Host-Mimicking Conditions Promote Pseudomonas aeruginosa PA14 Virulence Gene Expression

Amber Grace ¹ Rajnish Sahu ¹ Donald R Owen ² Vida A Dennis ^1*

• ¹ Alabama State University, Montgomery, United States
• ² Owen Biosciences Inc., Baton Rouge, Louisiana, United States

Pseudomonas aeruginosa is a ubiquitous, opportunistic bacterium whose highly plastic genome and adaptable phenotype have yielded serious treatment challenges for immunocompromised patients. Its characteristic drug resistance has produced a vacuum for potent antibiotics. Antibiotic alternatives, such as anti-virulence therapeutics, have gained interest because they disable bacterial virulence mechanisms, thereby improving the efficacy of host immunity or traditional antibiotics. Identifying successful anti-virulence therapeutics may require a paradigm shift from decades-old antimicrobial susceptibility testing (AST) in Mueller Hinton broth to media that foster optimal virulence expression. For the first time, our study reveals that culturing the highly virulent P. aeruginosa PA14 in host-mimicking media enhances expression of multiple virulence therapeutic targets critical to host colonization and infection. RNA sequencing showed multiple Type III Secretion (T3SS), Type I Secretion (T1SS), pyoverdine biosynthesis, uptake and efflux, and Type IV pili (T4P) initiation genes were promoted when PA14 transitioned into host-mimicking conditions, represented by Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS), but remained unchanged when transitioned into standard AST conditions. Moreover, quantitative polymerase chain reaction (qPCR) results disclosed that human serum (HuS) and FBS delivered differential effects on the expression of membrane-associated virulence genes involved in host colonization. HuS was more effective than FBS in increasing the expression of select T3SS, pyoverdine transport, and T4P membrane-associated genes. However, T1SS (Has) genes were more highly expressed in FBS than in HuS. To determine if enhanced gene expression translates to enhanced virulence activity, we evaluated twitching motility, pyoverdine production and biofilm formation. Our macroscopic PA14 twitching motility results aligned more closely with PA14 growth patterns than with virulence gene expression patterns. Our biofilm assay, however, revealed earlier biofilm formation in DMEM 0 than in AST conditions and both showed inhibited twitching motility in serum conditions. Pyoverdine production aligned with our gene expression data, showing higher pyoverdine production in serum conditions for planktonic PA14. Overall, our findings support using host-mimicking conditions to improve the expression of candidate targets for anti-virulence therapeutics against P. aeruginosa PA14 in a planktonic state. These recommendations may be broadly applicable for anti-virulence therapeutic screening against multiple bacterial species at large.