The present study using the human system thus systematically addressed the question of whether or not virulent Brucella spp. are internalized via conventional or special uptake mechanisms and are located in conventional or special intracellular compartments. The uptake of B. suis and B. melitensis by freshly isolated or subcultivated human monocytes under opsonic and nonopsonic conditions was investigated in detail by means of electron and fluorescence microscopy and by counting of internalized as well as surviving brucellae. The uptake of B. suis by epithelioid HeLa and CHO cells was included for comparison. The novel results obtained reveal that brucellae enter their host cells via conventional phagocytosis but locate in conventional and special compartments in parallel, with only the latter one allowing for intraphagosomal survival.
MATERIALS AND METHODS
Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and vitamin D3(VD3) were kindly provided by Jacques Dornand, thapsigargin was kindly provided by Michel Vignes, and neuraminidase (Behring catalog no. ORKD 04) was kindly provided by Virginie Lafont (all at the University of Montpellier, Montpellier, France). Dextran 500 (Nycograde 500) was obtained from Pharmacia Biotech Europe, Saclay, France; Ficoll-Hypaque (1.077 g/liter) was from Eurobio, Les Ulis, France; and tryptic soy broth (TSB) was from Difco, Detroit, Mich. All cell culture reagents were purchased from Gibco BRL, Cergy-Pontoise, France, or Sigma-Aldrich, St. Quentin Fallavier, France; the latter company also supplied the chemical compounds, except for theN-hydroxysuccinimidyl esters of 5- and 6-carboxyfluorescein (CF), 5- and 6-carboxytetramethylrhodamine (Rho), and LysoTracker Red DND-99, which were purchased from Molecular Probes, Eugene, Oreg.
Peripheral blood monocytes (PBM) were isolated from buffy coats of healthy donors obtained from the Etablissement Transfusion Sanguine Languedoc-Roussillon, Montpellier, France, using established methods resulting in low activation (12
). Briefly, red blood cells (RBC) were sedimented with Nycograde 500; mononuclear cells were isolated and platelets were removed by buoyant-density-based centrifugation using MSL and heat-inactivated fetal calf serum (hiFCS), respectively, as separation media; CD2+
lymphocytes were rosetted with neuraminidase-desialinated RBC (BAG, Lich, Germany) on ice; and the remaining RBC were osmotically lysed by exposing them to distilled water. The remaining cells were resuspended in RPMI 1640 cell culture medium supplemented with 10% hiFCS (RPMI+). Trypan blue-excluding cells were counted and split into aliquots. One buffy coat usually would give a yield of 60 × 106
to 100 × 106
viable monocytes among about 20% of other cells, mainly B lymphocytes (28
Some monocytes were maintained for up to 7 days in RPMI+ with or without addition of 500 U of GM-CSF per ml or 100 nM VD3
to further promote differentiation into monocyte-derived macrophages (MDM). Murine macrophage-like J774.A1 cells (ATCC TIB 67) and human cervix HeLa cells (ATCC CCL-2) were grown in RPMI+, and Chinese hamster ovary (CHO) cells were grown in α-MEM supplemented with 10% hiFCS (α-MEM+). CHO cells expressing the human complement receptor type 3 (CHO/Mac1+
) were maintained in α-MEM without ribo- and deoxyribonucleotides but with 0.1 mM l
-glutamine, 18 mM thymidine, and 10% FCS dialyzed against a cutoff of 10,000 molecular weight. Both types of CHO cells were a kind gift from David Mosser, Philadelphia, Pa., to Marina Cinco, Trieste, Italy, and were used as part of a joint study. Culture took place in T75 polystyrene Falcon flasks in a humidified incubator at 37°C and 5% CO2
in the presence of 100 U of penicillin G per ml and 100 mg of streptomycin per ml.
Growth conditions, opsonization, and killing of bacteria.
strains B. melitensis
16 M (ATCC 23456), B. suis
1330 (ATCC 23444), B. suis
p/sog (a green fluorescent protein [GFP]-expressing mutant of strain 1330) (33
), and B. suis
D1 (a GFP-expressingvirB9
mutant of strain 1330) (32
) were maintained at 4°C on plates containing agar-solidified TSB and appropriate antibiotics. The evening before an experiment, brucellae were transferred to liquid TSB and grown with agitation in an incubator at 37°C overnight to stationary phase. Immediately prior to the experiments, the optical density of the bacterial solutions was adjusted using a spectrophotometer at 600 nm. For studying the effect of opsonins, washed bacterial aliquots were resuspended in RPMI supplemented or not with 10% hiFCS, fresh FCS, or hiFCS containing 5 μl of heat-inactivated human B. suis
-specific antiserum per ml (all at room temperature for 30 min). Brucellae were killed by exposing aliquots either to a temperature of 60°C or to 4% freshly prepared paraformaldehyde for 1 h; successful killing was confirmed by the absence of bacterial growth on plates.
