Borrelia burgdorferi needs chemotaxis to establish infection in mammals and to accomplish its enzootic cycle

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, can be recovered from different organs of infected animals and patients, indicating that the spirochete is very invasive. Motility and chemotaxis contribute to the invasiveness of B. burgdorferi and play important roles in the process of the disease. Recent reports have shown that motility is required for establishing infection in mammals. However, the role of chemotaxis in virulence remains elusive. Our previous studies showed that cheA₂, a gene encoding a histidine kinase, is essential for the chemotaxis of B. burgdorferi. In this report, the cheA₂ gene was inactivated in a low-passage-number virulent strain of B. burgdorferi. In vitro analyses (microscopic observations, computer-based bacterial tracking analysis, swarm plate assays, and capillary tube assays) showed that the cheA₂ mutant failed to reverse and constantly ran in one direction; the mutant was nonchemotactic to attractants. Mouse needle infection studies showed that the cheA₂ mutant failed to infect either immunocompetent or immunodeficient mice and was quickly eliminated from the initial inoculation sites. Tick-mouse infection studies revealed that although the mutant was able to survive in ticks, it failed to establish a new infection in mice via tick bites. The altered phenotypes were completely restored when the mutant was complemented. Collectively, these data demonstrate that B. burgdorferi needs chemotaxis to establish mammalian infection and to accomplish its natural enzootic cycle.

Fig 1

Detection of CheA₂ under various culture conditions. The wild-type A3-68 Δbbe02 or B31A3 (15, 45) strain was cultivated under various conditions: UF conditions (23°C and pH 7.6), 34°C and pH 7.6, 37°C and pH 6.8, or FT conditions (switch from 23°C and pH 7.6 to 37°C and pH 6.8) (A) or in the presence of DMC (B) (1). Similar amounts of whole-cell lysates were analyzed by SDS-PAGE and then probed with CheA₂, DnaK (a loading control), and OspC antibodies as previously described (57). Increased OspC expression was used as a marker for cells cultured in DMC.

Fig 2

Detection of CheA₂ in the cheA₂^mut strain and the complemented cheA₂^com strain. (A) Construction of CheA₂/pBBE22G for complementation of cheA₂^mut. The fused P_flgB-cheA₂ fragment was released from pFlgBA₂com, a previously constructed vector for complementing an avirulent cheA₂ mutant (3), and cloned into pBBE22G (62), a shuttle vector of B. burgdorferi, yielding CheA₂/pBBE22G. (B) Western blot analysis of cheA₂^mut and cheA₂^com strains. The same amounts of wild-type, cheA₂^mut, and cheA₂^com whole-cell lysates were analyzed by SDS-PAGE and then probed with CheA₂ and DnaK antibodies as previously documented (28, 29).

Fig 3

Detection of plasmid contents in the cheA₂^mut (A) and cheA₂^com (B) strains by PCR. The primers for PCR were described previously (15).

Fig 4

The B. burgdorferi cheA₂ mutant is nonchemotactic. (A) Swarm plate assay. The assay was carried out on 0.35% agarose containing 1:10-diluted BSK-II medium as described previously (35). The flaB mutant, a previously constructed nonmotile mutant (35), was included to determine the sizes of the inocula. The data are presented as mean diameters (in millimeters) of rings ± SEM for four plates. (B) Capillary tube chemotaxis assay. Assays were carried out using 100 mM N-acetyl-d-glucosamine as an attractant according to a previous report (3). The results are expressed as fold increases in cell number entering the capillary tubes containing the attractant relative to the number entering control tubes without attractant (buffer alone). Results are expressed as means ± SEM for five tubes. A 2-fold increase is considered the threshold for a positive response. *, significant difference (P < 0.01).

Fig 5

Detection of spirochete burdens in needle-infected SCID mice by qRT-PCR. Groups of three mice were needle inoculated with 10⁵ cells of the wild-type, cheA₂^mut, and cheA₂^com strains. Infected animals were sacrificed at 24, 48, and 72 h. Skin samples from areas around the inoculation sites were collected and subjected to qRT-PCR. The bacterial burdens in these samples were measured by determining the number of copies of flaB mRNA compared to the number of copies of mouse β-actin transcript as previously documented (41, 66). The data are expressed as the means of relative levels of flaB transcript ± SEM for three independent experiments. At 72 h, the flaB mRNA was undetectable in the skin tissues infected by cheA₂^mut. *, significant difference (P < 0.01). Bb, B. burgdorferi.

Fig 6

Detection of spirochete burdens in microinjected nymphal ticks before and after feeding, using qRT-PCR. RNA samples were extracted from whole unfed ticks (10 days after injection) and fed ticks (after repletion; 5 to 7 days) and subjected to PCR analysis. The bacterial burdens in ticks were measured by the number of copies of flaB mRNA compared to the number of copies of tick β-actin transcript as previously described (23, 41). The data are presented as the means of relative levels of flaB transcript ± SEM for three groups (each group containing 3 unfed ticks or 4 replete ticks) for each strain (wild type, cheA₂^mut, and cheA₂^com).

Fig 7

Detection of spirochete burdens in C3H mice infected via tick bite, using qRT-PCR. At day 14 after tick feeding, mice were sacrificed and tissues (skin, heart, joint, and bladder) collected for RNA isolations and then subjected to qRT-PCR analysis as described in the legend to Fig. 4. The data shown here are the results for skin and heart tissues. No trace of flaB message was detected in the mouse tissues infected by cheA₂^mut; since its PCR cycle thresholds (C_T) are higher than that of the negative controls (no transcriptase added), it is considered negative.