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.