O Antigen Protects Bordetella parapertussis from Complement

Bacterial strains and growth. B. parapertussis strain CN2591 and an isogenic mutant lacking O antigen, CN2591Δwbm, have previously been described (42) and were maintained on Bordet-Gengou agar (Difco, Sparks, MD) containing 10% sheep's blood (Hema Resources, Aurora, OR) with 20 μg/ml streptomycin. For inoculation, the bacteria were grown overnight at 37°C in Stainer-Scholte broth (26, 54) to mid-log phase and diluted in phosphate-buffered saline (PBS; OmniPur, Gibbstown, NJ) to a concentration of 1.0 × 10⁷ CFU/ml (23, 28, 60, 61).

Animal experiments.Four- to 6-week-old C57BL/6, CD11b^−/−, μMT, RAG1^−/−, and C5^−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were bred in a Bordetella-free environment. C3 knockout (C3^−/−) mice were a kind gift from Rick Wetsel and have previously been described (15). All mice were maintained in housing facilities approved by Pennsylvania State University and were closely monitored in accordance with institutional policies and IACUC regulations. For inoculation, mice were lightly sedated with 5% isofluorane in oxygen and 50 μl of PBS containing 5 × 10⁵ CFU of the indicated bacteria was pipetted onto the tips of the external nares (28, 62). Groups of three or four animals were sacrificed via CO₂ inhalation on the indicated days for postmortem dissection of the lungs, trachea, nasal cavity, spleen, liver, and/or kidneys, as indicated. We quantified the bacterial colonization by homogenizing each tissue in PBS, serial plating, and subsequently counting colonies (22). Serum was collected via postmortem cardiac puncture from μMT mice. Survival curves were generated by infection of groups of 15 to 20 RAG1^−/− mice with either CN2591 or CN2591Δwbm with or without cobra venom factor (CVF) treatment, as indicated (50). Mice suffering from lethal bordetellosis, as determined by severe hunched posture, ruffled fur, extremely labored breathing, and apathy, were euthanized to prevent unnecessary suffering (31, 62).

Depletion of immune factors and quantification of leukocytes.For neutrophil depletion, mice were intraperitoneally injected with 1 mg αLy-6G antibody from the hybridoma RB6-8C5 48 h prior to inoculation (27, 51). The depletion of 99% of neutrophils was confirmed via blood smear. For complement depletion, mice were intraperitoneally injected with 5 U of CVF in PBS (Sigma, St. Louis, MO) at 26 and 24 h prior to inoculation, and every 5 days thereafter until the completion of the experiment (50). For alveolar macrophage depletion, mice were given an intranasal dose of 100 μl of clodronate liposomes (Roche Diagnostics, Mannheim, Germany) 48 h prior to inoculation (11). The presence or absence of alveolar macrophage or neutrophils in the lungs was determined via visual identification of cells from bronchoalveolar lavage fluid spun onto slides via Cytospin and then stained with modified Wright-Giemsa stain (Fisher Scientific, Kalamazoo, MI).

C3 deposition assay.Approximately 4 × 10⁸ CFU of mid-log-phase CN2591 and CN2591Δwbm bacteria were harvested by centrifugation and incubated at 37°C for 30 min with 20% naïve, antibody-deficient, complement-active or heat-inactivated serum collected from μMT mice. Samples were washed twice with PBS, incubated for 30 min on ice with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse C3 (MP Biomedicals), washed twice with cold PBS to remove unbound antibody and then analyzed by flow cytometry. Samples were run in triplicate, and the experiment was repeated independently.

Serum killing assay.Naïve C57BL/6 and C3^−/− mice were bled, and the pooled blood was placed on ice for 30 min. Samples were spun, and the serum was extracted and diluted to the indicated concentrations. One thousand CFU of mid-log-phase CN2591 and CN2591Δwbm in 5 μl of PBS were incubated with 45 μl of diluted serum for 1 h at 37°C, followed by subsequent plating and colony count. Samples were run in triplicate, and the experiment was repeated independently.

In vivo bioluminescence imaging of bacterial colonization.The B. parapertussis strains C2591 and C2591Δwbm were rendered bioluminescent by the chromosomal insertion of pSS4266 to produce strains BPS1766 (B. parapertussis::luciferase) and BPS1768 (B. parapertussis Δwbm::luciferase), respectively. Briefly, pSS4266 is a derivative of the chromosomally integrating promoter assay vector pSS3110 (57) in which the lacZYA operon has been replaced by the luxCDABE operon of Photorhabdus luminescens, derived from pUTminiTn5kmlux (21). Luciferase expression in pSS4266 is driven by the fha promoter of Bordetella pertussis. BPS1766 and BPS1768 were used to inoculate littermate RAG1^−/− mice intranasally as described above. Mice were subsequently imaged daily in an IVIS-100 apparatus (Xenogen Corp.) according to the manufacturer's instructions.

