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*
Institut de Pharmacologie et de Biologie Structurale–Centre National de la Recherche Scientifique, Unité Propre de Recherche 9062, Toulouse, France; and
The Netherlands Cancer Institute, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogenic mycobacteria are facultative intracellular parasites that are able to survive and replicate within macrophages (2). In addition to the well-known pathogenic species Mycobacterium tuberculosis and Mycobacterium leprae, opportunistic pathogenic mycobacteria have attracted epidemiologic interest since they cause infections in immunocompromised people. Two major opportunistic species, Mycobacterium avium and Mycobacterium kansasii, have frequently been isolated from pulmonary and disseminated infections in AIDS patients (3, 4). In macrophages, Mycobacterium microti, Mycobacterium marinum, M. avium, and M. tuberculosis inhibit the fusion between lysosomal granules and their phagosomes (5, 6, 7, 8, 9, 10), leading to the proposal that these properties favor their survival in macrophages (2).
Macrophages have long been regarded as the key phagocytic cells in mycobacterial infections. However, neutrophils have emerged as playing a significant protective role in tuberculosis (11, 12, 13). In contrast to macrophages, which contain only lysosomes (14), neutrophils contain at least two types of secretory granules, the azurophil and specific granules (15). Azurophil granules are a special class of lysosomes that, in addition to typical lysosomal enzymes, store bactericidal proteins and neutral proteases. Specific granules also contain proteins implicated in the bactericidal function of neutrophils, and they are a reservoir of plasma membrane receptors and cytochrome b558, a component of the NADPH oxidase complex (15). Proteins controlling the process of mobilization/fusion of these granules with the phagosomal or the plasma membrane have not yet been identified. Several reports suggest that a tyrosine kinase of the Src family, Hck, the expression of which is restricted to phagocytes (16), could be involved in this process: 1) it is mainly associated with the membrane of azurophil granules (17); 2) in the course of phagocytosis of opsonized zymosan, azurophil granules fuse with phagosomes and Hck translocates to the phagosomal membrane (17); 3) during this process, Hck is activated in the granular fraction and, to a lesser extent, in the phagosomal fraction (18); and 4) tyrosine kinase inhibitors inhibit the degranulation process (16).
Although neutrophils play a defensive role during mycobacterial infections (11, 12, 13), few data regarding bactericidal responses of neutrophils toward mycobacteria (19, 20, 21, 22) are available, e.g., mobilization of granules in response to mycobacteria has not been studied. In this paper, the release of azurophil and specific granule content was determined in human neutrophils during the phagocytosis of pathogenic (M. avium and M. kansasii) and nonpathogenic (Mycobacterium phlei and Mycobacterium smegmatis) mycobacteria. The tyrosine kinase activity of Hck was also studied.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ficoll and PMSF were purchased from Eurobio (Les Ullis, France). MEM and HEPES were purchased from Life Technologies (Cergy Pontoise, France). Leupeptin, aprotinin, pepstatin, diisopropylfluorophosphate, and FITC were purchased from Sigma (St. Louis, MO). Anti-myeloperoxidase Abs (23) were from CLB (Amsterdam, The Netherlands); anti-lactoferrin was from Cappel Laboratories (Cochranville, PA); and anti-rabbit Abs gold conjugates were from Amersham Nederlands (s’Hertogenbosch, The Netherlands).
Human neutrophils
Neutrophils were isolated from the blood of healthy donors,
separated by the Dextran-Ficoll method as previously described (24),
resuspended in MEM, 10 mM HEPES, pH 7.4, and maintained for 20 min at
37°C before stimulation. The final suspensions contained 
95%
neutrophils.
