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The Journal of Immunology, 2008, 181, 2651 -2663
Copyright © 2008 by The American Association of Immunologists, Inc.
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NF-{kappa}B Activation Controls Phagolysosome Fusion-Mediated Killing of Mycobacteria by Macrophages1

Maximiliano Gabriel Gutierrez2,3,*, Bibhuti B. Mishra2,{dagger}, Luisa Jordao{dagger}, Edith Elliott{ddagger}, Elsa Anes{dagger} and Gareth Griffiths*

* European Molecular Biology Laboratory, Heidelberg, Germany; {dagger} Unidade de Retrovírus e Infecções Associadas-Centro de Patogénese Molecular Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal; and {ddagger} School of Biochemistry, Genetics, Microbiology and Plant Pathology, University of KwaZulu-Natal, Pietermaritzburg, South Africa


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-{kappa}B in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-{kappa}B that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-{kappa}B activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-{kappa}B during infection. Our results argue that NF-{kappa}B activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-{kappa}B inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-{kappa}B in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mycobacterium tuberculosis is the causative agent of tuberculosis that infects more than one-third of the world’s population (1). Only 5–10% of these infected individuals will develop tuberculosis at some point in their life (2). A major factor in determining whether the disease is contained or progresses is the macrophage, which hosts mycobacteria in membrane-enclosed phagosomes. When macrophages are activated, phagosomes enclosing the pathogen can fuse with lysosomes and the bacteria are killed. However, under steady-state conditions, the phagosome becomes programmed to prevent its maturation. As a consequence the bacteria survive and may grow. The mechanisms by which lysosomal factors kill mycobacteria are poorly understood (3). In this study, we focused on the nonpathogenic Mycobacterium smegmatis as a model system and addressed the intracellular mechanisms that are responsible for killing mycobacteria in mature phagosomes.

M. smegmatis is phagocytosed by macrophages and is killed within phagosomes by a combination of factors delivered from lysosomes, reactive nitrogen intermediates, and likely other still unidentified components (4). In the latter study, endocytosis of a mixture of lysosomal enzyme inhibitors significantly retarded M. smegmatis killing, providing functional evidence that lysosomal enzymes delivered to the phagosome contribute to this process. In contrast, pathogenic mycobacteria such as M. tuberculosis and Mycobacterium avium block phagolysosome fusion and NO release and consequently survive and grow within phagosomes (5). However, when the proinflammatory response of macrophages infected with pathogenic mycobacteria is stimulated by some specific lipids, even pathogenic mycobacteria may be efficiently killed (6).

The family of NF-{kappa}B transcription factors plays a crucial role in the regulation of the inflammatory process (7). This family of proteins is composed of two subfamilies: the NF-{kappa}B proteins and the Rel proteins. NF-{kappa}B transcription factors are present in the cytoplasm as heterodimers, most commonly of p65 and p50 subunits in a complex with an inhibitor, I{kappa}B (8). When proinflammatory signaling occurs via activation of cell surface receptors (e.g., TLRs), the I{kappa}B becomes phosphorylated, leading to its degradation by the proteosome system. This allows the active subunits to enter the nucleus where they up-regulate the transcription of two to three hundred genes (9).

The products characteristic of early events of the early immune response mediated by NF-{kappa}B include the release of ILs and inducible NO synthase-mediated NO production (10). However, reactive nitrogen intermediate production only plays a partial role in mycobacterial clearance (6, 11). It is clear that some ILs such us IFN-{gamma} and TNF-{alpha} increase the maturation of mycobacterial phagosomes as well as killing of mycobacteria. However, the intracellular mechanisms by which ILs boost mycobacterial killing are poorly understood (12, 13, 14).

The fusion of phagosomes with late endocytic organelles (often abbreviated as lysosomes) is usually considered to be one, perhaps the main effector of mycobacterial killing. This is based on extensive correlative data associating killing with the late fusion events that deliver the proton ATPase and lysosomal enzymes to phagosomes (15, 16, 17). For example, mutants of both M. tuberculosis and bacillus Calmette-Guérin that fail to block phagosome maturation are killed more effectively than wild-type bacteria (18, 19). As already mentioned, a role for lysosomal enzymes in killing is supported by our earlier studies (4).

Phagocytosis and inflammation are strongly correlated (20). Binding of mycobacteria to macrophage surface receptors, especially TLR2 and TLR4, initiates a rapid proinflammatory response which is more robust for nonpathogenic than for pathogenic mycobacteria (21). This proinflammatory response is mediated by MAPKs and by proinflammatory transcription factors, including NF-{kappa}B (21). In addition, the signaling from TLR has also been implicated in the regulation of phagosome maturation and bacterial killing (22).

The data regarding the activation of NF-{kappa}B by pathogenic mycobacteria are conflicting. A number of studies have described inhibition of NF-{kappa}B activation by M. tuberculosis components (23, 24). Other studies showed an initial, transient NF-{kappa}B activation but afterward the system is inhibited (25, 26). Other studies reported that M. avium and M. tuberculosis activate NF-{kappa}B under some conditions (27, 28).

In this study, we focus on the role of NF-{kappa}B during the proinflammatory response to M. smegmatis infection. Our results show that the NF-{kappa}B pathway is required for mycobacterial killing since blocking this pathway prevents killing and can even allow the bacteria to grow in macrophages. In addition, we show that the pathogen M. avium is indeed able to initiate the activation of NF-{kappa}B in the first minutes after infection, but thereafter the pathogen strongly represses NF-{kappa}B activation. Activation of NF-{kappa}B during infection leads to the synthesis of a set of lysosomal enzymes as well as a large number of membrane-trafficking regulators, whose increased synthesis is likely important for efficient phagosome maturation and to allow the macrophages to kill M. smegmatis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

SC-514, SN-50, NF-{kappa}B activation inhibitor (6-amino-4-(4-phenoxyphenylethylamino)quinazoline) and MG-132 were obtained from Calbiochem. The following Abs were used: polyclonal rabbit anti-p65, polyclonal rabbit anti-I{kappa}B, polyclonal goat Ab anti-cathepsin (Cts) Z (Santa Cruz Biotechnology). Rat monoclonal anti-LAMP-2 was purchased from the Iowa Hybridoma Bank. The polyclonal rabbit anti-v-ATPase Ab was previously described (29). Rabbit polyclonal Ab anti-CtsH was raised as described previously (30). Rabbit affinity-purified Ab anti-Rab34 was purchased from Genemed Synthesis and generated using a specific peptide. Secondary Abs were conjugated with Alexa Fluor 555, Alexa Fluor 488 (Molecular Probes), or HRP (Amersham Biosciences).

