Metagenome diversity illuminates the origins of pathogen effectors.

Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus Rickettsia and delineated a sister lineage (the novel genus Tisiphia) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages illuminates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role in shaping the rvh effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.IMPORTANCEWhile rickettsioses are deadly vector-borne human diseases, factors distinguishing Rickettsia pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The rvh type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen rvh effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of Rickettsia pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between Rickettsia and Legionella species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.

Dating Alphaproteobacteria evolution with eukaryotic fossils.

Elucidating the timescale of the evolution of Alphaproteobacteria, one of the most prevalent microbial lineages in marine and terrestrial ecosystems, is key to testing hypotheses on their co-evolution with eukaryotic hosts and Earth's systems, which, however, is largely limited by the scarcity of bacterial fossils. Here, we incorporate eukaryotic fossils to date the divergence times of Alphaproteobacteria, based on the mitochondrial endosymbiosis that mitochondria evolved from an alphaproteobacterial lineage. We estimate that Alphaproteobacteria arose ~1900 million years (Ma) ago, followed by rapid divergence of their major clades. We show that the origin of Rickettsiales, an order of obligate intracellular bacteria whose hosts are mostly animals, predates the emergence of animals for ~700 Ma but coincides with that of eukaryotes. This, together with reconstruction of ancestral hosts, strongly suggests that early Rickettsiales lineages had established previously underappreciated interactions with unicellular eukaryotes. Moreover, the mitochondria-based approach displays higher robustness to uncertainties in calibrations compared with the traditional strategy using cyanobacterial fossils. Further, our analyses imply the potential of dating the (bacterial) tree of life based on endosymbiosis events, and suggest that previous applications using divergence times of the modern hosts of symbiotic bacteria to date bacterial evolution might need to be revisited.

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle.

The Rickettsiales are a group of obligate intracellular vector-borne Gram-negative bacteria that include many organisms of clinical and agricultural importance, including Anaplasma spp., Ehrlichia chaffeensis, Wolbachia, Rickettsia spp. and Orientia tsutsugamushi. This Review provides an overview of the current state of knowledge of the biology of these bacteria and their interactions with host cells, with a focus on pathogenic species or those that are otherwise important for human health. This includes a description of rickettsial genomics, bacterial cell biology, the intracellular lifestyles of Rickettsiales and the mechanisms by which they induce and evade the innate immune response.

Molecular identification of protozoal and bacterial organisms in domestic animals and their infesting ticks from north-eastern Algeria.

A molecular survey was undertaken to determine the presence of protozoal and bacterial organisms in 120 ticks and 87 blood samples collected from mammals in north-eastern Algeria. Eight tick species were morphologically identified including 70 Rhipicephalus (Boophilus) annulatus, 23 Rhipicephalus bursa, five Rhipicephalus sanguineus sensu lato, 11 Hyalomma impeltatum, five Hyalomma scupense, two Hyalommma marginatum, one Hyalomma anatolicum and three Ixodes ricinus. Quantitative PCR screening of the ticks showed that Theileria annulata, "Candidatus Ehrlichia urmitei", Theileria buffeli and Anaplasma platys were detected in Rh. annulatus. Rickettsia massiliae and Anaplasma ovis were detected in Rh. sanguineus s.l. and Rh. bursa. Rickettsia aeschlimannii was detected in Hy. marginatum, Hy. scupense and Hy. impeltatum. Finally, "Candidatus Rickettsia barbariae" was detected in Rh. bursa. In the screening blood samples, Theileria equi, T.annulata, T. buffeli, Babesia bovis, Anaplasma marginale, A. ovis and Borrelia spp. were detected in cattle. Theileria ovis, T. annulata, and A. ovis were detected in sheep. In addition, A. ovis and T. equi were detected in goats and equidea respectively. In this study, T. equi and "Candidatus Rickettsia barbariae" were identified for the first time in Algeria as well as potential new species of Ehrlichia and Anaplasma. Although molecular detection does not indicate vector/reservoir competence when investigating ticks removed from animals, this study expands the knowledge of the microorganisms detected in ticks in north-east of Algeria.

Insight into diversity of bacteria belonging to the order Rickettsiales in 9 arthropods species collected in Serbia.

Rickettsiales bacteria in arthropods play a significant role in both public health and arthropod ecology. However, the extensive genetic diversity of Rickettsiales endosymbionts of arthropods is still to be discovered. In 2016, 515 arthropods belonging to 9 species of four classes (Insecta, Chilopoda, Diplopoda and Arachnida) were collected in Serbia. The presence and genetic diversity of Rickettsiales bacteria were evaluated by characterizing the 16S rRNA (rrs), citrate synthase (gltA) and heat shock protein (groEL) genes. The presence of various Rickettsiales bacteria was identified in the majority of tested arthropod species. The results revealed co-circulation of five recognized Rickettsiales species including Rickettsia, Ehrlichia and Wolbachia, as well as four tentative novel species, including one tentative novel genus named Neowolbachia. These results suggest the remarkable genetic diversity of Rickettsiales bacteria in certain arthropod species in this region. Furthermore, the high prevalence of spotted fever group Rickettsia in Ixodes ricinus ticks highlights the potential public health risk of human Rickettsia infection.

Epidemiology and Diversity of Rickettsiales Bacteria in Humans and Animals in Jiangsu and Jiangxi provinces, China.

Diseases caused by Rickettsiales bacteria are a global public health problem. To better understand the diversity and origins of Rickettsiales infection in humans and animals, we sampled 134 febrile patients, 173 rodents and 43 shrews, as well as 358 ticks, from two cities in Jiangsu and Jiangxi provinces, China. Our data revealed a relatively high prevalence of scrub typhus cases in both localities. In addition, both serological tests and genetic analysis identified three patients infected with Anaplasma bovis, Rickettsia monacensis, and Orientia tsutsugamushi bacteria. Molecular epidemiological investigation revealed the co-circulation of multiple species of Rickettsiales bacteria in small mammals and ticks in both provinces, potentially including novel bacterial species. In sum, these data demonstrate the ongoing importance of Rickettsiales infection in China and highlight the need for the regular surveillance of local arthropods, mammals and humans.

Phylogenetic, genomic, and biogeographic characterization of a novel and ubiquitous marine invertebrate-associated Rickettsiales parasite, Candidatus Aquarickettsia rohweri, gen. nov., sp. nov.

Bacterial symbionts are integral to the health and homeostasis of invertebrate hosts. Notably, members of the Rickettsiales genus Wolbachia influence several aspects of the fitness and evolution of their terrestrial hosts, but few analogous partnerships have been found in marine systems. We report here the genome, phylogenetics, and biogeography of a ubiquitous and novel Rickettsiales species that primarily associates with marine organisms. We previously showed that this bacterium was found in scleractinian corals, responds to nutrient exposure, and is associated with reduced host growth and increased mortality. This bacterium, like other Rickettsiales, has a reduced genome indicative of a parasitic lifestyle. Phylogenetic analysis places this Rickettsiales within a new genus we define as "Candidatus Aquarickettsia." Using data from the Earth Microbiome Project and SRA databases, we also demonstrate that members of "Ca. Aquarickettsia" are found globally in dozens of invertebrate lineages. The coral-associated "Candidatus A. rohweri" is the first finished genome in this new clade. "Ca. A. rohweri" lacks genes to synthesize most sugars and amino acids but possesses several genes linked to pathogenicity including Tlc, an antiporter that exchanges host ATP for ADP, and a complete Type IV secretion system. Despite its inability to metabolize nitrogen, "Ca. A. rohweri" possesses the NtrY-NtrX two-component system involved in sensing and responding to extracellular nitrogen. Given these data, along with visualization of the parasite in host tissues, we hypothesize that "Ca. A. rohweri" reduces coral health by consuming host nutrients and energy, thus weakening and eventually killing host cells. Last, we hypothesize that nutrient enrichment, which is increasingly common on coral reefs, encourages unrestricted growth of "Ca. A. rohweri" in its host by providing abundant N-rich metabolites to be scavenged.

Tick-borne pathogens Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. may trigger endocarditis.

BACKGROUND: Infections caused by tick-borne pathogens such as Bartonella spp., Borrelia burgdorferi s.l., Coxiella burnetii, and Rickettsia spp. are capable of causing serious lesions of the mitral and aortic valves, leading to a need for valve replacement. OBJECTIVES: The aim of the study was to determine whether such cases are sporadic or frequent. An additional goal was to establish effective diagnostic methods to detect these infections. MATERIAL AND METHODS: The study involved 148 patients undergoing valve replacement. Blood samples were drawn for serological testing. Samples of the removed mitral and aortic valves were tested with polymerase chain reaction and immunohistochemical staining. RESULTS: Specific antibodies to Bartonella spp. were detected in 47 patients (31.7%) and in 1 of the healthy controls (1%) (p < 0.05). Antibodies to B. burgdorferi spirochetes were found in 18 of the patients (12.2%) and in 6 blood donors from the control group (5.8%) (p < 0.1). Antibodies to Rickettsia spp. were detected in 12 (8.1%) and to C. burnetii phase I and II antigens in the serum of 1 patient. All the participants in the control group were seronegative to C. burnetii and Rickettsia spp. antigens. Polymerase chain reaction (PCR) tests for detection of Bartonella spp., B. burgdorferi s.l., C. burnetii and Rickettsia spp. DNA in the valve samples were all negative. Inflammation foci with mononuclear lymphoid cells in the aortic and mitral valves were seen in sections stained with hematoxiline and eozine. In sections dyed using the indirect immunofluorescence method with hyperimmune sera, Bartonella spp. and Rickettsia spp. were found. CONCLUSIONS: The results obtained indicate that laboratory diagnostics for patients with heart disorders should be expanded to include tests detecting tick-borne zoonoses such as bartonelloses, Lyme borreliosis, rickettsioses and Q fever.