Bacteria strains and growth conditions
M. phlei (ATCC 11758), M. smegmatis (ATCC 607), and M. kansasii (ATCC 124478) (all from American Type Culture Collection (ATCC), Manassas, VA) were grown at 37°C as surface pellicles in 250-ml flasks containing 100 ml of Sauton broth medium. The medium was discarded, and the pellicles were disrupted by gentle shaking with glass beads (4-mm diameter) for 30 s and resuspended in PBS, pH 7.4. When another method to individualize mycobacteria was used, by passing bacteria through a narrow gauge syringe needle (0.45-mm diameter), the phagocyte responses studied herein were similar. In contrast to the other mycobacteria, it was not necessary to use beads for M. avium (IP140310013), which was cultured in Middlebrook 7H9 medium (Difco, Bonneuil sur Marne, France) with agitation in a New Brunswick incubator (125 rpm; New Brunswick Scientific, Edison, NJ). The culture was centrifuged at 10,000 x g for 10 min, and the pellet was resuspended in PBS. To remove clumps, the bacterial suspensions were sedimented for 10 min, and the supernatants were collected and centrifuged for 10 min at 200 x g. Supernatants were collected, counted under a microscope in a Thoma chamber, supplemented with 5% glycerol, and stored at -80°C until use. At least 90% of the mycobacteria were individualized, and the remaining 10% formed small aggregates containing two or three bacilli. Bacillus subtilis (ATCC 6633) was grown overnight in Tryptase-Soja broth (Biomerieux, Lyon, France) at 37°C, washed in PBS, and stored under the same conditions as the mycobacteria. All experiments were performed using a bacteria:phagocyte ratio of 50:1.
FITC staining of bacteria
Bacteria were stained with FITC as previously described (25). Briefly, 109 bacteria were added to 1 ml 0.01% FITC in 0.2 M Na2CO3/NaHCO3 buffer, pH 10.2, for 10 min. The bacteria were then washed twice in PBS, pH 7.4.
Opsonization of bacteria and zymosan
Bacteria or zymosan were incubated in pooled human sera for 20 min at 37°C, washed twice with PBS, pH 7.4, and resuspended in the same buffer containing 1 mM CaCl2 and 0.5 mM MgCl2 (24). Opsonization was checked by incubating bacteria or zymosan with FITC-coupled rabbit Abs directed against human IgG. Fluorescence was measured by FACScan analysis (Becton Dickinson, San Jose, CA).
Phagocytosis measurement and indirect immunofluorescence
Neutrophils (7 x 105/ml) adhering on glass coverslips were incubated at 37°C for 20 min in MEM-HEPES as previously described (26) and exposed to FITC-stained mycobacteria. Cells were then washed and fixed with 3.7% paraformaldehyde in PBS containing 15 mM sucrose, pH 7.4, for 30 min at room temperature. After neutralizing with 50 mM NH4Cl, slides were washed with PBS, pH 7.4, and mounted with 5 mg/ml trypan blue (27) to quench FITC-coupled bacteria remaining in the extracellular medium. Cells containing fluorescent mycobacteria were counted by alternately viewing them by phase contrast and fluorescence microscopy. For each condition, at least 100 cells were counted.
For immunolocalization of Hck, adherent neutrophils were fixed and permeabilized by plunging the glass coverslips into methanol at -20°C for 6 min, washed in PBS containing 0.1% Tween-20, then exposed to affinity-purified anti-Hck Abs (Santa Cruz Biotechnologies, Santa Cruz, CA), and then to FITC- or rhodamine-conjugated secondary Abs as previously described (17).
Immuno-electron microscopy
Neutrophils were fixed in 0.5% (v/v) glutaraldehyde and 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.2, for 2 h and then pelleted in 10% (w/v) gelatin in PBS. Ultrathin frozen sections were incubated at room temperature with mouse monoclonal anti-human myeloperoxidase (1:200) (23) or rabbit anti-human lactoferrin (1:400) followed by incubation with secondary Abs linked to 10-nm gold particles (dilution 1:40). All incubations were performed for 1 h. Ultrathin cryosections incubated with irrelevant control rabbit or murine Abs under the same conditions produced negligible background labeling. After immunolabeling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM10 electron microscope (Eindhoven, The Netherlands).
O2- production
The production of O2- was measured using the superoxide dismutase-inhibitable ferricytochrome c reduction method, as previously described (28).
Immunoblots
Control or stimulated neutrophils (5 x 106/ml) were disrupted and fractionated as previously described (28). Membrane (1 x 107 cell equivalents) and cytosolic proteins (5 x 106 cell equivalents) were loaded on a 12% SDS-PAGE gel, transferred onto a nitrocellulose membrane, blotted with p47phox and p67phox polyclonal Abs (kindly provided by A. Segal, London, U.K.), and revealed by enhanced chemiluminescence.
Protein exocytosis
Control or stimulated neutrophils (5 x 106/ml) were pelleted and the supernatants were centrifuged (10,000 x g for 10 min) to eliminate bacteria. Cell pellets lysed in Triton X100 and cell supernatants were stored at -80°C until used. Lysozyme was measured in the extracellular medium and in the cells by following the hydrolysis of Micrococcus sp at 450 nm, as previously described (29). Myeloperoxidase and lactoferrin were measured by ELISA as previously described (17). The enzyme activity of ß-glucuronidase was measured at 405 nm as previously described (30).
In some experiments, for quantification of cell death, release of the cytosolic enzyme lactate dehydrogenase was measured using the colorimetric assay kit from Boehringer (Meylan, France) according to the manufacturer’s instructions.
Kinase assays
Proteins were solubilized from neutrophils with a buffer
containing 2% Nonidet P-40 and cytosol from NB4 cells to avoid
proteolysis, as previously described (17). Kinases were
immunoprecipitated and assayed for in vitro kinase activity in the
presence of acid-treated enolase as exogenous substrate, 10 mM
MnCl2, 10 µM MgCl2, and 10 µCi of
[
-32P]ATP (6000 mCi/mmol) as described (17, 31). The
specificity of the rabbit anti-Hck antiserum has been characterized
previously (17), and affinity-purified anti-Fgr and anti-Lyn
IgG (Santa Cruz Biotechnology) were used as described previously (31).
The radioactivity incorporated in enolase was quantified using the
ImageQuant program on the Storm840 imager, Molecular Dynamics
(Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of neutrophils to engulf pathogenic and nonpathogenic
mycobacteria was examined. Human neutrophils internalized both
pathogenic and nonpathogenic mycobacteria under nonopsonic conditions
(Fig. 1
). Opsonization of mycobacteria in
human serum enhanced the percentage of cells with engulfed bacteria
(Fig. 1).
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We first examined whether neutrophils degranulate when exposed to
mycobacteria. The release of lysozyme, a hydrolase present
predominantly in specific but also in azurophil granules (1), was
measured. In these experiments and the following, opsonized zymosan was
used as a positive control. Pathogenic as well as nonpathogenic
mycobacteria induced the exocytosis of lysozyme to similar extents
(Fig. 2). Next, we studied whether the
release of lysozyme could be modified when mycobacteria were serum
opsonized. Under these conditions, the percentage of cells engulfing
opsonized mycobacteria had approximately doubled (data not shown), but
the exocytosis of lysozyme was comparable with that obtained with
nonopsonized mycobacteria (Fig. 2).
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, while the
release of lactoferrin was stimulated in response to pathogenic or
nonpathogenic mycobacteria, the exocytosis of myeloperoxidase was
insignificant except for opsonized M. smegmatis (Fig. 3),
the effect of which was, however, negligible. This could not be
attributed to a defective capacity of neutrophils to release
myeloperoxidase, as attested to by the cell response to opsonized
zymosan. The possibility that serum opsonization of mycobacteria could
elicit the release of myeloperoxidase was tested. As shown in Figure 3, neither myeloperoxidase nor lactoferrin secretion was enhanced in
response to opsonized mycobacteria. To test the possibility that this
phenomenon was restricted to mycobacteria, B. subtilis,
another Gram-positive bacterium, was used in parallel. We observed that
B. subtilis was ingested by neutrophils with an efficiency
similar to mycobacteria (data not shown), but in contrast, it triggered
the release of both myeloperoxidase and lactoferrin (Fig. 4). Opsonized or nonopsonized B.
subtilis induced similar responses (Fig. 4). The low level of
release of azurophil granule content in the presence of mycobacteria
was confirmed using another marker: the lysosomal enzyme
ß-glucuronidase (15). Like myeloperoxidase, ß-glucuronidase was
efficiently released by neutrophils in response to zymosan or B.
subtilis, but only a limited and insignificant release was
observed in response to pathogenic and nonpathogenic mycobacteria (Fig. 5). During the course of these
experiments, cell viability was measured by the release of the
cytosolic enzyme lactate dehydrogenase. In control cells or cells
exposed to zymosan, the release was 4% and in neutrophils
exposed to mycobacteria, 6 to 8%. It is possible that the low level of
release of azurophil markers from mycobacteria-infected neutrophils
results from the higher percentage of dead neutrophils.
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Secretion of granule contents to the external milieu may occur
subsequent to granule fusion with unsealed phagosomes. To determine
whether the absence or low release of azurophil granule markers in the
presence of mycobacteria correlated with a limited fusion with
phagosomes, immuno-electron microscopy was performed on neutrophils
infected by M. smegmatis and M. kansasii,
compared with B. subtilis and zymosan. Figure 6 shows that myeloperoxidase and
lactoferrin were present in phagosomes containing zymosan or B.
subtilis. In contrast, phagosomes containing mycobacteria were
very poorly stained by anti-myeloperoxidase Abs (Fig. 6), whereas
they were strongly lactoferrin positive. This finding demonstrates that
phagosomes containing mycobacteria are permissive for specific granule
fusion but do not fuse with azurophil granules.
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The possibility that mycobacteria could alter another function
contributing to the bactericidal response of neutrophils was
investigated. The production of O2- was therefore
examined in cells infected by mycobacteria or exposed to zymosan.
O2- generation, continuously measured in response to
mycobacteria (Fig. 7A),
increased linearly until a plateau was reached between 90 and 120 min
depending on the blood donor. Next, experiments were performed at 40
min to measure the production of O2- at the initial
rate. When neutrophils were exposed to pathogenic or nonpathogenic
mycobacteria, the O2- generation was elicited to a
similar magnitude, and again, serum opsonization of mycobacteria did
not affect the results (Fig. 7B). We verified the functional
assembly of the O2--producing enzyme NADPH oxidase,
which requires translocation of cytosolic components to the plasma
membrane (32). The presence of the cytosolic components
p47phox and p67phox in the membrane fraction of
opsonized zymosan-stimulated neutrophils is shown as a positive control
(Fig. 7C). When neutrophils were exposed to M.
smegmatis, p47phox and p67phox became
membrane associated (Fig. 7C), indicating that NADPH oxidase
is assembled in cells exposed to mycobacteria.
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The Src family tyrosine kinase Hck is associated with the membrane
of azurophil granules, is activated during the process of exocytosis,
and translocates to the phagosomal membrane (16, 17, 18). Therefore, we
investigated whether the translocation of Hck toward phagosomes and its
activation occurred during the phagocytosis of mycobacteria. When
indirect immunofluorescence was performed, no Hck staining was
detectable at the phagosomal membrane around FITC-stained mycobacteria
(Fig. 8, middle panel), while
Hck was clearly present at the phagosomal membrane in neutrophils that
had internalized zymosan (Fig. 8, upper panel). The
possibility that Hck could be present at the phagosomal membrane but
hidden by the fluorescence of mycobacteria was ruled out, because
similar results were obtained with nonfluorescent M. phlei
(data not shown) or when Hck was stained with rhodamine-coupled Abs and
mycobacteria stained with FITC (Fig. 8, lower panels).
When mycobacteria were serum opsonized, Hck did not translocate to
the phagosomal membrane either (data not shown).
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). Again, there
was no difference between opsonized and nonopsonized M.
phlei (results not shown). To determine whether this inhibition
was Hck specific, the kinase activity of another Src family tyrosine
kinase, Fgr, the expression of which is also restricted to phagocytes
(16), was measured. We have previously reported that Fgr is activated
during phagocytosis of opsonized zymosan (31). Here, we observed that,
during phagocytosis of nonopsonized zymosan, M. phlei, or
M. kansasii, Fgr was activated (Fig. 9), indicating that not
all of the members of the Src family are affected during phagocytosis
of mycobacteria. This conclusion was further supported by the
observation that Lyn was activated in neutrophils exposed to zymosan or
M. phlei (data not shown). Taken together, these results
show that, during their phagocytosis, mycobacteria interfere with
translocation of Hck to the phagosomes and its activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neutrophils contain granules that are secreted during the host’s defense against microbial pathogens. Under physiopathologic conditions, whereas exocytosis of specific granules is initiated by soluble or particulate stimuli, azurophil granules are mobilized when cells perform phagocytosis of particles (1, 14). Once neutrophils start to phagocytose, specific granules fuse with phagosomes and the plasma membrane (14, 34). Azurophil granules are also promptly mobilized and fuse with nascent, unclosed phagosomes (35, 36). Therefore, the presence of azurophil granule markers in the extracellular medium could reflect their fusion with unsealed phagosomes (14, 36). This is in contrast with macrophages in which maturation of phagosomes is required to make them competent for fusion with lysosomes (37, 38). In the present report, we have investigated the mobilization of neutrophil granules in response to both pathogenic and nonpathogenic mycobacteria. Regardless of the mycobacterial species, the specific granule content was released, whereas azurophil granules were very poorly or not at all mobilized. When B. subtilis and mycobacteria were used at the same bacteria:cell ratio, they were internalized with similar efficiency, but exocytosis of azurophil granule markers was detected only in response to B. subtilis. Immuno-electron microscopy confirmed the lack of azurophil granule fusion, showing that phagosomes containing either pathogenic or nonpathogenic mycobacteria were essentially myeloperoxidase negative, whereas phagosomes containing zymosan or B. subtilis were myeloperoxidase positive. In macrophages, inhibition of the fusion between lysosomes and phagosomes has been essentially studied with pathogenic species (M. microti, M. marinum, M. avium, and M. tuberculosis) (5, 6, 7, 8, 9, 10). A report on the nonpathogenic species Mycobacterium aurum shows that lysosome-phagosome fusions are comparable with that of M. avium after a 24-h infection (7). In the light of our results on neutrophils, it would be important to further investigate the release of lysosomal markers in macrophages infected with nonpathogenic mycobacteria to establish whether this inhibition is restricted to pathogenic species. This knowledge would be helpful in the understanding of mycobacterial pathogenicity.
Attempts have been made to identify membrane-associated proteins that could differentially regulate the mobilization and fusion of specific and azurophil granules. We and others have shown that several proteins, due to their unique localization on azurophil or specific granule membranes, may be implicated in these regulations (14, 15, 17, 24, 26). Until now, only two proteins that could play a role in exocytosis have been identified on the surface of azurophil granules: leukophysin, a protein related to synaptophysin (39), and the tyrosine kinase Hck (17). The exocytosis of azurophil granules is inhibited by tyrosine kinase inhibitors (16). Moreover, Hck, which is mainly expressed in phagocytes, is associated with the membrane of azurophil granules, translocates to the phagosomal membrane during internalization of either opsonized zymosan (17, 18) or zymosan (this report), and is concomitantly activated (18). When neutrophils are activated by FMLP or PMA, which induce activation of NADPH oxidase and the release of specific but not of azurophil granules, Hck is not activated (18). In contrast to soluble stimuli, which do not or only poorly mobilize azurophil granules, phagocytosis of particles is accompanied by secretion of this granule type. Here we show that when mycobacteria are phagocytosed by neutrophils, there is almost no release of azurophil granule markers in the extracellular space or inside phagosomes. Under these consitions, Hck does not translocate to the phagosomal membrane, and its level of tyrosine kinase activity remains very low. Taken together, these results support the hypothesis that Hck might play a critical role in the mobilization of azurophil granules toward phagosomes. In HL60 cells differentiated in macrophages and in human monocytes, Hck is also present on cytoplasmic granules, probably lysosomes (17). It is possible, therefore, that Hck is also implicated in the regulation of lysosome mobilization in these cells. Protein phosphorylation on tyrosine residues during phagolysosome biogenesis has been observed on phagosomes isolated from murine macrophages (40). Interestingly, we have previously shown that during the mobilization of azurophil granules in response to phagocytosis of opsonized zymosan, Hck is activated in the azurophil granule fraction but also in the fraction enriched in phagosomes. Therefore, these data strongly suggest that Hck could phosphorylate substrates on phagosomes and/or on azurophil granules. Identification of these proteins will help the understanding of the function of Hck.
To explain why Hck is not activated when neutrophils internalize mycobacteria, one could postulate that membrane receptors involved in their phagocytosis do not evoke signalization for Hck activation. However, this seems unlikely because zymosan and opsonized zymosan use distinct receptors, possibly the lectin site of CR3 (41) and a putative ß-glucan receptor (42) for the former and complement and IgG receptors (43) for the latter; but in both cases, similar Hck activation is observed (see Ref. 18 and this report). Moreover, opsonized or nonopsonized mycobacteria use distinct receptors (2, 44) but do not trigger Hck activation, suggesting that the control of Hck tyrosine kinase activity during phagocytosis might be independent of the receptor used. Alternatively, mycobacteria could either interfere with downstream receptor degranulation signals or directly inhibit Hck. Recently, a factor located in the periplasmic compartment of Escherichia coli and secreted in the culture medium, which is inhibitory for the Src family tyrosine kinase p56lck, has been identified (45). A similar activity, with a tropism for Hck, given that Fgr and Lyn were not affected, could also exist in mycobacteria. This possibility is currently under investigation in our laboratory.
