Pseudomonas aeruginosa biofilm-associated homoserine lactone C12 rapidly activates apoptosis in airway epithelia

Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum-sensing molecules including N-(3-oxododecanoyl)-l-homoserine lactone (C12). C12 (10-100 μM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ(mito)) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca(2+) and ER-targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1-2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu-3 (wtCFTR, bronchial gland) cells, showing that C12-triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ(mito) and reduction of redox potential in the ER) in JME and CFTR-corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12-triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ(mito) and increases in Ca(cyto) like 10-50 μM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.     

Modeling the effect of acylated homoserine lactone antagonists in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious illnesses, particularly in immunocompromised individuals, often with a fatal outcome. The finding that the acylated homoserine lactone quorum sensing (QS) system controls the production of virulence factors in P. aeruginosa makes this system a possible target for antimicrobial therapy. It has been suggested that an N-(3-oxododecanoyl)-homoserine lactone (3O-C12-HSL) antagonist, a QS blocker (QSB), would interfere efficiently with the quorum sensing system in P. aeruginosa and thus reduce the virulence of this pathogen. In this work, a mathematical model of the QS system in P. aeruginosa has been developed. The model was used to virtually add 3O-C12-HSL antagonists that differed in their affinity for the receptor protein and for their ability to mediate degradation of the receptor. The model suggests that very small differences in these parameters for different 3O-C12-HSL antagonists can greatly affect the success of QSB based inhibition of the QS system in P. aeruginosa. Most importantly, it is proposed that the ability of the 3O-C12-HSL antagonist to mediate degradation of LasR is the core parameter for successful QSB based inhibition of the QS system in P. aeruginosa. Finally, this study demonstrates that QSBs can shift the system to a low steady state, corresponding to an uninduced state and thus, suggests that the use of 3O-C12-HSL antagonists may constitute a promising therapeutic approach against P. aeruginosa involved infections.     

N‐(3‐Oxo‐acyl)‐homoserine lactone induces apoptosis primarily through a mitochondrial pathway in fibroblasts

N‐(3‐Oxododecanoyl)‐l ‐homoserine lactone (C12) is produced by Pseudomonas aeruginosa to function as a quorum‐sensing molecule for bacteria–bacteria communication. C12 is also known to influence many aspects of human host cell physiology, including induction of cell death. However, the signalling pathway(s) leading to C12‐triggered cell death is (are) still not completely known. To clarify cell death signalling induced by C12, we examined mouse embryonic fibroblasts deficient in “initiator” caspases or “effector” caspases. Our data indicate that C12 selectively induces the mitochondria‐dependent intrinsic apoptotic pathway by quickly triggering mitochondrial outer membrane permeabilisation. Importantly, the activities of C12 to permeabilise mitochondria are independent of activation of both “initiator” and “effector” caspases. Furthermore, C12 directly induces mitochondrial outer membrane permeabilisation in vitro. Overall, our study suggests a mitochondrial apoptotic signalling pathway triggered by C12, in which C12 or its metabolite(s) acts on mitochondria to permeabilise mitochondria, leading to activation of apoptosis.     

Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone

Abstract A range of Pseudomonas spp. and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor l - N -(3-oxohexanoyl)homoserine lactone. In one of these, P. aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner. The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 μg/ml of the autoregulator. In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.     

N-Acylated homoserine lactone production and involvement in the biodegradation of aromatics by an environmental isolate of Pseudomonas aeruginosa

N-Acyl homoserine lactone (AHL) is a widespread quorum sensing signal molecule in Gram-negative bacteria and has an important role in many biological processes. However, it is still poorly understood whether or not AHL is present in pollutant treatment processes and further, what its role is in biodegradation processes. In this work, an environmental isolate of Pseudomonas aeruginosa CGMCC 1.860 that is an aromatic degrader and AHL producer was selected. The AHL plate bioassay indicated that AHL was produced by this strain during biodegradation of aromatic compounds including phenol, benzoate, p-hydroxy-benzoate, salicylate, and naphthalene. The AHLs were identified as N-butyryl-l-homoserine lactone (BHL) and N-hexanoyl-l-homoserine lactone (HHL) by using thin layer chromatography (TLC) and high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (HPLC–APCI-MS/MS) analyses. Furthermore, phenol biodegradation was improved by exogenously added AHL extracts or by endogenously over-produced AHLs, repressed by abolishment of AHLs production, and not affected by the addition of extracts without AHLs. The results indicated that AHL was involved in the process of biodegradation of pollutants.     

Role of the mitochondrial permeability transition and cytochrome C release in hydrogen peroxide-induced apoptosis

We investigated the role of the mitochondrial inner membrane permeability transition and subsequent release of cytochrome c into the cytosol during oxidative stress-evoked apoptosis. Sublethal oxidative stress was applied by treating L929 cells with 0.5 mM H2O2 for 90 min. Then the cellular localization of cytochrome c was examined by immunofluorescent staining and Western blotting. H2O2 treatment caused the permeability transition and pore formation, resulting in membrane depolarization and translocation of cytochrome c from the mitochondria into the cytosol. Pretreatment with cyclosporin A and aristolochic acid (to inhibit pore formation) significantly attenuated a reduction of the mitochondrial membrane potential, as well as signs of apoptosis such as DNA fragmentation, increased plasma membrane permeability, and chromatin condensation. Therefore, exposure to H2O2 caused the opening of permeability transition pores in the inner mitochondrial membrane. An essential role of cytosolic cytochrome c in the execution of apoptosis was demonstrated by its direct microinjection into the cytosol, thus bypassing the need for cytochrome c release from the mitochondrial intermembrane space. Microinjection of cytochrome c caused caspase-dependent apoptosis.     

MEK kinase 1 induces mitochondrial permeability transition leading to apoptosis independent of cytochrome c release.

Induction of apoptosis often converges on the mitochondria to induce permeability transition and release of apoptotic proteins into the cytoplasm resulting in the biochemical and morphological alteration of apoptosis. Activation of a serine threonine kinase MEK kinase 1 (MEKK1) is involved in the induction of apoptosis. Expression of a kinase-inactive MEKK1 blocks genotoxin-induced apoptosis. Upon apoptotic stimulation, MEKK1 is cleaved into a 91-kDa kinase fragment that further induces an apoptotic response. Mutation of a consensus caspase 3 site in MEKK1 prevents its induction of apoptosis. The mechanism of MEKK1-induced apoptosis downstream of its cleavage, however, is unknown. Herein we demonstrate that full-length and cleaved MEKK1 leads to permeability transition in the mitochondria. This permeability transition occurs through opening of the permeability transition (PT) pore. Inhibiting PT pore opening and reactive oxygen species production effectively reduced MEKK1-induced apoptosis. Overexpression of MEKK1, however, failed to release cytochrome c from the mitochondria or activate caspase 9. Since Bcl2 regulates changes in mitochondria and blocks MEKK1-induced apoptosis, we determined that Bcl2 blocks MEKK1-induced apoptosis when targeted to the mitochondria. This occurs downstream of MEKK1 cleavage, since Bcl2 fails to block cleavage of MEKK1. In mouse embryonic fibroblast cells lacking caspase 3, the cleaved but not full-length MEKK1 induces apoptosis and permeability transition in the mitochondria. Overall, this suggests that cleaved MEKK1 leads to permeability transition contributing to MEKK1-induced apoptosis independent of cytochrome c release from the mitochondria.     

The evolutionary stability of cytochrome c-551 in Pseudomonas aeruginosa and Pseudomonas fluorescens biotype C

Cytochrome c-551 was prepared from nine different strains of Pseudomonas aeruginosa and six of Pseudomonas fluorescens biotype C, and their amino acid sequences were compared with the sequences previously determined for the cytochromes of type strains of each species. The standard of sequence examination was such that all single amino acid substitutions, delections or insertions ought to have been detected. Balanced double changes in sites in the same part of the sequence might have escaped detection. The standard of some of the quantitative amino acid analyses was not as high as would be required for the investigation of completely unknown sequences. Eight of the Ps. aeruginosa sequences could not be distinguished from the type sequence, whereas the ninth had a single amino acid substitution. The sequences from Ps. fluorescens biotype C were more varied, differing in from zero to four substitutions from the type sequence, with the most diverse sequences differing in seven positions. The results for Ps. aeruginosa are interpreted as evidence that neutral mutations are not responsible for much molecular evolution. The superficially paradoxical differences in the results for the two species are discussed.     

Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak-) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak-) was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.     

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation

The mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53(-/-) and bax(-/-) mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-x(L) expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53(+/+), but not in p53(-/-) MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.     

Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis

Bif-1, a member of the endophilin B protein family, interacts with Bax and promotes interleukin-3 withdrawal-induced Bax conformational change and apoptosis when overexpressed in FL5.12 cells. Here, we provide evidence that Bif-1 plays a regulatory role in apoptotic activation of not only Bax but also Bak and appears to be involved in suppression of tumorigenesis. Inhibition of endogenous Bif-1 expression in HeLa cells by RNA interference abrogated the conformational change of Bax and Bak, cytochrome c release, and caspase 3 activation induced by various intrinsic death signals. Similar results were obtained in Bif-1 knockout mouse embryonic fibroblasts. While Bif-1 did not directly interact with Bak, it heterodimerized with Bax on mitochondria in intact cells, and this interaction was enhanced by apoptosis induction and preceded the Bax conformational change. Moreover, suppression of Bif-1 expression was associated with an enhanced ability of HeLa cells to form colonies in soft agar and tumors in nude mice. Taken together, these findings support the notion that Bif-1 is an important component of the mitochondrial pathway for apoptosis as a novel Bax/Bak activator, and loss of this proapoptotic molecule may contribute to tumorigenesis.     

Microcin J25 triggers cytochrome c release through irreversible damage of mitochondrial proteins and lipids

We previously showed that the antimicrobial peptide microcin J25 induced the over-production of reactive oxygen species with the concomitant release of cytochrome c from rat heart mitochondria via the opening of the mitochondrial permeability transition pore. Here, we were able to demonstrate that indeed, as a consequence of the oxidative burst, MccJ25 induces carbonylation of mitochondrial proteins, which may explain the irreversible inhibition of complex III and the partial inhibition of superoxide dismutase and catalase. Moreover, the peptide raised the levels of oxidized membrane lipids, which triggers the release of cytochrome c. From in silico analysis, we hypothesize that microcin would elicit these effects through interaction with heme c1 at mitochondrial complex III. On the other hand, under an excess of l-arginine, MccJ25 caused nitric oxide overproduction with no oxidative damage and a marked inhibition in oxygen consumption. Therefore, a beneficial anti-oxidative activity could be favored by the addition of l-arginine. Conversely, MccJ25 pro-oxidative–apoptotic effect can be unleashed in either an arginine-free medium or by suppressing the nitric oxide synthase activity.     

Herpes simplex virus blocks apoptosis by precluding mitochondrial cytochrome c release independent of caspase activation in infected human epithelial cells

Expression of HSV-1 genes leads to the induction of apoptosis in human epithelial HEp-2 cells but the subsequent synthesis of infected cell protein prevents the process from killing the cells. Thus, viruses unable to produce appropriate prevention factors are apoptotic. We now report that the addition of either a pancaspase inhibitor or caspase-9-specific inhibitor prevented cells infected with an apoptotic HSV-1 virus from undergoing cell death. This result indicated that HSV-1-dependent apoptosis proceeds through the mitochondrial apoptotic pathway. However, the pancaspase inhibitor did not prevent the release of cytochrome c from mitochondria, implying that caspase activation is not required for this induction of cytochrome c release by HSV-1. The release of cytochrome c was first detected at 9 hpi while caspase-9, caspase-3 and PARP processing were detected at 12 hpi. Finally, Bax accumulated at mitochondria during apoptotic, but not wild type HSV-1 infection. Together, these findings indicate that HSV-1 blocks apoptosis by precluding mitochondrial cytochrome c release in a caspase-independent manner and suggest Bax as a target in infected human epithelial cells.     

