PMID- 36409075 OWN - NLM STAT- MEDLINE DCOM- 20230306 LR - 20231128 IS - 2150-7511 (Electronic) VI - 13 IP - 6 DP - 2022 Dec 20 TI - The Obligate Intracellular Bacterial Pathogen Anaplasma phagocytophilum Exploits Host Cell Multivesicular Body Biogenesis for Proliferation and Dissemination. PG - e0296122 LID - 10.1128/mbio.02961-22 [doi] LID - e02961-22 AB - Anaplasma phagocytophilum is the etiologic agent of the emerging infection, granulocytic anaplasmosis. This obligate intracellular bacterium lives in a host cell-derived vacuole that receives membrane traffic from multiple organelles to fuel its proliferation and from which it must ultimately exit to disseminate infection. Understanding of these essential pathogenic mechanisms has remained poor. Multivesicular bodies (MVBs) are late endosomal compartments that receive biomolecules from other organelles and encapsulate them into intralumenal vesicles (ILVs) using endosomal sorting complexes required for transport (ESCRT) machinery and ESCRT-independent machinery. Association of the ESCRT-independent protein, ALIX, directs MVBs to the plasma membrane where they release ILVs as exosomes. We report that the A. phagocytophilum vacuole (ApV) is acidified and enriched in lysobisphosphatidic acid, a lipid that is abundant in MVBs. ESCRT-0 and ESCRT-III components along with ALIX localize to the ApV membrane. siRNA-mediated inactivation of ESCRT-0 and ALIX together impairs A. phagocytophilum proliferation and infectious progeny production. RNA silencing of ESCRT-III, which regulates ILV scission, pronouncedly reduces ILV formation in ApVs and halts infection by arresting bacterial growth. Rab27a and its effector Munc13-4, which drive MVB trafficking to the plasma membrane and subsequent exosome release, localize to the ApV. Treatment with Nexinhib20, a small molecule inhibitor that specifically targets Rab27a to block MVB exocytosis, abrogates A. phagocytophilum infectious progeny release. Thus, A. phagocytophilum exploits MVB biogenesis and exosome release to benefit each major stage of its intracellular infection cycle: intravacuolar growth, conversion to the infectious form, and exit from the host cell. IMPORTANCE Anaplasma phagocytophilum causes granulocytic anaplasmosis, a globally emerging zoonosis that can be severe, even fatal, and for which antibiotic treatment options are limited. A. phagocytophilum lives in an endosomal-like compartment that interfaces with multiple organelles and from which it must ultimately exit to spread within the host. How the bacterium accomplishes these tasks is poorly understood. Multivesicular bodies (MVBs) are intermediates in the endolysosomal pathway that package biomolecular cargo from other organelles as intralumenal vesicles for release at the plasma membrane as exosomes. We discovered that A. phagocytophilum exploits MVB biogenesis and trafficking to benefit all aspects of its intracellular infection cycle: proliferation, conversion to its infectious form, and release of infectious progeny. The ability of a small molecule inhibitor of MVB exocytosis to impede A. phagocytophilum dissemination indicates the potential of this pathway as a novel host-directed therapeutic target for granulocytic anaplasmosis. FAU - Read, Curtis B AU - Read CB AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Lind, Mary Clark H AU - Lind MCH AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Chiarelli, Travis J AU - Chiarelli TJ AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Izac, Jerilyn R AU - Izac JR AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Adcox, Haley E AU - Adcox HE AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Marconi, Richard T AU - Marconi RT AUID- ORCID: 0000-0002-6035-7306 AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. FAU - Carlyon, Jason A AU - Carlyon JA AUID- ORCID: 0000-0003-2778-5066 AD - Department of Microbiology and Immunology, Virginia Commonwealth University Medical Center, School of Medicine, Richmond, Virginia, USA. LA - eng GR - R01 AI072683/AI/NIAID NIH HHS/United States GR - R01 AI139072/AI/NIAID NIH HHS/United States GR - R37 AI072683/AI/NIAID NIH HHS/United States PT - Journal Article DEP - 20221121 PL - United States TA - mBio JT - mBio JID - 101519231 RN - 0 (Endosomal Sorting Complexes Required for Transport) SB - IM MH - Animals MH - *Anaplasma phagocytophilum/pathogenicity/physiology MH - *Anaplasmosis/metabolism/microbiology MH - *Cell Proliferation MH - Endosomal Sorting Complexes Required for Transport/metabolism MH - *Multivesicular Bodies/metabolism MH - Protein Transport MH - *Organelle Biogenesis PMC - PMC9765717 OTO - NOTNLM OT - Anaplasma phagocytophilum OT - ESCRT OT - Munc13-4 OT - Rab27a OT - exocytosis OT - exosome OT - intralumenal vesicle OT - multivesicular body OT - multivesicular endosome OT - nutritional virulence OT - obligate intracellular bacterium OT - rickettsia OT - vacuolar pathogen COIS- The authors declare no conflict of interest. EDAT- 2022/11/22 06:00 MHDA- 2023/03/03 06:00 PMCR- 2022/11/21 CRDT- 2022/11/21 09:03 PHST- 2022/11/21 09:03 [entrez] PHST- 2022/11/22 06:00 [pubmed] PHST- 2023/03/03 06:00 [medline] PHST- 2022/11/21 00:00 [pmc-release] AID - 02961-22 [pii] AID - mbio.02961-22 [pii] AID - 10.1128/mbio.02961-22 [doi] PST - ppublish SO - mBio. 2022 Dec 20;13(6):e0296122. doi: 10.1128/mbio.02961-22. Epub 2022 Nov 21.