Role of ExoU island accessory genes in P. aeruginosa virulence

Abstract Pseudomonas aeruginosa is a leading cause of blinding corneal infections worldwide. The type III secretion system (T3SS), a molecular syringe that allows the bacterium to inject effector proteins into host cells, is critical for P. aeruginosa virulence. P. aeruginosa isolates do not produce many effector proteins and even

more curiously, genes for the effectors ExoS and ExoU are distributed in a mutually exclusive fashion. This distribution materially affects the virulence of the strains. ExoS producing strains can invade epithelial cells in

vitro, are slower to elicit overt lysis of host cells, and cause a less fulminant infections with less tissue destruction in vivo. ExoU producing strains, on the other hand, tend to cause more severe disease, and are linked to increased antibiotic resistance. While ExoS producing strains predominate in the environment and in patients

with cystic fibrosis or ventilator-associated pneumonia, ExoU-producing strains are significantly enriched among corneal isolates from keratitis patients. The correlation between production of ExoU, increased disease severity in the clinic, and elevated frequency of multidrug resistant isolates has been documented epidemiologically for decades. However, the

molecular underpinnings of this correlation are not well understood. The exoU gene is encoded on a pathogenicity island, which can vary greatly in size, ranging from as little as 4 kb to 81 kb. The role of the accessory genes present on the ExoU pathogenicity island is unclear, but our preliminary work demonstrates

that they play an important role in pathogenesis. However, which ExoU-island genes are important for virulence and whether these pathogenicity island genes can also help explain the increased frequency of antibiotic resistant isolates is unclear. The goal of this proposal is to test the hypothesis that ExoU islands encode virulence

functions other than ExoU and that, at least in some instances, they also contribute to the increased antibiotic resistance associated with this class of P. aeruginosa strains. The proposal is divided into two aims, that will dissect the virulence role of the two largest ExoU islands: the 30kb, type B ExoU island of strain 19660 (Aim 1),

and the 81 kB, type A ExoU island of strain 6077 (Aim 2). Strain 6077 is a multidrug resistant corneal isolate that belongs to the globally distributed, high-risk ST235 sequence type. We will also examine the role of the type A ExoU island as it relates to antibiotic resistance. Taken together the proposed program of research will uncover new virulence functions which will expand

our understanding of the molecular toolbox available to bacterial pathogens. The proposal is aimed squarely at understanding the virulence of the most pathogenic and difficult to treat P. aeruginosa infections encountered in the clinic.