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Development of genetic system to inactivate a Borrelia turicatae surface protein selectively produced within the salivary glands of the arthropod vector.

Lopez JE, Wilder HK, Hargrove R, Brooks CP, Peterson KE, Beare PA, Sturdevant DE, Nagarajan V, Raffel SJ, Schwan TG - PLoS Negl Trop Dis (2013)

Bottom Line: We then applied a novel genetic system for B. turicatae to inactivate brpA and examined the role of the gene product for vector colonization and the ability to establish murine infection.These results demonstrate the complexity of protein production in a population of spirochetes within the tick.Additionally, the development of a genetic system is important for future studies to evaluate the requirement of specific B. turicatae genes for vector colonization and transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Mississippi State University, Starkville, Mississippi, United States of America.

ABSTRACT

Background: Borrelia turicatae, an agent of tick-borne relapsing fever, is an example of a pathogen that can adapt to disparate conditions found when colonizing the mammalian host and arthropod vector. However, little is known about the genetic factors necessary during the tick-mammalian infectious cycle, therefore we developed a genetic system to transform this species of spirochete. We also identified a plasmid gene that was up-regulated in vitro when B. turicatae was grown in conditions mimicking the tick environment. This 40 kilodalton protein was predicted to be surface localized and designated the Borrelia repeat protein A (brpA) due to the redundancy of the amino acid motif Gln-Gly-Asn-Val-Glu.

Methodology/principal findings: Quantitative reverse-transcriptase polymerase chain reaction using RNA from B. turicatae infected ticks and mice indicated differential regulation of brpA during the tick-mammalian infectious cycle. The surface localization was determined, and production of the protein within the salivary glands of the tick was demonstrated. We then applied a novel genetic system for B. turicatae to inactivate brpA and examined the role of the gene product for vector colonization and the ability to establish murine infection.

Conclusions/significance: These results demonstrate the complexity of protein production in a population of spirochetes within the tick. Additionally, the development of a genetic system is important for future studies to evaluate the requirement of specific B. turicatae genes for vector colonization and transmission.

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Related in: MedlinePlus

Spirochete densities within murine blood after feeding ticks infected with ΔbrpA mutants or wild type spirochetes.The white (mutant infected mice) and grey (wild type infected mice) shaded box and whisker plots represent the lower and upper quartiles, median, and lower and upper extremes of spirochete densities on a given day. The number below each plot and the zero values above the x-axis represents the number of animals infected with mutant or wild type spirochetes on the given day.
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pntd-0002514-g008: Spirochete densities within murine blood after feeding ticks infected with ΔbrpA mutants or wild type spirochetes.The white (mutant infected mice) and grey (wild type infected mice) shaded box and whisker plots represent the lower and upper quartiles, median, and lower and upper extremes of spirochete densities on a given day. The number below each plot and the zero values above the x-axis represents the number of animals infected with mutant or wild type spirochetes on the given day.

Mentions: Cohorts of uninfected O. turicata were fed to repletion on mice infected by needle inoculated with wild type or mutant spirochetes, and salivary gland colonization was confirmed on a portion of ticks by IF-LSCM (data not shown). Ticks infected with wild type or mutant spirochetes were fed on naïve mice and transmission was detected by microscopy and qPCR in two animals within three days (Figure 8). All animals were infected by the fourth day after tick bite, followed by a day of quiescence, and spirochetes repopulated the blood on the eight day. Both wild type and ΔbrpA mutant spirochetes attained similar densities within the blood. One animal infected with wild type spirochetes was euthanized before the completion of the experiment, for causes unrelated to this study. Similar results were obtained when transmission experiments were repeated with cohorts of ticks that had molted, which indicated that the mutant spirochetes were maintained transstadially (data not shown). Overall, these results demonstrated that brpA was dispensable for tick colonization and establishing infection in the murine model we used. Given the absence of a phenotype in the mutant, complementation was not performed.


