scholarly journals Motility Characteristics Are Altered for Rickettsia bellii Transformed To Overexpress a HeterologousrickAGene

2013 ◽  
Vol 80 (3) ◽  
pp. 1170-1176 ◽  
Author(s):  
Jonathan D. Oliver ◽  
Nicole Y. Burkhardt ◽  
Roderick F. Felsheim ◽  
Timothy J. Kurtti ◽  
Ulrike G. Munderloh
Keyword(s):  
Host Cell ◽  
Spotted Fever ◽  
Shuttle Vector ◽  
Negative Control ◽  
Spotted Fever Group ◽  
Content Type ◽  
Shuttle Vectors ◽  
Rickettsia Monacensis ◽  
Species Specific ◽  
Rickettsia Bellii

ABSTRACTThe rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. TherickAgene sequence and protein vary substantially betweenRickettsiaspecies, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of threeRickettsia belliivariants. These included two shuttle vector-transformedR. belliistrains and the wild-type isolate from which they were derived,R. belliiRML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one containedRickettsia monacensis-derivedrickA, and the other lacked therickAgene.Rickettsia belliitransformed to expressR. monacensis rickAhighly overexpressed this transcript in comparison to its nativerickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion ofR. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques.

scholarly journals Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

2014 ◽  
Vol 83 (3) ◽  
pp. 1048-1055 ◽  
Author(s):  
Natthida Petchampai ◽  
Piyanate Sunyakumthorn ◽  
Kaikhushroo H. Banajee ◽  
Victoria I. Verhoeve ◽  
Michael T. Kearney ◽  
...  
Keyword(s):  
Host Cell ◽  
Tyrosine Kinases ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Spotted Fever ◽  
Dermacentor Variabilis ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Bacterial Survival ◽  
Content Type

Tick-borne spotted fever group (SFG)Rickettsiaspecies are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake ofRickettsiainto mammalian andDrosophilacells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFGRickettsiabetween vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved inRickettsia montanensisinfection of tick-derived cells from a natural host,Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3′-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals A streamlined method for transposon mutagenesis ofRickettsia parkeriyields numerous mutations that impact infection

2018 ◽  
Author(s):  
Rebecca L. Lamason ◽  
Natasha M. Kafai ◽  
Matthew D. Welch
Keyword(s):  
Host Cell ◽  
Virulence Factors ◽  
Vascular Endothelial Cells ◽  
Spotted Fever ◽  
Transposon Mutagenesis ◽  
Host Cells ◽  
Spotted Fever Group ◽  
Loss Of Function ◽  
Rickettsia Parkeri ◽  
Obligate Intracellular

AbstractThe rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria,Rickettsiaare highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improvedmariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group speciesRickettsia parkeri. These mutants targeted genes implicated in a variety of pathways, including bacterial replication and metabolism, hypothetical proteins, the type IV secretion system, as well as factors with previously established roles in host cell interactions and pathogenesis. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.

scholarly journals Detection of tick-borne pathogens in questing Ixodes ricinus in the French Pyrenees and first identification of Rickettsia monacensis in France

Parasite ◽  
2019 ◽  
Vol 26 ◽  
pp. 20 ◽  
Author(s):  
Toufic Akl ◽  
Gilles Bourgoin ◽  
Marie-Line Souq ◽  
Joël Appolinaire ◽  
Marie-Thérèse Poirel ◽  
...  
Keyword(s):  
Ixodes Ricinus ◽  
Pathogen Detection ◽  
Spotted Fever ◽  
Spotted Fever Group ◽  
Rupicapra Pyrenaica ◽  
Rickettsia Monacensis ◽  
Animal Pathogens ◽  
Questing Ticks ◽  
Pyrenean Chamois ◽  
First Time

Ticks are important vectors of several human and animal pathogens. In this study, we estimated the prevalence of important tick-borne infections in questing ticks from an area in Southwestern France (Hautes-Pyrénées) inhabited by Pyrenean chamois (Rupicapra pyrenaica pyrenaica) experiencing high tick burden. We examined adult and nymph ticks collected by the flag dragging method from 8 to 15 sites in the Pic de Bazès during the years 2009, 2011, 2013 and 2015. PCR assays were conducted on selected ticks for the detection of Borrelia burgdorferi s.l., Babesia spp., Rickettsia spp., spotted fever group (SFG) Rickettsia and Anaplasma phagocytophilum. Randomly selected positive samples were submitted for sequence analysis. A total of 1971 questing ticks were collected including 95 males, 101 females and 1775 nymphs. All collected ticks were identified as Ixodes ricinus. Among them, 696 ticks were selected for pathogen detection and overall prevalence was 8.4% for B. burgdorferi s.l.; 0.4% for Babesia spp.; 6.1% for A. phagocytophilum; 17.6% for Rickettsia spp.; and 8.1% for SFG Rickettsia. Among the sequenced pathogens, we detected in this population of ticks the presence of Babesia sp. EU1 and Rickettsia helvetica, as well as Rickettsia monacensis for the first time in France. The detection of these pathogens in the Pic de Bazès highlights the potential infection risks for visitors to this area and the Pyrenean chamois population.

