A unique characteristic of the species
Streptococcus pneumoniae is its auxotrophic requirement for choline (
14). The bacterium takes up choline from the growth medium and incorporates this amino alcohol into the cell wall teichoic acid (
2,
18) and the membrane-anchored glycolipid lipoteichoic acid polymers (
1) that are located on the pneumococcal cell surface (
7). A choline-independent strain, R6Cho
−, capable of growing in choline-free medium acquired heterologous genetic elements during transformation of the
S. pneumoniae strain R6 with DNA from
Streptococcus oralis (
10,
17), a streptococcal species that contains choline in its cell wall but does not require choline for growth (
9). The choline independence of another, recently isolated mutant, R6Chip, is based on a single point mutation in the
tacF gene, which encodes a teichoic acid flippase (
4). However, these mutant strains were still able to utilize environmental choline. In order to prevent this, mutant derivatives of R6Cho
− and R6Chip were prepared in which genes in the
lic1 operon responsible for the cellular uptake and metabolism of choline were inactivated (
11). Such mutants, for instance, R6Cho
−licA64, were able to maintain a choline-free phenotype in the choline-containing in vivo environment. R6Cho
−licA64 grew in long autolysis-defective chains both in choline-containing and choline-free medium, and D39Cho
−licA64, a derivative of the strain expressing capsular polysaccharide II, could grow both in vitro and also in vivo in a murine model of pneumococcal infection (
11).
In a previous study it was demonstrated that the choline-free strains D39Cho
−licA64 and D39ChiplicB31, expressing the polysaccharide capsular type II, were virtually avirulent in the mouse model of sepsis (
4,
11). Nevertheless, following intraperitoneal inoculation, D39Cho
−licA64 was able to invade the bloodstream and replicate for a limited time, after which the bacteria were cleared from the blood. The purpose of the study described here was to determine to what extent these avirulent strains have the capacity to engage the host immune system during their transient period of growth within the host.
MATERIALS AND METHODS
Cultivation of bacteria and preparation of inocula.
For murine in vivo assays the
S. pneumoniae strains D39Cho
−, D39Cho
−licA64, and D39ChiplicB31 (
4) were grown in choline-free Cden medium at 37°C without aeration (
19). By using choline-free Cden medium all strains showed a chain-forming phenotype just prior to intraperitoneal inoculation into the mice. Strains D39 and SV36 (the latter producing a capsular polysaccharide type III) (
13) were grown in C+Y medium at 37°C without aeration (
9). For inoculum preparation exponentially growing cultures of the strains were back-diluted in the respective growth medium and were allowed to grow to an optical density at 590 nm of 0.6. Bacterial pellets were washed twice with 0.9% NaCl. The cultures were further diluted in 0.9% NaCl and adjusted to the desired inoculum concentrations.
Murine intraperitoneal sepsis model.
For the majority of the experiments 8-week-old female CD1 mice (Charles River Laboratories, Wilmington, MA) were used. Time course studies on the in vivo growth of bacteria were repeated three times. Mice were injected in the peritoneal cavity with 0.5 ml of the prepared inocula containing 106 CFU of bacteria. In each experiment blood from two mice per time point was collected by cardiac puncture and pooled. For bacterial titer determinations serial dilutions of the blood samples were plated on 3% sheep blood agar plates supplemented with 5 μg/ml gentamicin and incubated at 37°C in a 5% CO2 atmosphere. Prior to the assay of serum cytokine levels, 10 μg/ml mitomycin C was added to the blood samples followed by incubation for 1 h at 37°C, in order to kill the bacteria. Control experiments showed that mitomycin C did not trigger cytokine production in uninfected blood samples. After centrifugation the serum was collected and stored at −80°C.
For experiments studying the effect of Toll-like receptor 2 (TLR-2) on the infection, 8-week-old male B6.129-Tlr2tm1Kir/J mice (The Jackson Laboratories, Bar Harbor, ME) were used. For survival curves five B6.129-Tlr2tm1Kir/J mice were compared to five C57BL/6 mice, which had the appropriate genetic background to serve as controls. Both groups (n = 5 animals) were infected with 105 CFU D39Cho−, and survival of the mice was monitored.
Cytokine determinations.
