PLANT MICROBIOTA-DRIVEN FACILITATION OF THE PERSISTENCE, GROWTH AND INTERNALIZATION OF HUMAN PATHOGENS ON LEAFY GREENS
My research focuses on the survival of enteropathogenic E. coli O157:H7 in the leaf environment. I hypothesize that this survival is a function of the microbiota that naturally associate with plant leaves. More specifically, I will test the idea that E. coli benefits from the ability of foliar bacteria to produce the plant hormone indole 3-acetic acid (IAA) as follows: 1) IAA makes E. coli resistant to leaf-related stresses, 2) IAA stimulates leaching of nutrients to the leaf surface, and 3) IAA interferes with normal stomatal function, allowing for internalization of E. coli. I have begun identifying and characterizing bacterial isolates from leafy greens grown in the Salinas Valley for their IAA production in culture. Using a newly developed GFP-based bacterial bioreporter I will test if, where, and how much IAA is produced by these isolates after inoculation onto lettuce leaves. In preliminary experiments, I was able to show that pre-exposure of E. coli to IAA resulted in increased survival under non-nutritive conditions in vitro. To identify the genetic mechanism that underlies this phenomenon, I will use a genome-wide transposon screening of E. coli mutants for loss of IAA-induced survival. Additional experiments will assess IAA-induced survival in response to bleach and on lettuce leaves. I will also determine the outcome of co-inoculating E. coli with bacterial IAA producers on lettuce leaves; if my hypothesis holds, such co-inoculation should favor foliar persistence of E. coli. I will also use the leaf co-inoculation approach to ask whether proximity to bacterial IAA producers increases E. coli’s chances of gaining access to leaf surface nutrients or to the leaf apoplast. My research will reveal whether IAA is a risk factor in the persistence of E. coli on leafy greens and a target for future strategies towards minimizing the potential of E. coli contamination.
The increasing public demands for healthy food choices and fresh produce brings to the forefront the importance of food safety research, especially as it relates to leafy greens where food-borne illness outbreaks have historically been a public threat. Numerous instances of human pathogenic bacteria detected in the phyllosphere have occurred recently and should serve as reminder of the need to uncover the mechanisms under which they have been able to persist throughout production and processing. Microbial communities on the phyllosphere may function cooperatively to allow certain species to remain by producing exogenous secondary metabolites, such as IAA. Bacterial derived IAA on the leaf surface influencing E. coli’s survival, persistence and internalization calls for further understanding. By studying this effect we may be able to manipulate agricultural settings to deter establishment of enteric pathogens.
Fluorescent microscopy of E. coli ATCC700728 (O157:H7 that does not have the capability to produce the Shiga toxin) that has been labeled with GFP and dsRED. Seen at 100X.