A simple model host for identifying Gram-positive virulence factors

Abstract

We demonstrate the use of the nematode Caenorhabditis elegans as a facile and inexpensive model host for several Gram-positive human bacterial pathogens. Enterococcus faecalis, Streptococcus pneumoniae, and Staphylococcus aureus, but not Bacillus subtilis, Enterococcus faecium, or Streptococcus pyogenes, kill adult C. elegans. Focusing our studies on the enterococcal species, we found that both E. faecalis and E. faecium kill C. elegans eggs and hatchlings, although only E. faecalis kills the adults. In the case of adults, a low inoculum of E. faecalis grows to a high titer in the C. elegans intestine, resulting in a persistent infection that cannot be eradicated by prolonged feeding on E. faecium. Interestingly, a high titer of E. faecium also accumulates in the nematode gut, but does not affect the longevity of the worms. Two E. faecalis virulence-related factors that play an important role in mammalian models of infection, fsr, a putative quorum-sensing system, and cytolysin, are also important for nematode killing. We exploit the apparent parallels between Gram-positive infection in simple and more complex organisms by using the nematode to identify an E. faecalis virulence factor, ScrB, which is relevant to mammalian pathogenesis. Gram-positive pathogens in the genera Streptococcus, Staphylococcus, and Enterococcus are leading causes of human infections, causing diseases such as pneumonia, meningitis, bacteremia, endocarditis, and necrotizing fasciitis. The genus Enterococcus, the main focus of this article, is particularly problematic because of multidrug resistance, including resistance to vancomycin, often the antibiotic of last resort. Nevertheless, only a limited number of enterococcal virulence-related factors have been described, including cytolysin (Cyl), a factor called aggregation substance (AS), a zinc metalloprotease (gelatinase), and fsr, a putative quorum-sensing system thought to be involved in gelatinase and͞or serine protease regulation (1-5). As the development of antibiotic resistance continues to erode one of the greatest advances in modern health care, it is crucial to identify bacterial targets that can form the basis of novel anti-infective therapies.One reason that relatively little is known about enterococcal virulence factors is that the mammalian models used to study enterococcal infections are cumbersome and expensive. Using a mammalian host to screen enterococcal mutant libraries for avirulent mutants, for example, would be prohibitively time consuming and expensive because of the large number of animals involved. Therefore, we have sought to develop alternative nonvertebrate hosts for Enterococcus and other Grampositive human pathogens.Previously, our laboratory and others have shown not only that the Gram-negative human pathogens Pseudomonas aeruginosa and Salmonella enterica kill the nematode Caenorhabditis elegans, but also that P. aeruginosa and S. enterica virulence factors required for mammalian pathogenesis also are required for efficient killing of C. elegans (6-10). In...

A Caenorhabditis elegans model of Yersinia infection: biofilm formation on a biotic surface

“…These include a variety of plant and animal pathogens such as Erwinia chrysanthemi, Agrobacterium tumefaciens, Shewanella frigidimarina, Photorhabdus luminescens, Xenorhabdus nematophilus (Couillault & Ewbank, 2002) and 'Microbacterium nematophilum' (Hodgkin et al, 2000). They also include bacteria pathogenic to humans, including the Gramnegative bacteria Pseudomonas aeruginosa, which kills the worm by one of three mechanisms -fast, slow or neurotoxin-mediated (Tan et al, 1999;Darby et al, 1999), Burkholderia pseudomallei, which kills via a neuromuscular endotoxin (O'Quinn et al, 2001), and Serratia marcescens, which also kills via a (different) toxin (Kurz & Ewbank, 2000); and the Gram-positive bacteria Enterococcus faecalis, Streptococcus pneumoniae and Staphylococcus aureus (Garsin et al, 2001).…”

Caenorhabditis elegans as a Model To Determine Fitness of Antibiotic-Resistant Salmonella enterica Serovar Typhimurium

“…Although mouse models have been used for these purposes, simpler invertebrates such as Caenorhabditis elegans have recently become more attractive for assessing the in vivo biological costs of antibiotic resistance (23,24). Many bacterial genes known to be required for mammalian pathogenesis are needed also in the nematode (1,9,11,15,16,27,28). Some bacterial pathogens, such as Salmonella enterica serovar Typhimurium are able to establish a persistent infection in the intestine of C. elegans, reducing the life span of the host.…”

Evaluation of Caenorhabditis elegans as the Host in an Infection Model for Food-borne Pathogens

“…In all the previous reports mentioned, the nematocidal assays were performed by introducing the worms onto NGM agar plates containing peptone, which would allow the pathogens to proliferate. The composition of the NGM was reported to influence the virulence of the pathogens 11) and metabolites produced by the bacteria from tryptophan in the medium were found to be fatal to the nematodes 2) . To exclude the e#ects of nutrients in the medium, the present study was performed on mNGM plates, which contained no bacterial nutrients.…”

“…To recover internal bacteria from the nematodes, the method of Garsin et al (2001) was used with some modifications 11) . Five worms were picked and the surface bacteria were removed by washing 4 times in 4-ml drops of M9 bu#er on agar plates.…”

“…C. elegans has also been reported as a successful model for the investigation of virulence-associated factors of human pathogens such as Burkholderia pseudomallei 10) , Cryptococcus neoformans 19) , Enterococcus faecalis 27) , enteropathogenic Escherichia coli 2,16) , Listeria monocytogenes 34) , P. aeruginosa 32) , Serratia marcescens 14) , Shigella flexneri 6) , Staphylococcus aureus 11,28) , and Vibrio vulnificus 9) .…”