Abstract
BackgroundMost bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Due to specific metabolic requirements and symbiotic relationships, these bacteria cannot survive on typical laboratory media. Many economically and medically important bacteria are unculturable; including phloem-limited plant pathogens like Candidatus Liberibacter asiaticus (CLas). CLas is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks an effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive.ResultsWe developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized tissue from CLas-infected ACP and a propidium monoazide (PMA) assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between intact and disrupted CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent PCR interference and identified a suitable positive control for nearly complete CLas disruption (0.1% Triton-X 100). Isolation buffer components were optimized with 72 mM salt mixture, 1 mM phosphate buffer and 1% glycerol serving to minimize unwanted interactions with treatment and PMA chemistries and to maximize recovery of intact CLas cells. The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity and identify which were most effective.ConclusionThis psyllid homogenate assay allows for a quick assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms. Use of an intact vs. disrupted assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.
Highlights
Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies
If PMAxx dye remains active in the sample after light treatment, it may interfere with the polymerase chain reactions (PCR) by binding deoxyribonucleic acid (DNA) released in the DNA extraction protocol or by interfering with template amplification by DNA polymerase [33, 34]
Full reaction of free PMAxx prior to PCR is necessary for accurate v-quantitative polymerase chain reactions (qPCR) analysis
Summary
Most bacteria are not culturable, but can be identified through molecular methods such as metagenomics studies. Many metabolites and gene products are released into the environment where they can be absorbed and utilized by other species. These interactions favor species with streamlined genomes that omit the cellular machinery to produce already available resources [12]. Advances are being made to grow such microbes through use of genomic data to tailor media to the microbes’ metabolic needs [15] This analysis has begun in CLas with the report of a full genome sequence and analysis to identify deficient metabolic pathways [16, 17]. Other efforts with mutually dependent strains, that are not individually amenable to pure cultures, include being grown through co-culture or the maintenance of communities in biofilms [18, 19]
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