on locomotive ability of larvae of three eastern North American tick species: Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. We conducted two different experiments on flat or inclined surfaces to measure tick motility. In each experiment, a moderately heated surface was used and placed at a selected incline. The individual's path across the surface was traced for a period of 2 min, or until the larval tick had moved off the surface. Following the microbial identification of Arsenophonus and Rickettsia, a mixed model analysis of variance (ANOVA) indicated that clutch microbial infection status had a significant effect on tick motility with Rickettsia increasing and Arsenophonus decreasing motility averaged over tick species and inclines. There was also a significant difference in motility among tick species and a highly significant effect of the Species*Incline interaction where Dermacentor had higher motility than Ixodes on the flat surface.

Modelling spatial concordance between Rocky Mountain spotted fever disease incidence and habitat probability of its vector Dermacentor variabilis (American dog tick)

The spatial distribution of Dermacentor variabilis, the most commonly identified vector of the bacterium Rickettsia rickettsii which causes Rocky Mountain spotted fever (RMSF) in humans, and the spatial distribution of RMSF, have not been previously studied in the south central United States of America, particularly in Texas. From an epidemiological perspective, one would tend to hypothesise that there would be a high degree of spatial concordance between the habitat suitability for the tick and the incidence of the disease. Both maximum-entropy modelling of the tick's habitat suitability and spatially adaptive filters modelling of the human incidence of RMSF disease provide reliable portrayals of the spatial distributions of these phenomenons. Even though rates of human cases of RMSF in Texas and rates of Dermacentor ticks infected with Rickettsia bacteria are both relatively low in Texas, the best data currently available allows a preliminary indication that the assumption of high levels of spatial concordance would not be correct in Texas (Kappa coefficient of agreement = 0.17). It will take substantially more data to provide conclusive findings, and to understand the results reported here, but this study provides an approach to begin understanding the discrepancy.

Actinobacteria as essential symbionts in firebugs and cotton stainers (Hemiptera, Pyrrhocoridae)

Actinobacteria engage in defensive symbioses with several insect taxa, but reports of nutritional contributions to their hosts have been exceptionally rare. Cotton stainers (Dysdercus fasciatus) and red firebugs (Pyrrhocoris apterus) (both Hemiptera, Pyrrhocoridae) harbour the actinobacterial symbionts Coriobacterium glomerans and Gordonibacter sp. as well as Firmicutes (Clostridium sp. and Lactococcus sp.) and Proteobacteria (Klebsiella sp. and a Rickettsiales bacterium) in the M3 region of their mid-gut. We combined experimental manipulation with community-level analyses to elucidate the function of the gut symbionts in both pyrrhocorid species. Elimination of symbionts by egg-surface sterilization resulted in significantly higher mortality and reduced growth rates, indicating that the microbial community plays an important role for host nutrition. Fitness of symbiont-deprived bugs could be completely restored by re-infection with the original microbiota, while reciprocal cross-infections of microbial communities across both pyrrhocorid species only partially rescued fitness, demonstrating a high degree of host-symbiont specificity. Community-level analyses by quantitative PCRs targeting the dominant bacterial strains allowed us to link the observed fitness effects to the abundance of the two actinobacterial symbionts. The nutritional mutualism with Actinobacteria may have enabled pyrrhocorid bugs to exploit Malvales seeds as a food source and thereby possibly allowed them to occupy and diversify in this ecological niche.

Rickettsia parkeriandCandidatusRickettsia andeanae in Gulf Coast Ticks, Mississippi, USA

<i>Rickettsia parkeri</i>and<i>Candidatus</i>Rickettsia andeanae in Gulf Coast Ticks, Mississippi, USA

Geographical and ecological stability of the symbiotic mid‐gut microbiota in European firebugs, Pyrrhocoris apterus (Hemiptera, Pyrrhocoridae)

Symbiotic bacteria often play an essential nutritional role for insects, thereby allowing them to exploit novel food sources and expand into otherwise inaccessible ecological niches. Although many insects are inhabited by complex microbial communities, most studies on insect mutualists so far have focused on single endosymbionts and their interactions with the host. Here, we provide a comprehensive characterization of the gut microbiota of the red firebug (Pyrrhocoris apterus, Hemiptera, Pyrrhocoridae), a model organism for physiological and endocrinological research. A combination of several culture-independent techniques (454 pyrosequencing, quantitative PCR and cloning/sequencing) revealed a diverse community of likely transient bacterial taxa in the mid-gut regions M1, M2 and M4. However, the completely anoxic M3 region harboured a distinct microbiota consisting of facultative and obligate anaerobes including Actinobacteria (Coriobacterium glomerans and Gordonibacter sp.), Firmicutes (Clostri-dium sp. and Lactococcus lactis) and Proteobacteria (Klebsiella sp. and a previously undescribed Rickettsiales bacterium). Characterization of the M3 microbiota in different life stages of P.apterus indicated that the symbiotic bacterial community is vertically transmitted and becomes well defined between the second and third nymphal instar, which coincides with the initiation of feeding. Comparing the mid-gut M3 microbial communities of P.apterus individuals from five different populations and after feeding on three different diets revealed that the community composition is qualitatively and quantitatively very stable, with the six predominant taxa being consistently abundant. Our findings suggest that the firebug mid-gut microbiota constitutes a functionally important and possibly coevolved symbiotic community.

GC content dependency of open reading frame prediction via stop codon frequencies

A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to have the same sequence as a stop codon. In contrast, the open reading frames (ORFs) of most protein-coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting protein-coding ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50%. However, many genomes show significant deviations from that value. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of potentially coding ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80%. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions. We have adapted the method for predicting coding ORFs by stop codon frequency to the case of GC contents different from 50%. Usually, several methods for gene finding need to be combined. Thus, our results concern a specific part within a package of methods. Interestingly, for genomes with low GC content such as that of R. prowazekii, the presented method provides remarkably good results even when applied alone.

A Rickettsia parkeri-like agent infecting Amblyomma calcaratum nymphs from wild birds in Mato Grosso do Sul, Brazil

In total, 142 birds, mostly passerines, belonging to 42 species were examined for the presence of ticks in 3 locations in Mato Grosso do Sul Brazil during 2006. Seven birds (5%) were infested with 4 nymphs of Amblyomma calcaratum (Ramphocelus carbo, 3 infested/12 examined) and 5 larvae of Amblyomma sp. (Furnarius rufus, 2/5; Turdus leucomelas, 1/6; and Paroaria capitata, 1/8). All 4 nymphs of A. calcaratum tested by polymerase chain reaction targeting rickettsial genes gltA and ompA and by amplicon sequencing were found to be infected with a Rickettsia sp. strain NOD, a Rickettsia parkeri-like agent. A. calcaratum infected with a rickettsial bacterium was found for the first time.

Surface Proteome Analysis and Characterization of Surface Cell Antigen (Sca) or Autotransporter Family of Rickettsia typhi

Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1–3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.

Polymorphism of mitochondrial DNA and distribution of cytoplasmic symbionts in the populations of two-spot ladybird beetle Adalia bipunctata

In geographically distant populations of ladybird beetle Adalia bipunctata from Eurasia mitotypes and infection with symbiotic bacteria Spiroplasma and Rickettsia were determined. All populations examined demonstrated mtDNA polymorphism and striking differences in prevalence of bacteria (from about 50% of individuals infected with Spiroplasma in St.-Petersburg population and 50% of the Rickettsia prevalence in Kem' population to complete absence of bacteria in the population from Archangelsk). In the populations studied a total of 14 mitotypes were discovered, including two mitotypes that were remarkably different from the others in nucleotide composition. Mitotype 10, which was the most different from all the others, was found in all populations from Germany to Transbaikalia, excluding the population from Tashkent. Linkage disequilibrium between mitotype 10 and the Rickettsia infection was confirmed. Infection with the Spiroplasma bacteria was typical of the individuals with haplotype 1 and relative to it. The results obtained supported the conclusion on the association between infection with Spiroplasma and Rickettsia and certain mitotype of A. bipunctata, which was the consequence of either absence or rare horizontal transfer of symbionts and ancientness of the first contact between the bacteria and A. bipunctata ladybird beetles.

GC content dependency of open reading frame prediction

Motivations. A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to be equivalent to a stop codon. In contrast, the open reading frames (ORFs) of most protein coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50 %. However, many genomes show significant deviations from that value. Methods and Results. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80 %. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions.

