Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

mutation Background: Cystic fibrosis is characterized by defective autophagy and increased Burkholderia cenocepacia infection. Results: The depletion of SQSTM1/p62 from (cid:1) F508 macrophages improves bacterial clearance via autophagy. Conclusion: p62 expression level determines the fate of B. cepacia infection in (cid:1) F508 macrophages. Significance: Our study reveals the role of p62 in diseases characterized by protein aggregates that compromise autophagy by consuming essential autophagy molecules.


Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ⌬F508
Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery * tribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ⌬F508 macrophages.
Cystic fibrosis (CF) 3 is the most common inherited lethal disease among Caucasians, which is caused by mutations in the cftr gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common CFTR mutation results in a deletion of phenylalanine at position 508 (⌬F508), which affects the processing of the CFTR protein in such way that it cannot reach the epithelial cell surface. This mutation results in an aggresome-prone protein that forms intracellular aggregates (1)(2)(3)(4).
Autophagy is a conserved physiological process that eliminates non-functional organelles and recycles cytosolic components to generate nutrients during periods of stress or starvation (5,6). Autophagy also targets cytosolic long-lived proteins and organelles for lysosomal degradation in eukaryotic cells and plays a role in innate immunity (7). Loss of autophagy in murine tissues is accompanied by accumulation of protein aggregates and disordered organelles, leading to life-threatening diseases (8). Autophagy plays a key role in protecting the cytosol from bacterial infection. The mechanisms of bacterial recognition by this pathway are starting to be elucidated. Some cellular cargos are marked for autophagy by acquiring adaptor * This work was supported by a doctoral fellowship from the Egyptian Bureau of Education (to B. A. A.). This work was supported, in whole or in part, by the National Institutes of Health (Grant R01HL094586 (to A. O. A.)) and a Cystic Fibrosis Canada grant (to M. A. V). 1  proteins such as Calcoco2 (also known as NDP52) and neighbor of BRCA1 gene product (NBR1) (9 -14). In addition, SQSTM1 (also known as p62) is required for targeting Salmonella enterica serovar Typhimurium (Salmonella typhimurium), intracytosolic Shigella, and Listeria to the autophagic pathway (9,10). The adaptor molecule p62 is a ubiquitously expressed cellular protein that is conserved in metazoa but not in plants or fungi (15,16). The quantity of p62 is critical for cell viability and is strictly controlled (17). p62 has multiple protein-protein interaction domains, including the ubiquitin-associated domain for ubiquitinated cargo binding and the LC3 interaction region for binding LC3 (10). Accordingly, impaired autophagy is accompanied by accumulation of p62 followed by formation of aggregates containing p62 and ubiquitinated proteins. This accumulation occurs because of the nature of both self-oligomerization and ubiquitin binding of p62 (18,19).
Burkholderia cenocepacia (B. cepacia) is an opportunistic Gram-negative bacterium that infects CF patients and leads to severe lung inflammation and lung tissue destruction. Occasionally, this infection results in a lethal necrotizing pneumonia (20 -22). Unfortunately, B. cepacia is resistant to most known antibiotics and, thus, is nearly impossible to treat. B. cepacia adopts an extracellular or intracellular lifestyle (23,24). This bacterium can survive within a variety of eukaryotic cells such as amoebae, epithelial cells, and macrophages (25)(26)(27)(28).
We have demonstrated previously that in WT macrophages, the majority of B. cepacia-containing vacuoles slowly acquire the specific autophagy marker LC3 within 2 h of infection. Subsequently, these vacuoles fuse with the lysosomes, and the bacterium is degraded. In ⌬F508 macrophages, B. cepacia-containing vacuoles do not acquire autophagosome markers and do not fuse with the lysosomes.
Here, we demonstrate that in WT macrophages, p62 is required for targeting B. cepacia to the autophagosome. Upon p62 down-regulation, bacterial growth increases, whereas the overexpression of p62 results in a significant decrease in B. cepacia replication. On the contrary, down-regulation of p62 in ⌬F508 macrophages is associated with decreased bacterial growth, and p62 overexpression results in increased B. cepacia replication. p62 down-regulation in ⌬F508 macrophages releases the trapped BECN1 from CFTR aggregates, allowing its recruitment to the B. cepacia vacuole. BECN1 acquired by the B. cepacia-containing vacuole subsequently attracts LC3, thereby mediating the fusion of the maturing autophagosome containing B. cepacia with the lysosome via the autophagic machinery. These data provide mechanistic insight on how B. cepacia persists in ⌬F508 macrophages. This report also suggests that p62 may be an attractive drug target to improve B. cepacia clearance by autophagic machinery.

