Survival, replication, and antibody susceptibility of Ehrlichia chaffeensis outside of host cells

Abstract

Ehrlichia chaffeensis, an obligate intracellular, tick-transmitted bacterium, is susceptible to antibody-mediated host defense, but the mechanism by which this occurs is not understood. One possible explanation is that antibodies directly access the bacteria in the extracellular environment of the host, perhaps during bacterial intercellular transfer. Accordingly, we investigated whether bacteria could be found outside of host cells during infection. Host cell-free plasma obtained from infected mice was found to contain ehrlichiae, and the host cell-free ehrlichiae readily transferred disease to susceptible SCID recipients. The host cell-free ehrlichiae were found during infection of both immunocompetent BALB/c and immunocompromised BALB/c-scid mice and reached levels as high as 10(8)/ml in plasma during persistent infection in SCID mice. Approximately 10% of the blood-borne bacteria were found outside of host cells. Although it is generally accepted that replication of ehrlichiae occurs only within host cells, the cell-free bacteria were shown to undergo DNA replication and cell division in vitro for 3 to 5 days when incubated at 37 degrees C in plasma. Paradoxically, both infectivity and virulence were lost after 24 h of ex vivo culture. The data indicate that E. chaffeensis is exposed to the extracellular milieu during infection, presumably during intercellular transfer, and reveal that these intracellular bacteria do not require the environment of the host cell for replication. Our findings reveal a possible mechanism by which antibodies can access the intracellular bacteria upon their release into the extracellular milieu and mediate host defense and also have implications for understanding the replication and transmission of this vector-borne pathogen.

FIG. 1.

Transfer of plasma from E. chaffeensis-infected mice caused disease in recipient SCID mice. (a) Groups of three BALB/c-scid mice were uninfected or injected with either 200 μl of plasma, 10⁶ PBMCs isolated from 21-day-infected SCID mice, or 10⁶ E. chaffeensis-infected DH82 cells via the peritoneum. Body weight changes were monitored at weekly intervals. The average body weight and standard deviation of groups of three mice are shown. Statistical comparison of each group of infected mice with the uninfected control group revealed P values of 0.05, as determined with the Mann-Whitney test. (b) Quantitation of bacterial colonization in the livers of mice from panel a on day 22 posttransfer. The data are representative of three independent experiments.

FIG. 2.

Detection of cell-free ehrlichiae by direct immunofluorescence assay. Freshly isolated plasma from infected mice was centrifuged onto microscope slides with a Cytospin apparatus and air dried. The slides were blocked and incubated with the E. chaffeensis-specific FITC-conjugated MAb Ec56.5 in the absence of permeabilization or fixation. The cell-free ehrlichiae ranged in size from 0.5 to 1 μm (scale bar, 5 μm). No staining was observed with an isotype-matched control antibody (not shown).

FIG. 3.

Detection of cell-free ehrlichiae in plasma during infection. BALB/c and BALB/c-scid mice were inoculated with 10⁶ E. chaffeensis-infected, sex-matched BALB/c-scid splenocytes (approximately 10⁸ genome units) via the peritoneum. Groups of three mice were harvested at various times postinfection, and the bacterial numbers in plasma and liver tissues were measured by quantitative PCR. The kinetics of the appearance of cell-free ehrlichiae in plasma are shown in a, and colonization of the liver is shown in b. The BALB/c-scid mice became moribund and succumbed to infection within 21 days postinfection. The experiments were repeated at least two times with similar results. Mean and standard deviation are indicated.

FIG. 4.

Replication of cell-free ehrlichiae. (a) Plasma was isolated from infected BALB/c-scid mice and incubated at 37°C, and the copy number of the cell-free ehrlichiae was determined by quantitative PCR at various intervals. On day 22 postincubation, an aliquot of the plasma was treated with DNase I prior to DNA extraction (open circle). (b) Freshly isolated cell-free ehrlichiae were incubated in plasma at 37°C in the presence of [³H]thymidine for 5 days, and incorporation of [³H]thymidine was measured. Cell-free ehrlichiae that had been heat treated at 65°C for 30 min and normal mouse plasma incubated under the same conditions were used as controls. The experiment shown is representative of two separate experiments. The error bars indicate the standard deviation observed from triplicate samples. (c) Freshly isolated cell-free ehrlichiae were isolated and stained with SYTO9 and propidium iodide, and the relative numbers of viable bacteria were enumerated by epifluorescence at various intervals during ex vivo incubation at 37°C. The data represent the mean and standard deviation of counts obtained from three randomly chosen fields at 400× magnification. (d) Plasma samples isolated from infected BALB/c-scid mice were incubated at the indicated temperatures, and the ehrlichial copy number was measured at various intervals. Data are representative of four independent experiments.

FIG. 5.

Immunofluorescence analysis of cell-free ehrlichiae after ex vivo incubation. Cell-free plasma obtained from infected SCID mice was incubated at 37° for 5 days, and the immunofluorescence assay for E. chaffeensis was performed with FITC-conjugated MAb Ec56.5. Some bacteria appeared to have undergone cell division by binary fission (arrows). A scale bar is indicated.

FIG. 6.

Infectivity and virulence of cell-free ehrlichiae. (a) Cell-free ehrlichiae in plasma were incubated in vitro at the indicated temperatures for 12 to 72 h. At various times postincubation, infectious bacteria were enumerated in vitro at limiting dilution with an in vitro infection assay. (b) Virulence of freshly isolated cell-free ehrlichiae was demonstrated by their ability to cause disease in the susceptible SCID mice. The minimal infectious dose, which is inversely correlated with virulence, was determined in vivo by injection of serial dilutions of cell-free ehrlichiae to BALB/c-scid mice via the peritoneum, and weight change was monitored in the recipient mice. The legend indicates the approximate copy number of ehrlichiae used for inoculation. (c) Infected cell-free plasma was incubated for 0 to 24 h at the indicated temperatures, and the minimal infectious dose required to cause disease and morbidity in SCID recipients was determined. The experiments were repeated two times with similar results.

FIG. 7.

Antibody-mediated bacterial clearance. Groups of three to four BALB/c-scid mice were infected with 10⁶ splenocytes from infected BALB/c-scid mice. On days 6, 12, and 18 postinfection, the infected mice were either left untreated or injected with 200 μg of Ec56.5 or the isotype-matched irrelevant antibody (KJ1-26) via the peritoneum. Bacterial copy number in the liver, PBMCs, and cell-free plasma was measured, as indicated. The experiment was repeated two times with similar results. Statistical comparisons were made between groups of three to four mice treated with either PBS,or the isotype control antibody and mice treated with the specific antibody Ec56.5. In all cases P values were less than 0.05, as determined with the Mann Whitney test.

FIG. 8.

Cell-free ehrlichiae were detected after ex vivo culture of infected peritoneal cells. (a) Peritoneal exudate cells (PECs) isolated from 21-day-infected BALB/c-scid mice (>90% infected) were washed and cultured in Eagle's MEM containing 10% fetal bovine serum and 2 mM l-glutamine, with or without 10 μg of the E. chaffeensis-specific MAb Ec56.5 per ml at a concentration of 10⁶ cell/ml at 37°C for 48 h. Bacterial copy number was measured in the supernatant. No significant differences were observed between the untreated and antibody-treated groups. (b) The viability of the infected PECs was monitored with the trypan blue exclusion method. Also shown is the viability of thioglycolate-elicited PECs from uninfected BALB/c-scid mice cultured under identical conditions (control). Data are representative of four independent experiments. The difference observed between the uninfected control group and the infected groups (untreated and antibody treated) at 6 h was statistically significant (P < 0.05).