Theriot et al. used caged-resorufin bound to G-actin which was micro-injected into potoroo kidney epithelial cells to investigate actin filament dynamics within the actin tail formed by Listeria moncytogenes during infection of these cells. How was caged-resorufin used in their study and what observation was made?

• Caged resorufin becomes fluorescent when exposed to UV light of 360 nm. Caged resorufin conjugated to G-actin was incorporated into the F-actin tails formed behind the Listeria. By activating a discrete spot of caged-resorufin in the actin tail using a laser, the researchers were able to determine that the actin tail remained stationary in the cytoplasm while the bacterium moved.

What unique cell wall associated feature does Listeria monocytogenes possess that is not found associated with the cell walls of other gram positive bacteria?

Listeria monocytogenes is an intracellular pathogen that is capable of multiplying inside resting macrophages. How does Listeria avoid the phagolysosomal killing mechanism of this phagocytic cell?

In the study conducted by Bielecki et al., the hlyA gene of L. monocytogenes that encodes listeriolysin O was mutated. Describe how this gene was mutated, the resulting 2 phenotypes that were looked at following mutation, and the conclusions based upon these data.

Listeriolysin O is a member of a family that contains several other cholesterol-dependent cytolysins secreted by gram positive pathogens. However, Listeriolysin O is the only one that functions within the vacuole whereas these other cytolysins function extracellularly. Name 2 properties of Listeriolysin O that are different from the properties of these other cytolysins and how is this important to successful Listeria pathogenesis?

When Kocks et al. compared infection in J774A.1 macrophages using wild type L. monocytogenes strain 104035, a B. subtilis wild type strain, and this B. subtilis strain containing a plasmid expressing the L. monocytogenes hlyA gene what did they find with regard to a) hemolytic activity, and how was this determined b) the ability to grow intracellularly c) the intracellular location of the bacteria as assessed by electron microscopy d) what conclusions were drawn from this experiment.

• Both Lm 104035 and B. subtilis phlylA, but not wild type B. subtilis, possessed hemolytic activity on blood agar plates
• Both Lm 10435 and B. subtilis phlyA were found to grow in the macrophages, but not wild type B. subtilis
• Both Lm 104035 and B. subtilis phlyA were found free in the cytoplasm – not enclosed within phagosomes.
• The conclusions was a single gene product, hlyA, is sufficient to confer upon B. subtilis the ability to escape the phagosome and grow in the cytoplasm.

What do Listeria monocytogenes and Shigella flexneri have in common with regard to their pathogenic strategies?

• Both are able to escape the phagocytic vesicle, to multiply in the cytoplasm of infected cells, to polymerize actin, and to spread directly from cell to cell.

Because of this pathogenic strategy, what type of immune response is required for control of both of these infections?

• A cytotoxic T-lymphocyte (CTL) type immune response is required to clear these infections.

Theriot et al. used a phalloidin-rhodamine conjugate to observe intracellular Listeria monocytogenes. What does the phalloidin-rhodamine conjugate bind to, what was the observation made in this study, and what was the conclusion drawn from the data?

Listeria produces a toxin called listeriolysin O (LLO). LLO is a UNIQUE member of a family of pore-forming toxins called cholesterol dependent cytolysins (CDC’s). Another CDC, streptolysin O (SLO), is produced by Streptococcal species. In an experiment using Yersinia, it was determined that YopB and YopD produce SMALL pores in the plasma membranes of target-cells in the specific instance when Yop effectors are deleted. The size of the pores was determined to be small by showing that a small dye (Lucifer yellow) could enter cells while a larger dye (Texas red phalloidin) could not. As a control, researchers used SLO to show that Texas red phalloidin could enter cells. What property of LLO would make this toxin a pore choice to replace SLO in this experiment

In Listeria monocytogenes, actin polymerization mediated by the gene product ActA has been shown to be polar resulting in unidirectional movement. Design an experiment to show that actin polymerization is polar because ActA is polar.