Abstract 1479 Investigating Regulation of Biofilm Deficiency in Lon Protease-Mutant Acinetobacter baumannii

Acinetobacter baumannii is a Gram-negative nosocomial opportunistic pathogen capable of robust biofilm formation providing it with the concerning ability to persist in harsh environmental conditions. Previous research has found that deletion of the energy dependent Lon Protease in A. baumannii produces a striking decrease in biofilm formation as well as a large transcriptional upregulation of two genes: surA1 and A1S_2230, a protein of unknown function we have re-named surA2, as both are hypothesized to localize to the cell surface. Intriguingly, joint, but not individual, disruption of these genes in the Δlon strain-the triple knockout (TKO)-shows substantially restored surface attachment and biofilm formation. To investigate whether Lon's DNA binding domain is needed to prevent the Δlon observed phenotypes, a genomic Lon mutant with impaired DNA-binding capability (LonDNAbind-) was created. This strain phenocopies the Δlon strain in single cell morphology, motility, and overproduction of SurA1 and SurA2, while partially restoring biofilm formation. These results suggest SurA1 and SurA2 are dysregulated in the Δlon strain due to a Lon-sensitive transcriptional activator of the surA genes for which the Lon DNA binding motif is needed for proteolysis. To identify the activator(s), we created a plasmid encoding a LonTrap where a FLAG-tagged and catalytically dead Lon mutant is pulled down with any bound proteins. This approach is yet to produce high potential targets but successfully collects the LonTrap protein. While the regulatory system at hand remains to be fleshed out, the interplay between upregulated SurA proteins and altered biofilm formation aligns with changes in the cellular envelope of these strains and led us to predict cell envelope modification as a mediator of the observed phenotypes. Considering A. baumannii biofilm formation is influenced by the membrane bound Csu pilus, a working hypothesis attributes the high levels the SurA proteins in Lon deficient strains with potential to interfere with Csu externalization. Investigation into the expression of the Csu pilus revealed increased Csu expression levels in both the TKO and LonDNAbind- strains compared to Δlon and even wild-type. Despite increased expression levels, Csu externalization determined by western blot of purified surface pili samples from strains lacking the Lon Protease showed Csu detection for only the TKO strain, and at rates lower than wild-type. Put together, these results suggest a role for the SurA protein's overproduction in altering surface Csu levels and contributing biofilm alterations in Lon deficient strains. Continued research to fully uncover the regulatory network controlling both the expression and externalization of the surface proteins described here will be critical for understanding A. baumannii biofilm development and ultimately aiding eradication efforts. Funding for this work was provided by the Northeastern University Office of Undergraduate Research and Fellowships Spring 2023 AJC Merit Research Scholarship and Fall 2023 PEAK Experiences Summit Award.