Inoculation of host cells.
To infect nonadherent monocytes, aliquots of freshly isolated PBM were challenged withBrucella in tubes, washed once in a large volume of medium to remove extracellular bacteria, seeded, and chased. To infect adherent monocytes, aliquots of freshly isolated PBM were seeded first, rinsed after 30 min to remove nonadherent cells, challenged withBrucella, rinsed thoroughly to remove extracellular bacteria, and chased. For studying the effect of prolonged culture of PBM and resulting differentiation into MDM, aliquots of adherent monocytes were infected after various periods of culture. The cell lines were grown to semiconfluency before being challenged withBrucella. RPMI+ containing 60 μg of gentamicin per ml was used as the culture medium during the chasing period in order to kill remaining extracellular bacteria.
Uptake of Brucella and intracellular survival.
For the quantitative evaluation of infection, aliquots of monocytes (2 × 105 each) were challenged with GFP-expressing B. suis in a total of 0.2 ml of medium at a bacterium/host cell ratio (multiplicity of infection) (MOI) of 500, with a pulse time of 20 min and a chase time of 30 min. The monocytes were seeded in Lab-Tek eight-well chamber slides (Nunc Inc., Naperville, Ill.). At the end of the chasing period, the cells were thoroughly rinsed and fixed with 2% paraformaldehyde. Slides with coverslips were viewed with a Leica DM IRB epifluorescence microscope. Both the percentage of infected monocytes for at least 100 monocytes (relative infection index) and the mean number of GFP-expressing bacteria for at least 50 infected monocytes (phagocytosis index) were evaluated in duplicate chambers, screening cells from different sites of the chambers. For statistical analysis, two-way analysis of variance was performed.
For the determination of intracellular bacterial survival, aliquots of monocytes (5 × 105 each) were challenged with Brucella in a total of 0.5 ml of medium at an MOI of 500 with a pulse time of 20 min and a chase time of 30 min. The monocytes were seeded in Falcon Primaria 24-well tissue culture plates. At the end of chasing periods of 1 to 36 h, the wells were carefully rinsed, the monocytes were osmotically lysed with 0.5 ml of Triton X-100 per well in distilled water, TSB agar plates were inoculated in duplicate with 100 μl (each) of supernatant in serial dilutions, and the CFU from duplicate wells were evaluated after 3 to 5 days of growth.
The phagosomal pH was measured by fluorescence microscopy as described in full detail before (37
). Briefly, B. suis
which had been labeled with CF and Rho and opsonized with Brucella
-specific human antiserum was used to infect J774 cells in Lab-Tek chamber coverslides with an MOI of 100, a pulse time of 45 min, and a chase time of 90 min. An in situ calibration curve of the CF/Rho emission ratio versus pH was obtained by exposing the infected J774 cells to nigericin-containing buffers of defined pH. Alternatively, J774 cells which had been preinfected with opsonized GFP-expressingBrucella
were incubated with 0.1 or 1 μM LysoTracker Red. Fluorescence was measured with a Cool View camera (Photonic Science, Robertsbridge, United Kingdom) and an image processor linked to a Leica DM IRB epifluorescence microscope. For each reading, usually four paired images were acquired at probe-specific wavelengths and analyzed by the VISIOLAB 1000 (Biochem, Les Ulis, France) imaging system.
Ultrastructural analysis of infected cells.
Aliquots of nonadherent PBM (106
each) were challenged withBrucella
in a total of 1 ml of medium at an MOI of 20 to 500 with a pulse time of 2 to 30 min and a chase time of up to 36 h. The infected PBM were either kept in 15-ml Falcon tubes (for chasing periods of less than 2 h) or seeded in T25 Falcon flasks (for periods exceeding 2 h). Aliquots of adherent PBM, MDM, and the semiconfluent epithelioid cells (about 4 × 106
cells/flask with 5 × 106
) were challenged in T25 Falcon flasks in a total of 5 ml of medium; some of the flasks containing epithelioid cells were gently centrifuged (400 × g
for 10 min). The chase was stopped by adding an excess amount of cold Ito's fixative (22
) to the tubes or flasks, and fixation was continued overnight at 4°C. Adherent cells were then gently scraped off the flasks and transferred to tubes.
The samples were further processed according to established protocols (22
). Briefly, they were postfixed with ferricyanide-reduced osmium tetroxide, embedded in agarose, en bloc stained with an alcoholic mixture of phosphotungstic acid and uranyl acetate, physically dehydrated with a graded series of alcoholic solutions followed by pure acetone, and embedded in Epon 812 resin. Ultrathin sections were on-grid stained with a mixture of uranyl acetate and lead citrate and viewed with a Zeiss type 906 transmission electron microscope. The Brucella
-containing phagosomes of at least 100 infected monocytes from different areas of two nonconsecutive sections were classified according to the intraphagosomal space as tight, loose, or other, with the last group comprising all equivocal phagosomes and macropinosomes.
