BackgroundBacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known.ResultsSuperimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of ε-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells.ConclusionWe present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model.

Recent studies aimed at elucidating the rickettsia-tick interaction have discovered that the spotted fever group rickettsia Rickettsia montanensis, a relative of R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, induces differential gene expression patterns in the ovaries of the hard tick Dermacentor variabilis. Here we describe a new defensin isoform, defensin-2, and the expression patterns of genes for three antimicrobials, defensin-1 (vsnA1), defensin-2, and lysozyme, in the midguts and fat bodies of D. variabilis ticks that were challenged with R. montanensis. Bioinformatic and phylogenetic analyses of the primary structure of defensin-2 support its role as an antimicrobial. The tissue distributions of the three antimicrobials, especially the two D. variabilis defensin isoforms, are markedly different, illustrating the immunocompetence of the many tissues that R. montanensis presumably invades once acquired by the tick. Antimicrobial gene expression patterns in R. montanensis-challenged ticks suggest that antimicrobial genes play a role during the acquisition-invasion stages in the tick.In natural transmission cycles, the vector-pathogen interaction is of central importance with respect to sylvatic epizootic and enzootic cycles (vector-pathogen-nonhuman animal), as well as zoonotic cycles that involve humans as incidental hosts (vector-pathogen-human). Vector-pathogen interactions are studied in many contexts, including how vectors respond to microbial challenge. Investigating vector innate immunity addresses the broad question of what factors intrinsic to the arthropod underlie vector competence.The innate immune system of ticks is less well studied than those of insects. Most reports deal with antimicrobial blood meal digestion by-products (9, 32, 44) and differential patterns of expression of antimicrobials such as lectins (22), lysozymes (21, 42), and defensins (4,18,23,(33)(34)(35)39).Defensin expression is reported to occur in the midguts, fat bodies, hemolymph, and hemocytes of both argasid (soft) and ixodid (hard) ticks as well as the synganglia of ixodid ticks. Before this study, only one defensin isoform, functional against gram-positive bacteria, was isolated from the plasma of the hard tick Dermacentor variabilis (18). Further research implicated hemocytes as one source of the soluble peptide (4). Two nonionic defensin isoforms, ADP1 and ADP2, that originate from synganglia of the hard tick Amblyomma hebraeum have been identified and found to possess activity against grampositive and gram-negative bacteria but not against the fungal pathogens Candida albicans and Candida glabrata (23). Additionally, there are numerous studies reporting defensin-like genes in Ixodes sp. (13,39,47).From previous work, we know that D. variabilis is capable of expressing antimicrobial genes in response to Borrelia burgdorferi (16,19), that the abundance of transcripts of immune responsive factor D (43) and lysozyme (42) genes increases upon challenge with Escherichia coli, and that a pattern of diff...

