an-electroporation-method-to-transform-rickettsia-spp-with

An Electroporation Method to Transform <em>Rickettsia</em> spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts ...

University of Minnesota

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts- and -both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing  and  will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions.

 subsp.  is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of  The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between  ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely,  ticks were not susceptible to infection by the  Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A  transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks. Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the  subsp. , a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.

 Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

 is the principal symbiotic bacterium of the medically significant tick  This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of , it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of  naturally occurring in  ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating  from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing  from the majority of progeny. This paper describes the growth of symbiotic  within  through the life cycle of the tick and provides methods to eliminate  from  ticks.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

Apoptosis is an innate immune response induced by infection in eukaryotes that contributes significantly to protection from pathogens. However, little is known about the role of apoptosis in the interactions of arthropod vectors with the rickettsiae that they transmit.  spp. are vector-borne obligately intracellular bacteria and display different degrees of virulence in their eukaryotic hosts. In this study, we found that infection with  () activated the apoptosis pathway in an  tick cell line (AAE2), as evidenced by the loss of phospholipid membrane asymmetry and DNA fragmentations. Additionally, infection with  also led to apoptosis activation in cell lines of different tick species. Interestingly, suppressing apoptosis decreased  infection and replication, while the activation of apoptosis increased  accumulation at the early stage of infection. Moreover, mitochondrion-dependent apoptosis was essential for  infection and replication in vector cells, and apoptosis induction required intracellular rickettsia replication. We further showed that  utilizes two different survival strategies to modulate apoptosis in the arthropod vectors and mammalian host cells. There was no direct correlation between apoptosis activation in vector cells and rickettsial pathogenicity. These novel findings indicate a possible mechanism whereby apoptosis facilitates infection and replication of a  sp. in an arthropod vector. These results contribute to our understanding of how the vector's responses to pathogen infection affect pathogen replication and therefore transmission. Rickettsioses, infections caused by the genus , are among the oldest known infectious diseases. Ticks are essential arthropod vectors for rickettsiae, and knowledge about the interactions between ticks, their hosts, and pathogens is fundamental for identifying drivers of tick-borne rickettsioses. Despite the rapid development in apoptosis research with rickettsiae, little is known regarding the role of apoptosis in the interactions between  spp., vertebrate hosts, and arthropod vectors. Here, we demonstrated that mitochondrion-dependent apoptosis is essential for rickettsial infection and replication in vector cells and that apoptosis induction requires intracellular rickettsial replication. However, rickettsial pathogenicity is not linked with apoptosis activation in tick cells. Our findings improve understanding of the apoptosis mechanism in arthropods exploited by rickettsiae and also the potential to discover specific targets for new vaccines and drugs to prevent or treat rickettsial infections.

Mitochondrion-Dependent Apoptosis Is Essential for Rickettsia parkeri Infection and Replication in Vector Cells.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

 is the primary vector of tick-borne pathogens in North America but notably does not transmit pathogenic  species. This tick harbors the transovarially transmitted endosymbiont , which is widespread in  populations, suggesting that it confers a selective advantage for tick survival such as providing essential nutrients. The  genome includes genes with similarity to those involved in antibiotic synthesis. There are two gene clusters not found in other Rickettsiaceae, raising the possibility that these may be involved in excluding pathogenic bacteria from the tick. This study explored whether the  antibiotic genes might exert antibiotic effects on pathogens associated with . Markedly reduced infectivity and replication of the tick-borne pathogens , and  were observed in IRE11 tick cells hosting . Using a fluorescent plate reader assay to follow infection dynamics revealed that the presence of  in tick cells, even at low infection rates, inhibited the growth of  by 86-100% relative to -free cells. In contrast, presence of the low-pathogenic species  or the endosymbiont  only partially reduced the infection and replication of . Addition of host-cell free , cell lysate of infected IRE11, or supernatant from infected IRE11 cultures had no effect on  infection and replication in IRE11, nor did these treatments show any antibiotic effect against non-obligate intracellular bacteria  and . However, lysate from infected IRE11 challenged with  showed some inhibitory effect on  infection of treated IRE11, suggesting that challenge by pathogenic rickettsiae may induce the antibiotic effect of . This research suggests a potential role of the endosymbiont in preventing other rickettsiae from colonizing  and/or being transmitted transovarially. The confirmation that the observed inhibition is linked to 's antibiotic clusters requires further investigation but could have important implications for our understanding of rickettsial competition and vector competence of  for rickettsiae.

Nicole Y. Burkhardt

Department of Entomology

University of Minnesota

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines