Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3′(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.

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Faculty Opinions recommendation of Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001041.17564 ◽

2001 ◽

Author(s):

Ted Hackstadt

Keyword(s):

Phagosome Maturation ◽

Phosphatidylinositol 3 Kinase ◽

Maturation Arrest ◽

Phosphatidylinositol 3

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Faculty Opinions recommendation of Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001041.14506 ◽

2001 ◽

Author(s):

David Russell

Keyword(s):

Phagosome Maturation ◽

Phosphatidylinositol 3 Kinase ◽

Maturation Arrest ◽

Phosphatidylinositol 3

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Tuberculosis Toxin Blocking Phagosome Maturation Inhibits a Novel Ca2+/Calmodulin-PI3K hVPS34 Cascade

Journal of Experimental Medicine ◽

10.1084/jem.20030527 ◽

2003 ◽

Vol 198 (4) ◽

pp. 653-659 ◽

Cited By ~ 214

Author(s):

Isabelle Vergne ◽

Jennifer Chua ◽

Vojo Deretic

Keyword(s):

Mycobacterium Tuberculosis ◽

Membrane Trafficking ◽

Intracellular Signaling ◽

Antigen Processing ◽

Phagosome Maturation ◽

Maturation Arrest ◽

Phagosomal Maturation

The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.

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IRAP Endosomes Control Phagosomal Maturation in Dendritic Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.585713 ◽

2020 ◽

Vol 8 ◽

Author(s):

Mirjana Weimershaus ◽

François-Xavier Mauvais ◽

Irini Evnouchidou ◽

Myriam Lawand ◽

Loredana Saveanu ◽

...

Keyword(s):

Dendritic Cells ◽

Immune Surveillance ◽

Phagosome Maturation ◽

Cross Presentation ◽

Phagosomal Maturation ◽

Histocompatibility Complex ◽

Key Factor ◽

Intermediate Compartment ◽

Specialized Population

Dendritic cells (DCs) contribute to the immune surveillance by sampling their environment through phagocytosis and endocytosis. We have previously reported that, rapidly following uptake of extracellular antigen into phagosomes or endosomes in DCs, a specialized population of storage endosomes marked by Rab14 and insulin-regulated aminopeptidase (IRAP) is recruited to the nascent antigen-containing compartment, thereby regulating its maturation and ultimately antigen cross-presentation to CD8+ T lymphocytes. Here, using IRAP–/– DCs, we explored how IRAP modulates phagosome maturation dynamics and cross-presentation. We find that in the absence of IRAP, phagosomes acquire more rapidly late endosomal markers, are more degradative, and show increased microbicidal activity. We also report evidence for a role of vesicle trafficking from the endoplasmic reticulum (ER)–Golgi intermediate compartment to endosomes for the formation or stability of the IRAP compartment. Moreover, we dissect the dual role of IRAP as a trimming peptidase and a critical constituent of endosome stability. Experiments using a protease-dead IRAP mutant and pharmacological IRAP inhibition suggest that IRAP expression but not proteolytic activity is required for the formation of storage endosomes and for DC-typical phagosome maturation, whereas proteolysis is required for fully efficient cross-presentation. These findings identify IRAP as a key factor in cross-presentation, trimming peptides to fit the major histocompatibility complex class-I binding site while preventing their destruction through premature phagosome maturation.

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Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. Vol. 278 (2003) 49239-49245

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)74962-8 ◽

2004 ◽

Vol 279 (7) ◽

pp. 6204

Author(s):

Parimal Sheth ◽

Shyamali Basuroy ◽

Chunying Li ◽

Anjaparavanda P. Naren ◽

Radhakrishna K. Rao

Keyword(s):

Oxidative Stress ◽

Tight Junctions ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Regulation of clathrin coat assembly by Eps15 homology domain–mediated interactions during endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0380 ◽

2012 ◽

Vol 23 (4) ◽

pp. 687-700 ◽

Cited By ~ 7

Author(s):

Ryohei Suzuki ◽

Junko Y. Toshima ◽

Jiro Toshima

Keyword(s):

Functional Diversity ◽

Protein Interaction ◽

Interaction Domain ◽

Protein Protein Interaction ◽

Molecular Events ◽

Biological Studies ◽

Eh Domain ◽

Actin Patch ◽

Lines Of Evidence

Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein–protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain–containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain.

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Novel Role of Phosphatidylinositol 3-Kinase in CD28-mediated Costimulation

Journal of Biological Chemistry ◽

10.1074/jbc.m005051200 ◽

2000 ◽

Vol 276 (12) ◽

pp. 9003-9008 ◽

Cited By ~ 40

Author(s):

Yohsuke Harada ◽

Eri Tanabe ◽

Ryosuke Watanabe ◽

Bonnie D. Weiss ◽

Akira Matsumoto ◽

...

Keyword(s):

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria

Open Biology ◽

10.1098/rsob.170087 ◽

2017 ◽

Vol 7 (7) ◽

pp. 170087 ◽

Cited By ~ 10

Author(s):

Yi Ting Tsai ◽

Valentina Salzman ◽

Matías Cabruja ◽

Gabriela Gago ◽

Hugo Gramajo

Keyword(s):

Cell Envelope ◽

Phospholipid Composition ◽

Mycolic Acid ◽

Transcriptional Level ◽

Direct Role ◽

Conditional Mutant ◽

In The Wild ◽

Fas Ii

One of the dominant features of the biology of Mycobacterium tuberculosis , and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc 2 155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and P fasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C 18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

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