For the cytochemical demonstration of reactive oxygen intermediates, the method of Briggs et al. was used (7
). Briefly, 1 mg of 3,3′-diaminobenzidine (DAB) was dissolved in RPMI+. After the pH of the medium was readjusted to 7.4 with 0.2 N NaOH, PBM were challenged with antibody-opsonized or nonopsonized bacteria in RPMI+–DAB for 15 min, chased in RPMI+ for another 120 min, and fixed and further processed for electron microscopy as described above, except that the on-grid staining was done at half strength or was totally omitted. Since monomeric DAB will be oxidatively linked to highly osmiophilic polymers in presence of endogenously produced reactive oxygen intermediates, thus revealing sites of oxidative burst (26
), the different types of Brucella
-bearing phagosomes were qualitatively screened for the presence of electron-dense precipitates.
For the demonstration of phagosome-endosome fusion, a combination of the methods described by Rabinowitz et al. (38
) and Strasser et al. (44
) was used. To load early endosomal compartments, PBM were pulsed either with bovine serum albumin (BSA)-conjugated colloidal 10-nm-diameter gold particles (BSA-gold) at an optical density at 520 nm of 10, with 0.5 mg of cationized ferritin per ml, or with 0.1% ruthenium red for 10 min and chased for another 10 min before incubation with Brucella
for another 20 min. To load late endosomal compartments, PBM were pulsed with BSA-gold overnight, chased for 6 h, pulsed with Brucella
for 20 min, and chased again for another 6 h. The different types ofBrucella
-bearing phagosomes were qualitatively screened for the presence of electron-dense markers.
The present study systematically addressed the uptake of virulentB. suis and B. melitensis by human monocytes, MDM, and epithelioid cells and their subsequent intracellular localization. This work proves evidence that brucellae are engulfed via regular zipper-type mechanisms by both professional and nonprofessional phagocytes, resulting in two types of phagosomes: regular SP representing the killing compartment and special TP representing the survival-permitting compartment. Whereas these observations were qualitatively the same for all host cells and experimental conditions investigated, the kinetics of uptake, the receptors involved, and the relative frequency of the survival-permitting compartment varied according to type of host cell and the experimental protocol used for infection. These results likely explain the variety of results obtained in previous studies and argue for great care when comparing results obtained under different experimental conditions.
In our system, Brucella
organisms were readily internalized by human monocytes in both the absence and presence of complement and antibodies, and the ultrastructural features for opsonic and nonopsonic uptake were similar. It has long been known that in phagocytosis both the engulfing pseudopods and the phagosomal walls may have more or less continuous contact with the particles and that the engulfing pseudopods may be more or less extended; these two conditions have been classified as FcR- and CR-type or type I and II phagocytosis, respectively, according to the two most prominent opsonins (for a detailed discussion, see references 39
). The present results, however, indicate that (i) these ultrastructural features occur under nonopsonic conditions as well and (ii) discontinuous contact may be seen with extended pseudopods and vice versa, arguing for greater care with this popular classification scheme.
As to the receptors promoting the uptake of Brucella
, opsonization with antibodies contributed considerably to the uptake ofB. suis
. The increased uptake, however, did not result in increased survival, which is in line with results from previous studies (5
). FcR-mediated phagocytosis intrinsically leads to the assembly of the NADPH oxidase and generation of reactive oxidative intermediates. Chemiluminescence assays for measuring the overall oxidative burst during uptake of Brucella
gave contradictory results (5
), whereas the ultracytochemical approaches of the present study and an early publication (21
) reveal a rare oxidative activity inBrucella
-bearing phagosomes, suggesting that intracellular killing mechanisms other than the oxidative burst mediate the eradication of internalized Brucella.