The concentrations of cytokines from mouse sera were measured by using Luminex (Millipore Corporation, St. Charles, MO), according to the manufacturer's protocol: 25-μl serum samples were incubated for 15 min with 25 μl of a serum diluent, followed by a 2-h incubation with Beadmates coated with anticytokine monoclonal antibodies. The plate was washed once, and biotin-conjugated anticytokine monoclonal antibodies were added for 1.5 h, followed by 30 min of incubation with Beadlyte streptavidin-phycoerythrin. Samples were measured in duplicate with Luminex and analyzed using Beadview software (Millipore Corporation, St. Charles, MO).
Maturation of murine splenic dendritic cells.
To analyze the activation state of dendritic cells in mice infected with the different bacterial strains, one mouse spleen per bacterial strain was collected at 6 and 9 h postinfection. The tissues were transferred to petri dishes containing Hank's medium supplemented with collagenase. After flushing with medium using a syringe, the spleen tissues were homogenized and incubated at 37°C for 25 min. Next, 100 μl of 0.5 M EDTA was added followed by incubation at 37°C for 5 min, after which the cell suspensions were strained through a mesh and centrifuged. ACK lysing buffer (1.5 ml; BioSource, Rockville, MD) was added and mixtures were incubated for 4 min at room temperature to allow erythrocyte lysis. To stop lysis 13 ml of fluorescence-activated cell sorting buffer (phosphate-buffered saline [PBS] plus 5% fetal calf serum) was added and cells were centrifuged for 10 min. CD11c+ CD8+ and CD11c+ CD8− dendritic cells were analyzed by fluorescence-activated cell sorting for the presence of cell surface maturation markers CD80 and CD86. Two independent experiments were performed.
Maturation of human monocyte-derived dendritic cells.
Buffy coats purchased from the New York Blood Center were used as a source of mononuclear cells from healthy donors. Peripheral blood mononuclear cells were isolated from the peripheral blood by density gradient centrifugation (Ficoll-Paque Plus; GE Healthcare). Monocytes were separated from the peripheral blood mononuclear cells by using CD14 microbeads (Miltenyi Biotech). Dendritic cells (DCs) were generated from purified blood monocytes as previously described (
5). The CD14
+ monocytes were cultured in RPMI 1640 (Biowhittaker) supplemented with 1% plasma in the presence of 800 U/ml of granulocyte-macrophage colony-stimulating factor (Immunex) and 1,000 U/ml of interleukin-4 (IL-4; R&D Systems). The cultures were supplemented with cytokines on days 1, 3, and 5 of culture. On day 5, immature DCs were allowed to mature overnight with 100 ng/ml lipopolysaccharide (LPS; Sigma-Aldrich).
Various bacteria as well as bacterial cell wall preparations were tested for their potential to induce DC maturation after incubation for 36 h. Cultures of the choline-containing strains D39Cho− and the isogenic choline-free derivative D39Cho−licA64 were grown in C+Y medium at 37°C. When the cell concentration in the cultures reached about 107 CFU per ml the cultures received 10 μg/ml mitomycin C and were incubated for 1 h at 37°C to kill the bacteria, which was confirmed by plating on blood agar. CFU equivalents of 108 of the nonviable bacterial suspensions were applied to assay dendritic cell maturation.
Choline-free and choline-containing cell walls were prepared and purified by a published procedure (
17) from the nonencapsulated strain R6Cho
− grown in Cden medium that was either free of choline or was supplemented with 5 μg/ml choline. Suspensions of cell walls were adjusted to the desired concentration in PBS, and approximately 10
7 CFU equivalents were used in the assays.
Dendritic cell maturation was monitored by flow cytometry (FACSCalibur; BD Biosciences) based on the presence of various maturation markers, CD80, CD83, and CD86 (BD Biosciences). Each experiment was repeated three times using fresh batches of dendritic cells and freshly prepared mitomycin C-killed bacteria.
Induction of protective immunity.
Mice were injected intraperitoneally (i.p.) with 103 CFU of the avirulent D39Cho−licA64 strain expressing the type II capsular polysaccharide (n = 4 animals per group). Control mice (n = 5 animals) were injected with saline. On day 10 after inoculation with the avirulent strain the animals were challenged with 104 CFU of the highly virulent strain, D39Cho−, and survival was monitored.
