scholarly journals Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Pedro Escoll ◽  
Lucien Platon ◽  
Mariatou Dramé ◽  
Tobias Sahr ◽  
Silke Schmidt ◽  
...  

Legionella pneumophila, the causative agent of Legionnaires'; disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

2021 ◽  
Author(s):  
Pedro Escoll ◽  
Lucien Platon ◽  
Mariatou Drame ◽  
Tobias Sahr ◽  
Silke Schmidt ◽  
...  

Legionella pneumophila, the causative agent of Legionnaires disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the mitochondrial membrane potential, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the mitochondrial membrane potential, but not LncP and MitF. The inhibition of the L. pneumophila-induced reverse mode of the FOF1-ATPase collapsed the mitochondrial membrane potential and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the mitochondrial membrane potential and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.


2006 ◽  
Vol 203 (9) ◽  
pp. 2177-2189 ◽  
Author(s):  
Vicki P. Losick ◽  
Ralph R. Isberg

Legionella pneumophila, the causative agent of Legionnaires' disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low-dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of antiapoptotic genes positively controlled by the transcriptional regulator nuclear factor κB (NF-κB). Consistent with this finding, L. pneumophila triggered nuclear localization of NF-κB in human and mouse macrophages in a Dot/Icm-dependent manner. The mechanism of activation at low-dose infections involved a signaling pathway that occurred independently of the Toll-like receptor adaptor MyD88 and the cytoplasmic sensor Nod1. In contrast, high multiplicity of infection conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-κB. Inhibition of NF-κB translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one antiapoptotic protein, plasminogen activator inhibitor–2, host cell death increased in response to L. pneumophila infection, indicating that induction of antiapoptotic genes is critical for host cell survival.


2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Sekar Natesampillai ◽  
Nathan W. Cummins ◽  
Zilin Nie ◽  
Rahul Sampath ◽  
Jason V. Baker ◽  
...  

ABSTRACTHIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.IMPORTANCEThe Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.


2014 ◽  
Vol 82 (10) ◽  
pp. 4021-4033 ◽  
Author(s):  
Stephanie Dolinsky ◽  
Ina Haneburger ◽  
Adam Cichy ◽  
Mandy Hannemann ◽  
Aymelt Itzen ◽  
...  

ABSTRACTLegionellaspp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed theLegionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different “effector” proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays inLegionella longbeachaelysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)Pbinding of SidC] fragment. Isothermal titration calorimetry revealed that SidC ofL. longbeachae(SidCLlo) binds PtdIns(4)Pwith aKd(dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue ofLegionella pneumophila(SidCLpn). Upon infection of RAW 264.7 macrophages withL. longbeachae, endogenous SidCLloor ectopically produced SidCLpnlocalized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. AnL. longbeachaeΔsidCdeletion mutant was impaired for calnexin recruitment to LCVs inDictyostelium discoideumamoebae and outcompeted by wild-type bacteria inAcanthamoeba castellanii. Calnexin recruitment was restored by SidCLloor its orthologues SidCLpnand SdcALpn. Conversely, calnexin recruitment was restored by SidCLloinL. pneumophilalackingsidCandsdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlois anL. longbeachaeeffector that binds through a P4C domain with high affinity to PtdIns(4)Pon LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.


2018 ◽  
Author(s):  
Tatiana M. Clemente ◽  
Minal Mulye ◽  
Anna V. Justis ◽  
Srinivas Nallandhighal ◽  
Tuan M. Tran ◽  
...  

AbstractCoxiella burnetiiis an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires theCoxiellaType IVB Secretion System (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets ofCoxiellaT4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with aCoxiellaT4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild type (WT) bacteria, suggestingCoxiellaT4BSS effector proteins downregulate expression of these genes. In addition, the IL-17 signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 duringCoxiellainfection is unknown. We found that IL-17 kills intracellularCoxiellain a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT or mock-infected cells, including the pro-inflammatory cytokinesI11a, Il1bandTnfa, the chemokinesCxcl2andCcl5, and the antimicrobial proteinLcn2. We further confirmed that theCoxiellaT4BSS downregulates macrophage CXCL2/MIP-2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest thatCoxielladownregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.


mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charles L. Larson ◽  
Kelsi M. Sandoz ◽  
Diane C. Cockrell ◽  
Robert A. Heinzen

ABSTRACTThe Q fever agentCoxiella burnetiiis a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify theCoxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report thatC. burnetiiinhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However,C. burnetiidid not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 byC. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulatesC. burnetiiintracellular growth.IMPORTANCECoxiella burnetiiis an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of theCoxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report thatC. burnetiigrowth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefitC. burnetiigrowth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate thatC. burnetiiinhibition of mTORC1 without accelerated autophagy promotes bacterial growth.


