Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

Abstract

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.

FIG. 1

Complementation of the killing defect in hcnC mutant MP507. (A) Restriction map of the hcnABC region, showing the locations and orientations of known genes hcnA, hcnB, hcnC, and exoY and of putative genes (unlabeled arrows), including a homologue of the conserved hypothetical E. coli protein gene ycnB. The solid triangle indicates the location of the mTn5-Tc transposon insertion in the hcnC mutant MP507. (B and C) Maps of the insertion regions in recombinant plasmids carrying the hcnABC region. The results of nematode killing assays for hcnC mutant MP507 carrying these plasmids are also shown. The open triangles indicate the orientations of the P_lac promoter in the pUCP18 vector. The killing percentages are averages based on three separate assays. MP507 carrying only the vector plasmid pUCP18 exhibited less than 1% killing.

FIG. 2

Direct exposure of C. elegans to cyanide gas. Wild-type (□) or egl-9 (○) nematodes were exposed to 1 μmol (A) or 4 μmol (B) of cyanide gas in sealed 10-cm-diameter petri plates. Worms were considered dead if they did not respond detectably when the assay plate was tapped repeatedly against the microscope stage. Each datum point represents the average level of killing based on three separate assays.

FIG. 3

Killing of C. elegans by hydrogen cyanide gas with and without exposure to bacteria. Wild-type (squares) or egl-9 (circles) nematodes placed on plates containing either no bacteria (open symbols) or hcnC mutant MP507 (solid symbols) were exposed to 1 μmol of hydrogen cyanide in sealed 10-cm-diameter petri plates. Each datum point represents the average based on triplicate experiments.

FIG. 4

Killing of C. elegans by hydrogen cyanide gas when bacteria were present but not in contact with the nematodes. Wild-type nematodes were exposed to 2 μmol (A) or 6 μmol (B) of hydrogen cyanide in sealed 10-cm-diameter petri plates when plates containing either no bacteria (□), a 24-h lawn of hcnC mutant MP507 (○), or a 24-h lawn of proC mutant MP506 (▵) were also present in the chamber but were not in contact with the nematodes.