Rickettsia Sca4 Reduces Vinculin-Mediated Intercellular Tension to Promote Spread.

Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.

Critical roles of Rickettsia parkeri outer membrane protein B (OmpB) in the tick host.

Rickettsia parkeri is a pathogen of public health concern and transmitted by the Gulf Coast tick, Amblyomma maculatum. Rickettsiae are obligate intracellular bacteria that enter and replicate in diverse host cells. Rickettsial outer membrane protein B (OmpB) functions in bacterial adhesion, invasion, and avoidance of cell-autonomous immunity in mammalian cell infection, but the function of OmpB in arthropod infection is unknown. In this study, the function of R. parkeri OmpB was evaluated in the tick host. R. parkeri wild-type and R. parkeri ompBSTOP::tn (non-functional OmpB) were capillary fed to naïve A. maculatum ticks to investigate dissemination in the tick and transmission to vertebrates. Ticks exposed to R. parkeri wild-type had greater rickettsial loads in all organs than ticks exposed to R. parkeri ompBSTOP::tn at 12 h post-capillary feeding and after 1 day of feeding on host. In rats that were exposed to R. parkeri ompBSTOP::tn-infected ticks, dermal inflammation at the bite site was less compared to R. parkeri wild-type-infected ticks. In vitro, R. parkeri ompBSTOP::tn cell attachment to tick cells was reduced, and host cell invasion of the mutant was initially reduced but eventually returned to the level of R. parkeri wild-type by 90 min post-infection. R. parkeri ompBSTOP::tn and R. parkeri wild-type had similar growth kinetics in the tick cells, suggesting that OmpB is not essential for R. parkeri replication in tick cells. These results indicate that R. parkeri OmpB functions in rickettsial attachment and internalization to tick cells and pathogenicity during tick infection.

Rickettsia parkeri Rickettsiosis in Kidney Transplant Recipient, North Carolina, USA, 2023.

Spotted fever rickettsiosis is rarely observed in solid organ transplant recipients, and all previously reported cases have been associated with tick bite months to years after transplantation. We describe a kidney transplant recipient in North Carolina, USA, who had a moderately severe Rickettsia parkeri infection develop during the immediate posttransplant period.

Evaluation of a mimotope of the Rickettsia outer membrane protein A (OmpA) as an antigen in enzyme-linked immunosorbent assay to detect rickettsiosis in capybaras (Hydrochoerus hydrochaeris), horses (Equus caballus), and opossums (Didelphis sp.).

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever, which is an important tick-borne zoonosis and, in Brazil, it causes Brazilian spotted fever, which has high lethality rate. This study aimed to evaluate a synthetic peptide corresponding to a segment of the outer membrane protein A (OmpA) as an antigen in a serological test for the diagnosis of rickettsial infections. The amino acid sequence of the peptide was selected by predicting B cell epitopes using B Cell Epitope Prediction (Immune Epitope Database and Analysis Resource) and Epitopia and OmpA sequences of Rickettsia rickettsii strain 'Brazil' and Rickettsia parkeri strains 'Maculatum 20' and 'Portsmouth'. A peptide with amino acid sequence common to both Rickettsia species was synthesized and arbitrarily named OmpA-pLMC. To evaluate this peptide in enzyme-linked immunosorbent assay (ELISA), serum samples of capybara (Hydrochoerus hydrochaeris), horse (Equus caballus), and opossum (Didelphis albiventris) that had been previously tested by indirect immunofluorescence assay (IFA) for rickettsial infection were separated into IFA-positive and IFA-negative groups and used in the assay. There were no significant differences in ELISA optical density (OD) values between IFA-positive and IFA-negative groups with horse samples. The mean OD values were significantly higher in the IFA-positive capybara serum samples (IFA-pos vs. IFA-neg = 2.389 ± 0.761 vs. 1.760 ± 0.840). However, receiver operating characteristic (ROC) curve analysis did not show significant diagnostic parameters. On the other hand, 12 out of 14 (85.7%) opossum samples of the IFA-positive group showed reactivity in ELISA, and this was significantly higher than of the IFA-negative group (0.7196 ± 0.440 vs. 0.2318 ± 0.098, respectively; 85.7% sensitivity, 100% specificity). Therefore, our results show that OmpA-pLMC has a potential to be used in immunodiagnostic assays to detect spotted fever group rickettsial infections.

