PMID- 35130452 OWN - NLM STAT- MEDLINE DCOM- 20220420 LR - 20240109 IS - 1098-5522 (Electronic) IS - 0019-9567 (Print) IS - 0019-9567 (Linking) VI - 90 IP - 3 DP - 2022 Mar 17 TI - The Pseudomonas aeruginosa Type III Secretion System Exoenzyme Effector ExoU Induces Mitochondrial Damage in a Murine Bone Marrow-Derived Macrophage Infection Model. PG - e0047021 LID - 10.1128/IAI.00470-21 [doi] LID - e00470-21 AB - Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A(2) (PLA(2)) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1beta (IL-1beta), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A(2) enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1beta. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1beta were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1beta activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed. FAU - Hardy, Kierra S AU - Hardy KS AD - Department of Microbiology and Immunology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. AD - Center for Lung Biology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Tuckey, Amanda N AU - Tuckey AN AD - Department of Microbiology and Immunology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. AD - Center for Lung Biology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Housley, Nicole A AU - Housley NA AD - Department of Microbiology and Immunology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. AD - Center for Lung Biology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Andrews, Joel AU - Andrews J AD - Mitchell Cancer Institute, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Patel, Mita AU - Patel M AD - Department of Pharmcology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Al-Mehdi, Abu-Bakr AU - Al-Mehdi AB AD - Department of Pharmcology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Barrington, Robert A AU - Barrington RA AD - Department of Microbiology and Immunology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. AD - Center for Lung Biology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. FAU - Cassel, Suzanne L AU - Cassel SL AD - Women's Guild Lung Institute, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA. FAU - Sutterwala, Fayyaz S AU - Sutterwala FS AD - Women's Guild Lung Institute, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA. FAU - Audia, Jonathon P AU - Audia JP AUID- ORCID: 0000-0002-0022-4800 AD - Department of Microbiology and Immunology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. AD - Center for Lung Biology, University of South Alabamagrid.267153.4 College of Medicine, Mobile, Alabama, USA. LA - eng GR - R01 HL118334/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20220207 PL - United States TA - Infect Immun JT - Infection and immunity JID - 0246127 RN - 0 (Inflammasomes) RN - 0 (Type III Secretion Systems) RN - EC 3.1.- (Phospholipases) RN - EC 3.4.22.36 (Caspase 1) SB - IM MH - Animals MH - Caspase 1/metabolism MH - Inflammasomes/metabolism MH - Inflammation/metabolism MH - Macrophages/metabolism MH - Mice MH - Phospholipases/metabolism MH - *Pseudomonas Infections MH - *Pseudomonas aeruginosa/physiology MH - Type III Secretion Systems/metabolism PMC - PMC8929383 OTO - NOTNLM OT - ExoT OT - ExoU OT - NLRC4 inflammasome OT - Pseudomonas aeruginosa OT - caspase-1 OT - mitochondria OT - pneumonia OT - sepsis COIS- The authors declare no conflict of interest. EDAT- 2022/02/08 06:00 MHDA- 2022/04/21 06:00 PMCR- 2022/03/17 CRDT- 2022/02/07 20:04 PHST- 2022/02/08 06:00 [pubmed] PHST- 2022/04/21 06:00 [medline] PHST- 2022/02/07 20:04 [entrez] PHST- 2022/03/17 00:00 [pmc-release] AID - 00470-21 [pii] AID - iai.00470-21 [pii] AID - 10.1128/IAI.00470-21 [doi] PST - ppublish SO - Infect Immun. 2022 Mar 17;90(3):e0047021. doi: 10.1128/IAI.00470-21. Epub 2022 Feb 7.