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.

Figure 1

C. elegans killing by Gram-positive pathogens. (A) Survival of C. elegans fed on clinical isolates of S. pneumoniae (SP003), S. aureus (A003), E. faecalis (E002), S. pyogenes (GAS002), E. faecium (E007), and nonpathogenic B. subtilis (PY79). (B) Survival of C. elegans fed on lawns of E. faecalis (E001, E002, E006, and E009) and E. faecium (E003 and E007) clinical isolates. (C) Survival of C. elegans fed on lawns of Escherichia coli strain OP50 grown on BHI medium or nematode growth (NG) medium.

Figure 2

E. faecalis proliferates in the nematode gut. (A) Various ratios of E. faecalis V583 to E. faecium E007 were plated to form bacterial lawns. The survival of C. elegans fed on these lawns was measured. (B) C. elegans were placed on a lawn of E. faecalis V583 and E. faecium E007 in the ratio of 1:1,000. After 12 hr, the C. elegans were washed and moved to a plate of E007 (time 0). At the times indicated, nematodes were washed and ground, and dilutions of the resulting suspension were plated on selective media. The number of colony-forming units (cfu) of V583 (⋄) and E007 (♦) per worm was calculated. (C) The same protocol as described for B was followed except that two E. faecium strains with different drug sensitivities were used. C. elegans were initially plated on a lawn of E003 (▵) and E007 (▴) in a ratio of 1:1,000. Note that the error bars correspond to the standard deviations calculated from three experiments and are present on only the upper side of the curves because of the logarithmic scale.

Figure 3

Intact bacteria and gut distension visualized in C. elegans feeding on E. faecalis and E. faecium but not Escherichia coli or B. subtilis. Micrographs were taken of worms fed on lawns of bacteria on BHI plates for 3 days. Arrows point to the borders of the intestinal lumen. (A, E, C, and G) Beginning of intestinal tracts of worms fed on Escherichia coli, E. faecalis, E. faecium, and B. subtilis, respectively. In the upper right-hand corner of each image, a round structure is visible that corresponds to the grinder organ, which functions to disrupt ingested bacteria. (B, F, D, and H) Middle of the intestinal tracts.

Figure 4

Mammalian virulence factors enhance E. faecalis killing of C. elegans. (A) Survival of C. elegans placed on lawns of E. faecalis OG1X containing no plasmids (Cyl⁻, AS⁻), OG1X + pAM9058 (Cyl⁻ AS⁺), OG1X + pAM944 (Cyl⁺ AS⁻), and OG1X + pAM714 (Cyl⁺ AS⁺). (B) Survival of nematodes feeding on lawns of OG1RF or TX5266 (fsrBΔ). (C) Survival of mice infected peritoneally with 1.3 × 10⁸ cfu of OG1RF or 2.1 × 10⁸ cfu of TX5266 (fsrBΔ). (D) Survival of mice infected peritoneally with 2.20 × 10⁸ cfu of OG1RF or 2.86 × 10⁸ cfu of 1C-C9.