RESEARCH PLAN

A. SPECIFIC AIMS

This proposal aims to provide insights regarding organ-specific immunity through the novel antimicrobial defense mechanisms specific to plant roots. We investigate the roots of plants rather than the leaves, the classical organ of choice for this type of study, because information developed from our research suggests that unique defense pathways operate in roots. Our research has demonstrated that plant roots can be used as an alternative host for E. faecalis infections (2, 3). The work envisaged through this R21 grant proposal intends to utilize the untapped potential of plant root exudates as a source of small molecular weight compounds exhibiting potential antimicrobial or anti-infective activities against E. faecalis. The soil presents a dynamic environment in which roots are constantly attacked by different foreign organisms, and where they must be in continual chemical communication to discern friendly from pathogenic microbes. Concomitantly, plant roots are involved in continuous secretion of small molecular weight compounds into the rhizosphere as a mode of underground chemical communication. This communication allows the roots to obtain and respond to stress/stimuli signals from other roots and microbes (4). On a similar line, our recent work demonstrates that the root exudate composition of A. thaliana determines the general resistance in plants towards compatible and incompatible bacterial interactions against Pseudomonas syrinage (5). Further, we found that some of the secondary metabolites defined this interaction by exhibiting a direct antimicrobial effect on incompatible P. syringae pathovars.

It is now known that several potential opportunistic human pathogens do, infect plants and distinct bacterial virulence factors have been shown to be conserved in mammalian, nematode and plant species (2, 3). Our recent studies showed that a pathogenic isolate of E. faecalis (OG1RF) and E. faecium (GE-1) infects A. thaliana, causing systemic infection to the roots, resulting in death of the infected plant (2, 3; Figure 2 in the preliminary study section). To dissect the involvement of mammalian virulence-related factors in plant pathogenicity, we also tested E. faecalis mutant strains, which corresponded to different virulence factors involved in pathogenesis of various animal models (2). Our published results showed that two E. faecalis virulence-related factors play an important role in mammalian and nematode models of infection. A putative quorum-sensing system and a serine protease were also found to be important for plant pathogenesis (2). This result highlights the development of an E. faecalis-plant model system that could potentially be used to circumvent certain inherent limitations that an animal model imposes for the identification and study of virulence factors.

Similarly, utilizing both the bioactive potential of secondary metabolites present in root exudates and a live interaction between a given plant and a bacterial system (i.e. direct interaction of a plant species against E. faecalis/E. faecium), we are developing a simple and methodical screen to chem-mine potential anti-microbial and anti-infective compounds.  This screen will enable the elicitation of compounds that may not typically be present in the root exudates in the absence of the given pathogen under sterile conditions. E. faecalis, an opportunistic human pathogen, is an ideal candidate for this type of screening system due to its ability to infect plants under laboratory conditions, and because it is a natural inhabitant of the soil (6). Three possible interactions may occur when the pathogen directly challenges the plant root systems. First, the plant may develop disease symptoms as a result of infection between the roots and the pathogen. Alternatively, the plant will survive at the expense of the bacteria; this indicates the exudation or presence of an bactericidal compound. Lastly, both the plant and bacteria will survive, indicating the possible presence of an anti-infective compound, which is capable of inhibiting bacterial virulence but not harming the bacteria. The occurrence of the last two possibilities would be much more interesting from a drug discovery point of view and would allow for further investigation.

In recent, unpublished preliminary data, we have found that the root exudates from a distinct plant species exhibit diverse antimicrobial and anti-infective properties against Pseudomonas aeruginosa infections. When grown in a co-culture with the different plants species, P. aeruginosa inflicts disease symptoms on the roots of most of the plant species. In contrast, a few plant species showed resistance in this interaction. The exudates collected from these resistant plant species showed anti-infective and cure-infection properties in the C. elegans pathogenicity system. We know that these plant root based screens would be much more effective in the case of E. faecalis and E. faecium, as both of these Enterococcus species are known to cause persistent infections unlike P. aeruginosa in both plant and worm model (1, 6-7). These results also suggest the potential of root exudates as a chemical-genetic screening pool for the investigation of host-microbe interactions. Although the majority of noscomical Enterococcus infections are caused by E. faecalis (85-90%), the remaining infections are due to E. faecium pathogenesis and there is an immediate upsurge to discover compounds/antibiotics that could potentially inhibit the growth and virulence of E. faecium. The curing of infections inflicted by E. faecalis and E. faecium using both a direct and indirect plant challenge would open a novel frontier to screen plant root products for development of alternate therapeutics against E. faecalis and E. faecium infectivity. Therefore, since both E. faecalis and E. faecium are capable of infecting plant models under laboratory conditions, we hypothesize that there is an exciting possibility that the portion of plant species that are not infected with E. faecalis and E. faecium exude anti-infective and anti-microbial compounds. This novel approach could easily be exploited to look for potential root-derived therapeutic agents against E. faecalis and E. faecium pathogenicity.   

The studies currently proposed are:

Aim 1: To apply a simple root screening method to test E. faecalis and E. faecium pathogenicity.  Identify plant species that resist infection by E. faecalis and E. faecium and collect their root exudates. Determine bioactivity of root exudates in both antimicrobial (micro-titer and agar-diffusion based assays) and anti-infective (virulence factors and C. elegans infection model) assays.

Aim 2: To identify and chemically characterize compound(s) from the root exudates of resistant and tolerant plant species that exhibit antimicrobial or anti-infective properties against E. faecalis and E. faecium. To use classical microbiological and nematode infection assays to characterize the effectiveness of the identified compound as a therapeutic agent. Analyze bioactivity of a pure compound by measuring minimum inhibitory concentrations (MIC), colony-forming units, spectrum of activity, rates of resistance, mode of growth inhibition and testing anti-infective properties in a nematode infection model.

1.      Moy, T.I., Ball, A.R., Anklesaria, Z., Casadei, G., Lewis K. and Ausubel, F.M. 2006.  Identification of novel antimicrobials using a live-animal infection model. Proc Natl Acad Sci USA. 103:10414.

2.      Jha, A.K., Bais, H.P. and Vivanco, J.M. 2005. Enterococcus faecalis mammalian virulence-related factors exhibit potent pathogenicity in the Arabidopsis thaliana plant model. Infect Immun. 73: 464.

3.      Prithiviraj, B., Weir, T., Bais, H.P., Schweizer, H.P. and Vivanco, J.M. 2005. Plant models for animal pathogenesis. Cell Microbiol. 7: 315.

4.      Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. and Vivanco, J.M. 2006.  The role of root exudates in rhizosphere interactions with plants and other organisms. Annu Rev Plant Biol. 57:233.