Cell lines and bacterial culture conditions

The mouse macrophage cell lines J774A.1 and RAW264.7 were cultured as described previously (4). M. smegmatis mc2155 harboring a p19 (long-lived)-EGFP plasmid and M. avium MAC101 were grown as previously described (6).

Macrophage infection

Bacterial cultures in exponential growth phase were pelleted, washed in PBS, and resuspended in DMEM to reach a multiplicity of infection of 10. Clumps of bacteria were removed by ultrasonic treatment of bacteria suspensions in an ultrasonic water bath for 15 min followed by a low-speed centrifugation for 2 min. Cells were seeded onto 24-well tissue culture plates at 70% confluence. In each experiment, after 1 h of infection, cells were washed and medium plus gentamicin (10 µg/ml) were added to kill extracellular bacteria. NF-{kappa}B inhibitors were added concomitant with the infection (t0) or after 1 h of infection (t1).

CFU assay

Macrophages were plated in 24-well plates and infected with M. smegmatis at different time points. Cells were washed with PBS and lysed with sterilized water. Quantitative cultures for M. smegmatis or M. avium were performed by 10-fold serial dilutions inoculated on 7H10 agar plates. Five microliters was plated by triplicate and the number of colonies was counted after 48 h and referred as number of colonies (CFU) per milliliter.

Indirect immunofluorescence

Cells were fixed with 3% paraformaldehyde solution in PBS for 10 min and quenched by incubating with PBS/50 mM NH4Cl. Subsequently, cells were permeabilized with 0.05% saponin in PBS containing 0.2% BSA and then incubated with the primary and secondary Abs. Cells were mounted with Permafluor mounting medium (DAKO) and analyzed by confocal microscopy (Zeiss LSM510).

ELISA

The p65 activation was assayed using a multiwell assay TransAM NF-{kappa}B Kit (Active Motif). Briefly, J774-infected macrophages were scraped and nuclear extracts were isolated. Five micrograms of total protein from each sample was incubated in 96-well plates coated with NF-{kappa}B consensus oligonucleotide sequence (5'- AGTTGAGGGGACTTTCCCAGGC-3') for 1 h and then with primary anti-NF-{kappa}B Ab and subsequently with secondary HRP-conjugated Ab. After a chemiluminescent reaction, the luminescence produced was recorded. Competition experiments were conducted with the 22-bp dsDNA, either wild type (see above) or mutated: 5'- AGTTGAGCTCACTTTCCCAGGC.

Western blot

Cells under the different conditions were washed with PBS, scraped in lysis buffer, and processed as described before (4). Briefly, 50 µg of protein extracts was subjected to electrophoresis in 10% SDS-PAGE gels, transferred to a nitrocellulose membrane, and blocked with 0.1% Tween 20/5% of milk TBS. The nitrocellulose membrane was then incubated with primary Abs, washed, and incubated with secondary HRP-conjugated Abs. The bands were visualized with a chemiluminescent reagent (Amersham Biosciences).

Small interfering RNA (siRNA)4 knockdown

RAW264.7 macrophages were grown to a final density of 40,000 cells/well in 24-well plates. Cells were transfected using HiPerFect (Qiagen) with 25 nM siRNA against p65 (Qiagen) according to the manufacturer’s instructions. Cells were left for 48 h before analysis of knockout phenotype by Western blot. Scrambled siRNA were used as nonsilencing control.

RNA extraction

Total RNAs were isolated from J774 (uninfected and infected with M. smegmatis) at 1 and 4 h postinfection (with and without NF-{kappa}B inhibitors SC-514 and SN-50) using a Qiagen RNeasy kit.

Microarray analysis

Sample preparation and hybridization to CodeLink Mouse Whole Genome Bioarray (~36 000 mouse gene targets) were performed at the European Molecular Biology Laboratory (EMBL) Genecore Facility. The complete data set has been submitted to the GEO database as accession number GSE8999.

RNA samples isolated as described in Materials and Methods were subjected to quality control tests using a Bioanalyzer (Agilent Technologies) at the Genomics Core Facility, EMBL-Heidelberg. Samples with a 28S/18S rRNA > 1 were used for microarray template preparation. Briefly, 2 µg of total RNA diluted bacterial mRNA controls and T7 oligo(dT) primers were incubated for 10 min at 70°C. All of the components were mixed and incubated for 2 h at 42°C for first-strand cDNA synthesis. The first-strand components were then added to second-strand reaction mix and incubated for 2 h at 16°C. The double-stranded cDNAs were purified and concentrated. This was then used for the in vitro transcription reaction in a mix of biotinylated UTP, dNTPs, and 10x T7 enzyme mix at 37°C for 14 h. The cRNAs were purified and assessed for quality using a Bioanalyzer.

Array hybridization

cRNA probes were subjected to fragmentation to generate 40–60 bp long oligonucleotide RNAs in a standard fragmentation reaction. These oligonucleotide RNAs were then used for hybridization onto a Mouse CodeLink Mouse Whole Genome Bioarray (~36 000 mouse gene targets). Briefly, 10 µg of cRNAs and 5 µl of 5x fragmentation buffer were incubated at 94°C for 20 min. Ten micrograms of fragmented cRNA, 78 µl of hybridization buffer component A, 130 µl of component B to a final volume of 260 µl were incubated at 90°C for 5 min to denature the samples followed by immediate cooling on ice for 30 min for annealing. Two hundred fifty microliters of hybridization reaction mixture was loaded onto the array input port and sealed with strips to avoid dehydration. The slides were incubated for 20 h at 37°C on a shaker at 300 rpm. Then bioarrays were transferred to a bioarray rack containing 0.75x TNT (0.10 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20) followed by the transfer of the racks to a preheated 0.75x TNT-containing chamber. It was then incubated at 46°C for 1 h. Each bioarray was then transferred to a chamber containing 3.4 ml of streptavidin-Cy5 solution and incubated at room temperature for 30 min. The arrays were then washed four times (5 min each) with 1x TNT followed by a brief rinsing of the same with 0.1x SSC (0.05% Tween 20) for 30 s. Bioarrays were dried by centrifugation at 9000 rpm for 10 min. Then they were scanned with GenePix 4000B (Genomics Core Facility, EMBL-Heidelberg) and scanned files were analyzed by CodeLink Expression Analysis software.

Statistical analysis and calculation of fold change

The GenePix-scanned files were analyzed using the CodeLink Expression Analysis software Codelink EXP version 4.0 (http://www4. gelifesciences.com/aptrix/upp01077.nsf/Content/codelink_soft). The signal and background fluorescence intensities were calculated for each spot to average the intensities of every pixel inside the target region. The intensity of each spot was calculated as the difference between mean signal intensity and mean local background intensity. Each time point/condition tested was repeated two to three times as biological replicates. The output Code Link files were analyzed by using GeneSpring GX 7.3 Expression analysis software (Agilent Technologies), which filters those genes which are absent in all of the samples and we considered those genes which exhibit a signal intensity above the background level in at least one of the samples using the "Filtering on Flags" tool. All of the fold changes in signal intensity of the test samples were calculated by subtracting the mean of the signal intensity of the controls (uninfected macrophages) using the "Filtering on Fold Changes" tool. Genes showing 1.5-fold higher or lower expressions against the controls were considered significant. All analyses of the fold changes were performed using a p value cutoff <0.01.

Sample preparation and hybridization to CodeLink Mouse Whole Genome Bioarray (~36 000 mouse gene targets) were performed at the EMBL Genecore Facility. The complete data set has been submitted to the GEO database as accession number GSE8999.

qPCR

A total of 1 µg of RNA was used for random hexamer-primed cDNA synthesis (Superscript II reverse transcriptase; Invitrogen) according to the manufacturer’s protocol. Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System using a SYBR Green PCR master mix (Applied Biosystems) and different sets of primers (Qiagen) at a final concentration of 0.3 µM, with a slight modification of the PCR steps: 95°C for 15 min for 1 cycle, 95°C for 15 s, 55°C for 30 s, and 60°C for 32 s for 40 cycles. Fluorescence data were collected at the amplification step. The mRNA expression profiles were normalized with respect to β-actin. Fold increase of each gene was calculated using the –2–{Delta}{Delta}Ct method.

Statistical analysis

Data are presented as means ± SEM of at least three independent experiments; p values (two-way ANOVA) are relative to the control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
M. smegmatis infection induces NF-{kappa}B activation in macrophages

To monitor NF-{kappa}B activation during infection, we first used fluorescence microscopy to evaluate the translocation to the nucleus of p65 upon infection of J774 cells with M. smegmatis-GFP or rhodamine-M. avium. In uninfected cells, we detected a low basal level of translocation (Fig. 1B), in agreement with the observations of constitutive p65 shuttling in resting cells (31). In contrast, with LPS (10 µg/ml), a classical activator of NF-{kappa}B, the number of cells with positive nuclear staining increased steadily, reaching 80% of the cells by 24 h. Similar results were obtained with heat-killed M. smegmatis (Fig. 1B). When macrophages were infected with live M. smegmatis, p65 was translocated to the nucleus in a fraction (20%) of the cells already at 30 min postinfection, reaching 34% by 1 h after infection (Fig. 1, A and B). Surprisingly, this translocation was transient, peaking at 50 min postinfection and switching to a cytoplasmic distribution of p65 after 1 h of infection that reached basal levels by 24 h (Fig. 1, A and B). To confirm that these results are not restricted to J774 macrophages, the same set of experiments was performed in bone marrow macrophages (BMM). In BMM, the same pattern of p65 activation in response to the different conditions was observed (data not shown).


Figure 1
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  		FIGURE 1. M. smegmatis infection activates NF-{kappa}B in macrophages. A, J774 macrophages were infected with GFP-M. smegmatis or rhodamine-stained M. avium MAC101 (depicted in red) at the indicated time points and subjected to indirect immunofluorescence against p65 (green). Pictures show representative confocal images. N, Nucleus. B, Quantitative kinetic analysis of p65 translocation in macrophages. Cells were infected with the indicated strains or incubated with LPS (10 µg/ml). Data represent the means ± SEM from at least three independent experiments. At least 100 cells were counted in each experiment. **, p ≤ 0.001 relative to control in the same conditions. MS, M. smegmatis; MS-HK, M. smegmatis heat killed; MAC101, M. avium. C, J774 cells were infected with M. smegmatis or M. avium MAC101 at the indicated time points. Uninfected cells were used as a control. Nuclear fractions were isolated and a DNA-binding assay of induced NF-{kappa}B activation was determined. NF-{kappa}B-binding activity was effectively competed for by the wild-type consensus oligonucleotide but not mutated oligonucleotide. **, p ≤ 0.001 relative to control in the same conditions. D, Western blot analysis of I{kappa}B degradation in cytoplasm. J774 cells were infected with M. smegmatis at the indicated time points and cytoplasmic fractions were isolated and I{kappa}B was detected by Western blotting. A loading control detecting tubulin is shown.

 
When cells were infected with M. avium, we observed NF-{kappa}B translocation in ~30% of the cells after 30 min of infection; this level lowered to 5% by 1 h and 3% by 24 h after infection (Fig. 1, A and B). Similar results were obtained in BMM (data not shown). These data argue that, although NF-{kappa}B is activated in a fraction of M. smegmatis and, to a lesser extent, in M. avium-infected cells, in both cases the live bacteria are able to suppress the activation by 30–60 min of infection. In agreement with previous reports, M. avium induced a less potent and more transient activation of NF-{kappa}B in macrophages (26).

To corroborate these observations, we used ELISA to monitor the active form of NF-{kappa}B in nuclear extracts of M. smegmatis-infected cells. For this we used an Ab that recognizes an epitope on p65 that is accessible only when NF-{kappa}B is activated and bound to its target DNA. The phosphorylated p65 can be detected in isolated nuclei from infected cells at 10 min after infection (Fig. 1C). This confirmed that p65 was translocated to the nucleus in its active form. In agreement with the immunofluorescence data, the signal after 60 min was lower than at 30 min. Moreover, infection with M. avium induced a less potent activation of NF-{kappa}B compared with M. smegmatis (Fig. 1C). As expected, the wild-type consensus oligonucleotide used as a competitor for NF-{kappa}B binding inhibited the binding and a mutated oligonucleotide, unable to bind p65, had no effect on NF-{kappa}B binding activity (Fig. 1C); this provides strong evidence of specificity of the positive reaction. In addition, Western blot analysis showed an increase in degradation of I{kappa}B in cytoplasm during the first hour of infection (Fig. 1D).

Taken together, these data show clearly that both live and killed M. smegmatis induced an early proinflammatory response mediated by NF-{kappa}B. However, the live bacteria are able to repress the activation of NF-{kappa}B after the first hour of infection.

Activation of the transcription factor NF-{kappa}B is essential for macrophages to kill the nonpathogen M. smegmatis

NF-{kappa}B is activated during the first hour after infection with M. smegmatis. In the subsequent 3 h, the bacteria are subjected to a robust killing attack by the macrophage and the majority are killed by 4 h (4). To investigate the role of NF-{kappa}B in the intracellular killing of M. smegmatis, J774 macrophages were infected in the presence or absence of inhibitors of NF-{kappa}B activation. For this, bacteria were isolated from infected cells at different time points and CFU were determined. The addition of SN-50, an inhibitor of NF-{kappa}B transport to the nucleus (32), significantly increased the survival of M. smegmatis when it was added at the start of the infection (Fig. 2A). However, when the inhibitor was added after 1 h of infection, bacterial killing was not affected, arguing that the killing potential of the NF-{kappa}B system is restricted to the first hour of infection.


Figure 2
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  		FIGURE 2. NF-{kappa}B is required for killing of M. smegmatis by macrophages. A, J774 cells were infected with M. smegmatis in the presence or absence of the NF-{kappa}B inhibitor SN-50 at the indicated times after infection and CFU were determined. B, Survival of M. smegmatis in the presence of several inhibitors of the NF-{kappa}B pathway added concomitant with the infection. C, RAW264.7 cells were knocked down for p65 using specific siRNA or scrambled RNA as a control. After 2 days, cells were infected with M. smegmatis and CFU were determined. Inset shows a representative knockdown of p65 using siRNA p65 compared with treatment with scrambled (scram) RNA (scrRNA). Tubulin was used as an internal control. D, J774 cells were infected with M. avium MAC101 in the presence or absence of the NF-{kappa}B inhibitor SN-50 at the indicated times after infection and CFU were determined. E, Survival of M. avium in the presence of several inhibitors of the NF-{kappa}B pathway added concomitant with the infection. F, RAW264.7 cells were knocked down for p65 using specific siRNA or scrambled RNA as a control. After 2 days, cells were infected with M. avium and CFU were determined. Inset shows a representative knockdown of p65 using siRNA p65 compared with treatment with scrambled RNA (scrRNA). Tubulin was used as an internal control. **, p ≤ 0.001 relative to control in the same conditions.

 
Similar results were seen with SC-514, a highly selective inhibitor of IKK-2 (Fig. 2B). This compound specifically blocks NF-{kappa}B-dependent gene expression but not MAPK pathways (33). Moreover, the addition of NF-{kappa}B activation inhibitor, a cell-permeable compound that acts as an inhibitor of NF-{kappa}B transcriptional activation, also increased bacterial survival (Fig. 2B). Since NF-{kappa}B activation requires proteosome function, we also observed that MG-132, a reversible and cell-permeable proteasome inhibitor, completely blocked bacterial killing (Fig. 2B). Treatment of cells with different inhibitors had no effect on the phagocytic uptake process for M. smegmatis or latex beads (data not shown). In agreement, in BMM-treated with either SN-50 or SC-514, we also observed an increase in the number of viable intracellular bacteria only when these inhibitors were added coincident with infection (data not shown). In addition, there was no significant effect on overall macrophage viability under any conditions using these drugs, as monitored by trypan blue staining (data not shown).

To complement these data using chemical inhibitors, we used interference RNA. The siRNA treatment led to an almost complete removal of p65 from macrophages, as revealed by Western blot analysis (Fig. 2C, inset). In agreement with the previous results, knock-down of p65 in macrophages also increased the survival of M. smegmatis (Fig. 2C). After the treatment of cells with siRNA, some killing still occurred, possibly due to a residual pool of p65 being synthesized.

We next performed the same set of experiments using inhibitors of NF-{kappa}B activation and siRNA against p65 to evaluate the survival of M. avium. In contrast to what we observed with M. smegmatis, there were no significant differences in survival of M. avium when NF-{kappa}B activation was blocked or p65 knocked down (Fig. 2, D–F). Altogether, these results argue strongly that proteins regulated by the NF-{kappa}B system induced relatively early in infection, especially during the first hour, are required for the process by which macrophages kill nonpathogenic intracellular mycobacteria. In contrast, although an early transient activation of NF-{kappa}B was seen with M. avium, this activation was not sufficiently robust to have any significant effect on the killing of this pathogen.

M. smegmatis infection modulates the expression of membrane-trafficking regulators and several lysosomal enzymes

We next focused on the identification of factors that were regulated during infection that could contribute to killing. For this, global gene expression analysis was performed. We compared the gene expression profile in noninfected vs M. smegmatis-infected macrophages at 1 and 4 h. We scored a significant up- or down-regulation when the degree of difference was at least 1.5-fold and statistically significant (p ≤ 0.01).

Overall, the analysis showed that after 1 h of infection, when infected cells were compared with uninfected cells, 1145 genes were up-regulated and 1435 down-regulated. We categorized this set of genes into different subsets of genes of interest. Numerous IL genes were up-regulated upon 1 h of infection such as IL-6, IL-1β, IL-10, and TNF-{alpha}. From the MAPK signaling pathway, MAPK13 was 2.2-fold up-regulated. The expression of some metalloproteinases (MMP) was elevated, such as MMP10, MMP12, and MMP13. The mRNA levels for TLRs TLR2 and TLR5 were also higher. As expected from our data, proteins associated with the NF-{kappa}B signaling pathway were also up-regulated, such as RelA and IKK{epsilon} (Table I).


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  		Table I. Representative genes regulated during M. smegmatis infection in the presence or absence of NF-{kappa}B inhibitors (INH)

 
Since the most intense phase of killing occurs between 1 and 4 h after infection, we next monitored gene expression in live M. smegmatis-infected cells at 4 h after infection. IL-6, IL-1β, IL-10, and TNF-{alpha} were also up-regulated at this time but the mRNA levels were considerably higher compared with 1 h of infection. In contrast to the 1-h infected cells, other ILs were additionally up-regulated, such us IL-1{alpha} and IL-19. Besides MMP10, the other related metalloproteinases MMP3 and MMP12, MMP13, MMP17, and MMP21 were also higher at 4 h after infection. TLR2 and TLR5 were up-regulated to a higher level at 4 h than at 1 h after infection (Table I).

Although the functional classes of proteins described above were not unexpected, two sets of genes associated with lysosome-mediated killing were more surprising. These two sets comprise those for lysosomal enzymes and vesicular trafficking regulators. After 4 h, the expression of several lysosomal enzymes such as hexosaminidase A, prosaposin, dipeptidilpeptidases 7 and 10, mannosidase 1{alpha}, and {alpha}-galactosidase (Gla) was up-regulated (Table II). However, not all lysosomal enzyme genes were up-regulated. Smpd1 and Lpl were both down-regulated after 4 h of infection (Table II). Rab and syntaxin proteins are regulators of vesicle budding, vesicle delivery, tethering, and fusion (34). Many genes from both families were highly up-regulated after infection, including syntaxin 6, syntaxin 7, Rab5b, Rab20, Rab24, and Rab12 (Table III).


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  		Table II. Lysosomal hydrolases regulated during M. smegmatis infection in the presence or absence of NF-{kappa}B inhibitors (INH)

 

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  		Table III. Membrane-trafficking proteins regulated during M. smegmatis infection in the presence or absence of NF-{kappa}B inhibitors (INH)

 
Inhibition of NF-{kappa}B leads to a change in the expression of genes connected to lysosomal enzymes and intracellular trafficking

The above list could now provide us with a reference for asking which genes up-regulated by M. smegmatis infection are under the control of NF-{kappa}B. For this, we conducted an expression analysis in the presence and absence of NF-{kappa}B inhibitors. Those genes which display differences in their activation when the NF-{kappa}B system was blocked would be considered to be potentially under the control (directly or indirectly) of NF-{kappa}B (Fig. 3A). From the set of genes that were differentially regulated when the NF-{kappa}B system is blocked, some new interesting targets of lysosomal-mediated killing were identified. In the presence of the NF-{kappa}B inhibitor, some lysosomal enzymes were down-regulated at 1 h postinfection such as pro-CtsC, mannosidase 1{alpha}, and lysozyme. A larger set of hydrolytic enzymes were down-regulated at 4 h after infection in the presence of the inhibitor; including pro-CtsB, pro-CtsC, pro-CtsH, pro-CtsZ, lysozyme, and Gla (Table II and Fig. 3B).


Figure 3
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  		FIGURE 3. Identification of genes associated to lysosomal-mediated killing by microarray. A, Classification of the gene expression data according to function. +, Up regulated; –, down regulated. B, Lysosomal enzymes regulated during infection with M. smegmatis in a NF-{kappa}B dependent manner. C, Membrane- trafficking regulators regulated during infection with M. smegmatis in a NF-{kappa}B dependent manner.

 
When NF-{kappa}B activation was blocked, a number of genes involved in the regulation of intracellular trafficking processes were significantly down-regulated, including Rab5a, Rab10, Rab20, and Rab34 (Table III and Fig. 3C). Moreover, the expression of LAMP-2, a protein implicated recently in phagolysosome fusion (35), also decreased.

These data argue that the NF-{kappa}B system regulates gene expression of both lysosomal enzymes and proteins that control membrane trafficking during M. smegmatis infection. These findings also suggest that the activation of NF-{kappa}B could provide a link between the proinflammatory response and lysosome-mediated killing of intracellular bacteria.

M. smegmatis infection up-regulates genes for lysosomal enzymes and membrane-trafficking regulators in an NF-{kappa}B-dependent manner

We tested some selected genes of interest by quantitative real-time PCR (qRT-PCR) after 1 and 4 h of infection. We were especially interested in selected lysosomal enzymes and membrane-trafficking regulators. To address the lysosomal enzymes, we selected pro-CtsB, pro-CtsH, pro-CtsZ, and Gla. At the level of transcripts, none of the genes was significantly altered after 1 h of infection (Fig. 4A). In addition, CtsZ, CtsB, CtsH, and Gla were markedly up-regulated at 4 h after infection. However, the expression of CtsZ, CtsH, and Gla was markedly down-regulated in M. smegmatis-infected cells treated with the NF-{kappa}B inhibitor. This is consistent with these genes being normally activated by NF-{kappa}B. In contrast, CtsB was up-regulated at 1h but no differences relative to uninfected controls were observed at 4 h (Fig. 4A). These results suggest that NF-{kappa}B activation is a negative regulator of the expression of CtsB.


Figure 4
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  		FIGURE 4. Analysis of the expression pattern of genes connected to intracellular trafficking and lysosomal enzymes during M. smegmatis infection. A and B, qRT-PCR of the indicated genes were measured by SYBR Green incorporation. Results are shown as the average absolute gene regulation ± SD of at least three independent experiments. **, p ≤ 0.001 relative to control in the same conditions. C and D, J774 cells were infected for 1 and 4 h in the presence or absence of SC-514. Western blot analysis of CtsH and CtsZ (C) or LAMP-2 and Rab34 (D) in the treated cell lysates. Right panel of each figure indicates the representative quantitation of one of three experiments that showed similar results. Results are represented as normalized to the untreated control levels, arbitrarily put as 1.

 
We next focused on four interesting membrane-trafficking genes, Rab10, Rab20, Rab34, and LAMP-2. These genes were all significantly up-regulated during infection with M. smegmatis, compared with noninfected cells (Fig. 4B). The pattern of regulation at the transcriptional level during infection was different for these proteins. In the case of Rab10, Rab34, and LAMP-2, the levels of transcripts were higher at 4 h after infection but not significantly different from the uninfected cells at 1 h after infection. In contrast, Rab20 was highly up-regulated already from 1 h after infection. Strikingly, the up-regulation of these four genes was strongly NF-{kappa}B dependent (Fig. 4B). Some discrepancies were observed between the microarray and qRT-PCR data. The microarray approach was not able to detect the up-regulation of Rab34, LAMP-2, Rab10, and cathepsins during infection observed by qRT-PCR. The lack of detection of the mRNA-relative signals for these proteins was probably because of the lower sensitivity of this technique in detecting low-abundance messages, as previously reported (36). Altogether, these results indicate that the expression of many genes involved in hydrolytic activity and intracellular transport is up-regulated during M. smegmatis infection by a NF-{kappa}B-dependent process.

Expression of Rab34, LAMP-2 but not mature cathepsins is regulated by NF-{kappa}B during the first 4 h of M. smegmatis infection

After infection, the activation of NF-{kappa}B led to increased mRNA levels for some lysosomal enzymes. However, Western blot analysis showed that the total levels of mature Cts H and Z were not significantly affected at 1 and 4 h after infection (Fig. 4C). Since lysosomal enzymes are known to be long-lived proteins, it is likely that the newly made RNAs for procathepsins will result in higher amounts of the active enzymes at later times of infection. Actually, we observed an increase in the levels of these enzymes after 12 and 24 h of infection; therefore, the increased synthesis may contribute to later stages of killing (data not shown and Ref. 4).

The delivery of lysosomal enzymes to the phagosome depends on vesicular trafficking machinery. According to our data, some of the proteins that control trafficking are up-regulated upon infection. Therefore, we performed experiments to evaluate the expression of membrane-trafficking proteins. Rab34 has been associated to lysosome positioning within the cell (37). However, the role of this protein in phagosome maturation is unknown. The role of LAMP-2 in phagolysosome fusion and organelle motility has been recently shown. Cells lacking both LAMP-1 and LAMP-2 showed impaired phagolysosome fusion without loss of lysosomal membrane integrity (35). We therefore selected Rab34 and LAMP-2 for further analysis. Western blot analysis showed that M. smegmatis infection up-regulates the level of both LAMP-2 and Rab34 proteins at 1 and 4 h after infection (Fig. 4D). However, both proteins were markedly down-regulated when NF-{kappa}B was blocked (Fig. 4D, see quantitation, right panels). Altogether, these data show that NF-{kappa}B-regulated expression of CtsH and CtsZ does not significantly contribute to the overall amounts of these proteins up to 4 h of infection with M. smegmatis. In contrast, the elevated expression of LAMP-2 and Rab34 proteins in macrophages is strongly dependent on NF-{kappa}B activation early during infection.

Regulation of the expression of genes for lysosomal enzymes and membrane-trafficking regulators during M. avium infection

We next analyzed the same set of selected genes in the context of M. avium infection. Focusing on CtsB, CtsH, CtsZ, and Gla1 expression, we observed an up-regulation of CtsB but not CtsH, CtsZ, and Gla1 between 1 and 4 h after infection that were similar to uninfected cells (Fig. 5A). These findings with CtsH and CtsZ were supported by Western blot (Fig. 5C). Surprisingly, two of the enzymes, CtsB and Gla1, were significantly up-regulated when the cells were infected in the presence of SC-514; CtsB at both 1 and 4 h, whereas up-regulation of Gla1 was only seen at 1 h. As for CtsB during M. smegmatis infection, this provides further evidence that NF-{kappa}B activation represses CtsB expression. A similar mechanism can be invoked for Gla1 in M. avium-infected cells.


Figure 5
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  		FIGURE 5. Analysis of the expression pattern of genes connected to intracellular trafficking and lysosomal enzymes during M. avium infection. A and B, qRT-PCR of the indicated genes were measured by SYBR Green incorporation. Results are shown as the average absolute gene regulation ± SD of at least three independent experiments. **, p ≤ 0.001 relative to control in the same conditions. C and D, J774 cells were infected for 1 and 4 h in the presence or absence of SC-514. Western blot analysis of CtsH and CtsZ (C) or LAMP-2 and Rab34 (D) in the treated cell lysates. Right panel of each figure indicates the representative quantitation of one of three experiments that showed similar results. Results are represented as normalized relative to the untreated control levels, arbitrarily put as 1.

 
When we investigated the expression of Rab10, Rab20, Rab34, and LAMP-2 in M. avium-infected cells, no easily discernable pattern could be seen that was general for all of these mRNAs (Fig. 5B). The levels of Rab10 were not altered by the infection with or without inhibitor. The levels of Rab20 were up-regulated by 1 and 4 h after infection but not when NF-{kappa}B was blocked. This pattern is similar to that seen during M. smegmatis infection. Rab34 was up-regulated after 1 h in presence of the NF-{kappa}B inhibitor but no differences in expression were observed at 4 h of infection. A similar increase in the expression of LAMP-2 was seen in the presence of the inhibitor at the 1-h time point. These observations were confirmed by Western blot analysis (Fig. 5D). Taken together, this argues that in the first hour of infection Rab34 and LAMP-2 are negatively regulated by NF-{kappa}B in M. avium-infected cells. However, no difference in the expression of these proteins was observed at 4 h after infection.

Block of NF-{kappa}B activation alters the normal fusion of mycobacterial phagosomes with lysosomes/late endosomes

Our results collectively argue that infection with M. smegmatis results in a significant NF-{kappa}B-dependent regulation of membrane-trafficking molecules (e.g., Rab34 and LAMP-2). Since M. smegmatis is effectively killed by macrophages when NF-{kappa}B is activated, this led to the hypothesis that some of these molecules are necessary for trafficking events that allow phagosomes to fuse with late endosomes/lysosomes. To address this hypothesis, we monitored by microscopy the acquisition of different markers of late endosomes/lysosomes by M. smegmatis phagosomes.

We first focused on the proton pumping v-ATPase. As in our recent study (4), this marker localized to 15–30% of live M. smegmatis phagosomes between 1 and 4 h after infection. Treatment of macrophages with the NF-{kappa}B inhibitor SC-514 significantly decreased the fraction of mycobacterial phagosomes labeled for this marker at 4 h after infection (Fig. 6A). When heat-killed mycobacteria were internalized, the v-ATPase-labeled fraction was higher than with live bacteria. In this case, inhibition of NF-{kappa}B after 1 and 4 h of internalization also led to a decrease in colocalization with this marker (Fig. 6B). Similar results were obtained using another NF-{kappa}B inhibitor, SN-50 (data not shown).


Figure 6
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  		FIGURE 6. NF-{kappa}B inhibition impairs fusion of M. smegmatis phagosomes with lysosomes/late endosomes. A, J774 cells were infected with live M. smegmatis in the presence or absence of SC-514. After 4 h of infection, cells were fixed and LAMP-2 localization was determined. B, Quantitative analysis of the colocalization under different conditions. C, Cells were infected and processed as in A and v-ATPase was detected by indirect immunofluorescence. D, Quantitative analysis of the percentage of colocalization of v-ATPase with M. smegmatis phagosomes. Results are shown as means ± SEM of at least three independent experiments. **, p ≤ 0.001 relative to control (CTRL) in the same conditions. At least 100 phagosomes were counted in each experiment. Bars, 10 µm.

 
As shown previously (4), live M. smegmatis phagosomes acquired LAMP-2 such that by 4 h the majority were positive for this marker (Fig. 6C). In contrast, J774 cell treated with SC-514 showed a significant reduction in the fraction of LAMP-2-positive phagosomes. In agreement, the number of phagosomes positive for LAMP-2 was lower in BMM treated with SC-514 compared with the control (data not shown). Thus, when the NF-{kappa}B activation is blocked, the efficiency of phagosome maturation is significantly retarded.

NF-{kappa}B had no effect on the overall macrophage protein content of CtsZ and CtsH during the first 4 h of infection (Fig. 4C). The observation that blocking NF-{kappa}B activation inhibited phagolysosomal fusion suggested that the delivery of these enzymes to phagosomes might be lowered in the absence of NF-{kappa}B activation. To investigate this, we monitored the fraction of M. smegmatis phagosomes that could be labeled for these markers. At 4 h after infection, the colocalization of live M. smegmatis-containing phagosomes with both CtsH and CtsZ was significantly decreased after treatment with SC-514 (Fig. 7, A–C) and SN-50 (data not shown). When macrophages were incubated with heat-killed bacteria, the fraction of phagosomes that labeled for CtsH and CtsZ was also significantly reduced in the presence of the NF-{kappa}B inhibitor (Fig. 7, B–D). This argues that NF-{kappa}B regulates processes that control the delivery of CtsH and CtsZ to phagosomes enclosing both live and killed M. smegmatis.


Figure 7
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  		FIGURE 7. NF-{kappa}B inhibition impairs the delivery of CtsH and CtsZ to M. smegmatis phagosomes. A, J774 cells were infected with M. smegmatis in the presence or absence of SC-514. After 4 h of infection, cells were fixed and subjected to indirect immunofluorescence or CtsH. B, Quantitative analysis of the percentage of colocalization under different conditions. C, Cells were infected and processed as in A for CtsZ. D, Quantitative analysis of the percentage of colocalization of CtsZ with M. smegmatis phagosomes. Results are shown as means ± SEM of at least three independent experiments. **, p ≤ 0.001 relative to control in the same conditions. At least 100 phagosomes were counted in each experiment. Bars, 10 µm.

 
To test whether this effect is general to all lysosomal enzymes, we analyzed CtsS. The expression of this enzyme was not altered in our microarray data. No difference in colocalization of M. smegmatis phagosomes with CtsS were observed in the presence or absence of the NF-{kappa}B inhibitor. Indeed, the percentage of colocalization for this marker was low from the beginning (data not shown). Since the total levels of CtsZ and CtsH were not affected at 4 h of infection, it seems that NF-{kappa}B is more important for the regulation of late endosome/lysosome delivery of these enzymes to M. smegmatis phagosomes than for up-regulating the levels of lysosomal enzymes. Taken together, these results confirm that NF-{kappa}B is required for processes leading to fusion of "lysosomes" and M. smegmatis phagosomes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Pathogenic mycobacteria such as M. tuberculosis or M. avium survive within macrophages, whereas nonpathogenic mycobacteria such as M. smegmatis are killed. This means that pathogenic mycobacteria block essential mechanisms of killing that are activated by nonpathogenic mycobacteria. We rationalized that by studying how nonpathogenic mycobacteria are killed, it would not only facilitate the identification of the general macrophage mechanisms involved in mycobacterial killing but it could also help to identify key targets that pathogenic mycobacteria subvert to survive.

In agreement with previous observations, we showed that M. smegmatis induced a potent activation of NF-{kappa}B after infection (26). In this study, we demonstrated that this NF-{kappa}B activation is essential for killing of M. smegmatis since several inhibitors and siRNA treatment targeting NF-{kappa}B prevent killing and even allowed M. smegmatis to grow in macrophages. For this effect, it was important that the inhibitors were added at the start of infection. However, when they were added after 1 h of infection, killing proceeded normally. This argues that a transient activation of NF-{kappa}B during the first hour of infection is essential for an efficient killing.

The infection with live M. avium also initiated the activation of NF-{kappa}B with the same kinetics as LPS or all other mycobacterial conditions. However, after 30 min, the system was blocked. Our results argue that this small transient peak of activation is not robust enough to activate genes that lead to the killing of M. avium since inhibition of NF-{kappa}B had no effect on M. avium survival. Thus, although both live M. avium and M. smegmatis allow an initial activation of NF-{kappa}B, the permissive period was more significant for M. smegmatis.

To identify which genes regulated by NF-{kappa}B were important for killing, we performed RNA microarray analysis. Many expected NF-{kappa}B-dependent genes were up-regulated during the infection including ILs and proteins involved in the known NF-{kappa}B signaling pathways. However, two classes of genes were identified that had previously not been associated either with the classical NF-{kappa}B pathway or with mycobacterial infection. The first class includes a large set of lysosomal enzymes while the second pinpoints several regulators of membrane trafficking whose expression is under the control of NF-{kappa}B.

It has been shown that NF-{kappa}B regulates the production of lysosomal enzymes in cancer cells (38). In some types of cancer, a continuous activation of NF-{kappa}B induces the overproduction of lysosomal enzymes that are secreted (39). Our results show that this phenomenon is more general since NF-{kappa}B controls the expression of several lysosomal enzymes in M. smegmatis-infected macrophages.

Our results show that CtsH and CtsZ are up-regulated after 4 h of infection and down-regulated when NF-{kappa}B was blocked. In contrast, Western blot analysis indicates that the total cellular content of some of the active enzymes was not affected by the 4-h time point. However, later during the infection, the observed up-regulation resulted in a higher amount of cathepsins that are potentially involved in later killing stages (data not shown).