[Autochthonous Rickettsia parkeri rickettsiosis in the Paranaense Forest, Misiones, Argentina]. / Caso autóctono de rickettsiosis por Rickettsia parkeri en la Selva Paranaense, Misiones, Argentina.

We report an autochthonous case of Rickettsia parkeri rickettsiosis occurred in June 2018 in a forested area of the Urugua-í Provincial Park, Misiones, Argentina. No previous records of this disease in humans have been previously reported in this region. The epidemiological, ecological, clinical, and laboratory features required for a proper diagnosis and adequate treatment are described here. The fact of considering rickettsiosis as a differential diagnosis in a patient with exanthematic acute febrile syndrome is highlighted, being the epidemiological history of exposure to the vector (ticks of the genus Amblyomma) an essential element.

The Ankyrin Repeat Protein RARP-1 Is a Periplasmic Factor That Supports Rickettsia parkeri Growth and Host Cell Invasion.

Rickettsia spp. are obligate intracellular bacterial pathogens that have evolved a variety of strategies to exploit their host cell niche. However, the bacterial factors that contribute to this intracellular lifestyle are poorly understood. Here, we show that the conserved ankyrin repeat protein RARP-1 supports Rickettsia parkeri infection. Specifically, RARP-1 promotes efficient host cell entry and growth within the host cytoplasm, but it is not necessary for cell-to-cell spread or evasion of host autophagy. We further demonstrate that RARP-1 is not secreted into the host cytoplasm by R. parkeri. Instead, RARP-1 resides in the periplasm, and we identify several binding partners that are predicted to work in concert with RARP-1 during infection. Altogether, our data reveal that RARP-1 plays a critical role in the rickettsial life cycle. IMPORTANCERickettsia spp. are obligate intracellular bacterial pathogens that pose a growing threat to human health. Nevertheless, their strict reliance on a host cell niche has hindered investigation of the molecular mechanisms driving rickettsial infection. This study yields much-needed insight into the Rickettsia ankyrin repeat protein RARP-1, which is conserved across the genus but has not yet been functionally characterized. Earlier work had suggested that RARP-1 is secreted into the host cytoplasm. However, the results from this work demonstrate that R. parkeri RARP-1 resides in the periplasm and is important both for invasion of host cells and for growth in the host cell cytoplasm. These results reveal RARP-1 as a novel regulator of the rickettsial life cycle.

Rickettsiosis Caused by Rickettsia parkeri, Mexico.

We report a human case of rickettsiosis caused by Rickettsia parkeri strain Atlantic Rainforest in Mexico in an adult woman from a small town in the north of Yucatan, Mexico. We confirmed diagnosis using conventional PCR and sequence analysis. Health providers should be aware of clinical manifestations of rickettsioses in this region.

Human Rickettsiosis Caused by Rickettsia parkeri Strain Atlantic Rainforest, Urabá, Colombia.

We describe the clinical, serologic, and molecular findings of a new human rickettsiosis in Colombia. Antibodies against Rickettsia spp. were detected. PCR showed amplification of genes for R. parkeri strain Atlantic Rainforest. This new rickettsiosis of minor virulence could explain some of the undifferentiated acute febrile diseases in Colombia.

Rickettsia parkeri and Candidatus Rickettsia andeanae in Amblyomma maculatum Group Ticks.

We determined prevalence of Rickettsia spp. in 172 ticks of the Amblyomma maculatum group collected from 16 urban sites in Oklahoma City, Oklahoma, USA, during 2017 and 2018. Most ticks (59.3%) were collected from 1 site; 4 (2.3%) were infected with Rickettsia parkeri and 118 (68.6%) with Candidatus Rickettsia andeanae.

Reproductive incompatibility between Amblyomma maculatum (Acari: Ixodidae) group ticks from two disjunct geographical regions within the USA.

