Elucidating the Protein-Protein Interaction Networks and Localizations of the Lytic Transglycoylases RlpA and Slt of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen that is the most prevalent in the hospital setting. Approximately 51,000 P. aeruginosa infections are reported in the U.S. alone each year, and more than 13% of these infections have been identified as multi-drug-resistant strains, according to the U.S. Centers of Disease Control and Prevention. The cell wall in pseudomonas is turned over by as much as 50% during homeostatic growth. Furthermore, it undergoes repair after exposure to cell-wall-active antibiotics. The health of the organism depends on maintenance of a functional cell wall, a process governed by a complex set of events involving dozens of enzymes. Lytic transglycosylases (LTs) are enzymes that facilitate cell-wall degradation and recycling by catalyzing the non-hydrolytic cleavage of the cell-wall peptidoglycan. It is understood that these periplasmic enzymes work in concert with many other proteins, an assembly that we refer to as the cell-wall degradome. Rare lipoprotein A (RlpA) and soluble lytic transglycosylase (Slt) are two of the 11 LTs of P. aeruginosa. RlpA is involved in the process of septation during bacterial growth, while Slt is distinguished for its endolytic capability. We have identified the binding partners of both RlpA and Slt using a pull-down strategy, followed by proteomics analysis. I have cloned and purified the putative recombinant partner proteins detected in the mass-spectrometry assays and have explored protein-protein interactions with RlpA and Slt using microscale thermophoresis to obtain dissociation constants (K_D). Additionally, I have assessed the rate constants of association (k_on)and of dissociation (k_off) for binary and tertiary complexes between the LTs and their respective binding partners by surface plasmon resonance. Moreover, uncovering the stoichiometry of select binding partners with RlpA was done through isothermal titration calorimetry. Finally, amber-codon suppression was utilized to uncover the spatial localization and in vivo photoaffinity-capture of both previously and newly identified binding partners for both LTs. In all, these analyses reveal a minimum of 20 partners for RlpA and of 22 partners for Slt, mediated through their precise localizations within the living cell. These findings substantially advance our understanding of the pseudomonal bacterial physiology and its close association to the mechanisms of resistance in which will aid in drug-discovery efforts.