Nevertheless, we clearly observed a reduction in the fraction of phagosomes that are labeled by CtsZ and CtsH in mycobacterial phagosomes when NF-{kappa}B activation was blocked. Moreover, the fraction of phagosomes that acquired LAMP-2 and v-ATPase was also reduced in the presence of NF-{kappa}B inhibitors. This argues that proteins induced by the NF-{kappa}B system regulate the overall processes leading to fusion of phagosomes with late endosome-lysosomes, which deliver lysosomal enzymes into the phagosome lumen. These observations could be rationalized by the finding that >20 known membrane-trafficking molecules are mostly up-regulated in a NF-{kappa}B-dependent fashion in infected macrophages.

Particularly interesting here were LAMP-2 and Rab34, two regulators of lysosome motility and function. The synthesis of these proteins was significantly increased in the first 4 h of infection of control macrophages but not when NF-{kappa}B was blocked. LAMP-2 has recently been shown to mediate lysosome motility that is important for phagolysosome fusion (35). Our results suggest that the macrophage protein level of LAMP-2 is rate limiting for this process and that its synthesis must be significantly enhanced to cope with the increased membrane trafficking to the phagosome in the infected macrophages.

Manipulation of Rab and SNARE proteins is a key component of the survival strategy of Mycobacterium (40, 41, 42). Moreover, expression of Rabs is down-regulated in patients with tuberculosis compared with healthy M. tuberculosis-infected donors (43).We found that the synthesis of Rab34 was elevated in a NF-{kappa}B dependent manner in both M. smegmatis- and M. avium-infected macrophages. However, the regulation of this protein was the opposite in the two systems. In M. smegmatis-infected cells, NF-{kappa}B was a positive regulator, whereas in M. avium-infected cells it behaved as a negative regulator. Although the mechanism of this difference is open, these data suggest that M. avium has the ability to switch the transcriptional response linked to NF-{kappa}B to a state more conducive for its survival. Rab34 has been shown to regulate lysosome positioning within the cell (37). Since lysosome positioning may be required for mycobacterial killing, we are testing the hypothesis that the regulation of Rab34 expression controls the ability of phagosomes to be positioned close to lysosomes (M. G. Gutierrez, W. Hong, and G. Griffiths, unpublished data). Indeed, we observed that the compartments positive for LAMP-2 are distributed more in the peripheral region in macrophages treated with the NF-{kappa}B inhibitor SC-514 (Fig. 6C).

Rab20 was also up-regulated in a NF-{kappa}B-dependent manner during both M. smegmatis and M. avium infection. This Rab was recently shown to be up-regulated in response to phagocytosis of Aspergillus fumigatus, a pathogen that also activates NF-{kappa}B (44). However, the role of Rab20 in the host response against intracellular pathogens has not been investigated. Syntaxins are another group of membrane-trafficking regulators that are up-regulated during the response against M. smegmatis infection. Stx6 is especially highly up-regulated at 1 and 4 h after infection but not when NF-{kappa}B is blocked (Table II). Syntaxin 6 has been associated with a proposed direct transport pathway from the Golgi to latex bead phagosomes, but it fails to reach phagosomes enclosing M. bovis bacillus Calmette-Guérin (40).

It is still open whether the proteins we identified are directly or indirectly regulated by NF-{kappa}B. Using MatInspector (Genomatrix) software, we identified a putative NF-{kappa}B binding site in the first 2-kb promoter region of Rab34 (data not shown). However, it is expected that some of the proteins identified here that regulate the fusion of mycobacterial phagosomes with lysosomal compartments may be indirectly regulated by NF-{kappa}B (M. G. Gutierrez et al., unpublished data). In fact, 4 h after infection is sufficient time for other transcription factors to be activated in response to the direct activation of NF-{kappa}B. Indeed, NF-{kappa}B- activated gene products may themselves be transcription factors (45).

Whether or not pathogenic mycobacteria activate NF-{kappa}B is not clearly established, with different groups reporting conflicting results (23, 24, 28, 46). Using confocal microscopy and ELISA, we observed a low and transient activation of NF-{kappa}B by M. avium. Similar results were reported before (26). In contrast, others observed that high and low virulence strains of the M. avium-intracellular complex (MAC) activated NF-{kappa}B (46). There are similar reports of NF-{kappa}B activation in macrophages infected with M. tuberculosis (27, 28). However, many studies argue that the pathogens strongly block the proinflammatory response in general (47, 48).

Based on our observations, we suggest the model in which pathogenic mycobacteria induce NF-{kappa}B activation upon contact with the macrophage. As soon as the mycobacteria start the synthesis and secretion of virulence effectors, the system may be repressed. This model is in agreement with previous studies of gene expression in M. avium-infected cells (49). A hitherto unappreciated benefit to the pathogen in inhibiting the NF-{kappa}B system is the consequence that the phagolysosome fusion events would also be blocked.

In summary, our results pinpoint a novel role of the NF-{kappa}B system to allow (directly or indirectly) the synthesis of molecules involved in intracellular trafficking. This regulation is linked to phagolysosome fusion, which facilitates killing of intracellular mycobacteria. In addition, our microarray screen identified a large number of potential new regulators of phagolysosome fusion; NF-{kappa}B is thus a key regulator of factors that facilitate the intracellular killing of mycobacteria.


   Acknowledgments
 
We are grateful to Dr. Luis Mayorga for his critical reading of this manuscript. We also thank Vladimir Benes and Tomi Ivacevic at the Genecore Facility and Stefan Terjung at Advance Light Microscopy Facility (EMBL-Heidelberg) for excellent technical assistance and advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
We have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 M.G.G. was supported by a Research Fellowship from Alexander von Humboldt Foundation and is currently funded by an European Molecular Biology Organization Fellowship. E.A. was supported by Fundação para a Ciência e a tecnologia Grant POCI/BIA-BCM/55327/2004 with coparticipation of the European Union fund FEDER Programme POCI2010. Back

2 M.G.G. and B.B.M. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Maximiliano Gabriel Gutierrez, European Molecular Biology Laboratory, Postfach 102209, 69117 Heidelberg, Germany. E-mail address: mgutierr{at}embl.de Back

4 Abbreviations used in this paper: siRNA, small interfering RNA; qRT-PCR, quantitative real-time PCR; BMM, bone marrow macrophage; Cts, cathepsin; Gla, {alpha}-galactosidase. Back

Received for publication February 28, 2008. Accepted for publication May 28, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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