Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.Published copyLockwood, S., D. E. Voth, K. A. Brayton, P. A. Beare, W. C. Brown, R. A. Heinzen, and S. L. Broschat, Identification of Anaplasma marginale type IV secretion system effector proteins, PLoS ONE, Vol. 6, No. 11, e7724, Nov. 2011. DOI: 10.1371/journal.pone.0027724

Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum

Citation: Nair, A. D. S., Cheng, C., Ganta, C. K., Sanderson, M. W., Alleman, A. R., Munderloh, U. G., & Ganta, R. R. (2016). Comparative Experimental Infection Study in Dogs with Ehrlichia canis, E. chaffeensis, Anaplasma platys and A. phagocytophilum. Plos One, 11(2), 21. doi:10.1371/journal.pone.0148239Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects

Transmission Electron Microscopy Reveals Distinct Macrophage- and Tick Cell-Specific Morphological Stages of Ehrlichia chaffeensis

Background: Ehrlichia chaffeensis is an emerging tick-borne rickettsial pathogen responsible for human monocytic ehrlichiosis. Despite the induction of an active host immune response, the pathogen has evolved to persist in its vertebrate and tick hosts. Understanding how the organism progresses in tick and vertebrate host cells is critical in identifying effective strategies to block the pathogen transmission. Our recent molecular and proteomic studies revealed differences in numerous expressed proteins of the organism during its growth in different host environments. Methodology/Principal Findings: Transmission electron microscopy analysis was performed to assess morphological changes in the bacterium within macrophages and tick cells. The stages of pathogen progression observed included the attachment of the organism to the host cells, its engulfment and replication within a morulae by binary fission and release of the organisms from infected host cells by complete host cell lysis or by exocytosis. E. chaffeensis grown in tick cells was highly pleomorphic and appears to replicate by both binary fission and filamentous type cell divisions. The presence of Ehrlichia-like inclusions was also observed within the nucleus of both macrophages and tick cells. This observation was confirmed by confocal microscopy and immunoblot analysis. Conclusions/Significance: Morphological differences in the pathogen’s progression, replication, and processing within macrophages and tick cells provide further evidence that E. chaffeensis employs unique host-cell specific strategies in support of adaptation to vertebrate and tick cell environments

Development of Shuttle Vectors for Transformation of Diverse Rickettsia Species

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 – 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae

Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line

Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system

Ehrlichia chaffeensis Transcriptome in Mammalian and Arthropod Hosts Reveals Differential Gene Expression and Post Transcriptional Regulation

BACKGROUND: Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. METHODOLOGY/PRINCIPAL FINDINGS: The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30-80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. CONCLUSIONS/SIGNIFICANCE: Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms

Occurrence Patterns of Afrotropical Ticks (Acari: Ixodidae) in the Climate Space Are Not Correlated with Their Taxonomic Relationships

Foci of tick species occur at large spatial scales. They are intrinsically difficult to detect because the effect of geographical factors affecting conceptual influence of climate gradients. Here we use a large dataset of occurrences of ticks in the Afrotropical region to outline the main associations of those tick species with the climate space. Using a principal components reduction of monthly temperature and rainfall values over the Afrotropical region, we describe and compare the climate spaces of ticks in a gridded climate space. The dendrogram of distances among taxa according to occurrences in the climate niche is used to draw functional groups, or clusters of species with similar occurrences in the climate space, as different from morphologically derived (taxonomical) groups. We aim to further define the drivers of species richness and endemism at such a grid as well as niche similarities (climate space overlap) among species. Groups of species, as defined from morphological traits alone, are uncorrelated with functional clusters. Taxonomically related species occur separately in the climate gradients. Species belonging to the same functional group share more niche among them than with species in other functional groups. However, niche equivalency is also low for species within the same taxonomic cluster. Thus, taxa evolving from the same lineage tend to maximize the occupancy of the climate space and avoid overlaps with other species of the same taxonomic group. Richness values are drawn across the gradient of seasonal variation of temperature, higher values observed in a portion of the climate space with low thermal seasonality. Richness and endemism values are weakly correlated with mean values of temperature and rainfall. The most parsimonious explanation for the different taxonomic groups that exhibit common patterns of climate space subdivision is that they have a shared biogeographic history acting over a group of ancestrally co-distributed organisms

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod