Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages

Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.     

The fungal pathogen Cryptococcus neoformans manipulates macrophage phagosome maturation

Phagocytosis by cells of the innate immune system, such as macrophages, and the subsequent successful maturation of the phagosome, is key for the clearance of pathogens. The fungal pathogen Cryptococcus neoformans is known to overcome killing by host phagocytes and both replicate within these cells and also escape via a non‐lytic process termed vomocytosis. Here we demonstrate that, during intracellular growth, cryptococci modify phagolysosome maturation. Live cryptococci, but not heat‐killed pathogens or inert targets, induce the premature removal of the early phagosome markers Rab5 and Rab11. In addition, significant acidification of the phagosome, calcium flux and protease activity is hindered, thus rendering the phagosome permissive for cryptococcal proliferation. Interestingly, several attenuated cryptococcal mutants retain this ability to subvert phagosomal maturation, suggesting that hitherto unidentified pathogen mechanisms regulate this process.     

Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching

During phagosome maturation, the late endosomal marker Rab7 and the lysosomal marker LAMP1 localize to the phagosomes. We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis. Rab7 was mobile between the phagosomal membrane and the cytosol in macrophages that ingested latex beads during phagosome maturation. The addition of interferon-γ (IFN-γ) restricted this mobility, suggesting that Rab7 is forced to bind to the phagosomal membrane by IFN-γ-mediated activation. Immobilization of LAMP1 on the phagosomes was observed irrespective of IFN-γ-activation. We further examined the mobility of Rab7 on the phagosomes containing Mycobacterium bovis BCG by FRAP analysis. The rate of fluorescence recovery for Rab7 on mycobacterial phagosomes was lower than that on the phagosomes containing latex beads, suggesting that mycobacteria impaired the mobility of Rab7 and arrested phagosome maturation.     

TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria

Tumour progression locus 2 (TPL‐2) kinase mediates Toll‐like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase‐independent regulatory function for TPL‐2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL‐2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL‐2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V‐ATPase proton pump subunits. Furthermore, TPL‐2 stimulated the phosphorylation of DMXL1, a regulator of V‐ATPases, to induce V‐ATPase assembly and phagosome acidification. Consistent with these results, TPL‐2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL‐2 therefore controls innate immune responses of macrophages to bacteria via V‐ATPase induction of phagosome maturation.     

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii‐containing vacuole

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal‐like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii‐containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP‐1 and LAMP‐2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP‐1/2 double‐deficient fibroblasts as well as in LAMP‐1/2 knock‐down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double‐deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP‐1 or LAMP‐2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead‐containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.     

Effective generation of reactive oxygen species in the mycobacterial phagosome requires K+ efflux from the bacterium

Efficient killing of mycobacteria by host macrophages depends on a number of mechanisms including production of reactive oxygen species (ROS) by the phagosomal NADPH oxidase, NOX2. Survival of pathogenic mycobacteria in the phagosome relies on the ability to control maturation of the phagosome such that it is biologically and chemically altered in comparison to phagosomes containing non‐pathogenic bacteria. In this study we show that the action of NOX2 to produce ROS in the mycobacterial phagosome is paradoxically dependent on a bacterial potassium transporter. We show that a Mycobacterium bovis BCG mutant (BCGΔkef), deficient in a Kef‐type K+ transporter, exhibits an increased intracellular survival phenotype in resting and activated macrophages, yet retains the ability to inhibit phagosome acidification, and does not show increased resistance to acidic conditions or ROS. Addition of a ROS scavenger replicates this phenotype in macrophages infected with wild‐type BCG, and the production of ROS by macrophages infected with BCGΔkef is substantially decreased compared with those infected with wild‐type BCG. Our results suggest that increased intracellular survival of BCGΔkef is mediated by inducing a decreased macrophage oxidative burst, and are consistent with Kef acting to alter the ionic contents of the phagosome and promoting NOX2 production of ROS.     

Spermine oxidase is a regulator of macrophage host response to Helicobacter pylori: enhancement of antimicrobial nitric oxide generation by depletion of spermine

The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have reported that in H. pylori-activated macrophages, nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill the bacterium, iNOS protein expression is dependent on uptake of its substrate l-arginine (l-Arg), the polyamine spermine can inhibit iNOS translation by inhibiting l-Arg uptake, and inhibition of polyamine synthesis enhances NO-mediated bacterial killing. Because spermine oxidase (SMO), which back-converts spermine to spermidine, is induced in macrophages by H. pylori, we determined its role in iNOS-dependent host defense. SMO shRNA knockdown in RAW 264.7 murine macrophages resulted in a marked decrease in H. pylori-stimulated iNOS protein, but not mRNA expression, and a 90 % reduction in NO levels; NO production was also inhibited in primary murine peritoneal macrophages with SMO knockdown. There was an increase in spermine levels after H. pylori stimulation that rapidly decreased, while SMO knockdown caused a greater increase in spermine that was sustained. With SMO knockdown, l-Arg uptake and killing of H. pylori by macrophages was prevented. The overexpression of SMO by transfection of an expression plasmid prevented the H. pylori-stimulated increase in spermine levels, and led to increased l-Arg uptake, iNOS protein expression and NO production, and H. pylori killing. In two human monocytic cell lines, U937 and THP-1, overexpression of SMO caused a significant enhancement of NO production with H. pylori stimulation. By depleting spermine, SMO can abrogate the inhibitory effect of polyamines on innate immune responses to H. pylori by enhancing antimicrobial NO production.     

Analysis of peripheral blood lymphocyte phenotypes and Th1/Th2 cytokines profile in the systemic immune responses of Helicobacter pylori infected individuals

H. pylori elicits specific humoral and cellular immune responses in the mucosal immune system. However, the type and extent of T lymphocyte response in the systemic immune system is not clear for H. pylori positive patients. In this study, peripheral blood T lymphocyte phenotypes and serum Th1/Th2 based cytokines of 32 H. pylori positive patients were analyzed and compared to those of healthy controls. While αβ TCR⁺ lymphocytes and their phenotype analysis were not significantly different to those of healthy controls, the percentage of pan γδ TCR⁺ lymphocytes was up to 2.4 times greater in the H. pylori positive group then in healthy controls. Furthermore, significant increases in IL‐10 concentrations in serum samples of H. pylori patients indicated that their immune systems had switched toward a Th2 type immune response. The correlation between phenotype and type of T cell response in the peripheral blood during H. pylori infection is discussed.     

PIKfyve Inhibition Interferes with Phagosome and Endosome Maturation in Macrophages

Macrophages eliminate pathogens and cell debris through phagocytosis, a process by which particulate matter is engulfed and sequestered into a phagosome. Nascent phagosomes are innocuous organelles resembling the plasma membrane. However, through a maturation process, phagosomes are quickly remodeled by fusion with endosomes and lysosomes to form the phagolysosome. Phagolysosomes are highly acidic and degradative leading to particle decomposition. Phagosome maturation is intimately dependent on the endosomal pathway, during which diverse cargoes are sorted for recycling to the plasma membrane or for degradation in lysosomes. Not surprisingly, various regulators of the endosomal pathway are also required for phagosome maturation, including phosphatidylinositol‐3‐phosphate, an early endosomal regulator. However, phosphatidylinositol‐3‐phosphate can be modified by the lipid kinase PIKfyve into phosphatidylinositol‐3,5‐bisphosphate, which controls late endosome/lysosome functions. The role of phosphatidylinositol‐3,5‐bisphosphate in macrophages and phagosome maturation remains basically unexplored. Using Fcγ receptor‐mediated phagocytosis as a model, we describe our research showing that inhibition of PIKfyve hindered certain steps of phagosome maturation. In particular, PIKfyve antagonists delayed removal of phosphatidylinositol‐3‐phosphate and reduced acquisition of LAMP1 and cathepsin D, both common lysosomal proteins. Consistent with this, the degradative capacity of phagosomes was reduced but phagosomes appeared to still acidify. We also showed that trafficking to lysosomes and their degradative capacity was reduced by PIKfyve inhibition. Overall, we provide evidence that PIKfyve, likely through phosphatidylinositol‐3,5‐bisphosphate synthesis, plays a significant role in endolysosomal and phagosome maturation in macrophages.      

Lipoprotein access to MHC class I presentation during infection of murine macrophages with live mycobacteria

Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8(+) T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.     

Expression of T-cell immunoglobulin- and mucin-domain-containing molecules-1 and -3 (Tim-1 and Tim-3) in Helicobacter pylori infection

Background: Th immune response plays an important role in Helicobacter pylori (H. pylori) infection. Tim‐1 and Tim‐3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim in H. pylori infection remains unclear. Materials and Methods: (1) Lymphocytes isolated from the spleen of BALB/c mice were co‐cultured with different concentrations of viable H. pylori. Alternatively, mice were challenged by viable H. pylori to set up the H. pylori infection model. (2) The expression of Tim‐1 and Tim‐3 on mRNA level in lymphocytes or spleen of mice was determined by RT‐PCR. The percentage of Tim‐3‐positive cells was determined by flow cytometric analysis. The production of cytokine in supernatants was measured by standard sandwich cytokine ELISA. Results: (1) Co‐culture: At 12 hours, there was markedly decreased production of Tim‐1 and increased production of Tim‐3 in lymphocytes co‐cultured with H. pylori compared with normal control. The change of Th2 cytokine had the similar tendency as that of Tim‐1 expression; alternatively, the change of Th1 cytokine had the similar tendency as that of Tim‐3 expression. (2) Infection: Tim‐1 expression was declined in infected mice compared with control group; in the contrast, Tim‐3 expression was increased. Furthermore, the expression of Tim‐1 and Tim‐3 mRNA in spleen was significantly positively correlated with the level of Th2 and Th1 cytokine in gastric homogenized supernatant, respectively. Conclusion: H. pylori could inhibit the differentiation of T lymphocytes toward Th2 cells, promote the Th1 cell differentiation, and induce Th1‐biased immune response. The expression of Tim‐1 and Tim‐3 could reflect Th2 and Th1 immune response, respectively, which provide evidence for the prevention and treatment of H. pylori infection and correlation diseases through regulation of Tim‐1 and Tim‐3.     

Yersinia pestis Requires Host Rab1b for Survival in Macrophages

Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV) and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.     

Proteomics approach to understand reduced clearance of mycobacteria and high viral titers during HIV–mycobacteria co‐infection

Environmental mycobacteria, highly prevalent in natural and artificial (including chlorinated municipal water) niches, are emerging as new threat to human health, especially to HIV‐infected population. These seemingly harmless non‐pathogenic mycobacteria, which are otherwise cleared, establish as opportunistic infections adding to HIV‐associated complications. Although immune‐evading strategies of pathogenic mycobacteria are known, the mechanisms underlying the early events by which opportunistic mycobacteria establish infection in macrophages and influencing HIV infection are unclear. Proteomics of phagosome‐enriched fractions from Mycobacterium bovis Bacillus Calmette–Guérin (BCG) mono‐infected and HIV–M. bovis BCG co‐infected THP‐1 cells by LC‐MALDI‐MS/MS revealed differential distribution of 260 proteins. Validation of the proteomics data showed that HIV co‐infection helped the survival of non‐pathogenic mycobacteria by obstructing phagosome maturation, promoting lipid biogenesis and increasing intracellular ATP equivalents. In turn, mycobacterial co‐infection up‐regulated purinergic receptors in macrophages that are known to support HIV entry, explaining increased viral titers during co‐infection. The mutualism was reconfirmed using clinically relevant opportunistic mycobacteria, Mycobacterium avium, Mycobacterium kansasii and Mycobacterium phlei that exhibited increased survival during co‐infection, together with increase in HIV titers. Additionally, the catalogued proteins in the study provide new leads that will significantly add to the understanding of the biology of opportunistic mycobacteria and HIV coalition.     

Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen

Cryptococcus is a potentially fatal fungal pathogen and a leading cause of death in immunocompromised patients. As an opportunistic and facultative intracellular pathogen of humans, Cryptococcus exhibits a complex set of interactions with the host immune system in general, and macrophages in particular. Cryptococcus is resistant to phagocytosis but is also able to survive and proliferate within the mature phagolysosome. It can cause the lysis of host cells, can be transferred between macrophages or exit non‐lytically via vomocytosis. Efficient phagocytosis is reliant on opsonization and Cryptococcus has a number of anti‐phagocytic strategies including formation of titan cells and a thick polysaccharide capsule. Following uptake, phagosome maturation appears to occur normally, but the internalized pathogen is able to survive and replicate. Here we review the interactions and host manipulation processes that occur within cryptococcal‐infected macrophages and highlight areas for future research.     

Helicobacter pylori-pulsed dendritic cells induce H. pylori-specific immunity in mice

Background: The growing concern over the emergence of antibiotic‐resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)‐based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow‐derived DCs pulsed with phosphate‐buffered saline (PBS‐DC) or live H. pylori SS1 (HP‐DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP‐DC or PBS‐DC immunized mice. The effect of regulatory T‐cell (Treg) depletion by anti‐CD25 antibody on HP‐DC vaccine efficacy was also evaluated. Results: HP‐DC induced a Th1‐dominant response in vitro. In vivo, HP‐DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP‐DC immunized mice expressed a higher level of IFN‐γ compared to PBS‐DC immunized mice; no difference was found for interleukin‐5 expressions in the stomach. A lower bacterial colonization post‐H. pylori challenge was observed in HP‐DC immunized mice compared to PBS‐DC immunized mice with no significant difference in gastritis severity. H. pylori‐specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti‐CD25 antibody. Conclusion: DC‐based anti‐H. pylori vaccine induced H. pylori‐specific helper T‐cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.     

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.     

Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection

As part of their strategy for intracellular survival, mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages. The molecular basis for this inhibition is only now beginning to emerge, by way of the molecular characterisation of the phagosome membrane when it encloses virulent mycobacteria. Our own work has shown that at 15 days after the phagocytic uptake of Mycobacterium avium by mouse bone marrow-derived macrophages, the phagosome membrane is depleted about 4-fold for cell surface-derived membrane glycoconjugates, labelled by exogalactosylation, in comparison to the membrane of early endosomes with which it continues to interact. Here we asked whether this depletion occurred at early or late stages after infection. We found that only about half of the depletion had occurred at about 5 hours after the beginning of phagocytic uptake, with the remainder becoming established thereafter, with a half-time of about 2.5 days. Phagosomes became depleted in relation to early endosomes with which they continued to exchange membrane constituents. Early endosomes themselves became gradually depleted by about 30% during the 15-day post-infection period. In contrast, late endosomes/lysosomes remained unchanged, with a concentration of surface-derived glycoconjugates between that of early endosomes and of phagosomes at day 15 post infection. In view of the slowness of the post-infection change of phagosome membrane composition, we proposed that this change did not play a role in preventing maturation immediately after phagosome formation, but rather correlated with the process of maintaining the phagosomes in an immature state.     

The innate immune molecule,NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides

The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI⁺H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1‐dependent regulation of human β‐defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI⁺ bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type‐specific induction of DEFB4 and DEFB103, we generated stable NOD1‘knockdown’ (KD) and control AGS cells. Reporter gene assay and RT‐PCR analyses revealed that only DEFB4 was induced in an NOD1‐/cagPAI‐dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI⁺H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human β‐defensin 2 (hBD‐2), but not hBD‐3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD‐2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI⁺H. pylori infection.