BiotecVisions 2012, February

BiotecVisions 2012, February

Distribution of Rickettsia rickettsii in ovary cells of Rhipicephalus sanguineus (Latreille1806) (Acari: Ixodidae).

BackgroundConsidering the fact that the dog tick, Rhipicephalus sanguineus, has a great potential to become the vector of Brazilian Spotted Fever (BSF) for humans, the present study aimed to describe the distribution of the bacterium Rickettsia rickettsii, the etiological agent of BSF, in different regions of the ovaries of R. sanguineus using histological techniques. The ovaries were obtained from positive females confirmed by the hemolymph test and fed in the nymph stage on guinea pigs inoculated with R. rickettsii.ResultsThe results showed a general distribution of R. rickettsii in the ovary cells, being found in oocytes in all stages of development (I, II, III, IV and V) most commonly in the periphery of the oocyte and also in the cytoplasm of pedicel cells.ConclusionsThe histological analysis of the ovaries of R. sanguineus infected females confirmed the presence of the bacterium, indicating that the infection can interfere negatively in the process of reproduction of the ticks, once alterations were detected both in the shape and cell structure of the oocytes which contained bacteria.

Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda

BackgroundThe rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda.ResultsA total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium.ConclusionsThe compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.

Rickettsia,Ehrlichia,Anaplasma, andBartonellain Ticks and Fleas from Dogs and Cats in Bangkok

Flea and tick specimens (5-10 fleas or ticks) on dogs and cats from various sites in Bangkok were tested by polymerase chain reaction and DNA sequencing to detect DNA of bacteria Rickettsia (gltA and 17 kDa genes), Anaplasmataceae (16S rRNA gene), and Bartonella (pap31 and its genes). We confirmed that Rickettsia sp. related to Rickettsia felis was detected in 66 of 98 (67.4%) flea specimens from dogs, whereas 8 Bartonella henselae and 2 Bartonella clarridgeiae were detected in 10 of 54 (18.5%) flea specimens from cats. Further, this work provides the first evidence of 10 Ehrlichia canis (3.3%), 7 Anaplasma platys (2.3%), and 2 Wolbachia spp. (0.66%) in 304 Rhipicephalus sanguineus tick specimens in Thailand.

Experimental infection of Amblyomma aureolatum ticks with Rickettsia rickettsii.

We experimentally infected Amblyomma aureolatum ticks with the bacterium Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever (RMSF). These ticks are a vector for RMSF in Brazil. R. rickettsii was efficiently conserved by both transstadial maintenance and vertical (transovarial) transmission to 100% of the ticks through 4 laboratory generations. However, lower reproductive performance and survival of infected females was attributed to R. rickettsii infection. Therefore, because of the high susceptibility of A. aureolatum ticks to R. rickettsii infection, the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates (<1%) usually reported among field populations of A. aureolatum ticks in RMSF-endemic areas of Brazil. Because the number of infected ticks would gradually decrease after each generation, it seems unlikely that A. aureolatum ticks could sustain R. rickettsii infection over multiple successive generations solely by vertical transmission.

Scrub Typhus in Adults – A Case Series from a Tertiary Care Hospital

Introduction: Scrub typhus is a zoonosis caused by the rickettsial bacteria Orientia tsutsugamushi. The disease in endemic in many parts of India and continues to be a public health problem. Materials and Methods: This study describes the clinical profile of eight consecutive scrub typhus positive cases admitted in a tertiary care hospital. After complete physical examination, complete blood count, liver function tests, renal function tests, urine analysis, blood & urine cultures were done in all cases. In all the cases, scrub typhus was diagnosed using a rapid single step immunochromatographic assay. Results: Of eight cases five were females and three were males. Fever was the chief present symptom in all the cases. Other signs and symptoms were headache, myalgia, cough, hepatosplenomegaly, abdominal pain and lymphadenopathy. Eschar was present in all the cases. Liver enzymes were elevated in all the patients. Hypotension, thrombocytopenia, hepatic dysfunction, hypoalbuminemia, hematuria and proteinuria were the complications observed. All cases were successfully treated with doxycycline and there was no mortality. Conclusion: Our report emphasizes the fact that a diagnosis of scrub typhus should be suspected when a patient presents with fever and laboratory evidence of liver dysfunction, as early detection of the disease could reduce morbidity and mortality.