Loss of iron triggers PINK1/Parkin‐independent mitophagy

In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation‐induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.     

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release

ATP depletion induced by hypoxia or mitochondrial inhibitors results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured rat proximal tubule cells. Translocated Bax undergoes further conformational changes to oligomerize into high molecular weight complexes (Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Venkatachalam, M. A., and Saikumar, P. (2001) J. Biol. Chem. 276, 18361-18374). Here we report that following Bax translocation in ATP-depleted rat proximal tubule cells, Bak, a proapoptotic molecule that normally resides in mitochondria, also reorganizes to form homo-oligomers. Oligomerization of both Bax and Bak occurred independently of Bid cleavage and/or translocation. Western blots of chemically cross-linked membrane extracts showed nonoverlapping "ladders" of Bax and Bak complexes in multiples of approximately 21 and approximately 23 kDa, respectively, consistent with molecular homogeneity within each ladder. This indicated that Bax and Bak complexes were homo-oligomeric. Nevertheless, each oligomer could be co-immunoprecipitated with the other, suggesting a degree of affinity between Bax and Bak that permitted co-precipitation but not cross-linking. Furthermore, dissociation of cross-linked complexes by SDS and renaturation prior to immunoprecipitation did not prevent reassociation of the two oligomeric species. Notably, expression of Bcl-2 prevented not only the oligomerization of Bax and Bak, but also the association between these two proteins in energy-deprived cells. Using Bax-deficient HCT116 and BMK cells, we show that there is stringent Bax requirement for Bak homo-oligomerization and for cytochrome c release during energy deprivation. Using Bak-deficient BMK cells we further show that Bak deficiency is associated with delayed kinetics of Bax translocation but does not affect either the oligomerization of translocated Bax or the leakage of cytochrome c. These results suggest a degree of functional cooperation between Bax and Bak in this form of cell injury, but also demonstrate an absolute requirement of Bax for mitochondrial permeabilization.     

Translocation of a Bak C-terminus mutant from cytosol to mitochondria to mediate cytochrome C release: implications for Bak and Bax apoptotic function

Background

One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear.

Methodology/Principal Findings

To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1.

Conclusions/Significance

Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax.     

ADP/ATP carrier is required for mitochondrial outer membrane permeabilization and cytochrome c release in yeast apoptosis

Adenine nucleotide translocator (ANT) is a mitochondrial inner membrane protein involved in the ADP/ATP exchange and is a component of the mitochondrial permeability transition pore (PTP). In mammalian apoptosis, the PTP can mediate mitochondrial outer membrane permeabilization (MOMP), which is suspected to be responsible for the release of apoptogenic factors, including cytochrome c. Although release of cytochrome c in yeast apoptosis has previously been reported, it is not known how it occurs. Herein we used yeast genetics to investigate whether depletion of proteins putatively involved in MOMP and cytochrome c release affects these processes in yeast. While deletion of POR1 (yeast voltage-dependent anion channel) enhances apoptosis triggered by acetic acid, H(2)O(2) and diamide, CPR3 (mitochondrial cyclophilin) deletion had no effect. Absence of ADP/ATP carrier (AAC) proteins, yeast orthologues of ANT, protects cells exposed to acetic acid and diamide but not to H(2)O(2). Expression of a mutated form of Aac2p (op1) exhibiting very low ADP/ATP translocase activity indicates that AAC's pro-death role does not require translocase activity. Absence of AAC proteins impairs MOMP and release of cytochrome c, which, together with other mitochondrial inner membrane proteins, is degraded. Our findings point to a crucial role of AAC in yeast apoptosis.     

Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.