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In vitro reconstitution and single cell analysis of the Shigella-septin cage.

Periodic Reporting for period 1 - INCAGE (In vitro reconstitution and single cell analysis of the Shigella-septin cage.)

Reporting period: 2017-08-01 to 2019-07-31

Autophagy is a highly conserved degradative process that breaks down cytosolic material inside double-membrane vesicles (called autophagosomes) by fusion with lysosomes. Selective autophagy is an important host defense mechanism that recognizes intracellular bacterial pathogens, such as Shigella flexneri, for targeting to degradation. To avoid autophagy, S. flexneri can polymerize host cell actin and form an actin tail that permits dissemination and cell-to-cell spread. Septins, a poorly understood component of the cytoskeleton that interacts with actin filaments and cellular membranes, entrap actin-polymerizing Shigella in cage-like structures for targeting to autophagy. However, the molecular determinants that underpin septin cage assembly are mostly unknown, and, the fate of septin cage-entrapped bacterium (replication, persistence, death) remains to be fully defined.

Hosted by the Mostowy lab my project entitled "In vitro reconstitution and single cell analysis of the Shigella-septin cage (INCAGE)", aims to study factors controlling septin cage assembly and bacterial metabolism inside septin cages. I am investigating septin cage biology using bottom-up (using purified septin complexes in vitro and cell-free extracts) and top-down (using epithelial cells and different types of cutting-edge microscopy) approaches. For my Marie Curie postdoctoral project, I propose that in depth investigation of the Shigella-septin cage can lead to new septin biology, and new roles for the cytoskeleton in cell-autonomous immunity.

INCAGE is an excellent platform to illuminate new concepts on the cell biology of Shigella that are difficult or impossible to address using whole cells or animal models. Understanding how our cells respond to bacterial infections can help to combat infectious diseases and control the spread of antibiotic resistance.
INCAGE focuses on two main Objectives that aim to further our knowledge on the cell-autonomous immune response to the bacterial pathogen Shigella flexneri.

Objective (i): In vitro reconstitution to identify minimal components essential for septin cage assembly.
- How do host cells recognize bacteria for septin cage entrapment?
• Using HeLa cells producing fluorescently labelled septins and fluorescence microscopy we have discovered that septins are preferentially recruited to sites of bacterial positive membrane curvature (i.e. the poles and the division site).
• Using purified septin complexes (SEPT2/6/7) and, dot blots and liposome flotation assays we have demonstrated that septins bind cardiolipin, an anionic phospholipid that accumulates on the poles and division site of Shigella.
• We have shown that Shigella mutants unable to synthesize cardiolipin are entrapped less efficiently into septin cages.
Taken together, these results suggest that septins recognize membrane curvature and cardiolipin on the poles of Shigella for septin cage entrapment. This work was published in Cell Host and Microbe 24: 866-874, 2018.

- What are the factors that modulate septin caging?
• Using an in vitro reconstitution assay, I have reconstituted septin cages for the first time. I have demonstrated that (as seen in vivo during infection of human epithelial cells): (a) purified septins recognize the poles and the division site of Shigella cells (in the absence of other host factors), and (b) Shigella growth is important for septin cage entrapment.
• Human SEPT6 may contain an amphipathic helix domain (AH) that permits the recognition of membrane curvature. Preliminary data suggest that recombinant SEPT6 mutants lacking AH domain recognize host cell and Shigella curved membranes less efficiently.
In vitro reconstitution assays have demonstrated that growth is an essential factor during septin recognition. These assays also provide an ideal platform to test the structural features of septins important for bacterial recognition / entrapment.

- How do septins assemble on bacterial membranes?
• In collaboration with ALBA synchrotron I am preforming correlative light and cryo-soft X-ray tomography (cryo-SXT) on Shigella-infected HeLa cells to understand how septins interact with autophagosomes.
• In collaboration with the lab of Prof. Martin Pilhofer at ETH Zurich I am using cryo-electron tomography (cryo-ET) to visualize in vitro septin cages.
These studies will illuminate unprecedent structural insights on septin biology during the cell-autonomous immune response to Shigella.

Objective (ii): Investigate the lethal action of septin caging and search for mechanisms that enable bacterial persistence / replication inside the septin cage.
- Lethal action of the septin cage:
Autophagy and septin cages are recognized to restrict bacterial replication. On the other hand, we have shown that ~50% of Shigella entrapped by septin cages are metabolically active. How a subpopulation of cage-entrapped bacteria can avoid autophagy and remain metabolically active is unknown. To address this, I am employing fluorescent markers to follow the physiology of individual bacterium during infection, including reporters for metabolic activity and bacterial membrane integrity.
- Host and bacterial factors modulating septin caging:
• It is not known if Shigella can use effectors to avoid septin caging. I am designing Shigella mutants for different effector proteins and testing these strains for septin cage entrapment.
• I tested the depletion of septins and the autophagy receptor p62 during Shigella infection. Surprisingly, septins (SEPT2, SEPT7) and p62 are required for the proliferation of intracellular bacteria (not entrapped in septin cages). We also discovered that in the absence of septins the glycolytic pathway is altered. This work was recently published in the journal Cytoskeleton, 76(1):163-172.
Shigella is a Gram negative enteropathogen that causes over 180 million illness episodes per annum, including more than 160,000 deaths. Based on the spread of antibiotic resistant strains, the World Health Organization (WHO) has listed Shigella as one of the most important bacterial pathogens for which the development of new antimicrobials is required. In this context, understanding how Shigella infect and replicate inside the host can illuminate new antimicrobial targets focused on directly attacking the pathogen and/or boosting cellular defense mechanisms.

The main goals of INCAGE project are to identify the minimum set of factors that permit efficient assembly of the septin cage and to understand how septin cages can restrict bacterial replication/dissemination. Shigella infection of Hela cells has proven to be a highly valuable system to uncover new cell-autonomous immune response mechanisms and septin biology. For example, guanylate-binding proteins (GBPs) have recently been shown to restrict the dissemination of cytosolic pathogens, and we have discovered that septins mediate mitochondrial fission. Using in vitro reconstitution assays and high-resolution microscopy, INCAGE will shed light on how septins recognize, entrap and restrict the replication of cytosolic pathogens, including Shigella. Results derived from this project will show (at the nanometer scale) how septins interact with bacterial membranes and assemble to form antibacterial cages, and what are the host / bacterial factors that modulate this process.
Shigella flexneri entrapment by host cell septins.