EXPERIMENTAL PROCEDURES
Bone Marrow-derived Macrophages-Animal experiments were performed according to protocols approved by the Animal Care and Use Committee of the Ohio State University College of Medicine. WT C57BL/6 were purchased from The Jackson Laboratory. ⌬F508 mice on a C57BL/6 background were obtained from Case Western University and housed in the Ohio State University vivarium. Bone marrow-derived macrophages were isolated from the femurs of 6-to 12-week-old mice and were cultured in Iscove's modified Dulbecco's medium (Invitrogen, catalog no. 12440) containing 10% heat-inactivated FBS (Invitrogen, catalog no. 16000), 20% L cell-conditioned medium, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, catalog no. 15140) at 37°C in a humidified atmosphere containing 5% CO 2 . Macrophages were infected with B. cepacia K56-expressing m-RFP or the corresponding gentamicin-sensitive strain MHK1 at a multiplicity of infection of 10.
Bacterial Strains and Culture-B. cenocepacia strain K56-2 is a clinical isolate from a CF patient. The corresponding gentamicin-sensitive strain MHK1 was described previously (29). All bacterial strains were grown in Luria-Bertani broth at 37°C overnight with high-amplitude shaking. To kill extracellular bacteria, Iscove's media (Invitrogen, catalog no. 12440) plus FBS (Invitrogen, catalog no. 16000) containing 50 g/ml gentamicin (Invitrogen, catalog no. 3564) were added for 0.5 h, as described previously (29). To enumerate intracellular bacteria, infected macrophages were lysed with ice-cold PBS (Invitrogen, catalog no. 14190) at designated times. Recovered bacteria were quantified by plating serial dilutions on Luria-Bertani agar plates and counting colonies using the Acolyte Colony Counter, 5710/SYN.
Real-time PCR-Total RNA was isolated from cells lysed in TRIzol (Invitrogen, catalog no. 15596-026) and then converted to cDNA. Gene expression was calculated as relative copy numbers, as described previously (30,33). Briefly, C t values of the p62 gene were subtracted from the average Ct of two housekeeping genes (GADPH and CAP1), and the resulting ⌬C t was used in the following equation: RCN ϭ (2 Ϫ⌬Ct ) 100. The relative copy number of a gene is represented as the number of copies relative to the 100 copies of average housekeeping genes (30,33).
Statistical Analysis-All experiments were performed at least three times independently and yielded similar results. Comparisons of groups for statistical difference were conducted using Student's two-tailed t test. p Յ 0.05 was considered significant.
Ethics Statement-This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and Ohio State University. The Institutional Animal Care and Use Committee has approved our protocol number 2007A0070. All efforts were made to minimize suffering.

More B. cepacia Colocalized with p62 in WT Macrophages
Than in ⌬F508 Macrophages-We demonstrated previously that B. cepacia is cleared by the autophagy machinery in WT macrophages but not in their ⌬F508 counterparts. To determine why the B. cepacia vacuole is not efficiently recognized by the autophagy machinery in ⌬F508 macrophages, we followed the trafficking within WT and ⌬F508 macrophages. Recent studies showed that p62 is required for targeting S. typhimurium, Shigella, and Listeria to the autophagic pathway (9, 10). Therefore, we examined the colocalization of B. cepacia with p62 in WT and ⌬F508 macrophages. The time course for infection was 0.5, 1.5, 2, and 4 h. In WT macrophages, a significant percentage of B. cepacia colocalized with p62 at 1.5 h postinfection. Colocalization then declined at later time points (Fig.  1, A and B). However, B. cepacia vacuoles in ⌬F508 macrophages did not colocalize with p62 at any time point throughout infection (Fig. 1, A and B). Together, these data show that p62 labels the B. cepacia vacuole in WT but not in ⌬F508 macrophages.