In contrast to the case for antibodies, the presence of complement did not increase the uptake of Brucella
. A major role for complement in the uptake of this pathogen, at least in nonimmune serum, has been questioned before (for a review, see reference29
). Pathogens invading the human body have developed a broad array of methods to avoid recognition or lysis by complement (48
). Future studies will have to show which mechanism is used by Brucella
to overcome this major effector system of innate immune defense and whether CR-mediated uptake ofBrucella
depends on the simultaneous presence of complement and Brucella
-specific antibodies. Still, Brucella
may bind to CR3 directly without complement components acting as bridging opsonins, since this receptor has multiple opsonic and nonopsonic binding sites apart from that for the main opsonic complement fragment iC3b (reviewed in reference 41
If the reduced bacterial uptake of adherent versus nonadherent monocytes is simply due to a reduced surface area, then there should be (i) a proportional decrease in uptake following adherence, which was not the case, and (ii) no differences between monocytes cultured for various periods of time, which was the case. Thus, a major portion of the nonopsonic receptors used for the uptake of B. suis
likely are adhesion molecules of two types, one (possibly an integrin) Ca2+
dependent and the other not. Nonopsonic entry of B. abortus
into bovine macrophages was found to be competitively inhibited by fibronectin, mannan, and lipopolysaccharide (8
). Whereas lipopolysaccharide and mannan bind mainly to nonintegrin receptors (4
), fibronectin is recognized by several integrins, such as the main vitronectin receptor αV
), and all three substances are ligands for CR3 (41
). In our hands, expression of the human CR3 on CHO cells did not enhance uptake of B. suis
in the presence of fresh or inactivated serum, indicating that this receptor either is not involved at all in the uptake of B. suis
or may depend on other factors such as the functional cooperation with CR1 (45
The present study showed that the uptake of Brucella
leads to two types of phagosomes, with only one permitting survival. Morphological heterogeneity of Brucella
-bearing phagosomes has also been observed in J774 cells before (3
), but without consideration of possible functional consequences. In nonprofessional phagocytes, on the other hand, brucellae persist within cisternae of the rough ER (1
) which are reached quite late (≥24 h postinfection [hpi]) (17
) via the phagosomal route. The autophagosome-like structures of B. abortus
-bearing phagosomes in HeLa cells at 24 hpi, which are thought to derive from a merging with autophagosomes (34
), were not observed in the present study up to 8 hpi. The same applies to the osmiophilic membrane remnants, possibly derived from autophagosomes, described for long-term-infected hamster kidney tissue cultures (25
). In a recent study on murine macrophage-like cells, only a minor proportion of intracellularB. abortus
organisms colocalized with markers for the ER and autophagosomes (3
), further supporting the view that the parasitism-permitting compartments of Brucella
are distinct in professional and nonprofessional phagocytes.
Several other intracellular parasites are known to lodge in tight phagosomes, and it was concluded that the tight membrane apposition is crucial for a reduced fusiogenicity of these compartments (14
). Despite similar morphology, the molecular characteristics of such tight phagosomes may differ according to the different microbial strategies used to interfere with the organelle trafficking of the host cell (11
). Our difficulties in labeling Brucella
-bearing phagosomes with chloroquine or LysoTracker Red but not with ammonium chloride indicate that brucellae selectively influence the accessibility of their intracellular compartment. Since these lysosomotropic weak bases have greatly different molecular weights (515.9, 399.25, and 53.49 for chloroquine, LysoTracker Red, and ammonium chloride, respectively), the size of the substrate may be the factor determining access. Still, the extremely narrow intraphagosomal space typical of the tight phagosomes also has to be kept in mind. The formation of tight phagosomes did not depend on bacterial viability or virulence, pointing to the effect of some yet-to-be determined membrane component(s) of Brucella
Early publications (19
) suggested thatBrucella
inhibited phagosome-lysosome fusion in murine macrophages. More recently (3
), it was reported that earlyBrucella
-bearing phagosomes fused neither with early nor with late endosomes, although the procedure used to label early endosomes raises some questions as to the character of these vesicles (for a critical comparison, see reference 38
). Fusion between Brucella
-bearing phagosomes and early endosomes obviously was not impaired in a recent study on murine macrophages (36
) and was a frequent event in our system. However, regarding the drastic decline in the number of surviving bacteria within the first 18 to 24 h of infection, only the few remaining truly nonfusiogenic phagosomes may represent the actual survival-permitting compartments.
Our results show that the Brucella
-bearing phagosomes in J774 cells have an acidic pH, which is in line with previous reports (3
). Since acidification and fusiogenicity of the phagosome are two differently regulated consecutive steps (6
), TP may have an acidic pH and, nevertheless, restricted fusiogenicity. This raises the question of what is more important for permitting the survival of Brucella
in this compartment, an acidic pH or the tight apposition of the phagosomal wall. Although the answer is not yet clear, our findings that (i) ammonium chloride leads to both a rise in the intraphagosomal pH and a swelling of the phagosome, (ii) brucellae survive in acidic TP but are killed in acidic SP, and (iii) acidic shock of Brucella
prior to phagocytosis does not increase subsequent intracellular survival suggest that an acidic pH alone is not sufficient to establish intracellular infection. Successful parasitism may be determined by the timely synthesis of bacterial factors, and macrophage-specific induction of protein synthesis has been demonstrated for B. abortus
) and B. suis
). Moreover, brucellae share type IV secretion and regulatory systems withAgrobacterium tumefaciens
, Rhizobium meliloti,
and Bordetella pertussis
). Thus, it is tempting to assume that internalized brucellae sense the intraphagosomal environment and assemble secretion systems in order to establish intracellular parasitism, with the sensing and/or the secretion being dependent on a tight apposition of the phagosomal and bacterial membranes.