The Amblyomma maculatum Koch group of ixodid ticks consists of three species: A. maculatum, A. triste, and A. tigrinum. However, since Koch described this group in 1844, the systematics of its members has been the subject of ongoing debate. This is especially true of A. maculatum and A. triste; recent molecular analyses reveal insufficient genetic divergence to separate these as distinct species. Further confounding this issue is the discovery in 2014 of A. maculatum group ticks in southern Arizona (AZ), USA, that share morphological characteristics with both A. triste and A. maculatum. To biologically evaluate the identity of A. maculatum group ticks from southern Arizona, we analyzed the reproductive compatibility between specimens of A. maculatum group ticks collected from Georgia (GA), USA, and southern Arizona. Female ticks from both Arizona and Georgia were mated with males from both the Georgia and Arizona Amblyomma populations, creating two homologous and two heterologous F1 cohorts of ticks: GA ♀/GA ♂, AZ ♀/AZ ♂, GA ♀/AZ ♂, and AZ ♀/GA ♂. Each cohort was maintained separately into the F2 generation with F1 females mating only with F1 males from their same cohort. Survival and fecundity parameters were measured for all developmental stages. The observed survival parameters for heterologous cohorts were comparable to those of the homologous cohorts through the F1 generation. However, the F1 heterologous females produced F2 egg clutches that did not hatch, thus indicating that the Arizona and Georgia populations of A. maculatum group ticks tested here represent different biological species.

Rickettsia parkeri and Candidatus Rickettsia andeanae in Tick of the Amblyomma maculatum Group, Mexico.

We report Rickettsia parkeri and Candidatus Rickettsia andeanae in ticks of the Amblyomma maculatum group collected from dogs in Sonora, Mexico. Molecular characterization of these bacteria was accomplished by DNA amplification and sequence analysis of portions of the rickettsial genes gltA, htrA, ompA, and ompB.

Molecular Confirmation of Rickettsia parkeri in Amblyomma ovale Ticks, Veracruz, Mexico.

We found Rickettsia parkeri in Amblyomma ovale ticks collected in Veracruz, Mexico, in 2018. We sequenced gene segments of gltA, htrA, sca0, and sca5; phylogenetic reconstruction revealed near-complete identity with R. parkeri strain Atlantic Rainforest. Enhanced surveillance is needed in Mexico to determine the public health relevance of this bacterium.

Amblyomma maculatum-associated rickettsiae in vector tissues and vertebrate hosts during tick feeding.

Rickettsia parkeri, a causative agent of spotted fever rickettsiosis, is transmitted by Amblyomma maculatum (Gulf Coast tick), a tick that may also carry a non-pathogenic spotted fever group Rickettsia, "Candidatus Rickettsia andeanae". Here, we evaluated R. parkeri and "Candidatus R. andeanae" in tissues from A. maculatum prior to, during, and after blood feeding on rabbits. Using colony-reared A. maculatum that were capillary-fed uninfected cells, R. parkeri, "Candidatus R. andeanae", or both rickettsiae, we detected higher levels of Rickettsia spp. in the respective treatment groups. Rickettsial levels increased during blood feeding for both R. parkeri and "Candidatus R. andeanae", with a greater increase in R. parkeri in co-infected ticks compared to singly-infected ticks. We detected transovarial transmission of "Candidatus R. andeanae" in egg and larval cohorts and confirmed vertical transmission of R. parkeri in one group of larvae. Rabbits from all Rickettsia-exposed groups seroconverted on immunofluorescent antibody testing using R. parkeri antigen. Visualization of "Candidatus R. andeanae" in tick salivary glands suggested potential transmission via tick feeding. Here, rickettsial levels in artificially infected ticks demonstrate changes during feeding and transovarial transmission that may be relevant for interpreting rickettsial levels detected in wild A. maculatum.

Serologic and molecular survey of Rickettsia spp. in dogs, horses and ticks from the Atlantic rainforest of the state of Bahia, Brazil.

