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.

Bordetella pertussis is the human adapted pathogen that causes the disease known a “whooping cough”. B. pertussis is a non-invasive, extracellular pathogen that colonizes the upper respiratory tract (URT) during pathogenesis.

• Name 2 innate immune mechanisms that B. pertussis must evade in order to successfully colonize the URT.
• Name 2 secreted products expressed by B. pertussis during URT colonization that knockout the above 2 innate immune mechanisms that you listed.

B. pertussis infection is viewed as a two stage pathogenesis process involving first colonization of the URT and second toxin mediated tissue damage. Name two virulence factors that are expressed by B. pertussis that are responsible for toxin mediated tissue damage.

BvgAS, the virulence gene regulation locus of pathogenic Bordetella, is a 2 component sensory transduction system. How was BvgAS implicated in phase variation that occurs with Bordetella pertussis with respect to the virulence phenotype?

In a study designed to determine whether BvgAS of B. pertussis is responsible for phenotypic modulation of virulence, Miller et al. designed an experimental approach to find mutants that were insensitive to environmental modulators of BvgAS.

• In the genetic construct generated for the above goal by Miller (strain VIR102 of B. pertussis), why was the pertussis encoding toxin gene fused with the gene encoding chloramphenicol acetyltransferase that confers resistance to the antibiotic chloramphenicol?
• Why was hemolytic activity also assayed along with chloramphenicol resistance?
• Why in the genetic construct that they generated (VIR102) did they delete the wild type BvgAS from the chomosome and then integrate back into the chromosome a plasmid vector containing wild type BvgAS? How did this allow them to prove that BvgAS mediates phenotypic modulation?

The apparent function of BvgAS in Bordetella pathogens is to determine if Bordetella is within or outside a mammalian host. The Bvg minus phase in B. bronchiseptica results in the expression of several products required for survival of B. bronchiseptica when it is free living in the environment. What is the function of the Bvg minus phase products expressed by B. pertussis

During B. bronchiseptica infection of rabbits, serum from the infected rabbits have antibodies against FHA, fimbriae, adenylcyclase, but not flagella. What conclusion can you draw from this finding with respect to BvgAS?

In the “skinny pig” (the ugly hairless guinea pig) aerosol transmission model it was found that B. bronchiseptica having a wild type BvgAS locus, but not those having a Bvgc (constitutive Bvg+ phase) locus were transmission competent. Given that Bvgc is as virulent as wild type, and Bvg- is avirulent, what conclusions were drawn from this observation?

The approach of “In vivo expression technology” (IVET) was designed to find genes that are expressed by the bacterial pathogen within the host during infection. The strategy is based on the assumption that some virulence genes would not be expressed under in vitro conditions. The plasmid used to find “in vivo induced (ivi) genes”, called pIVET1, contains a promoterless purA gene and in tandem a promoterless lacZYgene downstream of a BglII restriction endonuclease site. a) What was the purpose of including the promoterless purA gene on the plasmid, i.e. how was it used to find the ivi genes? b) What was the purpose of the promoterless lacZY genes, i.e. how were they used in the search for ivi genes? c) Why was a “promoter trap” approach used rather than a transposon? (hint: What was the consequence of plasmid integration on the expression of the gene into which the plasmid integrated?

• Since a purA defective Salmonella strain was used, the bacteria would survive passage through the animal only if the purA gene on the plasmid was expressed (in cis complementation). The purA gene therefore provided a way to select for bacteria carrying plasmids with fragments containing promoters that were expressed in vivo.
• The investigators were looking for genes that were NOT expressed in vitro (on lactose containing MacConkey medium to be specific). By screening the bacteria recovered from the mice for those that were Lac- (no beta galactosidase activity), they could determine which ones contained plasmids with promoters that were expressed in vivo, but NOT in vitro.
• Since Mahan et al. were looking for virulence genes, they reasoned that mutation of such genes would result in avirulence. When transposons insert into genes, they usually inactivate them. Integration of a plasmid by homologus recombination results in duplication of the homologus sequences and if these sequences contain the promoter region, expression and function of the gene should not be disrupted. This stratgey was used, therefore, so the putative virulence genes would NOT be inactivated.

IVET and STM methodologies failed to identify virulence genes in Staphylococcus aureus (rather only housekeeping genes were identified). However, mutants (generated via different techniques) with disruptions in a single genetic locus were found to be avirulent. a) Name this locus and list or briefly describe the functions of the proteins encoded by the genes at this locus. b) What is the physical form of the downstream regulator controlled by this locus? c) List two classes of genes that are regulated by the moiety in (b) and state whether regulation is positive or negative.

• Locus = Accessory gene regulation (Agr)
  AgrD: precursor of autoinducing peptide (AIP)
  AgrB: process and export AIP
  AgrC: receptor for AIP
  AgrA: response regulator
• mRNA (accept RNAIII or RNA)
• Toxin genes upregulated (positive) and Adhesin genes downregulated (negative).

Protein A provides an immune-evasion advantage to Staphylococcus aureus by binding the Fc region of IgG. What are three immune mechanisms that use Fc recognition, all of which are disrupted by Protein A?

• Phagocytosis mediated by high-affinity Fc receptors on activated phagocytes
• Activation of complement by clustered Fc regions
• Natural killer cell (NK cell) activation by clustered Fc via low affinity Fc receptors

Staphylococcus aureus secretes an exotoxin called alpha toxin. This protein is produced as a water-soluble monomer. Describe two changes to this structure that occur upon interaction with mammalian cells.