Discovery and Determination of the Mechanism of Action of Listeriolysin O

Discovery and Determination of the Mechanism of Action of Listeriolysin O



This series of papers covers the discovery and determination of the mechanism of action of Listeriolysin O (LLO). LLO is major a virulence factor for the pathogenic bacteria Listeria monocytogenes, which causes food borne gastroenteritis. This is a series of papers from the Portnoy Lab, and begins with a transposon screen that identifies a mutant that lacks hemolysis activity and therefore cannot survive being phagocytized by macrophages. In paper #2, the target gene in question is cloned, characterized in a different bacterial species, and activity of the gene is noted to by pH sensitive. In Paper #3, this pH sensitivity is investigated and found to be necessary for bacterial survival inside of cells without killing of the host cells themselves. Paper #4 continues this determines that the pH sensitivity of LLO is required for virulence because it does not kill the host cell- allowing the bacteria to proliferate (killing of the host cell is a bad thing!).


I initially chose these papers for my senior elective course in Bacterial Pathogenesis. This series of papers nicely demonstrated the concept of bacterial virulence, intracellular survival, and evasion of the immune system. Many students have the misconception that a virulent organism is one that always kills and causes damage to host cells. The final conclusion of paper#4 nicely demonstrated why this is NOT the case for Listeria. However I think elements, or all, of this module could be adapted for biochemistry, cell biology, and microbiology courses.

Applicable for Courses:

microbiology, bacterial pathogenesis, or cell biology

Educational Level:


Roadmap Objectives:

    • Article: Role of hemolysin for the intracellular growth of Listeria monocytogenes. Portnoy DA, Jacks PS, Hinrichs DJ. J Exp Med. 1988 Apr 1;167(4):1459-71.
    • Content area/major concepts: This paper uses a transposon mutagenesis screen to identify a hemolysin in Listeria. They then characterize this hemolysin as being necessary for survival of the bacteria inside of a macrophage.

      Using a bacterial mutant screen, hemolysis, phagocytosis, LD50, bacterial invasion of cells, membrane structure, reversion of mutation
    • Methods or technology used to obtain data: Tissue Culture, Transposon mutagenesis, SDS-PAGE, LD50 determination, gentamycin protection assay, detection of hemolysis, immunofluorescence
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Understanding how transposons are used to generate mutants and how a screen is performed. Understanding how the gentamycin assay distinguishes between intra- and extra-cellular bacteria. There is a key element of this discussion that states that the screen has not definitively identified the gene for this hemolysin (part of the next paper). This may be confusing to students. Understanding the purpose and importance of the generation of reversion mutants.
    • Article: Capacity of listeriolysin O, streptolysin O, and perfringolysin O to mediate growth of Bacillus subtilis within mammalian cells. Portnoy DA, Tweten RK, Kehoe M, Bielecki J. Infect Immun. 1992 Jul;60(7):2710-7.
    • Content area/major concepts: This paper clones the hemolysin described in paper #1 (listeriolysin O or LLO). This protein is then compared to other, better characterized hemolysins from other bacteria in terms of their ability to allow for bacterial survival in the macrophage.

      Gene cloning, Enzyme kinetics
    • Methods or technology used to obtain data: Tissue culture, hemolysis assay, gentamycin protection assay, SDS-PAGE Cloning and expression of gene in different bacteria, Western Blot, protein purification, Hemolysis kinetics assay
    • How the CREATE strategy was used:
    • Biggest teaching challenge: The assay for enzyme activity is an indirect measure and so you should have a discussion of what is actually being measured (lysis of cells). This is a good paper to talk about the influence of environmental conditions (such as pH), on enzyme activity. Good discussion of what can be concluded and what is pushing things too far- leads nicely to a next experiment assignment.
    • Article: The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells. Glomski IJ, Gedde MM, Tsang AW, Swanson JA, Portnoy DA. J Cell Biol. 2002 Mar 18;156(6):1029-38. Epub 2002 Mar 18.
    • Content area/major concepts: This paper investigates why LLO is active at an acidic pH. The acidic pH is encountered when the bacteria has been phagocytized, and allows the bacteria to escape into the cytoplasm without lysing the entire cell.

      Point mutations, Semi-permeable membrane, Protein domains
    • Methods or technology used to obtain data: Generation of mutants, Protein expression and purifications (more advanced methods than paper #2), LD50, Tissue culture Construction of point mutations and chimeric proteins, a new and different hemolysis assay (can discuss how this has evolved over the papers), allelic exchange, plaque assay (phagosomal escape), fluorescence microscopy, flow cytometry
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Will need to explain how the flow cytometry data is presented (dot plots vs, histograms).
    • Article: Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic, avirulent, and unable to evade host extracellular defenses. Glomski IJ, Decatur AL, Portnoy DA. Infect Immun. 2003 Dec;71(12):6754-65.
    • Content area/major concepts: This paper investigates why having a pH sensitive LLO is advantageous to Listeria as opposed to one that is active at a range of Ph. They find that if LLO is active at neutral pH, it causes lysis of the host macrophage, which lowers the rate of survival of the bacteria.

      Relative virulence, Phagocytic vesicle processing
    • Methods or technology used to obtain data: Generation of mutants, Gentamycin Protection assay, Flow cytometry, Mouse infections, Plaque Assay Competition assays
    • How the CREATE strategy was used:
    • Biggest teaching challenge: Figure 7 is a nice model diagram of the final conclusions of the paper. This would be great to have students generate by themselves before reading the discussion. Some students may find the finding of this paper to contradict their notion of what a virulent pathogen does to the host. This could lead to a nice discussion about the concept of virulence from the point of view of the human vs. the bacteria.

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