M. Sayeedur Rahman

Plasmids and Rickettsial Evolution: Insight from Rickettsia felis

Background The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG) or spotted fever group (SFG) rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF) that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria. Methodology/Principal Findings Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG) rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives) also occur in AG (but not SFG) rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFδ, is an artifact of the original genome assembly. Conclusion/Significance Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG) rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the likely origin of plasmids within the rickettsial tree.

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Background Bacterial 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. Results Superimposition 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. Conclusion We 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.

A novel and naturally occurring transposon, ISRpe1 in the Rickettsia peacockii genome disrupting the rickA gene involved in actin‐based motility

While 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.

New tick defensin isoform and antimicrobial gene expression in response to Rickettsia montanensis challenge

ABSTRACT 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.

Functional Characterization and Novel Rickettsiostatic Effects of a Kunitz-Type Serine Protease Inhibitor from the Tick Dermacentor variabilis

ABSTRACT 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. A number of KPIs have been isolated and characterized from tick salivary gland extracts. Interestingly, we observe little D. variabilis KPI gene expression in the salivary gland and abundant expression in the midgut. However, our demonstration of D. variabilis KPIs anticoagulant properties indicates that D. variabilis KPI may be important for blood meal digestion in the midgut. In addition to facilitating long-term attachment and blood meal acquisition, gene expression studies of Drosophila, legumes, and ticks suggest that KPIs play some role in the response to microbial infection. Similarly, in this study, we show that challenge of D. variabilis with the spotted fever group rickettsia, Rickettsia montanensis, results in sustained D. variabilis KPI gene expression in the midgut. Furthermore, our in vitro studies show that D. variabilis KPI limits rickettsial colonization of L929 cells (mouse fibroblasts), implicating D. variabilis KPI as a bacteriostatic protein, a property that may be related to D. variabilis KPIs trypsin inhibitory capability. This work suggests that anticoagulants play some role in the midgut during feeding and that D. variabilis KPI may be involved as part of the ticks defense response to rickettsiae.

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.

Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells

The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.

TolC-Dependent Secretion of an Ankyrin Repeat-Containing Protein of Rickettsia typhi

Rickettsia typhi, the causative agent of murine (endemic) typhus, is an obligate intracellular pathogen with a life cycle involving both vertebrate and invertebrate hosts. In this study, we characterized a gene (RT0218) encoding a C-terminal ankyrin repeat domain-containing protein, named Rickettsia ankyrin repeat protein 1 (RARP-1), and identified it as a secreted effector protein of R. typhi. RT0218 showed differential transcript abundance at various phases of R. typhi intracellular growth. RARP-1 was secreted by R. typhi into the host cytoplasm during in vitro infection of mammalian cells. Transcriptional analysis revealed that RT0218 was cotranscribed with adjacent genes RT0217 (hypothetical protein) and RT0216 (TolC) as a single polycistronic mRNA. Given one of its functions as a facilitator of extracellular protein secretion in some Gram-negative bacterial pathogens, we tested the possible role of TolC in the secretion of RARP-1. Using Escherichia coli C600 and an isogenic tolC insertion mutant as surrogate hosts, our data demonstrate that RARP-1 is secreted in a TolC-dependent manner. Deletion of either the N-terminal signal peptide or the C-terminal ankyrin repeats abolished RARP-1 secretion by wild-type E. coli. Importantly, expression of R. typhi tolC in the E. coli tolC mutant restored the secretion of RARP-1, suggesting that TolC has a role in RARP-1 translocation across the outer membrane. This work implies that the TolC component of the putative type 1 secretion system of R. typhi is involved in the secretion process of RARP-1.

A Kunitz Protease Inhibitor from Dermacentor variabilis, a Vector for Spotted Fever Group Rickettsiae, Limits Rickettsia montanensis Invasion

ABSTRACT A defining facet of tick-Rickettsia symbioses is the molecular strategy employed by each partner to ensure its own survival. Ticks must control rickettsial colonization to avoid immediate death. In the current study, we show that rickettsial abundance in the tick midgut increases once the expression of a Kunitz-type serine protease inhibitor from the American dog tick (Dermacentor variabilis) (DvKPI) is suppressed by small interfering RNA (siRNA). A series of in vitro invasion assays suggested that DvKPI limits rickettsial colonization during host cell entry. Interestingly, we observed that DvKPI associates with rickettsiae in vitro as well as in the tick midgut. Collectively, our data demonstrate that DvKPI limits host cell invasion by Rickettsia montanensis, possibly through an association with the bacterium.

Functional Characterization of a Phospholipase A2 Homolog from Rickettsia typhi

Phospholipase A(2) (PLA(2)) has long been proposed to be involved in rickettsial entry into host cells, escape from the phagosome to evade destruction by lysosomal exposure, and lysis of the host cells. However, the corresponding rickettsial gene(s) encoding a protein with PLA(2) activity has not been identified or functionally characterized. Here, we report that the Rickettsia typhi genome possesses two genes encoding patatin-like PLA(2) proteins, RT0590 and RT0522. Sequence analysis of RT0522 and RT0590 reveals the presence of the conserved motifs essential for PLA(2) activity. Transcriptional analysis indicates that RT0522, but not RT0590, is transcribed at all stages of intracellular growth of R. typhi in Vero cells. The differential gene expression pattern of RT0522 at various stages of growth suggests its potential role during R. typhi infection of host cells. In silico, RT0522 is predicted to be noncytoplasmic and its gene does not encode a recognizable signal peptide sequence. However, our data indicate that RT0522 is secreted into the host cytoplasm. In addition, we observe that RT0522 protein expression is cytotoxic to both yeast and Vero cells. Importantly, we demonstrate that recombinant RT0522 possesses phospholipase A activity that requires a eukaryotic host cofactor for activation. Both cytotoxicity and phospholipase A activity associated with RT0522 were reduced by PLA(2) inhibitors. Site-directed mutagenesis of predicted catalytic Ser/Asp residues of RT0522 also eliminates cytotoxicity and phospholipase A activity. To our knowledge, RT0522 is the first protein identified from Rickettsia typhi with functional phospholipase A activity.