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Rickettsiae are obligate intracellular pathogens that induce their uptake into nonphagocytic cells; however, the events instigating this process are incompletely understood. Importantly, diverse Rickettsia species are predicted to utilize divergent mechanisms to colonize host cells, as nearly all adhesins and effectors involved in host cell entry are differentially encoded in diverse Rickettsia species. One particular effector, RalF, a Sec7 domain-containing protein that functions as a guanine nucleotide exchange factor of ADP-ribosylation factors (Arfs), is critical for Rickettsia typhi (typhus group rickettsiae) entry but pseudogenized or absent from spotted fever group rickettsiae. Secreted early during R. typhi infection, RalF localizes to the host plasma membrane and interacts with host ADP-ribosylation factor 6 (Arf6). Herein, we demonstrate that RalF activates Arf6, a process reliant on a conserved Glu within the RalF Sec7 domain. Furthermore, Arf6 is activated early during infection, with GTP-bound Arf6 localized to the R. typhi entry foci. The regulation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which generates PI(4,5)P 2 , by activated Arf6 is instrumental for bacterial entry, corresponding to the requirement of PI(4,5)P 2 for R. typhi entry. PI(3,4,5)P 3 is then synthesized at the entry foci, followed by the accumulation of PI(3)P on the short-lived vacuole. Inhibition of phosphoinositide 3-kinases, responsible for the synthesis of PI(3,4,5)P 3 and PI(3)P, negatively affects R. typhi infection. Collectively, these results identify RalF as the first bacterial effector to directly activate Arf6, a process that initiates alterations in phosphoinositol metabolism critical for a lineage-specific Rickettsia entry mechanism.

Rickettsia species are obligate intracellular bacteria with both conserved and lineage-specific strategies for invading and surviving within eukaryotic cells. One variable component of Rickettsia biology involves arthropod vectors: for instance, typhus group rickettsiae are principally vectored by insects (i.e., lice and fleas), whereas spotted fever group rickettsiae are exclusively vectored by ticks. For flea-borne Rickettsia typhi , the etiological agent of murine typhus, research on vertebrate host biology is facilitated using cell lines and animal models. However, due to the lack of any stable flea cell line or a published flea genome sequence, little is known regarding R. typhi biology in flea vectors that, importantly, do not suffer lethality due to R. typhi infection. To address if fleas combat rickettsial infection, we characterized the cat flea ( Ctenocephalides felis ) innate immune response to R. typhi . Initially, we determined that R. typhi infects Drosophila cells and increases antimicrobial peptide (AMP) gene expression, indicating immune pathway activation. While bioinformatics analysis of the C. felis transcriptome identified homologs to all of the Drosophila immune deficiency (IMD) and Toll pathway components, an AMP gene expression profile in Drosophila cells indicated IMD pathway activation upon rickettsial infection. Accordingly, we assessed R. typhi -mediated flea IMD pathway activation in vivo using small interfering RNA (siRNA)-mediated knockdown. Knockdown of Relish and Imd increased R. typhi infection levels, implicating the IMD pathway as a critical regulator of R. typhi burden in C. felis . These data suggest that targeting the IMD pathway could minimize the spread of R. typhi , and potentially other human pathogens, vectored by fleas.

Reductive genome evolution has purged many metabolic pathways from obligate intracellular Rickettsia (Alphaproteobacteria; Rickettsiaceae). While some aspects of host-dependent rickettsial metabolism have been characterized, the array of host-acquired metabolites and their cognate transporters remains unknown. This dearth of information has thwarted efforts to obtain an axenic Rickettsia culture, a major impediment to conventional genetic approaches. Using phylogenomics and computational pathway analysis, we reconstructed the Rickettsia metabolic and transport network, identifying 51 host-acquired metabolites (only 21 previously characterized) needed to compensate for degraded biosynthesis pathways. In the absence of glycolysis and the pentose phosphate pathway, cell envelope glycoconjugates are synthesized from three imported host sugars, with a range of additional host-acquired metabolites fueling the tricarboxylic acid cycle. Fatty acid and glycerophospholipid pathways also initiate from host precursors, and import of both isoprenes and terpenoids is required for the synthesis of ubiquinone and the lipid carrier of lipid I and O-antigen. Unlike metabolite-provisioning bacterial symbionts of arthropods, rickettsiae cannot synthesize B vitamins or most other cofactors, accentuating their parasitic nature. Six biosynthesis pathways contain holes (missing enzymes); similar patterns in taxonomically diverse bacteria suggest alternative enzymes that await discovery. A paucity of characterized and predicted transporters emphasizes the knowledge gap concerning how rickettsiae import host metabolites, some of which are large and not known to be transported by bacteria. Collectively, our reconstructed metabolic network offers clues to how rickettsiae hijack host metabolic pathways. This blueprint for growth determinants is an important step toward the design of axenic media to rescue rickettsiae from the eukaryotic cell., IMPORTANCE A hallmark of obligate intracellular bacteria is the tradeoff of metabolic genes for the ability to acquire host metabolites. For species of Rickettsia, arthropod-borne parasites with the potential to cause serious human disease, the range of pilfered host metabolites is unknown. This information is critical for dissociating rickettsiae from eukaryotic cells to facilitate rickettsial genetic manipulation. In this study, we reconstructed the Rickettsia metabolic network and identified 51 host metabolites required to compensate patchwork Rickettsia biosynthesis pathways. Remarkably, some metabolites are not known to be transported by any bacteria, and overall, few cognate transporters were identified. Several pathways contain missing enzymes, yet similar pathways in unrelated bacteria indicate convergence and possible novel enzymes awaiting characterization. Our work illuminates the parasitic nature by which rickettsiae hijack host metabolism to counterbalance numerous disintegrated biosynthesis pathways that have arisen through evolution within the eukaryotic cell. This metabolic blueprint reveals what a Rickettsia axenic medium might entail.