Statistical analysis.Student's two-tailed t test was used to determine statistical significance between experimental groups. Values of P ≤ 0.05 were considered significant.

O antigen contributes to B. parapertussis colonization of the murine respiratory tract.Previous studies have shown that B. parapertussis lacking O antigen (B. parapertussis Δwbm) is nearly cleared from the lower respiratory tract within 3 days using a low-dose (1,000 CFU) inoculation regimen in BALB/c mice, while B. parapertussis is able to increase in number (9). To more thoroughly examine interactions between O antigen and the immune response, we used a high-dose inoculation regimen that distributes the bacteria throughout the respiratory tract in numbers that rigorously test the innate and adaptive immune responses (9, 22, 28, 62). C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. parapertussis or B. parapertussis Δwbm and sacrificed on day 0, 3, 7, 14, or 28 postinoculation. Mice dissected 10 min postinoculation had approximately 10⁵ CFU of B. parapertussis or B. parapertussis Δwbm in their lungs (Fig. 1). B. parapertussis levels increased over the first few days to 10⁶ CFU in the lungs and decreased steadily over the subsequent 3 weeks to approximately 10⁵ CFU, 10³ CFU, and 10¹ CFU by days 7, 14, and 28 postinoculation, respectively (Fig. 1). B. parapertussis Δwbm levels in the lungs were approximately 10-fold lower than that of B. parapertussis on days 3 and 7 postinoculation, but both B. parapertussis and B. parapertussis Δwbm were found at similar levels on days 14 and 28 postinoculation (Fig. 1). The 90% lower levels of the O antigen-deficient strain on only days 3 and 7 postinoculation, but not later days, suggest that the O antigen of B. parapertussis contributes to infection at relatively early stages but has little effect on eventual clearance.

• Open in new tab
• Download powerpoint

FIG. 1.

Colonization of mouse lungs by B. parapertussis and B. parapertussis Δwbm over time. Groups of three 4- to 6-week-old C57BL/6 mice were inoculated with 5 × 10⁵ CFU of B. parapertussis (filled diamonds) or B. parapertussis Δwbm (open diamonds). The number of CFU recovered from the lungs at each indicated time point is expressed as the log₁₀ means ± standard errors (error bars). *, P ≤ 0.05. The limit of detection is log₁₀1, indicated as the y intercept.

O antigen does not contribute to colonization in the absence of complement.In order to investigate the immune mechanism that O antigen protects against, we blocked various innate immune functions and examined bacterial numbers 3 days postinoculation. Neutrophils or alveolar macrophages were depleted from wild-type mice with αLy6G antibody or clodronate liposome treatment, respectively (11, 27, 51). These mice were then inoculated with B. parapertussis or B. parapertussis Δwbm as described above and dissected 3 days postinoculation. In neutrophil-depleted mice, B. parapertussis reached approximately 10^6.5 CFU, while B. parapertussis Δwbm reached levels of 10^5.5 CFU in the lungs, a 10-fold defect relative to wild-type bacteria (Fig. 2). In addition, there was no difference in neutrophil accumulation in the lungs of wild-type mice at 12 h and 24 h postinoculation with B. parapertussis or B. parapertussis Δwbm (data not shown). This result indicated that O antigen contributes to colonization even in the absence of neutrophils and therefore must protect B. parapertussis from some other immune function (Fig. 2). Similarly, the O antigen-deficient strain was also defective in its ability to colonize alveolar macrophage-depleted mice (Fig. 2). Unfortunately, mice depleted of both neutrophils and alveolar macrophages succumbed to sham inoculations consisting of sterile PBS and, therefore, the effect of O antigen on the ability of B. parapertussis to colonize doubly depleted hosts was not assessed. These results suggest that the early defect of the O antigen-deficient strain is not simply the result of increased susceptibility to neutrophil- or macrophage-mediated clearance alone, but is instead due to sensitivity to some other innate immune function.

• Open in new tab
• Download powerpoint

FIG. 2.

Colonization of mouse lungs by B. parapertussis and B. parapertussis Δwbm upon depletion of neutrophils, alveolar macrophages, or complement. Groups of three 4- to 6-week-old C57BL/6 mice were left untreated or were treated with α-Ly-6G antibodies, clodronate liposomes, or CVF prior to inoculation with 5 × 10⁵ CFU of B. parapertussis (filled bars) or B. parapertussis Δwbm (open bars), as indicated. The number of CFU recovered from mouse lungs at day 3 postinoculation is expressed as the log₁₀ means ± standard errors (error bars). The asterisk indicates P ≤ 0.05. The limit of detection is log₁₀1, indicated as the y intercept.

