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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

Temperature mediated production and surface localization of BrpA.A rabbit pre-immunization serum sample was used as a negative control (A), while hyperimmune serum generated against rBrpA was used to determine differential protein production at 22°C and 35°C (B). Immunoblots were also probed with chicken serum generated against B. turicatae rFlaB (B lower panel). Surface localization assays were performed by immunoblotting using the rabbit serum generated against rBrpA (C upper panel) and chicken serum generated against rFlaB (C lower panel). Molecular mass is depicted in kilodaltons on the left of each blot. The asterisk and arrowhead indicate the expected size of BrpA and FlaB, respectively.
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pntd-0002514-g002: Temperature mediated production and surface localization of BrpA.A rabbit pre-immunization serum sample was used as a negative control (A), while hyperimmune serum generated against rBrpA was used to determine differential protein production at 22°C and 35°C (B). Immunoblots were also probed with chicken serum generated against B. turicatae rFlaB (B lower panel). Surface localization assays were performed by immunoblotting using the rabbit serum generated against rBrpA (C upper panel) and chicken serum generated against rFlaB (C lower panel). Molecular mass is depicted in kilodaltons on the left of each blot. The asterisk and arrowhead indicate the expected size of BrpA and FlaB, respectively.

Mentions: Immunoblot analysis using serum samples from mice infected by tick bite indicated that rBrpA was not immunogenic (data not shown). Moreover, immunoblotting using serum samples from rabbits immunized with rBrpA demonstrated up-regulation of the native protein when spirochetes were grown at 22°C when compared to 35°C (Figure 2 A and B). Reactivity against FlaB with the samples indicated that similar amounts of spirochete lysates were electrophoresed (Figure 2B insert). The susceptibility of BrpA to digestion with proteinase K suggested that the protein was localized on the outer surface of the spirochetes (Figure 2 C). Detecting the periplasmic protein, FlaB, verified that the outer membrane of B. turicatae remained intact during the proteinase K treatments (Figure 2 C lower insert).


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)

Temperature mediated production and surface localization of BrpA.A rabbit pre-immunization serum sample was used as a negative control (A), while hyperimmune serum generated against rBrpA was used to determine differential protein production at 22°C and 35°C (B). Immunoblots were also probed with chicken serum generated against B. turicatae rFlaB (B lower panel). Surface localization assays were performed by immunoblotting using the rabbit serum generated against rBrpA (C upper panel) and chicken serum generated against rFlaB (C lower panel). Molecular mass is depicted in kilodaltons on the left of each blot. The asterisk and arrowhead indicate the expected size of BrpA and FlaB, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002514-g002: Temperature mediated production and surface localization of BrpA.A rabbit pre-immunization serum sample was used as a negative control (A), while hyperimmune serum generated against rBrpA was used to determine differential protein production at 22°C and 35°C (B). Immunoblots were also probed with chicken serum generated against B. turicatae rFlaB (B lower panel). Surface localization assays were performed by immunoblotting using the rabbit serum generated against rBrpA (C upper panel) and chicken serum generated against rFlaB (C lower panel). Molecular mass is depicted in kilodaltons on the left of each blot. The asterisk and arrowhead indicate the expected size of BrpA and FlaB, respectively.
Mentions: Immunoblot analysis using serum samples from mice infected by tick bite indicated that rBrpA was not immunogenic (data not shown). Moreover, immunoblotting using serum samples from rabbits immunized with rBrpA demonstrated up-regulation of the native protein when spirochetes were grown at 22°C when compared to 35°C (Figure 2 A and B). Reactivity against FlaB with the samples indicated that similar amounts of spirochete lysates were electrophoresed (Figure 2B insert). The susceptibility of BrpA to digestion with proteinase K suggested that the protein was localized on the outer surface of the spirochetes (Figure 2 C). Detecting the periplasmic protein, FlaB, verified that the outer membrane of B. turicatae remained intact during the proteinase K treatments (Figure 2 C lower insert).

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