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Pathogenic members of the Rickettsia genus are Gram- negative, obligate, intracellular bacteria that have a life cycle which involves both an arthropod vector and a vertebrate host1–3 (FIG. 1). Rickettsiae are classified into four groups based on their biological, genetic and anti- genic characteristics4: the spotted fever group (SFG), typhus group, transitional group and ancestral group. SFG rickettsiae include highly pathogenic organisms, such as tick-transmitted Rickettsia rickettsii (Rocky Mountain spotted fever (RMSF))5,6, Rickettsia conorii (Mediterranean spotted fever)1,2,7, Rickettsia africae (African tick-bite fever)8,9, Rickettsia parkeri (mild- to-moderate spotted fever rickettsiosis, found in North and South America)10,11, Rickettsia slovaca (tick-borne lymphadenopathy)12,13, Rickettsia sibirica (North Asian tick typhus and lymphangitis-associated rickettsiosis)14, Rickettsia honei (found in Australia and Southeast Asia)15, Rickettsia japonica (found in Japan and Korea)16,17 and the apparently harmless Rickettsia montanensis, Rickettsia peacockii and Rickettsia rhipicephali2,18,19. The typhus group includes the highly pathogenic Rickettsia prowazekii (epidemic typhus) and Rickettsia typhi (murine typhus)20–23. The ancestral group includes Rickettsia bellii24 and Rickettsia canadensis25; whether these species are pathogens is unknown. The transitional group comprises Rickettsia akari (rickett- sialpox)26, Rickettsia australis (Queensland tick typhus)27 and Rickettsia felis (flea-borne spotted fever)28 (TABLE 1). Rickettsia phylogeny has been addressed by sequence analyses of different genes, varying from housekeeping  genes, which are useful for distinguishing distinct strains, to genes that are under evolutionary pressure, such as

those that encode variable immunodominant outer- membrane proteins. This phylogenetic analysis has sub- stantially affected the proposed taxonomy of rickettsiae. However, rickettsial taxonomy remains a controversial subject, owing to the absence of a universal consensus on those criteria that should be used for the designation of species (Box 1).

Rickettsiosis can present with an array of clinical signs and symptoms6–9,11–16,29–31. Highly lethal RMSF29,30,32,33 is characterized by headache, fever, myalgia, nausea and vomiting early in the illness; however, if untreated, severe injury can develop that sometimes progress to multi-organ failure. Systemic vascular infection in RMSF results in encephalitis, which leads to stupor, coma and seizures, interstitial pneumonia, non-cardiogenic pul- monary oedema and adult respiratory distress syndrome. In severe cases, hypovolaemia and hypotensive  shock result in acute renal failure. Infection of a network of endothelial cells at the site of tick or mite inoculation of most SFG rickettsiae is followed by local dermal and epidermal necrosis that forms an eschar31. Disseminated infection, further injury to the vascular endothelium and infiltration of perivascular mononuclear cells leads to vasodilation, an increase in fluid leakage into the inter- stitial space and a characteristic rash. Epidemic typhus, which moulded world history for five centuries, is char- acterized by fever, headaches, mental confusion and a rash22,34 (Box 2). Similar to RMSF, epidemic typhus can develop into life-threatening conditions in previously healthy, immunocompetent individuals, unless they are treated early with an appropriate antibiotic. However, unlike RMSF, R. prowazekii causes latent infection in

Department of Pathology, University of Texas, Medical Branch, Galveston, 77555-0609 Texas, USA. Correspondence to D.H.W. e-mail: [email protected] doi:10.1038/nrmicro1866

Housekeeping gene A gene that is involved in the  basic functions that are  required for normal cell  metabolism and is  constitutively expressed.

Hypovolaemia Decreased blood volume —  more specifically, a decrease in  the volume of blood plasma.

Hypotensive shock Shock in which blood pressure  is lower than normal and does  not supply blood to the organs.

Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events David H. Walker and Nahed Ismail

Abstract | Rickettsiae cause some of the most severe human infections, including epidemic typhus and Rocky Mountain spotted fever. Substantial progress has been made in research into the genomics, vector relationships, pathogenesis and immunity of these obligate, intracellular, arthropod-transmitted bacteria. This Review summarizes our understanding of the early and late events in pathogenesis and immunity, modulation of the host response to rickettsial infection by the vector, host defence, virulence mechanisms and rickettsial manipulation of host cells.

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Infected human

Infected eggs

Infected nymphal tick

Uninfected ticks

Infected female, adult tick

Tick moults; trans-stadial maintenance

Infected larval tick

Transovarial transmission

Infected rodents

Uninfected rodents

Nature Reviews | Microbiology

convalescent individuals, and recrudescence of latent R. prowazekii infection results in Brill–Zinsser disease, which is characterized by fever, rash and less-severe ill- ness that nevertheless can infect feeding lice and ignite an epidemic35,36.

Interest in the pathogenesis of R. prowazekii and R. rickettsii has increased following their classifica- tion as select agents and as category B and C agents of potential bioterrorism, respectively, by the united States Centers for Disease Control and Prevention37 (see Further information). These pathogens are highly infectious agents that are easily disseminated and cause high morbidity and fatal disease, and thus require specific improvement in diagnostic tests and disease surveillance1,6 — the use of R. prowazekii as a biological weapon was initiated by the Soviet union in the 1930s and Japan during the Second world war21,38.

The body of knowledge of rickettsial pathogenesis and immunity is based on disseminated infection of endothelial cells, the principal target host cells for rick- ettsiae. Human infections have rarely been investigated until the middle or late, and often fatal, stages of the ill- ness. The best animal models for SFG rickettsioses use R. conorii and R. australis or typhus group rickettsiosis (using R. typhi) in susceptible mice that are inoculated intravenously: these manifest systemic endothelial-cell infection and characteristic pulmonary and cerebral lesions that recapitulate the clinical and pathological manifestations of the disease in humans. Infections of guinea pigs with R. rickettsii and R. prowazekii provide models of RMSF and epidemic typhus, respectively.

This Review highlights how the arthropod host acquires, maintains and transmits rickettsiae, the initial steps in pathogenesis and the subsequent interaction of the bacteria with cells in the endothelium, the main target cells. These events include: rickettsial entry, phagosomal escape, actin-based motility, cell-to-cell spread and the induction of cell injury. Regarding the host immune response to rickettsial infection, we will address innate and acquired immunity, with emphasis

on recent data that illustrate the interaction of rick- ettsiae with dendritic cells (DCs). we also highlight some of the potential immunomodulatory effects of tick saliva on host defences and the immune response against Rickettsia spp.

Acquisition, interference and immunomodulation Acquisition. vertebrate hosts are infected with rick- ettsiae via direct inoculation by a feeding tick or mite or by scratching infected louse or flea faeces into their skin. Ticks with hard exoskeletal chitin are vectors and reservoirs for SFG rickettsiae. The principal vectors of RMSF in the united States are Dermacentor variabilis and Dermacentor andersoni (TABLE 1), which are most active during the late spring and summer, when RMSF peaks. Epidemic typhus (Box 2) caused by R. prowazekii is associated with cold weather and lack of hygiene22, and has re-emerged in louse-infested populations. Humans in endemic regions, as well as the eastern flying squirrel Glaucomys volans volans20,39, and its flea and louse in the united States, and ticks in Mexico and Africa are the known reservoirs of R. prowazekii40,41.

Interference. Infection of a tick with one SFG rick- ettsial species seems to interfere with infection by a second SFG rickettsial species. It was suggested that rickettsial infection of tick ovaries might alter the molecular-expression profiles of the oocytes and cause interference or blocking of the second infection42,43. This process of rickettsial ‘interference’ might affect the frequency and distribution of different pathogenic rickettsiae, and could explain the limited distribution of virulent R. rickettsii in the eastern part of the Bitterroot valley, Montana, uSA, where they infect less than 1% of wood ticks42,44. The low infection rate of R. rickettsii is attributed to the high infection rate of female wood ticks (D. andersoni) in the eastern, but not western, Bitterroot valley with non-virulent rickettsiae, particu- larly R. peacockii (70% in the eastern compared with 4% in the western side of Bitterroot valley)19,44. In most

Figure 1 | The life cycle of tick-borne rickettsiae. Spotted-fever-group rickettsiae are maintained in nature by transovarial and trans-stadial transmission in ticks and horizontal transmission to uninfected ticks that feed on rickettsemic rodents and other animals.

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Superinfection Infection by a microorganism  of a cell that is already infected  by another microorganism.

Transovarial Passage of parasites or  infective agents from the  maternal body to eggs within  the ovaries and subsequently  to the larvae that hatch from  the eggs.

geographic locations, fewer than 0.1% of Dermacentor spp. ticks carry R. rickettsii45. These data correspond to the focality of RMSF in the west side of the valley, where most human cases result from exposure to west-side ticks (D. andersoni). unlike pathogenic R. rickettsii, which is lethal for ticks46 and highly virulent in guinea pigs47, infection with R. peacockii does not cause a reduction in tick viability, and might even be beneficial for tick hosts by antagonizing superinfection of ovarian tissues by R. rickettsii.

Ticks acquire SFG rickettsial species through trans­ ovarial transmission (adult female to egg) and trans­stadial

passage (egg to larva to nymph to adult), and by hori- zontal acquisition during feeding on a rickettsemic host. Most SFG rickettsiae are probably maintained in nature by all these mechanisms (FIG. 1). Therefore, the adverse effect of virulent R. rickettsii on the viability of adult ticks and maintenance of Rickettsia spp. in nature are probably balanced by the feeding of susceptible ticks on a rickettsemic host, which functions as an amplifying reservoir for rickettsiae. In fact, it has been shown that despite the high mortality of experimentally infected ticks, many larvae that acquire rickettsiae during feeding survive and are capable of transmitting the infection as

Table 1 | Rickettsial diseases in humans

Disease organism Arthropod vector

life cycle geographic area

Eschar rash regional lymph- adenopathy

Symptoms or fever

mortality rate*

Tick-transmitted spotted fevers

Rocky Mountain spotted fever

Rickettsia rickettsii

Dermacentor variabilis, Dermacentor andersoni, Rhipicephalus sanguineus, Amblyomma cajennense and Amblyomma aureolatum

Transovarian in ticks and rodent ticks

Western hemisphere

Rare Yes No Yes High

Boutonneuse fever

Rickettsia conorii

R. sanguineus and Rhipicephalus pumilio

Transovarian in ticks

Southern Europe, Africa and southern Asia

Frequent Maculo- papular

No Yes Mild to moderate

African tick- bite fever

Rickettsia africae

Amblyomma hebraeum and Amblyomma variegatum

Transovarian in ticks

Africa and the West Indies

Frequent and often multiple

Papular or vesicular; often sparse or absent

Yes Yes None reported

Maculatum disease

Rickettsia parkeri

Amblyomma maculatum and Amblyomma triste

Ticks Western hemisphere

Yes Often Yes Yes None reported

Flea-transmitted diseases

Flea-borne spotted fever

Rickettsia felis

Ctenocephalides felis

Transovarian in the cat flea

Worldwide Sometimes Sometimes No Yes None reported

Murine typhus

Rickettsia typhi

Xenopsylla cheopis and Ctenocephalides felis

Rat-flea for X. cheopis and Opossumflea for C. felis

Worldwide No Yes No Yes Low

Louse-transmitted disease

Epidemic typhus

Rickettsia prowazekii

Pediculus humanus humanus

Human louse Worldwide No Yes No Yes High

Epidemic typhus

R. prowazekii Fleas and lice of flying squirrels and Glaucomys volans volans

Flying- squirrel flea and louse ectoparasite

United States

No Yes No Yes Low

Mite-transmitted diseases

Rickettsialpox Rickettsia akari

Liponyssoides sanguinus

Transovarian in mites

Worldwide Yes Yes Yes Yes None reported

*High mortality is >15%; moderate mortality is 7–15%; mild-to-moderate mortality is 2–7% and low mortality is ≤1%.

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Trans-stadial Passage of a microorganism  from one developmental stage  (stadium) of the host to a  subsequent stage (or stages).

Haemostatic Stops blood flow.

nymphs. This suggests that nymphs are a crucial link for R. rickettsii maintenance and transmission between ver- tebrates. Importantly, colonies of R. rickettsii-infected ticks have been observed to maintain the infection with- out overt deleterious effects for several generations. The pathological effect of rickettsiae on ticks would explain the occurrence of RMSF in endemic regions, despite the low prevalence of naturally infected adult ticks45,46.

Immune modulation. Studies of the virulence of rickett- siae within their tick vector revealed that feeding ticks or incubating them at 37°C for 24–48 hours before their inoculation onto non-immune guinea pigs results in severe disease, compared with asymptomatic infection following inoculation of guinea pigs with infected ticks that were maintained at 4°C or starved for a prolonged period48. This observation, described as the reactivation phenomenon by Parker and Spencer44, refers to changes in the virulence of rickettsiae that are linked to the physiological status of the ticks.

As an immune evasion or modulation mechanism that allows the ticks to feed for several days or weeks, ticks inoculate their saliva with anti-haemostatic com- ponents that are crucial for the enhancement of blood feeding and salivary immunomodulatory components that enhance pathogen transmission and prevent the host from rejecting the ticks49–53. For example, the saliva of ticks inhibits neutrophil function52, interferes with the complement system49–51, natural killer (NK) cell and mac- rophage activity54, decreases the production of cytokines, such as interleukin-12 (Il-12) and interferon-γ (IFN-γ), and decreases T-cell proliferation55,56. Tick-infested mice do not develop resistance to further infestations with Rhipicephalus sanguineus, and the immune response in infested mice exhibits a T helper 2 (TH2)-type pattern

56,57. Tick saliva might influence T-cell-effector functions through its initial interaction with professional antigen- presenting cells, namely DCs58. Such initial interactions can subsequently influence the differentiation towards either a TH2-cell phenotype (an ineffective acquired immune response against intracellular pathogens such as Rickettsia spp.) or an immunosuppressive phenotype58,59. Indeed, the addition of tick saliva to bone-marrow- derived DCs inhibits their maturation by decreasing the expression of co-stimulatory (CD40, CD80 and CD86)

and adhesion (CD54) molecules58,59. Furthermore, the maturation of DCs that is stimulated by lipopolysac- charide in the presence of tick saliva results in reduced expression of co-stimulatory molecules and reduced production of Il-12, but not immunosuppressive Il-10. More importantly, DCs cultured with tick saliva are inefficient in the induction and activation of antigen- specific, cytokine-producing T cells58,59. As discussed below, fully mature DCs are crucial for induction of an effective TH1 response against Rickettsia spp. Therefore, it is possible that suppression of DC maturation by tick saliva during the initial stages of rickettsial infection could interfere with their co-stimulatory and antigen- presentation functions. Such suppression of DC matu- ration would adversely influence the acquired immune response against tick-transmitted Rickettsia spp., thereby leading to increased host susceptibility to severe and fatal rickettsial disease. However, because the tick host is an important component in the life cycle of rickettsiae, fur- ther studies are required to address important questions related to vector biology and disease pathogenesis, such as whether tick saliva enhances Rickettsia spp. infectivity during natural transmission and whether pre-exposure to saliva from uninfected ticks that generates immunity to saliva protects the vertebrate host, particularly in endemic areas, from natural tick-transmitted rickett- sial infection. If immunity to salivary components can

Box 1 | Rickettsial taxonomy

Despite the major advances in serotyping and molecular genotyping of rickettsial isolates from defined geographic locations, Rickettsia taxonomy is still an evolving field. Novel Rickettsia isolates have been described in recent years, with the overenthusiastic designation of many new species, which vary much less from one another than the species of other bacterial genera107. The issue is not whether the isolates can be distinguished from one another, but rather whether the differences merit designation at the taxonomic level of species or even subspecies. Historically, different species of prokaryotic pathogens were defined based on the diseases that they caused, regardless of other ecological or evolutionary considerations. However, the clinical manifestations of most rickettsioses are neither specific to a particular agent nor to a geographic distribution. Thus, a consensus of taxonomic criteria has yet to be achieved for Rickettsia. A proposal to adopt the genetic-diversity limits of previously named Rickettsia species for several convenient, but not uniformly appropriate, genes is an approach that has been specifically rejected by experts in prokaryotic taxonomy108. In our opinion, if the classification of Rickettsia were congruent with other intracellular bacteria, many of the current species names would be designated as subspecies and scientists would recognize important new isolates as distinct strains without needing a new species name.

Box 2 | Epidemic typhus

Epidemic typhus determined the outcome of European wars from the sixteenth century to the twentieth century. In Russia, during the First World War, the revolution and its aftermath, 30 million people suffered from typhus fever and 3 million of them died. The first description of typhus originated from the siege of Naples in 1528, but the role of the human body louse as a vector was not recognized until 1909, for which Charles Nicolle was awarded a Nobel Prize. Among the enigmas of typhus, two of the most intriguing questions are: in what cells and organs of the body does latent Rickettsia prowazekii reside during the period after recovery from the acute infection; and what factors and mechanisms are responsible for the reactivation of infection that leads to rickettsemia and potential louse-borne spread of another epidemic?

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d

b

a

e

Endothelial cells

Actin filaments

Actin monomers

Cdc42

Phagosome

Rickettsia OmpB

Lysis of phagosome

Cbl

Ku70

Ub

PI3-K

Cbl

N-WASP

RickA

Arp2/3

c

Vasoactive Causes constriction or dilation  of blood vessels.

protect against natural rickettsial infection, an effective anti-rickettsial vaccine could be designed that contains tick salivary proteins that act as an adjuvant with specific rickettsial antigens.

Rickettsia–endothelial cell interactions Rickettsial entry. R. conorii ompB binds specifically to Ku70 (FIG. 2), a component of the DNA-dependent pro- tein kinase60. The binding and recruitment of Ku70 to the plasma membrane are important events in the entry of R. conorii into non-phagocytic mammalian cells60. Although nuclear Ku70 is translocated to the cytoplasm and plasma membrane, where it inhibits apoptosis and mediates homologous and heterologous cell adhesion and fibronectin binding, it has been proposed that the presence of Ku70 within lipid rafts might have an impor- tant role in the signal transduction that leads to induced phagocytosis. The role of cholesterol as an essential component of the membrane receptor that binds to R. prowazekii was described previously61. Similar to other intracellular pathogens, such as Listeria monocytogenes, the entry of R. conorii into non-phagocytic cells is dependent on membrane cholesterol. Ku70 is present within lipid microdomains that are enriched in lipid-raft components. The association of Ku70 with lipid micro- domains and its binding to R. conorii suggest that Ku70 has an important role in cholesterol-dependent bacterial entry60. Although the exact mechanism by which Ku70 supports the entry of R. conorii into non-phagocytic cells remains unclear, the binding of R. conorii ompB to Ku70 might activate membrane Ku70, which is postulated to lead to the activation of a cascade of signalling events, including the small GTPase, Cdc42, phosphoinositidyl- 3-kinase, src-family tyrosine kinases and the tyrosine phosphorylation of focal adhesion kinase62. These signalling events are known to be strongly associated with β1-integrin activation and bacterial entry63 (FIG. 2). Similar to the entry of L. monocytogenes into its host cells, R. conorii infection stimulates the ubiquitination

of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii-entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination60,62. The binding of Ku70 to ompB and the role of Ku70 in bacterial entry into host cells correlate with the decreased expression of ompB that is associated with reduced virulence of R. rickettsii str. Iowa64 and the observation that anti-ompB antibodies protect animals from an otherwise lethal challenge of R. conorii65–67. However, it is possible that SFG rick- ettsial ompA or other unidentified rickettsial outer- membrane proteins also mediate adhesion by binding to unknown receptors68.

Rickettsial diseases and endothelial pathogenesis. Most of the clinical characteristics of rickettsial diseases are attributed to disseminated infection of the endothelium, where they grow and stimulate oxidative stress, thereby causing injury to the endothelial cells. Severe morbidity and mortality of RMSF are due to effects such as cerebral oedema and non-cardiogenic pulmonary oedema. The most prominent pathophysiological effects of rickettsial infection of endothelial cells include: an increase in vas- cular permeability; generalized vascular inflammation; oedema; increased leukocyte–endothelium interactions; and release of powerful vasoactive mediators that pro- mote coagulation and pro-inflammatory cytokines69,70. Evidence that supports a pro-coagulant and pro-inflam- matory phenotype of the host response is provided by studies of cultured endothelial cells in which rickettsial infection causes increased expression of tissue factor, thrombomodulin plasminogen-activator inhibitor 1, Il-1, Il-6, Il-8 and E-selectin70–73. Increased plasma levels of von willebrand factor that are associated with increased levels of inflammatory cytokines, such as Il-6, have also been detected in patients with African tick-bite fever and Mediterranean spotted fever69,74. Prostaglandins and leukotrienes are crucial vasoactive modulators of vas- cular tone and permeability that are potential mediators

Figure 2 | Host cell interactions of rickettsiae. a | Spotted-fever-group rickettsiae attach to Ku70 on the surface of human target cells (the endothelium) via outer-membrane-protein B (OmpB) and to an unknown receptor via outer- membrane-protein A. b | Cbl ubiquitinates Ku70 (REF. 61), and signal-transduction events that involve Cdc42, protein tyrosine kinase, phosphatidylinositol 3′-kinase (PI3-K) and Src-family kinases activate the Arp 2/3 complex to induce cytoskeletal actin to phagocytose the rickettsia62. c | Membranolytic phospholipase D and haemolysin C mediate rickettsial phagosomal escape83. d | RickA-stimulated activation of Arp2/3-mediated polymerization of host actin propels the bacterium through the cytosol and into filopodia. e | Rickettsiae are then either released from filopodia extracellularly or spread into the adjacent cell84–88. Cbl, family of ubiquitin ligases; N-WASP, neural Wiskott–Aldrich syndrome protein; Ub, ubiquitin.

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of microvascular injury and vasculitis in rickettsial infection75,76. These vasoactive substances are generally generated by an inducible isoenzyme cyclooxygenase (CoX)75. Transcriptional activation of host endothe- lial cells in response to stimulation with R. rickettsii or R. conorii involves rapid regulation of CoX2 expression and inhibition of CoX2 activity during infection, which leads to decreased levels of secreted prostaglandins. As a regulatory mechanism that prevents the development of extensive vascular injury, endothelial cells that are infected with R. rickettsii produce haem oxygenase, an antioxidant, anti-inflammatory and vasoprotective enzyme that controls CoX2 activity77. The production of this antioxidant mechanism by R. rickettsii-infected cells seems to be dependent on several factors, including: dose and kinetics of rickettsial infection; viability of the host cells; de novo protein synthesis by host cells; adhesion and entry of Rickettsia spp. to the host cell membrane; rickettsial replication; and viability77. viability is prob- ably influenced by the host immune status, as well as whether patients are treated with doxycycline at an early stage of infection. Nevertheless, the balance between the production of vasopro