The B. cepacia Vacuole Efficiently Acquires Ubiquitin in ⌬F508 Macrophages-Autophagy recognizes cargo for uptake and degradation when it becomes ubiquitinated and bound to an autophagy adaptor molecule (10). The lack of p62 acquisition by the B. cepacia vacuole in ⌬F508 macrophages could be due to defective ubiquitination of the B. cepacia-containing vacuole or because of lack of p62 expression in ⌬F508 macrophages. To differentiate between these possibilities, we first infected WT and ⌬F508 macrophages with B. cepacia-expressing m-RFP for 0.5 h or 2 h and examined the colocalization of B. cepacia with ubiquitin. There was no significant difference in the colocalization of B. cepacia with ubiquitin between WT and ⌬F508 macrophages (Fig. 2, A and B). These data demonstrate that equivalent numbers of B. cepacia vacuoles acquired ubiquitin in WT and ⌬F508 macrophages. Therefore, the lack of colocalization of B. cepacia with autophagosomes in ⌬F508 macrophages is not due to the absence of ubiquitin around the B. cepacia vacuole.
Next, to determine whether the failure of the autophagy machinery to target the B. cepacia vacuole is due to the lack of p62 expression in ⌬F508 macrophages, we examined the level of p62 within WT and ⌬F508 macrophages. Immunoblot analysis using an antibody against p62 revealed that murine macrophages harboring the ⌬F508 mutation exhibited a higher level of p62 compared with WT macrophages (Fig. 3A). Quantitative PCR (q-PCR) was performed to determine whether the increase in p62 protein level in ⌬F508 macrophages, compared with WT macrophages, is due to regulation of gene expression or accumulation of the p62 protein. There was no significant difference in the p62 mRNA level in both types of macrophages (Fig. 3B). Together, these data show that the increase in p62 level in ⌬F508 macrophages is due to accumulation of the protein inside the cell, suggesting defective autophagy activity.
B. cepacia Infection Elevates p62 Expression within WT and ⌬F508 Macrophages-p62 is well expressed in ⌬F508 macrophages. However, B. cepacia infection down-regulates autophagy in both WT and ⌬F508 macrophages. Thus, it is possible that B. cepacia infection is accompanied by depletion of p62 from ⌬F508 macrophages upon infection. To examine this possibility, we examined the effect of B. cepacia on p62 expression upon infection in WT and ⌬F508 macrophages by q-PCR and immunoblot analysis. At 4 h post infection, q-PCR analysis demonstrated increased expression of the p62 gene level in both WT and ⌬F508 macrophages compared with non-infected macrophages (Fig. 3C). Similarly, immunoblotting showed a higher p62 level in both types of macrophages (Fig.  3D). Together, these data show that B. cepacia infection increases the expression level of p62 in WT and ⌬F508 macrophages.
Overexpression of p62 Conversely Affects B. cepacia Replication in WT and ⌬F508 Macrophages-To determine the role of p62 in B. cepacia replication in WT and ⌬F508 macrophages, we examined B. cepacia survival in the presence of ectopically  expressed p62. WT and ⌬F508 macrophages were nucleofected with p62 plasmid or vector control and after 24 h, cells were infected with B. cepacia for 2, 4, and 6 h (Fig. 4G). In WT macrophages harboring the p62 plasmid, recovered B. cepacia CFUs decreased at 6 h post-infection compared with the cells harboring the vector alone (Fig. 4A). Confocal microscopy revealed significantly less bacterial accumulation upon overexpression of p62 (Fig. 4, B and C). In contrast, ⌬F508 macrophages harboring the p62 plasmid allowed significantly increased B. cepacia accumulation after 6 h post-infection (Fig. 4D). Confocal microscopy confirmed increased bacterial accumulation (Fig.  4, E and F). Together, these results demonstrate that the availability of p62 differentially determines the fate of B. cepacia in WT and ⌬F508 macrophages.
Down-regulation of p62 Decreases the Growth of B. cepacia in ⌬F508 Macrophages-To determine whether p62 targets B. cepacia vacuoles to autophagosomes for degradation, we nucleofected WT and ⌬F508 macrophages with p62 siRNA or scrambled siRNA (Fig. 5G). After 48 h, cells were infected with B. cepacia for 2, 4, and 6 h. In WT macrophages, B. cepacia CFUs significantly increased upon down-regulation of p62 (Fig.  5A). In addition, confocal microscopic analysis demonstrated significantly increased bacterial numbers at 2 h post-infection (Fig. 5, B and C). In contrast, ⌬F508 macrophages showed decreased B. cepacia CFUs upon down-regulation of p62 (Fig.  5D). Furthermore, confocal microscopy revealed significantly low bacterial accumulation 2 h after B. cepacia infection upon down-regulation of p62 (Fig. 5, E and F). Therefore, these data demonstrate that p62 controls B. cepacia infection in WT macrophages but not in ⌬F508 macrophages. The details of this differential role are not clear.