Tick-borne spotted fever in Brazil is known to be caused by two agents, Rickettsia rickettsii and Rickettsia parkeri. Nothing was known about these agents in one area of the Atlantic rainforest biome of Bahia state, where during March to June 2016, 356 rural dogs and 69 horses were sampled and their sera were processed through indirect immunofluorescence assay against antigens of R. rickettsii, R. parkeri, Rickettsia amblyommatis and Rickettsia bellii. Ticks collected from these dogs and horses were molecularly tested for the presence of rickettsial DNA. Overall, 16.4% (58/356) dogs and 24.6% (17/69) horses were seroreactive to Rickettsia spp. Five tick species, Rhipicephalus sanguineus sensu lato (s.l.), Amblyomma ovale, A. sculptum, R. microplus, and A. naponense, were collected from dogs, whereas horses were infested by A. sculptum and Dermacentor nitens. A total of 242 ticks from dogs and 62 from horses were analyzed by PCR targeting rickettsiae, which were detected in only 4/27 (14.8%) A. ovale. Fragments of the rickettsial gltA and ompA genes from these four ticks were 100% identical to the Atlantic rainforest strain of R. parkeri. The presence of A. ovale on dogs was positively associated with local canine seroreactivity to R. parkeri. Our results provide evidence for the transmission of R. parkeri strain Atlantic rainforest from A. ovale to domestic dogs within the rural area of Ilhéus, similarly to other areas of the Atlantic rainforest biome of Brazil, where human cases of R. parkeri-caused spotted fever have been reported.

Role of Sca2 and RickA in the Dissemination of Rickettsia parkeri in Amblyomma maculatum.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro, similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia-free Amblyomma maculatum, the natural vector of R. parkeri, was exposed to wild-type, R. parkeri rickA::tn, or R. parkeri sca2::tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeririckA::tn or R. parkerisca2::tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Rickettsia parkeri in Dermacentor parumapertus Ticks, Mexico.

During a study to identify zoonotic pathogens in northwestern Mexico, we detected the presence of a rickettsial agent in Dermacentor parumapertus ticks from black-tailed jackrabbits (Lepus californicus). Comparison of 4 gene sequences (gltA, htrA, ompA, and ompB) of this agent showed 99%-100% identity with sequences of Rickettsia parkeri.

Phylogenetic Evidence for the Existence of Multiple Strains of Rickettsia parkeri in the New World.

The bacterium Rickettsia parkeri has been reported to infect ticks of the "Amblyomma maculatum species complex" in the New World, where it causes spotted fever illness in humans. In South America, three additional rickettsial strains, namely, Atlantic rainforest, NOD, and Parvitarsum, have been isolated from the ticks Amblyomma ovale, Amblyomma nodosum, and Amblyomma parvitarsum, respectively. These three strains are phylogenetically closely related to R. parkeri, Rickettsia africae, and Rickettsia sibirica Herein, we performed a robust phylogenetic analysis encompassing 5 genes (gltA, ompA, virB4, dnaA, and dnaK) and 3 intergenic spacers (mppE-pur, rrl-rrf-ITS, and rpmE-tRNAfMet) from 41 rickettsial isolates, including different isolates of R. parkeri, R. africae, R. sibirica, Rickettsia conorii, and strains Atlantic rainforest, NOD, and Parvitarsum. In our phylogenetic analyses, all New World isolates grouped in a major clade distinct from the Old World Rickettsia species (R. conorii, R. sibirica, and R. africae). This New World clade was subdivided into the following 4 clades: the R. parkerisensu stricto clade, comprising the type strain Maculatum 20 and all other isolates of R. parkeri from North and South America, associated with ticks of the A. maculatum species complex; the strain NOD clade, comprising two South American isolates from A. nodosum ticks; the Parvitarsum clade, comprising two South American isolates from A. parvitarsum ticks; and the strain Atlantic rainforest clade, comprising six South American isolates from the A. ovale species complex (A. ovale or Amblyomma aureolatum). Under such evidences, we propose that strains Atlantic rainforest, NOD, and Parvitarsum are South American strains of R. parkeriIMPORTANCE Since the description of Rickettsia parkeri infecting ticks of the "Amblyomma maculatum species complex" and humans in the New World, three novel phylogenetic close-related rickettsial isolates were reported in South America. Herein, we provide genetic evidence that these novel isolates, namely, strains Atlantic rainforest, NOD, and Parvitarsum, are South American strains of R. parkeri. Interestingly, each of these R. parkeri strains seems to be primarily associated with a tick species group, namely, R. parkerisensu stricto with the "Amblyomma maculatum species group," R. parkeri strain NOD with Amblyomma nodosum, R. parkeri strain Parvitarsum with Amblyomma parvitarsum, and R. parkeri strain Atlantic rainforest with the "Amblyomma ovale species group." Such rickettsial strain-tick species specificity suggests a coevolution of each tick-strain association. Finally, because R. parkerisensu stricto and R. parkeri strain Atlantic rainforest are human pathogens, the potential of R. parkeri strains NOD and Parvitarsum to be human pathogens cannot be discarded.

