Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Pathology and Environmen
Non Technical Summary
Disruption of bacterial outer membranes (OM) by antimicrobial agents is a major area of promise for biocontrol and plant disease resistance. Examples include bacterially produced toxins, termed tailocins, which show excellent promise for specific and engineerable prophylaxis. Additionally, LPS targeting antimicrobial peptides (AMP) are the major difference between Huanglongbing (HLB) resistant and susceptible plants. Indeed, plants are known to produce an array of AMPs that kill microbes through membrane disruption. Despite this promise, we've shown that bacteria can enter temporary physiological states that make them tolerant to membrane-active treatments, a phenomenon termed persistence. Such persistence complicates the effectiveness of these treatments. Moreover, modeling has predicted for many antimicrobials, including those that are OM-targeting, that persistence can speed the evolution of resistance, further impeding our ability to deploy these agents for long-term control. Thus, understanding the prevalence and mechanisms of survival to OM-targeting treatments will be critical for determining under what conditions these treatments will be durable and effective, and for designing resistance mitigation strategies. We aim to 1) understand the physiology and genetics that underlie bacterial persistence to multiple OM-targeting agents, 2) determine whether there is overlap in persistence mechanisms to distinct OM-targeting agents, and 3) assess empirically whether increased persistence can result in increased rates of resistance, as well as how persistence contributes to treatment survival in a plant host environment. The results of this research will improve our ability to predict the potential outcomes of OM-targeting antimicrobials and modify treatment strategies to ensure their long-term effectiveness.
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
The goal of this project is to identify the extent, genetic mechanisms, and potential agricultural consequences of persistence to membrane-disrupting antimicrobials.The three aims will address three major questions:Q1:Do pathogenic bacteria have persistent subpopulations to multiple major classes of membrane disruptors, and are these populations distinct or overlapping?Q2:What genetic pathways mediate persistence to different membrane-disrupting antimicrobials?AndQ3:Could persistence to membrane disrupting compounds lead to treatment failure and resistance in agricultural contexts?We will address these questions in the modelPphsystem by characterizing the phenotypes of persisters to diverse treatments (Aim 1), characterizing gene expression patterns and mutations associated with persistence (Aim 2), and by determining the diversity of persistence levels across diverseP. syringaepathovars, as well as whether persistence likely contributes to fitness or leads to resistance in the host plant context (Aim 3). The project will result in an understanding of how pathogen populations withstand different types of membrane disruption, and in turn which treatments could be used in combination to mitigate these effects. Our long-term goal is, through understanding the prevalence and mechanisms of these survival strategies, to maximize the efficacy of membrane-disrupting antimicrobials and minimize the potential for resistance.?
Project Methods
The project will be conducted using a combination of microbiological, bioinformatic, and plant pathological approaches.Aim 1 will be carried out using a combination of laboratory culture, fluorescent redox labelling, and single cell microfluidics. Specifically, we will use culture- and plant-based bacterial enumeration to quantify the effect of antimicrobial resistance; redox reporters will be combined with microfluidic cell sorting to determine the cell physiology underlying persistence. Results of this research will be delivered to target audiences through peer-reviewed publication and scientific presentation. Success associated with this objective will be measured through the production of a peer-reviewed publicationAim 2 will combine microfluidic cell sorting with transcriptomic sequencing to characterize the transcriptional profiles of persisters cells, which will be compared to non-persister cells sorted from the same parent population. Additionally, we will select for and recover mutants exhibiting increased persistence against each of the membrane-targeting antimicrobials to gain deeper insight into the genetics underlying persistence. Results of this research will be delivered to target audiences through peer-reviewed publication and scientific presentation. Success associated with this objective will be measured through the production of a peer-reviewed publication, as well as generating a collection of 10+ mutant isolates that are increased in persistence against each antimicrobial.Aim 3 will combine laboratory and plant-based microbiological techniques to assess the diversity of natural levels of persistence acrossP. syringae, as well as the effect of persistence in in planta survival and proliferation. Additionally, this aim will use an experimental evolution approach to determine the effect of persistence on the acquisition of resistance. Results of this research will be delivered to target audiences through peer-reviewed publication and scientific presentation. Success associated with this objective will be measured through the production of a peer-reviewed publication.