Effect of Temperature on Host Preference in Two Lineages of the Brown Dog Tick, Rhipicephalus sanguineus.

Rhipicephalus sanguineus is a species complex of ticks that vector disease worldwide. Feeding primarily on dogs, members of the complex also feed incidentally on humans, potentially transmitting disease agents such as Rickettsia rickettsii, Rickettsia conorii, and Ehrlichia species. There are two genetic Rh. sanguineus lineages in North America, designated as the temperate and tropical lineages, which had occurred in discrete locations, although there is now range overlap in parts of California and Arizona. Rh. sanguineus in Europe are reportedly more aggressive toward humans during hot weather, increasing the risk of pathogen transmission to humans. The aim of this study was to assess the impact of hot weather on choice between humans and dog hosts among tropical and temperate lineage Rh. sanguineus individuals. Ticks in a two-choice olfactometer migrated toward a dog or human in trials at room (23.5°C) or high temperature (38°C). At 38°C, 2.5 times more tropical lineage adults chose humans compared with room temperature, whereas temperate lineage adults demonstrated a 66% reduction in preference for dogs and a slight increase in preference for humans. Fewer nymphs chose either host at 38°C than at room temperature in both lineages. These results demonstrate that risk of disease transmission to humans may be increased during periods of hot weather, where either lineage is present, and that hot weather events associated with climatic change may result in more frequent rickettsial disease outbreaks.

Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii.

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64-32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

Fiebre manchada en Argentina. Descripción de dos casos clínicos / Spotted fever in Argentina. Description of two clinical cases

Resumen Las rickettsiosis son enfermedades zoonóticas transmitidas por artrópodos vectores, que en Argentina presentan 2 escenarios epidemiológicos diferenciados. Uno, en las yungas de Salta y Jujuy, involucra vectores pertenecientes al «complejo Amblyomma cajennense¼ (A. sculptum y A. toneliae) y a Rickettsia rickettsii como agente etiológico. En este escenario la forma clínica de la enfermedad se conoce como fiebre manchada (FM) y se presenta con manifestaciones cutáneas y sistémicas graves. El otro escenario incluye 2 zonas: una la del Delta del Río Paraná y Bahía de Samborombón, donde Amblyomma triste actúa como vector; otra, las provincias de Córdoba, La Rioja, San Luis y La Pampa, donde el vector es Amblyomma tigrinum. En este segundo escenario Rickettsia parkeri es el agente causal, y la FM se manifiesta con un cuadro benigno y autolimitado. En este trabajo describimos un caso fatal de FM por R. rickettsii en El Tunal, Salta, y el primer caso de FM por R. parkeri en San Juan.

Abstract Rickettsioses are zoonotic tick-borne diseases. In Argentina, there are two epidemiological scenarios: jungle of Salta and Jujuy, involving vectors from the "Amblyomma cajennense Complex" (A. sculptum, and A. toneliae) and Rickettsia rickettsii as the main etiological agent; and the second scene to Delta del Rio Paraná and Samborombón Bay, where Amblyomma triste acts as a vector; and the provinces of Córdoba, La Rioja, San Luis and La Pampa where Amblyomma tigrinum is the vector. In this second scenario, Rickettsia parkeri is the causal agent. The spotted fever (SF) due to R. rickettsii is responsible for a severe cutaneous and systemic disease. Contrarily, R. parkeri produces benign and self-limited clinical manifestation. Here we describe a fatal SF case by R. rickettsii, in El Tunal, Salta and the first SF case due to R. parkeri in San Juan.

Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo.

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Hosts mobility and spatial spread of Rickettsia rickettsii.

There are a huge number of pathogens with multi-component transmission cycles, involving amplifier hosts, vectors or complex pathogen life cycles. These complex systems present challenges in terms of modeling and policy development. A lethal tick-borne infectious disease, the Brazilian Spotted Fever (BSF), is a relevant example of that. The current increase of human cases of BSF has been associated with the presence and expansion of the capybara Hydrochoerus hydrochaeris, amplifier host for the agent Rickettsia rickettsii and primary host for the tick vector Amblyomma sculptum. We introduce a stochastic dynamical model that captures the spatial distribution of capybaras and ticks to gain a better understanding of the spatial spread of the R. rickettsii and potentially predict future epidemic outcomes. We implemented a reaction-diffusion process in which individuals were divided into classes denoting their state with respect to the disease. The model considered bidirectional movements between base and destination locations limited by the carrying capacity of the environment. We performed systematic stochastic simulations and numerical analysis of the model and investigate the impact of potential interventions to mitigate the spatial spread of the disease. The mobility of capybaras and their attached ticks was significantly influenced by the birth rate of capybaras and therefore, disease propagation velocity was higher in places with higher carrying capacity. Some geographical barriers, generated for example by riparian reforesting, can impede the spatial spread of BSF. The results of this work will allow the formulation of public actions focused on the prevention of BSF human cases.

Exposure to Ticks and their Pathogens in Northeast Missouri.

While the prevalence of human pathogens has been quantified in ticks in Adair County, Missouri, the prevalence of residents acquiring tick-borne diseases and seeking medical treatment has not. A public survey (n=109) revealed that 96% of respondents reported finding attached ticks on their person; of these, 38% developed symptoms post tick bite; of these, 55% reported consultation with a health care provider. Overall, 89% of practitioners surveyed had treated at least one patient for tick-borne disease. Rocky Mountain spotted fever and Lyme disease were the most common illnesses diagnosed, however, the only confirmed cases reported by Missouri Department of Health and Senior Services from 2013-2017 were ehrlichiosis. Results from these surveys indicate that exposure to ticks is common and ehrlichiosis infections are likely underdiagnosed while Rocky Mountain spotted fever and Lyme disease are likely overdiagnosed.

The Rickettsial Ankyrin Repeat Protein 2 Is a Type IV Secreted Effector That Associates with the Endoplasmic Reticulum.

Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever, vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia ankyrin repeat protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system-dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith colocalizes with multilamellar membranous structures bearing markers of the endoplasmic reticulum (ER), whereas the Iowa protein shows no colocalization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith RARP-2 in R. rickettsii Iowa converts this avirulent strain's typically nonlytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith RARP-2 did not, however, restore the virulence of the Iowa strain in a guinea pig model, likely due to the multifactorial nature of rickettsial virulence.IMPORTANCE Members of the genus Rickettsia are obligate intracellular bacteria that exhibit a range of virulence from harmless endosymbionts of arthropods to the etiologic agents of severe disease. Despite the growing number of available genomes, little is known regarding virulence determinants of rickettsiae. Here, we have characterized an ankyrin repeat-containing protein, RARP-2, which differs between a highly virulent and an avirulent strain of R. rickettsii, the agent of Rocky Mountain spotted fever. RARP-2 is secreted by a type IV secretion system into the cytosol of the host cell, where it interacts with and manipulates the structure of the endoplasmic reticulum. RARP-2 from the avirulent strain is truncated by the loss of seven of 10 ankyrin repeat units but, although secreted, fails to alter ER structure. Recognition of those rickettsial factors associated with virulence will facilitate understanding of regional and strain-specific variation in severity of disease.

The Evaluation and Management of Rocky Mountain Spotted Fever in the Emergency Department: a Review of the Literature.

BACKGROUND: Rocky Mountain spotted fever (RMSF) is potentially deadly and can present subtly with signs and symptoms overlapping with other clinical conditions. Delayed diagnosis can be fatal. OBJECTIVE: This review provides an evidence-based summary of the current data for the evaluation and management of RMSF in the emergency department. DISCUSSION: RMSF occurs through transmission of Rickettsia rickettsii by an infected tick. Exposure in the United States occurs most commonly from April to September, and high-risk locations include wooded, shrubby, or grassy areas. Approximately half of patients with infection do not recall tick exposure. Symptoms can include fever, headache, photophobia, malaise, myalgias, and a petechial rash that begins on the wrists and ankles and spreads to the trunk. Rash may not occur in ≤15% of patients, and the classic triad of fever, headache, and rash is also not definitive. Laboratory evaluation may demonstrate hyponatremia, anemia, thrombocytopenia, abnormal liver enzymes, and elevated coagulation tests. Antibody testing can be helpful, but these results are not typically available to the emergency clinician. Doxycycline is the treatment of choice in adults, children, and pregnant patients. Patients should be advised about prevention strategies and effective techniques for removing ticks. CONCLUSIONS: RMSF is a potentially deadly disease that requires prompt recognition and management. Focused history, physical examination, and testing are important in the diagnosis of this disease. Understanding the clinical features, diagnostic tools, and proper treatment can assist emergency clinicians in the management of RMSF.

Microbial Invasion vs. Tick Immune Regulation.

Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016). Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host's natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdusek et al., 2013). Moreover, the tick's unique intracellular digestive processes allow the midgut to provide a relatively permissive microenvironment for survival of invading microbes. Although tick-host-pathogen interactions have evolved over more than 300 million years (Barker and Murrell, 2008), few microbes have been able to overcome the tick's innate immune system, comprising both humoral and cellular processes that reject them. Similar to most eukaryotes, the signaling pathways that regulate the innate immune response, i.e., the Toll, IMD (Immunodeficiency) and JAK-STAT (Janus Kinase/ Signal Transducers and Activators of Transcription) also occur in ticks (Gulia-Nuss et al., 2016). Recognition of pathogen-associated molecular patterns (PAMPs) on the microbial surface triggers one or the other of these pathways. Consequently, ticks are able to mount an impressive array of humoral and cellular responses to microbial challenge, including anti-microbial peptides (AMPs), e.g., defensins, lysozymes, microplusins, etc., that directly kill, entrap or inhibit the invaders. Equally important are cellular processes, primarily phagocytosis, that capture, ingest, or encapsulate invading microbes, regulated by a primordial system of thioester-containing proteins, fibrinogen-related lectins and convertase factors (Hajdusek et al., 2013). Ticks also express reactive oxygen species (ROS) as well as glutathione-S-transferase, superoxide dismutase, heat shock proteins and even protease inhibitors that kill or inhibit microbes. Nevertheless, many tick-borne microorganisms are able to evade the tick's innate immune system and survive within the tick's body. The examples that follow describe some of the many different strategies that have evolved to enable ticks to transmit the agents of human and/or animal disease.

Fatal Rocky Mountain Spotted Fever along the United States-Mexico Border, 2013-2016.

Rocky Mountain spotted fever (RMSF) is an emerging public health concern near the US-Mexico border, where it has resulted in thousands of cases and hundreds of deaths in the past decade. We identified 4 patients who had acquired RMSF in northern Mexico and subsequently died at US healthcare facilities. Two patients sought care in Mexico before being admitted to US-based hospitals. All patients initially had several nonspecific signs and symptoms, including fever, headache, nausea, vomiting, or myalgia, but deteriorated rapidly without receipt of a tetracycline-class antimicrobial drug. Each patient experienced respiratory failure late in illness. Although transborder cases are not common, early recognition and prompt initiation of appropriate treatment are vital for averting severe illness and death. Clinicians on both sides of the US-Mexico border should consider a diagnosis of RMSF for patients with rapidly progressing febrile illness and recent exposure in northern Mexico.

MicroRNA Signature of Human Microvascular Endothelium Infected with Rickettsia rickettsii.

MicroRNAs (miRNAs) mediate gene silencing by destabilization and/or translational repression of target mRNA. Infection of human microvascular endothelial cells as primary targets of Rickettsiarickettsii, the etiologic agent of Rocky Mountain spotted fever, triggers host responses appertaining to alterations in cellular gene expression. Microarray-based profiling of endothelial cells infected with R.rickettsii for 3 or 24 h revealed differential expression of 33 miRNAs, of which miRNAs129-5p, 200a-3p, 297, 200b-3p, and 595 were identified as the top five up-regulated miRNAs (5 to 20-fold, p ≤ 0.01) and miRNAs 301b-3p, 548a-3p, and 377-3p were down-regulated (2 to 3-fold, p ≤ 0.01). Changes in the expression of selected miRNAs were confirmed by q-RT-PCR in both in vitro and in vivo models of infection. As potential targets, expression of genes encoding NOTCH1, SMAD2, SMAD3, RIN2, SOD1, and SOD2 was either positively or negatively regulated. Using a miRNA-specific mimic or inhibitor, NOTCH1 was determined to be a target of miRNA 200a-3p in R. rickettsii-infected human dermal microvascular endothelial cells (HMECs). Predictive interactome mapping suggested the potential for miRNA-mediated modulation of regulatory gene networks underlying important host cell signaling pathways. This first demonstration of altered endothelial miRNA expression provides new insights into regulatory elements governing mechanisms of host responses and pathogenesis during human rickettsial infections.

The Distinct Transcriptional Response of the Midgut of Amblyomma sculptum and Amblyomma aureolatum Ticks to Rickettsia rickettsii Correlates to Their Differences in Susceptibility to Infection.

Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain Spotted Fever (RMSF). In Brazil, two species of ticks in the genus Amblyomma, A. sculptum and A. aureolatum, are incriminated as vectors of this bacterium. Importantly, these two species present remarkable differences in susceptibility to R. rickettsii infection, where A. aureolatum is more susceptible than A. sculptum. In the current study, A. aureolatum and A. sculptum ticks were fed on suitable hosts previously inoculated with R. rickettsii, mimicking a natural infection. As control, ticks were fed on non-infected animals. Both midgut and salivary glands of all positively infected ticks were colonized by R. rickettsii. We did not observe ticks with infection restricted to midgut, suggesting that important factors for controlling rickettsial colonization were produced in this organ. In order to identify such factors, the total RNA extracted from the midgut (MG) was submitted to next generation RNA sequencing (RNA-seq). The majority of the coding sequences (CDSs) of A. sculptum differentially expressed by infection were upregulated, whereas most of modulated CDSs of A. aureolatum were downregulated. The functional categories that comprise upregulated CDSs of A. sculptum, for instance, metabolism, signal transduction, protein modification, extracellular matrix, and immunity also include CDSs of A. aureolatum that were downregulated by infection. This is the first study that reports the effects of an experimental infection with the highly virulent R. rickettsii on the gene expression of two natural tick vectors. The distinct transcriptional profiles of MG of A. sculptum and A. aureolatum upon infection stimulus strongly suggest that molecular factors in this organ are responsible for delineating the susceptibility to R. rickettsii. Functional studies to determine the role played by proteins encoded by differentially expressed CDSs in the acquisition of R. rickettsii are warranted and may be considered as targets for the development of strategies to control the tick-borne pathogens as well as to control the tick vectors.

Predictive Factors for Fatal Tick-Borne Spotted Fever in Brazil.

In Brazil, two pathogenic Rickettsia species have been identified causing tick-borne spotted fever (SF). The aetiological agent Rickettsia rickettsii causes serious illness, particularly in the south-eastern region of the country. Moreover, the Rickettsia sp. strain Atlantic Rainforest cause milder clinical manifestations in south-eastern, south and north-east regions. This study has sought to analyse predictive factors for fatal SF. A case-control study was performed using disease notification records in Brazil. The cases included were individuals with laboratory confirmation and fatal progression of SF, while the controls included individuals with SF who were cured. A total of 386 cases and 415 controls were identified (1 : 1.1), and the cases and controls were similar in age. The factors identified as being protective against death were reported presence of ticks (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.41-0.88), residing in urban areas (OR, 0.47, 95% CI, 0.31-0.74) and presenting lymphadenopathy (OR, 0.43; 95% CI, 0.23-0.82). Males exhibited a greater chance of death (OR, 1.57; 95% CI, 1.13-2.18), as did patients who were hospitalized (OR, 10.82; 95% CI, 6.38-18.35) and who presented hypotension or shock (OR, 10.80; 95% CI, 7.33-15.93), seizures (OR, 11.24; 95% CI, 6.49-19.45) and coma (OR of 15.16; 95% CI, 8.51-27.02). The study demonstrates the severity profile of the SF cases, defined either as the frequency of hospitalization (even in cases that were cured) or as the increased frequency of the clinical complications typically found in critical patients. Opportune clinical diagnosis, a careful evaluation of the epidemiological aspects of the disease and adequate care for patients are determining factors for reducing SF fatality rates.

Comparative genome sequencing of Rickettsia rickettsii strains that differ in virulence.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii, the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

Community-based control of the brown dog tick in a region with high rates of Rocky Mountain spotted fever, 2012-2013.

Rocky Mountain spotted fever (RMSF) transmitted by the brown dog tick (Rhipicephalus sanguineus sensu lato) has emerged as a significant public health risk on American Indian reservations in eastern Arizona. During 2003-2012, more than 250 RMSF cases and 19 deaths were documented among Arizona's American Indian population. The high case fatality rate makes community-level interventions aimed at rapid and sustained reduction of ticks urgent. Beginning in 2012, a two year pilot integrated tick prevention campaign called the RMSF Rodeo was launched in a ∼ 600-home tribal community with high rates of RMSF. During year one, long-acting tick collars were placed on all dogs in the community, environmental acaricides were applied to yards monthly, and animal care practices such as spay and neuter and proper tethering procedures were encouraged. Tick levels, indicated by visible inspection of dogs, tick traps and homeowner reports were used to monitor tick presence and evaluate the efficacy of interventions throughout the project. By the end of year one, <1% of dogs in the RMSF Rodeo community had visible tick infestations five months after the project was started, compared to 64% of dogs in Non-Rodeo communities, and environmental tick levels were reduced below detectable levels. The second year of the project focused on use of the long-acting collar alone and achieved sustained tick control with fewer than 3% of dogs in the RMSF Rodeo community with visible tick infestations by the end of the second year. Homeowner reports of tick activity in the domestic and peridomestic setting showed similar decreases in tick activity compared to the non-project communities. Expansion of this successful project to other areas with Rhipicephalus-transmitted RMSF has the potential to reduce brown dog tick infestations and save human lives.

Modelling spatial concordance between Rocky Mountain spotted fever disease incidence and habitat probability of its vector Dermacentor variabilis (American dog tick).

The spatial distribution of Dermacentor variabilis, the most commonly identified vector of the bacterium Rickettsia rickettsii which causes Rocky Mountain spotted fever (RMSF) in humans, and the spatial distribution of RMSF, have not been previously studied in the south central United States of America, particularly in Texas. From an epidemiological perspective, one would tend to hypothesise that there would be a high degree of spatial concordance between the habitat suitability for the tick and the incidence of the disease. Both maximum-entropy modelling of the tick's habitat suitability and spatially adaptive filters modelling of the human incidence of RMSF disease provide reliable portrayals of the spatial distributions of these phenomenons. Even though rates of human cases of RMSF in Texas and rates of Dermacentor ticks infected with Rickettsia bacteria are both relatively low in Texas, the best data currently available allows a preliminary indication that the assumption of high levels of spatial concordance would not be correct in Texas (Kappa coefficient of agreement = 0.17). It will take substantially more data to provide conclusive findings, and to understand the results reported here, but this study provides an approach to begin understanding the discrepancy.

Complementation of Rickettsia rickettsii RelA/SpoT restores a nonlytic plaque phenotype.

Spotted fever group rickettsiae are known to produce distinct plaque phenotypes. Strains that cause lytic infections in cell culture form clear plaques, while nonlytic strains form opaque plaques in which the cells remain intact. Clear plaques have historically been associated with more-virulent species or strains of spotted fever group rickettsiae. We have selected spontaneous mutant pairs from two independent strains of Rickettsia rickettsii, the virulent R strain and the avirulent Iowa strain. A nonlytic variant of R. rickettsii R, which typically produces clear plaques, was isolated and stably maintained. A lytic variant of the Iowa strain, which characteristically produces opaque plaques, was also selected and maintained. Genomic resequencing of the variants identified only a single gene disrupted in each strain. In both cases, the mutation was in a gene annotated as relA/spoT-like. In the Iowa strain, a single mutation introduced a premature stop codon upstream from region encoding the predicted active site of RelA/SpoT and caused the transition to a lytic plaque phenotype. In R. rickettsii R, the nonlytic plaque phenotype resulted from a single-nucleotide substitution that shifted a tyrosine residue to histidine near the active site of the enzyme. The intact relA/spoT gene thus occurred in variants with the nonlytic plaque phenotype. Complementation of the truncated relA/spoT gene in the Iowa lytic plaque variant restored the nonlytic phenotype. The relA/spoT mutations did not affect the virulence of either strain in a Guinea pig model of infection; R strain lytic and nonlytic variants both induced fever equally, and the mutation in Iowa to a lytic phenotype did not cause them to become virulent.

Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility.

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.