Deianiraea, an extracellular bacterium associated with the ciliate Paramecium, suggests an alternative scenario for the evolution of Rickettsiales.

Rickettsiales are a lineage of obligate intracellular Alphaproteobacteria, encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novel Rickettsiales bacterium associated with Paramecium, displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesise amino acids, compared to all investigated Rickettsiales. Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction of Rickettsiales bacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity in Rickettsiales. According to our reconstruction, the Rickettsiales ancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity would have evolved later, in parallel and independently, in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria within Alphaproteobacteria.

Rickettsiales in Ticks Removed from Outdoor Workers, Southwest Georgia and Northwest Florida, USA.

We determined the prevalence of selected Rickettsiales in 362 ticks removed from outdoor workers in southwest Georgia and northwest Florida, USA. Persons submitted an average of 1.1 ticks/month. We found Ehrlichia chaffeensis in an Amblyomma maculatum tick, and Panola Mountain Ehrlichia sp. in 2 A. maculatum ticks and 1 Dermacentor variabilis tick.

Detection and geographic distribution of seven facultative endosymbionts in two Rhopalosiphum aphid species.

Study of the mutualistic associations between facultative symbionts and aphids are developed only in a few models. That survey on the situation and distribution of the symbionts in a certain area is helpful to obtain clues for the acquisition and spread of them as well as their roles played in host evolution. To understand the infection patterns of seven facultative symbionts (Serratia symbiotica, Hamiltonella defensa, Regiella insecticola, Rickettsia, Spiroplasma, Wolbachia, and Arsenophonus) in Rhopalosiphum padi (Linnaeus) and Rhopalosiphum maidis (Fitch), we collected 882 R. maidis samples (37 geographical populations) from China and 585 R. padi samples (32 geographical populations) from China and Europe. Results showed that both species were widely infected with various symbionts and totally 50.8% of R. maidis and 50.1% of R. padi were multi-infected with targeted symbionts. However, very few Rhopalosiphum aphids were infected with S. symbiotica. The infection frequencies of some symbionts were related to the latitude of collecting sites, suggesting the importance of environmental factors in shaping the geographic distribution of facultative symbionts. Also, R. maidis and R. padi were infected with different H. defensa strains based on phylogenetic analysis which may be determined by host ×symbiont genotype interactions. According to our results, the ubiquitous symbionts may play important roles in the evolution of their host aphid and their impacts on adaptation of R. padi and R. maidis were discussed as well.

First molecular detection of the human pathogen Rickettsia raoultii and other spotted fever group rickettsiae in Ixodid ticks from wild and domestic mammals.

Tick-borne rickettsioses are recognized as emerging vector-borne infections capable of infecting both human and animal hosts worldwide. This study focuses on the detection and molecular identification of species belonging to the genus Rickettsia in ticks sampled from human, vegetation, and domestic and wild vertebrates in Sardinia. Ticks were tested by PCR targeting gltA, ompA, and ompB genes, followed by sequencing analysis. The results provide evidences of a great variety of Rickettsia species of the Spotted fever group in Ixodid ticks and allow establishing for the first time the presence of R. raoultii in Rhipicephalus sanguineus s.l. and Dermacentor marginatus ticks in Sardinia island. Rickettsia massiliae was detected on R. sanguineus s.l. and R. aeschlimannii in Hyalomma marginatum and Hy. lusitanicum ticks. In addition, eight D. marginatus ticks were positive for R. slovaca. This study provides further evidence that different Rickettsia species are widespread in Sardinian ticks and that detailed investigations are required to understand the role these tick species play on spotted fever group rickettsiae circulation. More studies will provide new background on molecular epidemiology of zoonotic rickettsiae, the geographical distribution of tick-transmitted rickettsial pathogens, and the involvement of vertebrate hosts in propagation and maintenance of these bacteria in nature.

The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome Structure and Composition.

Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations. We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.