2019 ◽  
Vol 116 (8) ◽  
pp. 3221-3228 ◽  
Author(s):  
Asaf Sol ◽  
Erion Lipo ◽  
Dennise A. de Jesús-Díaz ◽  
Connor Murphy ◽  
Mildred Devereux ◽  
...  

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role this interference plays in promoting diseases is unclear. Previously, we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, whereas S phase provides a toxic environment for bacterial replication. In this study, we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that, early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila. Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.


2013 ◽  
Vol 81 (11) ◽  
pp. 4261-4270 ◽  
Author(s):  
Clare R. Harding ◽  
Corinna Mattheis ◽  
Aurélie Mousnier ◽  
Clare V. Oates ◽  
Elizabeth L. Hartland ◽  
...  

ABSTRACTThe Dot/Icm type IV secretion system (T4SS) ofLegionella pneumophilais crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of theLegionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P]in vitroand colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae ofGalleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-bindingL. pneumophilaeffector that has a role in intracellular bacterial replication.


mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Giulia Oliva ◽  
Tobias Sahr ◽  
Monica Rolando ◽  
Maike Knoth ◽  
Carmen Buchrieser

ABSTRACT Legionella pneumophila is an environmental bacterium that parasitizes protozoa, but it may also infect humans, thereby causing a severe pneumonia called Legionnaires’ disease. To cycle between the environment and a eukaryotic host, L. pneumophila is regulating the expression of virulence factors in a life cycle-dependent manner: replicating bacteria do not express virulence factors, whereas transmissive bacteria are highly motile and infective. Here we show that Hfq is an important regulator in this network. Hfq is highly expressed in transmissive bacteria but is expressed at very low levels in replicating bacteria. A L. pneumophila hfq deletion mutant exhibits reduced abilities to infect and multiply in Acanthamoeba castellanii at environmental temperatures. The life cycle-dependent regulation of Hfq expression depends on a unique cis -encoded small RNA named Anti-hfq that is transcribed antisense of the hfq transcript and overlaps its 5′ untranslated region. The Anti-hfq sRNA is highly expressed only in replicating L. pneumophila where it regulates hfq expression through binding to the complementary regions of the hfq transcripts. This results in reduced Hfq protein levels in exponentially growing cells. Both the small noncoding RNA (sRNA) and hfq mRNA are bound and stabilized by the Hfq protein, likely leading to the cleavage of the RNA duplex by the endoribonuclease RNase III. In contrast, after the switch to transmissive bacteria, the sRNA is not expressed, allowing now an efficient expression of the hfq gene and consequently Hfq. Our results place Hfq and its newly identified sRNA anti- hfq in the center of the regulatory network governing L. pneumophila differentiation from nonvirulent to virulent bacteria. IMPORTANCE The abilities of L. pneumophila to replicate intracellularly and to cause disease depend on its capacity to adapt to different extra- and intracellular environmental conditions. Therefore, a timely and fine-tuned expression of virulence factors and adaptation traits is crucial. Yet, the regulatory circuits governing the life cycle of L. pneumophila from replicating to virulent bacteria are only partly uncovered. Here we show that the life cycle-dependent regulation of the RNA chaperone Hfq relies on a small regulatory RNA encoded antisense to the hfq -encoding gene through a base pairing mechanism. Furthermore, Hfq regulates its own expression in an autoregulatory loop. The discovery of this RNA regulatory mechanism in L. pneumophila is an important step forward in the understanding of how the switch from inoffensive, replicating to highly virulent, transmissive L. pneumophila is regulated.


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