Decreased p62 Expression Promotes LC3 Acquisition by B. cepacia Vacuole in ⌬F508 Macrophages-LC3 is the main marker for autophagosomes. The conversion of LC3-I to LC3-II denotes autophagy stimulation and autophagosome formation (7,36). We have demonstrated previously that B. cepacia colocalization with LC3 is markedly decreased in ⌬F508 macrophages compared with WT macrophages (37,38). To determine the underlying mechanism, WT and ⌬F508 macrophages were nucleofected with either siRNA against p62 to down-regulate p62 or with scrambled siRNA, and after 48 h, nucleofected macrophages were infected with B. cepacia-expressing m-RFP for 0.5 and 2 h. Confocal microscopy showed that in WT macrophages, B. cepacia colocalization with LC3 decreased significantly when p62 was down-regulated compared with the siRNA control-treated cells (Fig. 6, A and C). In contrast, ⌬F508 macrophages allowed significantly more B. cepacia colocalization with LC3 after the down-regulation of p62 compared with the siRNA control-treated cells (Fig. 6, B  and D). Together, these data suggest that p62 is required for the delivery of B. cepacia to the autophagosomes in WT macro- phages, a role that is compromised for unknown reasons in ⌬F508 macrophages.

Depletion of p62 Liberates BECN1, Allowing Its Redistribution and Recruitment by the B. cepacia Vacuole in ⌬F508
Macrophages-A growing body of evidence indicates that BECN1 is sequestered within the mutant CFTR aggresomes (1,2). BECN1/Atg6 is a member of the class III PI3K complex and is essential for the early stages of autophagosome formation (5,39). Thus, its unavailability leads to defective autophagic activity (1,2). Mutant CFTR aggregates sequester autophagy molecules such as BECN1, depleting them from their storage areas, leading to defective autophagy. We examined the colocalization of B. cepacia with BECN1 in WT and ⌬F508 macrophages. Confocal microscopy showed that in WT macrophages, high numbers of B. cepacia colocalized with BECN1 compared with ⌬F508 macrophages (Fig. 7, A and B, arrows). In WT macrophages, BECN1 was distributed throughout the cytosol, whereas in ⌬F508 macrophages, BECN1 was condensed in patches ( Fig. 7 A, arrowheads).
Because the sequestration of BECN1 in CFTR aggregates requires p62 (1, 2), we examined the effect of p62 depletion on BECN1 distribution within the cytosol and around the B. cepacia vacuole inside ⌬F508 macrophages. ⌬F508 macrophages nucleofected with p62 siRNA showed significantly more colocalization of B. cepacia with BECN1 compared with the siRNA control (Fig. 7, C, arrows, and D). Additionally, within the ⌬F508 macrophages nucleofected with siRNA against p62, BECN1 was redistributed within the cytosol with the disappearance of BECN1-containing patches (Fig. 7C) after downregulation of p62 (B). Notably, our immunoblot analysis using antibody specific to BECN1 showed equal amounts of the total BECN1 in the ⌬F508 macrophages before and after p62 depletion (Fig. 7E). Together, these data show that depletion of p62 from ⌬F508 macrophages allows the redistribution of BECN1 throughout the cell and increases its availability for the B. cepacia-containing vacuole.
Together, these data suggest that depletion of p62 from ⌬F508 macrophages mediates B. cepacia clearance via recuper- ated autophagy. To confirm this conclusion, ⌬F508 macrophages were depleted of p62 and Atg7 (an essential autophagy molecule) (40) to disrupt the autophagy machinery and then infected with B. cepacia. Depletion of p62 alone from ⌬F508 macrophages improved B. cepacia clearance, yet concomitant depletion of Atg7 hindered bacterial clearance (Fig. 7, F and G). Thus, improved bacterial clearance upon depletion of p62 from ⌬F508 macrophages is mediated by autophagy.