Many prokaryotes utilize type IV secretion systems (T4SSs) to translocate substrates (e.g. nucleoprotein, DNA, protein) across the cell envelope, and/or to elaborate surface structures (i.e. pili or adhesins). Among eight distinct T4SS classes, P-T4SSs are typified by the Agrobacterium tumefaciens vir T4SS, which is comprised of 12 scaffold components (VirB1-VirB11, VirD4). While most P-T4SSs include all 12 Vir proteins, some differ from the vir archetype by either containing additional scaffold components not analogous to Vir proteins or lacking one or more of the Vir proteins. In a special case, the Rickettsiales vir homolog (rvh) P-T4SS comprises unprecedented gene family expansion. rvh contains three families of gene duplications (rvhB9, rvhB8, rvhB4): RvhB9,8,4-I are conserved relative to equivalents in other P-T4SSs, while RvhB9,8,4-II have evolved atypical features that deviate substantially from other homologs. Furthermore, rvh contains five VirB6-like genes (rvhB6a-e), which are tandemly arrayed and contain large N- and C-terminal extensions. Our work herein focuses on the complexity underpinned by rvh gene family expansion. Furthermore, we describe an RvhB10 insertion, which occurs in a region that forms the T4SS pore. The significance of these curious properties to rvh structure and function is evaluated, shedding light on a highly complex T4SS.

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.

Bacteria of the order Rickettsiales are Gram-negative, small, rod-shaped, and coccoid, with all described species existing as obligate intracellular parasites of a wide range of eukaryotic organisms. This chapter primarily focuses on the family Rickettsiaceae , which is currently comprised well-studied species in the genera Rickettsia and Orientia, as well as candidate species in several provisional genera that are poorly understood. The genus Rickettsiella , originally described as a member of Rickettsiales, comprises a group of intracellular pathogens of diverse arthropod species. Family Holosporaceae was first added to the Rickettsiales in 2001. The first sequenced genome of a rickettsial species, R. prowazekii , revealed numerous pseudogenes and a highly reduced genome relative to facultative intracellular and free-living bacterial species. Throughout modern history, the morbidity and mortality associated with rickettsial infections has been underestimated, primarily due to misdiagnosis. In the United States, a major concern is the changing ecology of endemic typhus, hereafter referred to as murine typhus.

The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’.

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., Author Summary Typhus Group (TG) rickettsiae include Rickettsia typhi and R. prowazekii, the etiological agents of murine and epidemic typhus, respectively. The rickettsial obligate intracellular life cycle complicates conventional approaches to genetic manipulation, impeding characterization of molecules underlying pathogenesis. Phospholipase A2 (PLA2) enzymes are factors that likely mediate such processes as host entry, phagosomal escape, and host lysis for cell-to-cell spread. Here, we demonstrate across 46 Rickettsia genomes that genes encoding two PLA2 enzymes (pat1 and pat2) have divergent evolutionary histories and discordant selective constraints, with pat2 deleted from most other non-TG rickettsiae genomes. As previously determined for Pat2, we show here that R. typhi Pat1 is secreted into the host cell cytoplasm during infection and requires a host cofactor for enzymatic activity. Like Pat2, recombinant Pat1 protein is cytotoxic to yeast cells. Pretreatment of R. typhi with anti-Pat1 or anti-Pat2 antibody results in a significant decrease in rickettsial infection, implicating roles for both Pat1 and Pat2 during the early stage of R. typhi host cell infection. Collectively, our work suggests that R. typhi utilizes two evolutionary divergent phospholipases during its intracellular life cycle, a mechanism distinguishing TG rickettsiae-associated cell biology and pathogenesis from other rickettsioses, particularly those associated with Spotted Fever Group pathogens.

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 R ickettsia a nkyrin r epeat p rotein 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.

International audience; In contrast to 15 or more validated and/or proposed tick-borne spotted fever group species, only three named medically important rickettsial species are associated with insects. These insect-borne rickettsiae are comprised of two highly pathogenic species, Rickettsia prowazekii (the agent of epidemic typhus) and R. typhi (the agent of murine typhus), as well as R. felis, a species with unconfirmed pathogenicity. Rickettsial association with obligate hematophagous insects such as the human body louse (R. prowazekii transmitted by Pediculus h. humanus) and several flea species (R. typhi and R. felis, as well as R. prowazekii in sylvatic form) provides rickettsiae the potential for further multiplications, longer transmission cycles and rapid spread among susceptible human populations. Both human body lice and fleas are intermittent feeders capable of multiple blood meals per generation, facilitating the efficient transmission of rickettsiae to several disparate hosts within urban/rural ecosystems. While taking into consideration the existing knowledge of rickettsial biology and genomic attributes, we have analyzed and summarized the interacting features that are unique to both the rickettsiae and their vector fleas and lice. Furthermore, factors that underlie rickettsial changing ecology, where native mammalian populations are involved in the maintenance of rickettsial cycle and transmission, are discussed.

This unit includes protocols for the laboratory maintenance of the obligate intracellular bacterium Rickettsia rickettsii, including propagation in mammalian cell cultures, as well as isolation, counting, and storage procedures. Regulations for working with R. rickettsii in biosafety level 3 containment are also discussed.

Vero cells are derived from the kidney of an African green monkey, and are one of the more commonly used mammalian continuous cell lines in microbiology, and molecular and cell biology research. This unit includes protocols for the growth and maintenance of Vero cell lines in a research laboratory setting.