Development of genetic system to inactivate a Borrelia turicatae surface protein selectively produced within the salivary glands of the arthropod vector.

Lopez JE, Wilder HK, Hargrove R, Brooks CP, Peterson KE, Beare PA, Sturdevant DE, Nagarajan V, Raffel SJ, Schwan TG - PLoS Negl Trop Dis (2013)

Spirochete densities within murine blood after feeding ticks infected with ΔbrpA mutants or wild type spirochetes.The white (mutant infected mice) and grey (wild type infected mice) shaded box and whisker plots represent the lower and upper quartiles, median, and lower and upper extremes of spirochete densities on a given day. The number below each plot and the zero values above the x-axis represents the number of animals infected with mutant or wild type spirochetes on the given day.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814808&req=5

pntd-0002514-g008: Spirochete densities within murine blood after feeding ticks infected with ΔbrpA mutants or wild type spirochetes.The white (mutant infected mice) and grey (wild type infected mice) shaded box and whisker plots represent the lower and upper quartiles, median, and lower and upper extremes of spirochete densities on a given day. The number below each plot and the zero values above the x-axis represents the number of animals infected with mutant or wild type spirochetes on the given day.
Mentions: Cohorts of uninfected O. turicata were fed to repletion on mice infected by needle inoculated with wild type or mutant spirochetes, and salivary gland colonization was confirmed on a portion of ticks by IF-LSCM (data not shown). Ticks infected with wild type or mutant spirochetes were fed on naïve mice and transmission was detected by microscopy and qPCR in two animals within three days (Figure 8). All animals were infected by the fourth day after tick bite, followed by a day of quiescence, and spirochetes repopulated the blood on the eight day. Both wild type and ΔbrpA mutant spirochetes attained similar densities within the blood. One animal infected with wild type spirochetes was euthanized before the completion of the experiment, for causes unrelated to this study. Similar results were obtained when transmission experiments were repeated with cohorts of ticks that had molted, which indicated that the mutant spirochetes were maintained transstadially (data not shown). Overall, these results demonstrated that brpA was dispensable for tick colonization and establishing infection in the murine model we used. Given the absence of a phenotype in the mutant, complementation was not performed.

Bottom Line: We then applied a novel genetic system for B. turicatae to inactivate brpA and examined the role of the gene product for vector colonization and the ability to establish murine infection.These results demonstrate the complexity of protein production in a population of spirochetes within the tick.Additionally, the development of a genetic system is important for future studies to evaluate the requirement of specific B. turicatae genes for vector colonization and transmission.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Mississippi State University, Starkville, Mississippi, United States of America.

ABSTRACT

Background: Borrelia turicatae, an agent of tick-borne relapsing fever, is an example of a pathogen that can adapt to disparate conditions found when colonizing the mammalian host and arthropod vector. However, little is known about the genetic factors necessary during the tick-mammalian infectious cycle, therefore we developed a genetic system to transform this species of spirochete. We also identified a plasmid gene that was up-regulated in vitro when B. turicatae was grown in conditions mimicking the tick environment. This 40 kilodalton protein was predicted to be surface localized and designated the Borrelia repeat protein A (brpA) due to the redundancy of the amino acid motif Gln-Gly-Asn-Val-Glu.

Methodology/principal findings: Quantitative reverse-transcriptase polymerase chain reaction using RNA from B. turicatae infected ticks and mice indicated differential regulation of brpA during the tick-mammalian infectious cycle. The surface localization was determined, and production of the protein within the salivary glands of the tick was demonstrated. We then applied a novel genetic system for B. turicatae to inactivate brpA and examined the role of the gene product for vector colonization and the ability to establish murine infection.

Conclusions/significance: These results demonstrate the complexity of protein production in a population of spirochetes within the tick. Additionally, the development of a genetic system is important for future studies to evaluate the requirement of specific B. turicatae genes for vector colonization and transmission.

Show MeSH
Related in: MedlinePlus