In a second experiment, animals were immunized i.p. with 104 CFU of the avirulent D39ChiplicB31 strain (n = 5 animals per group). Five control mice were injected with saline. On day 10 postinfection the animals were challenged with a potentially lethal dose (104 and 106 CFU) of either of the highly virulent strains D39 (capsule type II) or SV36 (capsular type III), and survival was monitored. All mice that survived this first challenge were next challenged on day 25 with a lethal dose of 104 CFU of strain SV36.
Detection of capsule-specific IgM antibodies with an enzyme-linked immunosorbent assay (ELISA).
Capsular-specific antibodies were quantified using a modified version of a previously described protocol (
3). Plates (96-well; Nunc) were coated with 1 μg/well of type II or type III capsular polysaccharide in carbonate buffer, pH 9.5, overnight at 4°C. Plates were washed with 0.05% Tween 20-PBS and blocked for 6 h with 1% bovine serum albumin in PBS. Next, plates were washed and sera of mice, collected at day 10 after infection with D39ChiplicB31, were analyzed. Samples were plated out in duplicate serial threefold dilutions, starting at a 1:6 dilution, and incubated overnight at 4°C. The plates were washed, and goat anti-mouse immunoglobulin M (IgM) antibodies conjugated to horseradish peroxidase (Bethyl) were plated at 0.2 μg/ml and incubated for 4 h at room temperature. The plates were washed and developed with 3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, MD) per the manufacturer's instructions, and the absorbance was read at 405 nm.
DISCUSSION
In a previous report we showed that the high degree of invasiveness of
S. pneumoniae strain D39Cho
− expressing the capsular polysaccharide type II could be radically reduced by genetic manipulations that resulted in the removal of choline residues from the pneumococcal surface (
11). The product of such a genetic manipulation, strain D39Cho
−licA64, continued to express the capsular polysaccharide II on its surface but is virtually avirulent in the murine model of pneumococcal infection.
In the studies presented here we explored several alternative mechanisms to explain the profound impact of teichoic acid choline residues on pneumococcal virulence.
During the first 6 hours after infection of the mice, the viable titers of both the choline-containing strain D39Cho− and the choline-free double mutant D39Cho−licA64 increased substantially, accompanied by expression of the proinflammatory cytokines IL-1β, IL-6, IL-12(p70), TNF-α, and IFN-γ. In contrast to the infection with choline-free bacteria, in which, past the 6-h time point, declining bacterial blood titers were paralleled by decreasing cytokine levels, the choline-containing pneumococci continued to multiply, accompanied by steadily increasing levels of proinflammatory cytokines.
These observations suggest that pneumococci have to reach a certain blood titer threshold (∼10
5 to 10
6 CFU/ml) to induce inflammation in the host. Once bacterial titers drop below this threshold due to the onset of a successful immune clearance mechanism, the inflammation ends. This hypothesis is also in line with several previous studies that claimed threshold bioactivities of various pneumococcal cell wall preparations in animal models (
12).
We considered the possibility that the choline residues are directly recognized by TLR-2. A recent publication suggested that pneumococcal lipoteichoic acid is detected by Toll-like receptor 2 in vitro, resulting in the expression of the cytokines IL-1, IL-8, IL-10, TNF-α, and IFN-γ (
6). This pathway could contribute to the observed overwhelming immune response induced by choline-containing bacteria as well as to their lethality. In turn, the lack of covalently attached phosphorylcholine residues may weaken the overall binding of lipoteichoic acid to TLR-2 and impair the virulence of the bacteria. If these assumptions were true, TLR-2 knockout mice would be less susceptible to choline-containing bacteria. We tested this hypothesis by infecting TLR-2 knockout mice with D39Cho
− and found that the mortality was identical to what we observed in the isogenic wild-type mice, indicating that the pathway via TLR-2 has no influence on the recognition of the choline moieties and the choline-dependent virulence of the strain tested.
We compared the maturation status of dendritic cells in CD1 mice that received the choline-free versus choline-containing bacteria. Spleens were removed from mice and costimulatory molecules on the surface of isolated splenic dendritic cells were determined as indicators of maturation. Remarkably, both the choline-free as well as choline-containing strains equally engaged dendritic cells, despite the different intensities of the induced cytokine patterns. Lymphoid CD11c+ CD8+ as well as myeloid CD11c+ CD8− DC subsets showed equal upregulation of costimulatory molecules CD80 and CD86. Although we cannot predict yet whether the initiated immune response shows a cellular or humoral profile, these results demonstrate that choline-free S. pneumoniae bacteria possess potent adjuvant properties.
We also tested the ability of the bacterial preparations to stimulate maturation of human monocyte-derived dendritic cells in vitro. Human dendritic cells were incubated with mitomycin C-killed suspensions of strains D39Cho
−licA64 and D39Cho
−. Both strains were able to trigger upregulation of costimulatory molecules, although the choline-containing strain D39Cho
− led to a stronger activation. To narrow down the immunogenic epitope on the bacterial surface, purified cell walls differing in the presence or absence of choline were also tested in the same assay and were found to have equal potencies to induce maturation of dendritic cells. Similarly, the same pair of cell wall preparations showed identical inflammatory potentials when applied intracisternally into the cerebrospinal fluid space of infant rats (
8).
These experiments suggest that the choline residues may not be directly recognized by the immune system, nor do they represent the immediate trigger of the immune response, since choline-free bacteria were still able to stimulate cytokine production and DC maturation in vivo and the cell wall preparations from both choline-containing and choline-free bacteria could activate DCs in vitro.
To understand whether the observed immunogenicity of choline-free bacteria was also sufficient to induce protective immunity, mice were immunized with a single dose of D39Cho
−licA64. After 10 days the same animals were reinfected with the isogenic virulent strain D39Cho
− and they showed a surprisingly strong protection. However, it was not clear from this experiment whether the induced protection was serotype specific or was possibly related to the large heterologous
S. oralis elements that have been shown to be present in strain D39Cho
− (
10).
To address these questions we repeated the “protection” experiment using the recently isolated and genetically well-defined choline-independent mutant D39ChiplicB31, which only differs from the wild-type D39 (besides the mutation in the
licB gene) by a single point mutation (
4) and is therefore free of any possibly interfering, heterologous genetic elements. Table
1 shows that animals surviving infection by the avirulent double mutant D39ChiplicB31 were protected against challenge by the highly virulent, isogenic wild-type strain D39, but the same animals were still susceptible to challenge by the capsular type III strain SV36. These findings make it unlikely that the protection effect originally observed was related to the strain or to the large heterologous DNA sequences carried in D39Cho
−licA64, the strain used in the original protection experiment. Instead, the results suggest that infection by the avirulent pneumococci was able to generate a capsule-specific protective immunity in the mice. Consistently, mice vaccinated with D39ChiplicB31 developed capsule II-specific IgM antibodies within 10 days. No capsule III-specific antibodies were detected in the very same sera.
These observations allow two major conclusions. (i) Despite its drastically reduced virulence, the choline-free double mutant expressing the capsular polysaccharide II was still able to engage the host immune system, as evidenced by the expression of proinflammatory cytokines, maturation of dendritic cells, and the capacity to induce a substantial degree of serotype specific protective immunity in the surviving animals. The chemical entity responsible for triggering the observed immune response appears to be a component common to both choline-containing and choline-free cell walls, most likely a component of the peptidoglycan or teichoic acid backbone.
(i) The mechanism of the strikingly different virulence of choline-containing and choline-free bacteria is not well understood. The choline-containing bacteria have a superior capacity to continue proliferation and reach higher titers in the bloodstream, thus providing a continuous stimulus for cytokine production and keeping the immune system in a highly activated state for a prolonged time. In sharp contrast, the clearance of the choline-free bacteria from the bloodstream and their decreasing titers, beyond the 6-h time point after infection, limit the pathological inflammation.
Our observations strongly suggest that the critical contribution of choline residues to the virulence potential of pneumococci may be the role that these amino alcohol residues play in a pneumococcal immune evasion strategy. Whether this mechanism is directly linked to the choline moieties or is mediated through the choline-binding proteins attached to the choline residues on the pneumococcal surface is unknown at the present time.