Since B. parapertussis Δwbm is susceptible to complement in vitro (9), we sought to determine whether complement was responsible for the defect of B. parapertussis Δwbm. CVF was given to deplete complement in wild-type mice 26 and 24 h prior to inoculation (50). Both B. parapertussis and B. parapertussis Δwbm were recovered at approximately 10^6.5 CFU on day 3 postinoculation in the lungs of CVF-treated mice, a level similar to that for untreated mice inoculated with wild-type B. parapertussis (Fig. 2). These results suggest that the lack of O antigen leaves the bacteria more susceptible to complement-mediated control in vivo. Furthermore, there appears to be no significant role for O antigen in the absence of complement during the first week of infection (Fig. 2).

O antigen prevents C3 deposition on the surface of B. parapertussis.Since O antigen appeared to protect B. parapertussis against complement-mediated control, we sought to examine the ability of C3 to bind to the surface of B. parapertussis and B. parapertussis Δwbm. Bacteria were incubated with 20% naïve, antibody-deficient serum that was either maintained as complement active or heat-inactivated. Surface bound C3 was detected by FITC-labeled anti-C3 antibodies. B. parapertussis incubated with serum showed a similar amount of FITC-positive staining as B. parapertussis incubated without serum or with heat-inactivated serum (Fig. 3), indicating that wild-type B. parapertussis was able to avoid complement deposition. Interestingly, B. parapertussis Δwbm showed a 50-fold increase in C3-positive cells (Fig. 3F) when incubated with complement active serum compared to the number of C3-positive B. parapertussis cells with similar treatment (Fig. 3E). C3 deposition on the O antigen mutant was not observed upon incubation with heat-inactivated serum (Fig. 3G and H). Together, these data indicate that the presence of O antigen prevents C3 deposition on B. parapertussis.

• Open in new tab
• Download powerpoint

FIG. 3.

Flow cytometry analysis of C3 deposition on B. parapertussis and B. parapertussis Δwbm. Approximately 4 × 10⁸ CFU of B. parapertussis (A, C, E, and G) or B. parapertussis Δwbm (B, D, F, and H) was harvested from mid-log-phase cultures, incubated at 37°C without serum (A to D), with 20% complement active naïve mouse serum (E and F), or with 20% heat-inactivated naive mouse serum (G and H) for 30 min. Samples either were left unstained (A and B) or were labeled with FITC-anti-mouse C3 antibodies (C to H) and analyzed by flow cytometry. The percentage of FITC-positive cells is indicated as the average of three replicates. (I) B. parapertussis (squares) or B. parapertussis Δwbm (triangles) were exposed to complement active (closed) or complement-deficient (open) mouse serum at the indicated concentrations for 1 h. The average percent survival of three replicates is shown. Error bars represent standard errors. *, P ≤ 0.05.

To determine whether the deposition of C3 caused subsequent complement-mediated killing of B. parapertussis Δwbm, 1,000 CFU of either B. parapertussis or B. parapertussis Δwbm was incubated with various concentrations of complement active serum from wild-type mice or complement-deficient serum from C3^−/− mice. B. parapertussis Δwbm was significantly more sensitive to wild-type serum at concentrations of 10% serum or higher than complement-deficient serum (Fig. 3I). These results indicate that O antigen protects B. parapertussis from C3 deposition and subsequent complement-mediated killing.

C3 causes the in vivo defect of the O antigen-deficient strain of B. parapertussis.In order to dissect the specific components of complement that limited B. parapertussis Δwbm levels by day 3 postinoculation, we inoculated mice lacking different aspects of the complement cascade. C3^−/− mice are unable to produce complement component C3, the protein required for positive feedback and amplification of both classical and alternative complement activation pathways as well as opsonization and subsequent formation of the membrane attack complex (MAC) on bacterial cells (15). C3^−/− mice harbored similar bacterial loads of B. parapertussis and B. parapertussis Δwbm, indicating that C3 is required to reduce the levels of B. parapertussis Δwbm (Fig. 4). C5^−/− mice lack complement component C5, which is required to form the MAC but not for the positive feedback, amplification, and opsonization via C3 deposition. CD11b^−/− mice are able to form the MAC, but lack the complement receptor type III (CR3), which is known to be important in host response to LPS and is found on macrophages, neutrophils, dendritic cells, and natural killer cells (37, 40, 46, 58). B. parapertussis Δwbm levels were 10-fold lower than those of wild-type bacteria in both C5^−/− and CD11b^−/− mice, indicating that individually, these complement factors are not required for the more efficient control of B. parapertussis Δwbm (Fig. 4). These results further indicate that the defect in colonization of the O antigen mutant is a result of its increased susceptibility to complement activities that require C3, but not C5 or CR3 individually.