In conclusion, our results show that when human neutrophils are exposed to mycobacteria, they exhibit the typical early bactericidal responses: phagocytosis, generation of O2-, and exocytosis of specific granules. In contrast, mobilization of azurophil granules is not triggered by mycobacteria even when they are opsonized. This is an important finding as it shows that phagocytosis and fusion of azurophil granule with phagosomes can be dissociated events that are uncoupled by mycobacteria. In addition, we demonstrate that neutrophils exhibit similar bactericidal responses to opsonized or nonopsonized, pathogenic and nonpathogenic mycobacteria. Finally, we propose that Hck could be one of the key elements of the secretory pathway that are altered during phagocytosis of mycobacteria.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. I. Maridonneau-Parini, CNRS, IPBS, 205 route de Narbonne, 31077 Toulouse Cedex, France. E-mail address:
Received for publication February 26, 1998. Accepted for publication June 25, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of neutrophil responses to particulate zymosan and glucan. J. Immunol. 144:2712.[Abstract]
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M. Majeed, E. Caveggion, C. A. Lowell, and G. Berton Role of Src kinases and Syk in Fc{gamma} receptor-mediated phagocytosis and phagosome-lysosome fusion J. Leukoc. Biol., November 1, 2001; 70(5): 801 - 811. [Abstract] [Full Text] [PDF]
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P. Hartmann, R. Becker, C. Franzen, E. Schell-Frederick, J. Römer, M. Jacobs, G. Fätkenheuer, and G. Plum Phagocytosis and killing of Mycobacterium avium complex by human neutrophils J. Leukoc. Biol., March 1, 2001; 69(3): 397 - 404. [Abstract] [Full Text]
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P. Peyron, C. Bordier, E.-N. N'Diaye, and I. Maridonneau-Parini Nonopsonic Phagocytosis of Mycobacterium kansasii by Human Neutrophils Depends on Cholesterol and Is Mediated by CR3 Associated with Glycosylphosphatidylinositol-Anchored Proteins J. Immunol., November 1, 2000; 165(9): 5186 - 5191. [Abstract] [Full Text] [PDF]
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V. Le Cabec, C. Cols, and I. Maridonneau-Parini Nonopsonic Phagocytosis of Zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) Involves Distinct Molecular Determinants and Is or Is Not Coupled with NADPH Oxidase Activation Infect. Immun., August 1, 2000; 68(8): 4736 - 4745. [Abstract] [Full Text] [PDF]
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C. Astarie-Dequeker, J. Nigou, G. Puzo, and I. Maridonneau-Parini Lipoarabinomannans Activate the Protein Tyrosine Kinase Hck in Human Neutrophils Infect. Immun., August 1, 2000; 68(8): 4827 - 4830. [Abstract] [Full Text] [PDF]
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M. Francois, V. Le Cabec, M.-A. Dupont, P. J. Sansonetti, and I. Maridonneau-Parini Induction of Necrosis in Human Neutrophils by Shigella flexneri Requires Type III Secretion, IpaB and IpaC Invasins, and Actin Polymerization Infect. Immun., March 1, 2000; 68(3): 1289 - 1296. [Abstract] [Full Text] [PDF]
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N. Perskvist, L. Zheng, and O. Stendahl Activation of Human Neutrophils by Mycobacterium tuberculosis H37Ra Involves Phospholipase C{gamma}2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase J. Immunol., January 15, 2000; 164(2): 959 - 965. [Abstract] [Full Text] [PDF]
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C. Astarie-Dequeker, E.-N. N'Diaye, V. Le Cabec, M. G. Rittig, J. Prandi, and I. Maridonneau-Parini The Mannose Receptor Mediates Uptake of Pathogenic and Nonpathogenic Mycobacteria and Bypasses Bactericidal Responses in Human Macrophages Infect. Immun., February 1, 1999; 67(2): 469 - 477. [Abstract] [Full Text] [PDF]
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S. Carreno, M.-E. Gouze, S. Schaak, L. J. Emorine, and I. Maridonneau-Parini Lack of Palmitoylation Redirects p59Hck from the Plasma Membrane to p61Hck-positive Lysosomes J. Biol. Chem., November 10, 2000; 275(46): 36223 - 36229. [Abstract] [Full Text] [PDF]
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P. Peyron, I. Maridonneau-Parini, and T. Stegmann Fusion of Human Neutrophil Phagosomes with Lysosomes in Vitro. INVOLVEMENT OF TYROSINE KINASES OF THE Src FAMILY AND INHIBITION BY MYCOBACTERIA J. Biol. Chem., September 14, 2001; 276(38): 35512 - 35517. [Abstract] [Full Text] [PDF]
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1 fluorescent bacterium was determined
by fluorescence microscopy (see Fig. 8