Here we report the novel bacteriostatic function of a five-domain Kunitz-type serine protease inhibitor (KPI) from the tick Dermacentor variabilis. As ticks feed, they release anticoagulants, anti-inflammatory and immunosuppressive molecules that mediate the formation of the feeding lesion on the mammalian host. The success of ticks as long-term arthropod hosts and vectors to Rickettsia spp. is due, in part, to the defense response elicited upon detection of the threat of rickettsial colonization. As ticks feed, saliva rich with immunosuppressants and anticoagulants is released at the bite site (8). The pharmacologically active saliva creates a feeding lesion on the host and promotes microbial transmission and acquisition by the tick (16). Once imbibed, rickettsiae must first evade the immunologically active tick midgut to establish themselves as endosymbionts. Irrespective of evasion and colonization, studies show that Rickettsia montanensis elicits antimicrobial gene expression in the midgut and fat body of Dermacentor variabilis (2). Related studies demonstrate that insect-derived antimicrobial peptides effectively reduce the viability of Rickettsia peacockii in vitro (1), alluding to the possibility that rickettsiae may be sensitive to tick-derived antimicrobials.Kunitz-type protease inhibitors (KPIs) are secreted with tick saliva into the feeding lesion where they prevent blood coagulation, helping to ensure acquisition of a blood meal (6,7,14). In addition to their anticoagulant properties, several studies of different model systems suggest that KPIs have a role as part of the response to microbial challenge. Stimulation of Drosophila melanogaster with bacteria or fungi results in an increase in gene expression for two KPIs (3). Also, KPIs are expressed in plants as part of the hypersensitive response (HR) activated toward both pathogenic and nonpathogenic endosymbionts (10,11,21). Interestingly, the HR is shown to control the growth and spread of nodulating endosymbionts (21). Recently, expression of a KPI from the southern cattle tick, Rhipicephalus (Boophilus) microplus, was found to be upregulated in response to Babesia bovis infection (18).Our studies reveal that Dermacentor variabilis KPI is highly expressed in the midgut and is induced upon feeding. Additionally, rickettsial challenge elicits sustained gene expression of D. variabilis KPI in the midgut. Results from our studies, as well as others, suggest that D. variabilis KPI may have bacteriostatic as well as anticoagulant properties. We tested the hypothesis that D. variabilis KPI is a bacteriostatic protease inhibitor that limits rickettsial colonization of host cells. Upon further experimentation, we observed that D. variabilis KPI limits rickettsial colonization of host cells. These findings indicate that rickettsiae must evade the rickettsiostatic effects of D. variabilis KPI to colonize the tick. MATERIALS AND METHODSTicks. Female D. variabilis ticks fed for 4 days were a generous gift from Daniel E. Sonenshine (Department of Biological Sci...

SummaryWhile examining the molecular basis for the lack of actin-based motility for the non-pathogenic spotted fever group (SFG) R. peacockii , we identified a novel insertion sequence (IS) element, ISRpe1, disrupting the coding sequence of rickA , demonstrated to induce actin-tail polymerization for the SFG rickettsiae. This rickettsial IS element appears to be active in that complete terminal inverted repeat and recombinase/transposase open reading frame sequences are present and the transposase is transcriptionally expressed. Phylogenetically, ISRpe1 belongs to a new IS family that is most closely related to those transposable elements of other intracellular bacteria like Wolbachia spp. ISRpe1 was demonstrated to be present in at least 10 locations throughout the R. peacockii genome, including one that disrupted the putative cell surface antigen encoding gene, sca1 considered to be involved in adhesion and virulence of the rickettsiae. Additionally, three IS sites demonstrated rearrangements/relocations of the R. peacockii genome when compared to those of other SFG rickettsiae. Our findings of the disruptions of rickA and sca1 along with the comparative genomic reassortments associated with ISRpe1 in the non-virulent R. peacockii provides opportunities to uncover molecular mechanisms underlying the pathogenesis and evolution of rickettsiae as well as its potential to be used in rickettsial transposon-based mutagenesis.

Murine typhus is a flea-borne febrile illness that is caused by the obligate intracellular bacterium, Rickettsia typhi. The cat flea, Ctenocephalides felis, acquires R. typhi by imbibing a bloodmeal from a rickettsemic vertebrate host. To explore which transcripts are expressed in the midgut in response to challenge with R. typhi, cDNA libraries of R. typhi-infected and uninfected midguts of C. felis were constructed. In this study, we examined midgut transcript levels for select C. felis serine proteases, GTPases and defence response genes, all thought to be involved in the fleas response to feeding or infection. An increase in gene expression was observed for the serine protease inhibitors and vesicular trafficking proteins in response to feeding. In addition, R. typhi infection resulted in an increase in gene expression for the chymotrypsin and rab5 that we studied. Interestingly, R. typhi infection had little effect on expression of any of the defence response genes that we studied. We are unsure as to the physiological significance of these gene expression profiles and are currently investigating their potential roles as it pertains to R. typhi infection. To our knowledge, this is the first report of differential expression of flea transcripts in response to infection with R. typhi.