Rickettsia parkeri infections diagnosed by eschar biopsy, Virginia, USA.

BACKGROUND: Infection with Rickettsia parkeri is an emerging tick-borne illness, often accompanied by fever and an eschar at the site of tick attachment. We present three cases of R. parkeri in Virginia residents. CASE PRESENTATIONS: Case 1 presented initially afebrile, failed to seroconvert to rickettsial antigens, and was diagnosed by DNA testing of the eschar. Case 2 presented febrile with eschar, no serologies were performed, and was diagnosed by DNA testing of the eschar. Case 3 presented febrile with eschar, serologies were negative for rickettsial antigens, and was diagnosed by DNA testing of the eschar. CONCLUSION: DNA testing of eschars represents an under-utilized diagnostic test and may aid in cases where the diagnosis is not made clinically.

Molecular survey of Rickettsia spp. in the Neotropical deer tick Haemaphysalis juxtakochi from Brazilian Pampa.

Spotted fever (SF) is a tick-borne disease associated with Rickettsia spp.. In the Pampa biome, Southern Brazil, cases of SF seem to be strongly linked with the practice of hunting wild animals. An investigation of rickettsiae in tick species found on wild animals could provide more information regarding the rickettsiosis enzootic cycle. The aim of this study is to describe the results of a molecular survey of Rickettsia spp. in the Neotropical deer tick, Haemaphysalis juxtakochi Cooley, 1946 (Acari: Ixodidae), from the Brazilian Pampa. Ticks were obtained from 14 road-killed gray brocket deer, Mazama gouazoubira (Artiodactyla: Cervidae), found in nine different municipalities of Rio Grande do Sul state, Southern Brazil. Ticks were processed individually to obtain genomic DNA, and then Rickettsia spp. was investigated using a set of PCR reactions that amplified the rickettsial fragments of the gltA, ompA, and htrA genes. Of the 24 tick samples tested, DNA of Rickettsia parkeri sensu stricto (s.s.) was found in 11 H. juxtakochi specimens collected in two different areas of the Brazilian Pampa. This is the first report of R. parkeri s.s. (the main agent associated with SF in the Uruguayan, Argentinian, and Brazilian Pampa) in H. juxtakochi ticks. These findings indicate that R. parkeri s.s. may be much more common and widely distributed in the Pampa biome than previously assumed. Moreover, H. juxtakochi ticks and gray brocket deer could participate in the potential spillover of R. parkeri s.s. from endemic to non-endemic areas in the South American Pampa.

[Autochthonous case of spotted fever caused by Rickettsia parkeri in Ensenada, Buenos Aires]. / Caso autóctono de fiebre manchada por Rickettsia parkeri en Ensenada, Buenos Aires.

We present a case of spotted fever occurred in an adult residing in Ensenada, Buenos Aires province in February 2016. The patient presented with an acute febrile syndrome associated with a skin necrotic lesion on the left leg secondary to a tick bite. The general symptoms were a maculopapular rash, headache, myalgia, and arthralgias. Seroconversion of anti-Rickettsia specific IgG antibodies confirmed recent infection. The nucleotidic and aminoacidic sequences of a gltA gen fragment matched 100% the sequences of R. parkeri strains from Argentina and other countries of America. The patient responded well to treatment with doxycycline.