NBR1 and NDP52 Contribute to the Delivery of B. cepacia to Autophagosomes after Down-regulation of p62 in ⌬F508 Macrophages-Thus, down-regulation of p62 in ⌬F508 macrophages improves B. cepacia clearance by restoring autophagy activity. However, what targets the B. cepacia vacuole for autophagy in the absence of adequate amounts of p62 is unknown. Recently, it has been shown that p62 and NDP52 act cooperatively to drive efficient antibacterial autophagy of Salmonella, Shigella, and Listeria (9,41). Furthermore, it was revealed that NBR1 and p62 mark ubiquitinated cargo for autophagy (11,42). To determine whether NDP52 or NBR1 contribute to the delivery of B. cepacia to autophagosomes, we nucleofected WT and ⌬F508 macrophages with siRNA against NDP52 or NBR1 prior to infection with B. cepacia. Our results showed that in WT macrophages, down-regulation of NDP52 did not affect B. cepacia recovered CFUs, whereas that of NBR1 resulted in a significant increase in B. cepacia growth. In ⌬F508 macrophages, the down-regulation of either NDP52 or NBR1 resulted in an increase in B. cepacia growth. This result demonstrates that both NDP52 and NBR1 facilitate the delivery of B. cepacia to autophagosomes in ⌬F508 macrophages. In WT macrophages, however, only NBR1 contributes to the delivery of B. cepacia to autophagosomes (Fig. 8, A, B, and D). To determine whether NDP52 and NBR1 mark the B. cepacia vacuole for autophagy uptake in p62-depleted ⌬F508 macrophages, we nucleofected ⌬F508 macrophages with siRNA against p62 alone or in combination with either NDP52 or NBR1. Simultaneous down-regulation of p62 and NDP52 or p62 and NBR1 in ⌬F508 macrophages resulted in a significant increase in B. cepacia growth (Fig. 8, C and E). Together, our data suggest that both NDP52 and NBR1 contribute to labeling the B. cepacia vacuole for autophagy uptake when p62 is unavailable.

DISCUSSION
Human and mouse CF macrophages and airway epithelial cells exhibit impaired autophagy that is associated with formation of aggregates, including p62 and ubiquitinated mutant CFTR protein. This is due to both the self-oligomerization and ubiquitin-binding nature of p62 (1,2,10,37,38). Similar aggregates have been identified in various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and cancer (1,2,43,44). Impaired turnover of p62 is a major cause of the pathogenic changes seen in the autophagy-deficient mice, as the loss of Atg7 in mouse livers results in severe p62 accumulation. Loss of p62 greatly attenuates liver injury resulting from autophagy deficiency (18).
Here, we found that ⌬F508 macrophages express more p62 than WT macrophages. This could be due to p62 accumulation FIGURE 6. Down-regulation of p62 decreases B. cepacia colocalization with LC3 in WT macrophages but increases the colocalization in ⌬F508 macrophages. A and B, confocal microscopy for WT macrophages and ⌬F508 macrophages infected with B. cepacia-expressing m-RFP for 0.5 or 2 h. LC3 stained green, whereas nuclei were stained with DAPI. The percentage of colocalization of B. cepacia with LC3 at the indicated time points was scored in both WT and ⌬F508 macrophages, respectively. C and D, more than 200 cells were scored. Data are presented as means Ϯ S.D. *, p Ͻ 0.05; **, p Ͻ 0.01. FIGURE 7. Colocalization of B. cepacia with BECN1 is increased in ⌬F508 macrophages upon depletion of p62. A, confocal microscopy for WT and ⌬F508 macrophages infected with B. cepacia-expressing m-RFP for 2 h. BECN1 stained green, and nuclei were stained with DAPI. The white arrows indicate B. cepacia, whereas the arrowheads indicate BECN1 aggregates. B, the percentage of colocalization of B. cepacia with BECN1 was scored by examining more than 400 cells. C, confocal microscopy for ⌬F508 macrophages nucleofected with siRNA against p62 (Si-p62) or scrambled siRNA control (si-CT) 48 h prior to infection. Nucleofected macrophages were infected with B. cepacia-expressing m-RFP for 2 h. BECN1 stained green, whereas nuclei were stained with DAPI. The arrows indicate B. cepacia, whereas the arrowheads indicate BECN1 aggregates. D, the percentage of colocalization of B. cepacia with BECN1 at the assigned time point was scored. More than 400 bacteria were scored. E, immunoblot for ⌬F508 macrophages nucleofected with siRNA against p62 or control siRNA for 48 h. Antibodies against p62 and BECN1 were used. F, ⌬F508 macrophages were nucleofected with siRNA against p62 or control siRNA or siRNA against p62 and Atg7 together (si-p62ϩsi-Atg7) for 48 h and then infected with B. cepacia for 2, 4, and 6 h. CFUs were enumerated. G, immunoblot analysis for ⌬F508 macrophages nucleofected with control siRNA, siRNA against p62, siRNA against Atg7 (si-Atg7), or siRNA against p62 and Atg7 together for 48 h. Antibodies specific to p62 And Atg7 were used to detect the down-regulation. Data in B, D, and F are presented as means Ϯ S.D. of three different experiments. B and D, **, p Ͻ 0.01; ***, p Ͻ 0.001; significant differences between both types of macrophages at the designated time point. because of reduced recycling in ⌬F508 macrophages as a consequence of compromised autophagosome formation and maturation. Alternatively, the accumulation of p62 could stimulate the formation of more ⌬F508 CFTR aggregates. This latter possibility agrees with the observation that depletion of p62 from ⌬F508 macrophages improves autophagy and decreases the BECN1-positive aggregates. Also, a previous study using CF epithelial cells showed that p62 promotes aggresome accumulation of misfolded or modified proteins (43,45). Recently, it has reported that reducing the levels of p62 can rescue ⌬F508-CFTR trafficking to the plasma membrane of CF airway epithelial cells (1,2,46).
