Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors

Abstract

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium isolated from soil, water, and plants (1), and is an opportunistic human pathogen that infects patients who are immunodeficient or otherwise compromised. A variety of P. aeruginosa virulence factors have been described, and the majority of these, such as exotoxin A, elastase, and phospholipase C, were first detected biochemically on the basis of their cytotoxic activity (2). Subsequently, the genes corresponding to these factors or genes that regulate the expression of these factors were identified. In general, most pathogenicity-related genes in mammalian bacterial pathogens were first detected by using a biochemical assay. In contrast to mammalian pathogens, simple systematic genetic strategies have been routinely employed to identify pathogenicity-related genes in plant pathogens. Following random transposon-mediated mutagenesis, thousands of mutant clones of the phytopathogen are inoculated separately into individual plants to determine if they contain a mutation that affects the pathogenic interaction with the host (3-8). Comparable experiments with wholeanimal mammalian pathogenicity models are not feasible because of the vast number of animals that must be subjected to pathogenic attack.Reports indicating similarities between plant and animal pathogens (9, 10) prompted us to search for a strain of P. aeruginosa that was capable of eliciting disease in both a well-defined plant pathogenesis model and a well-defined animal pathogenesis model. Recently, we described (11) a clinical isolate of P. aeruginosa, UCBPP-PA14, that is infectious in both an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin thermal burn model. We showed that mutations in three P. aeruginosa pathogenicity-related genes, toxA, plcS, and gacA, caused significant decrease in pathogenicity in both models (11). The utilization of common virulence-related genes by P. aeruginosa for infecting animals and plants led us to hypothesize that previously unknown virulence determinants required for P. aeruginosa pathogenesis in animals could be identified by screening randomly mutagenized UCBPP-PA14 clones for ones that exhibited decreased virulence in plants. A subset of these mutants would...

Biogenic synthesis of silver nanoparticles using guava (Psidium guajava) leaf extract and its antibacterial activity against Pseudomonas aeruginosa

“…P. aeruginosa is an opportunistic human pathogen, and the genus itself is a potent plant pathogen also. It produces soft rot disease in Arabidopsis thaliana (Walker et al 2004) and Lactuca sativa (Rahme et al 1997). The results of disc diffusion assays indicate that the antimicrobial activities of silver nanoparticles (ZoI = 7-8 mm) are as effective as silver ions (ZoI = 8-10 mm), whereas the raw leaf extract has no visible inhibitory effect on P. aeruginosa MTCC 741 strain.…”

Antibacterial Activity of Green Synthesized Silver Nanoparticles Using Vasaka (Justicia adhatoda L.) Leaf Extract

“…P. aeruginosa is an opportunistic human pathogen and the genus itself is a potent plant pathogen also. It produces soft rot disease in Arabidopsis thaliana [24] and Lactuca sativa [25]. The results of disc diffusion assays indicate that the antimicrobial activities of silver nanoparticles (ZoI = 7-9 mm) are as effective as silver ions (ZoI = 8-10 mm) whereas the raw leaf extract has no visible inhibitory effect on P. aeruginosa MTCC 741 strain.…”

Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica

“…As controls, midribs were inoculated with 10 mM MgSO 4 only (negative control) or control strain P. aeruginosa ATCC 27853 (rhamnolipid-positive/ elastase-negative). The assessment parameters according to Rahme et al (1997) were chlorosis, discolouration at site of inoculation and along the midrib, water soaking, necrotic lesions or decomposition of leaf tissues.…”

“…The virulence potential of P. aeruginosa isolates (using 20 isolates grouped into four categories based on the presence or absence of elastase production and rhamnolipid formation) was performed as described by Rahme et al (1997) and modified by Filiatrault et al (2006) using romaine lettuce as a model of infection. Romaine lettuce has been shown to be a suitable model system for assessment of virulence, and as noted by Rahme et al (1997), bacterial pathogenesis in P. aeruginosa might use an overlapping set of basic virulence genes for plant and animal hosts. Midribs were inoculated in triplicates with 10 ml of each strain (containing *10 6 bacteria).…”