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In vivo expression technology identifies a novel virulence factor critical for Borrelia burgdorferi persistence in mice.

Ellis TC, Jain S, Linowski AK, Rike K, Bestor A, Rosa PA, Halpern M, Kurhanewicz S, Jewett MW - PLoS Pathog. (2013)

Bottom Line: Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions.Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America.

ABSTRACT
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

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Spirochetes lacking bbk46 retain seroreactivity in mice.Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46::flaBp-aadA/pBSV2G (Δbbk46/vector) and bbk46::flaBp-aadA/pBSV2G-bbk46 (Δbbk46/bbk46+) at a dose of 1×104 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.
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ppat-1003567-g007: Spirochetes lacking bbk46 retain seroreactivity in mice.Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46::flaBp-aadA/pBSV2G (Δbbk46/vector) and bbk46::flaBp-aadA/pBSV2G-bbk46 (Δbbk46/bbk46+) at a dose of 1×104 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.

Mentions: In vitro growth analysis demonstrated that the bbk46 mutant and complemented clones had no detectable in vitro phenotypes (Figure 5C). Therefore, to examine the role of bbk46 in mouse infectivity, groups of five C3H/HeN female mice were needle inoculated intradermally under the skin of the upper back with 1×104 wild-type, Δbbk46/vector or Δbbk46/bbk46+ spirochetes. Three weeks post inoculation, mice were assessed for B. burgdorferi infection by serology and reisolation of spirochetes from the inoculation site, ear, bladder and joint tissues. All five mice from each infection group were seropositive for anti-B. burgdorferi antibodies (Figure 7, Table 4). Surprisingly however, no spirochetes were reisolated from all tissues examined from the five mice inoculated with the Δbbk46/vector clone (Table 4), whereas, all five mice inoculated with the wild-type or the Δbbk46/bbk46+ clone resulted in reisolation of spirochetes from all tissues analyzed (Table 4). Together these data demonstrated that spirochetes lacking the bbk46 gene transiently infected and elicited a humoral response in mice, but were unable to maintain a persistent infection in mouse tissues. To further define the contribution of the host immune response to the inability of spirochetes lacking the bbk46 genes to cause a persistent infection, groups of five severe combined immunodeficiency (scid) mice were inoculated with 1×104 wild-type, Δbbk46/vector or Δbbk46/bbk46+ spirochetes. Three weeks post inoculation the animals were assessed for infection by reisolation of spirochetes from the ear, bladder and joint tissues. Consistent with the hypothesized role of bbk46 in immune evasion and persistence, four out of five immunodeficient mice inoculated with the Δbbk46 mutant were positive for spirochete reisolation from all tissues examined (Table 4). These data demonstrated that a functional host immune response is required for the clearance of spirochetes lacking bbk46 from mouse tissues 3 weeks post infection, indicating that bbk46 is essential for the ability of B. burgdorferi to avoid killing by the host immune system in order to establish a persistent infection.


In vivo expression technology identifies a novel virulence factor critical for Borrelia burgdorferi persistence in mice.

Ellis TC, Jain S, Linowski AK, Rike K, Bestor A, Rosa PA, Halpern M, Kurhanewicz S, Jewett MW - PLoS Pathog. (2013)

Spirochetes lacking bbk46 retain seroreactivity in mice.Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46::flaBp-aadA/pBSV2G (Δbbk46/vector) and bbk46::flaBp-aadA/pBSV2G-bbk46 (Δbbk46/bbk46+) at a dose of 1×104 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757035&req=5

ppat-1003567-g007: Spirochetes lacking bbk46 retain seroreactivity in mice.Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46::flaBp-aadA/pBSV2G (Δbbk46/vector) and bbk46::flaBp-aadA/pBSV2G-bbk46 (Δbbk46/bbk46+) at a dose of 1×104 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.
Mentions: In vitro growth analysis demonstrated that the bbk46 mutant and complemented clones had no detectable in vitro phenotypes (Figure 5C). Therefore, to examine the role of bbk46 in mouse infectivity, groups of five C3H/HeN female mice were needle inoculated intradermally under the skin of the upper back with 1×104 wild-type, Δbbk46/vector or Δbbk46/bbk46+ spirochetes. Three weeks post inoculation, mice were assessed for B. burgdorferi infection by serology and reisolation of spirochetes from the inoculation site, ear, bladder and joint tissues. All five mice from each infection group were seropositive for anti-B. burgdorferi antibodies (Figure 7, Table 4). Surprisingly however, no spirochetes were reisolated from all tissues examined from the five mice inoculated with the Δbbk46/vector clone (Table 4), whereas, all five mice inoculated with the wild-type or the Δbbk46/bbk46+ clone resulted in reisolation of spirochetes from all tissues analyzed (Table 4). Together these data demonstrated that spirochetes lacking the bbk46 gene transiently infected and elicited a humoral response in mice, but were unable to maintain a persistent infection in mouse tissues. To further define the contribution of the host immune response to the inability of spirochetes lacking the bbk46 genes to cause a persistent infection, groups of five severe combined immunodeficiency (scid) mice were inoculated with 1×104 wild-type, Δbbk46/vector or Δbbk46/bbk46+ spirochetes. Three weeks post inoculation the animals were assessed for infection by reisolation of spirochetes from the ear, bladder and joint tissues. Consistent with the hypothesized role of bbk46 in immune evasion and persistence, four out of five immunodeficient mice inoculated with the Δbbk46 mutant were positive for spirochete reisolation from all tissues examined (Table 4). These data demonstrated that a functional host immune response is required for the clearance of spirochetes lacking bbk46 from mouse tissues 3 weeks post infection, indicating that bbk46 is essential for the ability of B. burgdorferi to avoid killing by the host immune system in order to establish a persistent infection.

Bottom Line: Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable.The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions.Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent.

View Article: PubMed Central - PubMed

Affiliation: Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America.

ABSTRACT
Analysis of the transcriptome of Borrelia burgdorferi, the causative agent of Lyme disease, during infection has proven difficult due to the low spirochete loads in the mammalian tissues. To overcome this challenge, we have developed an In Vivo Expression Technology (IVET) system for identification of B. burgdorferi genes expressed during an active murine infection. Spirochetes lacking linear plasmid (lp) 25 are non-infectious yet highly transformable. Mouse infection can be restored to these spirochetes by expression of the essential lp25-encoded pncA gene alone. Therefore, this IVET-based approach selects for in vivo-expressed promoters that drive expression of pncA resulting in the recovery of infectious spirochetes lacking lp25 following a three week infection in mice. Screening of approximately 15,000 clones in mice identified 289 unique in vivo-expressed DNA fragments from across all 22 replicons of the B. burgdorferi B31 genome. The in vivo-expressed candidate genes putatively encode proteins in various functional categories including antigenicity, metabolism, motility, nutrient transport and unknown functions. Candidate gene bbk46 on essential virulence plasmid lp36 was found to be highly induced in vivo and to be RpoS-independent. Immunocompetent mice inoculated with spirochetes lacking bbk46 seroconverted but no spirochetes were recovered from mouse tissues three weeks post inoculation. However, the bbk46 gene was not required for B. burgdorferi infection of immunodeficient mice. Therefore, through an initial IVET screen in B. burgdorferi we have identified a novel in vivo-induced virulence factor critical for the ability of the spirochete to evade the humoral immune response and persistently infect mice.

Show MeSH
Related in: MedlinePlus