The presence of intracellular bacteria such as B. cepacia increases the level of p62 expression in both WT and ⌬F508 macrophages. It is possible that p62 overexpression upon infection worsens the biology of ⌬F508 macrophages, providing an explanation for the deterioration of lung function and innate immune responses in the infected CF lung. There are several mechanisms by which B. cepacia may lead to the accumulation of p62. It is plausible that B. cepacia increases p62 accumulation by inhibiting autophagy in ⌬F508 macrophages, as we have published previously (37,38). Notably, B. cepacia infection increases p62 mRNA. Regardless of the mechanism of p62 accumulation, the p62 aggregates sequester essential autophagy molecules such as BECN1, making them unavailable for efficient autophagosome formation (48).
The sequestration of BECN1 occurs via transglutaminase 2 (TG2)-mediated cross-linking in aggresomes because the BECN1 protein sequence contains QP and QXXP motifs, which are specific target sites for TG2 activity (48), and TG2 is an autophagy inhibitor in pancreatic adenocarcinoma cells (49). Increased reactive oxygen species in CF epithelia sustain high TG2 levels through TG2 SUMOylation (48). Thus, BECN1, and not all autophagy molecules, is specifically recruited to aggresomes in CF cells.
Examining the sequential acquisition of autophagy molecules by the B. cepacia vacuole revealed that although ubiquitination is efficient in both WT and ⌬F508 macrophages, BECN1 acquisition is defective only in ⌬F508 macrophages. BECN1, also known as autophagy-related gene product 6 (Atg6), and its binding partner class III PI3K (also named Vps34) are required for the initiation of the autophagosome formation (47). Thus, supplementation of p62 alone to ⌬F508 macrophages will not improve the targeting of B. cepacia to autophagosomes. This conclusion is supported by the overexpression experiment of p62 in ⌬F508 macrophages, which actually leads to more bacterial growth. Therefore, to correct the trafficking defect of B. cepacia in ⌬F508 macrophages, "free" BECN1 is required, which is achieved by depletion of p62.
p62 targets several pathogens, such as S. typhimurium, Shigella, and Listeria to the autophagosome (9,10). Similarly, p62 associates with the B. cepacia vacuole in WT macrophages. However, depletion of p62 from ⌬F508 macrophages promotes B. cepacia uptake by autophagosomes and decreases the bacterial burden. It is possible that another adaptor molecule, such as NBR1, compensates for the loss of p62. The structure of NBR1 resembles that of p62. It can bind both LC3 and ubiquitinated proteins through the LC3 interaction region and ubiquitin-as-sociated domain, respectively (11,14). NDP52 is another cargo marker that drives certain bacteria to the autophagy machinery (9,41). In this study, we found that NDP52 facilitates autophagy uptake of B. cepacia in ⌬F508 macrophages but not in WT cells. NBR1, however, appears to contribute to the delivery of B. cepacia to the autophagy machinery in both WT and ⌬F508 macrophages. To our knowledge, this is the first demonstration of a role for NBR1 in bacterial targeting by autophagy.
We showed previously (37) that autophagy stimulation by rapamycin can overcome the down-regulating effect of B. cepacia on the ATG genes and can control the B. cepacia infection in the ⌬F508 mouse model both in vivo and in vitro. In this work, we demonstrate that p62 depletion from ⌬F508 mouse macrophages is another approach to improve autophagic control on B. cepacia infection.
Together, these data provide a molecular framework to better understand the emerging complexity of diseases related to autophagic defect such as CF and the ability of macrophages to defend against the bacterial infection. This study also indentifies p62 as a promising drug target for improving B. cepacia clearance in CF macrophages.