• Open in new tab
• Download powerpoint

FIG. 4.

C3 mediates in vivo control of B. parapertussis Δwbm. Groups of three 4- to 6-week-old C3^−/−, C5^−/−, or CD11b^−/− mice were inoculated with 5 × 10⁵ CFU of B. parapertussis (filled bars) or B. parapertussis Δwbm (open bars). The number of CFU recovered from the lungs at day 3 postinoculation is expressed as the log₁₀ means ± standard errors (error bars). *, P ≤ 0.05. The limit of detection is log₁₀1, indicated as the y intercept.

O antigen is required for the systemic spread of B. parapertussis.Since O antigen is known to facilitate systemic colonization in other infection models, we sought to determine the role of O antigen in the systemic spread of B. parapertussis (30, 49). RAG1^−/− mice were infected with either B. parapertussis::luciferase or B. parapertussis Δwbm::luciferase, and the colonization was visualized on day 21 postinoculation (Fig. 5A and B). Mice infected with B. parapertussis::luciferase showed high levels of colonization in the thoracic cavity and colonization in the upper abdominal cavity. In contrast, mice infected with B. parapertussis Δwbm::luciferase showed colonization only in the respiratory tract. To determine the numbers of B. parapertussis or B. parapertussis Δwbm isolates in systemic organs, groups of four to six RAG1^−/− mice were infected with either B. parapertussis or B. parapertussis Δwbm and then dissected on day 17 postinoculation. Significantly higher numbers of B. parapertussis isolates were found in the respiratory tract compared to the numbers of B. parapertussis Δwbm isolates. Additionally, B. parapertussis had spread systemically in all RAG1^−/− mice and was found in the spleen, liver, and kidneys, whereas no systemic colonization by B. parapertussis Δwbm was detected (Fig. 5C). Groups of 15 to 20 RAG1^−/− mice were inoculated with either B. parapertussis or B. parapertussis Δwbm and monitored for survival. Mice infected with B. parapertussis survived for 2 weeks before showing signs of lethal bordetellosis. By day 17 postinoculation, 50% of the mice had died and all mice succumbed to B. parapertussis infection by day 21 postinoculation, consistent with previously reported findings (62). In contrast, all mice inoculated with B. parapertussis Δwbm survived until the end of the experiment (day 100) without any overt signs of disease (Fig. 5D and data not shown).

• Open in new tab
• Download powerpoint

FIG. 5.

Role of O antigen in the lethality and systemic spread of B. parapertussis infections. RAG1^−/− mice were infected with (A) B. parapertussis::luciferase or (B) B. parapertussis Δwbm::luciferase, and the light production was visualized on day 21 postinoculation and is represented by heat plot. (C) Groups of three to six 4- to 6-week old RAG1^−/− mice were inoculated with B. parapertussis (closed bars) or B. parapertussis Δwbm (open bars) and dissected on day 17 postinoculation for bacterial enumeration in the indicated organs. CFU are expressed as the log₁₀ means ± standard errors (error bars). *, P ≤ 0.05. The limit of detection is log₁₀1, indicated as the y intercept. ND, no CFU were detected in the sample. (D) Groups of 15 to 20 4- to 6-week-old RAG1^−/− mice were inoculated with 5 × 10⁵ CFU of B. parapertussis (filled symbols) or B. parapertussis Δwbm (open symbols) or were sham inoculated (×) with (triangle or ×) or without (diamond) CVF treatment and were monitored for survival.

To determine whether complement-mediated control of B. parapertussis Δwbm prevented systemic spread and enabled the survival of B. parapertussis Δwbm-infected mice, groups of RAG1^−/− mice were treated with CVF to deplete complement. These mice were then either sham inoculated or inoculated with B. parapertussis Δwbm. CVF-treated, sham-inoculated animals showed no sign of deteriorating disease up to day 35. In contrast, CVF-treated, B. parapertussis Δwbm-inoculated animals survived for 2 weeks before showing signs of lethal bordetellosis, a survival curve similar to that of RAG1^−/− mice inoculated with B. parapertussis (Fig. 4D). By day 21 postinoculation, all CVF-treated, B. parapertussis Δwbm-inoculated mice had succumbed to infection (Fig. 4D). Therefore, the depletion of complement allowed the systemic spread of B. parapertussis Δwbm, further indicating that complement mediates the more efficient control of the O antigen-deficient strain.