scholarly journals Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Megan E. Reller ◽  
J. Stephen Dumler
Keyword(s):  
Quantitative Pcr ◽  
Scrub Typhus ◽  
Spotted Fever ◽  
Febrile Illness ◽  
Epidemiologic Studies ◽  
Qpcr Assay ◽  
Spotted Fever Group ◽  
Analytical Sensitivity ◽  
Content Type ◽  
Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

scholarly journals Spotted Fever GroupRickettsiaInfection and Transmission Dynamics inAmblyomma maculatum

2019 ◽  
Vol 87 (4) ◽  
Author(s):  
Chanakan Suwanbongkot ◽  
Ingeborg M. Langohr ◽  
Emma K. Harris ◽  
Wellesley Dittmar ◽  
Rebecca C. Christofferson ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Spotted Fever ◽  
Attachment Site ◽  
Spotted Fever Group ◽  
Tick Feeding ◽  
Tick Saliva ◽  
Content Type ◽  
Feeding Site ◽  
Vertebrate Hosts ◽  
Tick Salivary Glands

ABSTRACTTick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novelRickettsiaspecies or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG)Rickettsiaspecies is transmitted at higher levels during tick feeding. UsingAmblyomma maculatumcohorts infected withRickettsia parkerior “CandidatusRickettsia andeanae,” a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers ofR. parkerithan of “Ca. Rickettsia andeanae” rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localizedR. parkeri, but not “Ca. Rickettsia andeanae,” in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed toR. parkerithan in those exposed to “Ca. Rickettsia andeanae.” The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

scholarly journals Disruption of a Type II Endonuclease (TDE0911) Enables Treponema denticola ATCC 35405 To Accept an Unmethylated Shuttle Vector

2011 ◽  
Vol 77 (13) ◽  
pp. 4573-4578 ◽  
Author(s):  
Jiang Bian ◽  
Chunhao Li
Keyword(s):  
Southern Blot ◽  
Shuttle Vector ◽  
Cell Extract ◽  
Treponema Denticola ◽  
Type Ii ◽  
Restriction Digestion ◽  
Content Type ◽  
Shuttle Vectors ◽  
Genetic Manipulations ◽  
Dna Restriction

ABSTRACTThe oral spirocheteTreponema denticolais associated with human periodontal disease.T. denticolaATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared toT. denticolaATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence ofT. denticolaATCC 35405. In this report, we hypothesize thatT. denticolaATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome ofT. denticolaATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant ofTDE0911failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulateT. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.

Tick-borne rickettsial diseases

2011 ◽  
Author(s):  
Emmanouil Angelakis ◽  
Didier Raoult
Keyword(s):  
Spotted Fever ◽  
Reservoir Host ◽  
Spotted Fever Group ◽  
Rickettsia Species ◽  
The Family ◽  
Risk Areas ◽  
Vertebrate Hosts ◽  
Group 2 ◽  
Rickettsial Diseases ◽  
Rickettsia Bellii

Bacteria of the genus Rickettsia belong to the family Rickettsiaceae in the order Rickettsiales and have for long been described simply as short, Gram-negative, strict intracellular rods that retain basic fuchsin when stained by the method of Gimenez (Raoult and Roux 1997). These bacteria are associated with ticks, lice, fleas or mites. To date the Rickettsia genus contains 24 recognized species classified into three groups based on their antigenic, morphological, and ecologic patterns: 1) the typhus group, 2) the spotted fever group and 3) Rickettsia bellii (Fournier and Raoult 2007). Most spotted fever group (SFG) rickettsiae are closely associated with ticks belonging to the family Ixodidae (also called “hard” ticks) (Parola et al. 2005). Ticks can act as vectors, reservoirs, and/or amplifiers of SFG rickettsiae and require optimal environmental conditions which determine the geographic distribution of the vectors and consequently the risk areas for rickettsioses. Many Rickettsia species are strictly associated with one genus of ticks and the transmittion to people is made through the tick bite, which generally implies that the Rickettsia can localize to their salivary glands. Therefore, since larvae, nymphs, and adults may all be infective for susceptible vertebrate hosts, the ticks must be regarded as the main reservoir host of rickettsiae. Humans are not considered as good reservoirs for Rickettsiae, as they are seldom infested with ticks for long periods and rickettsiaemia has normally short duration, especially with antibiotic intervention.

scholarly journals Rickettsia monacensis sp. nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes ricinus) Collected in a European City Park

2002 ◽  
Vol 68 (9) ◽  
pp. 4559-4566 ◽  
Author(s):  
Jason A. Simser ◽  
Ann T. Palmer ◽  
Volker Fingerle ◽  
Bettina Wilske ◽  
Timothy J. Kurtti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ixodes Ricinus ◽  
Mammalian Cells ◽  
Citrate Synthase ◽  
Spotted Fever ◽  
Spotted Fever Group ◽  
Mammalian Cell Lines ◽  
Isolation And Characterization ◽  
Rickettsia Monacensis ◽  
City Park

ABSTRACT We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/MunichT) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

scholarly journals Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography

Diagnostics ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 897
Author(s):  
Lavel Chinyama Moonga ◽  
Kyoko Hayashida ◽  
Naoko Kawai ◽  
Ryo Nakao ◽  
Chihiro Sugimoto ◽  
...  
Keyword(s):  
Detection System ◽  
Spotted Fever ◽  
Simultaneous Detection ◽  
Febrile Illness ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Spotted Fever Group ◽  
Loop Mediated Isothermal Amplification ◽  
Rickettsia Monacensis ◽  
Sfg Rickettsia

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

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