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Previous Article | Next Article 
Journal of Bacteriology, November 2001, p. 6207-6214, Vol. 183, No. 21
Department of Genetics, University of
Washington, Seattle, Washington 98195-7360
Received 4 June 2001/Accepted 2 August 2001
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.
Pseudomonas aeruginosa is
a ubiquitous gram-negative bacterium that is virulent towards a wide
range of organisms, including bacteria, plants, nematodes, insects, and
mammals (5, 9, 17, 19, 35, 36, 41, 48, 49, 62). In humans,
P. aeruginosa chronically infects the lungs of most cystic
fibrosis patients, causes serious infections of burn wounds and eye
lesions, and causes systemic infections of immunocompromised
individuals (21, 29, 33, 39). The bacterium's pathogenic
versatility is reflected in its large arsenal of secreted and
surface-associated virulence factors and in the complexity of the
regulatory circuitry with which it controls these factors. Among the
specific virulence factors that it produces are adhesins, such as pili
and filamentous hemagglutinin (14, 39); protein toxins,
such as phospholipase, proteases, and ADP-ribosylating enzymes
(39, 64); and small-molecule poisons, such as phenazines,
rhamnolipid biosurfactant, and cyanide (4, 8, 44).
Additionally, the genome of P. aeruginosa boasts the highest
proportion of predicted regulatory genes of any of the bacterial
genomes sequenced to date (61), which is indicative of the
bacterium's remarkable ability to adapt and thrive in numerous
pathogenic and nonpathogenic environments.
Several model systems for Pseudomonas pathogenesis have been
developed recently, and numerous genes required for virulence towards
model hosts are also required for virulence towards mammals. For
example, mutants of P. aeruginosa PA-14 exhibiting reduced virulence towards Arabidopsis or Caenorhabditis
elegans also exhibit reduced virulence in a burned-mouse
infection model (49, 50, 62). In addition, a putative
Pseudomonas signal transduction gene cluster required for
full virulence towards Drosophila melanogaster also mediates
mammalian epithelial cell injury (19, 37). Such examples
help illustrate the value of using genetically tractable model
organisms to identify P. aeruginosa virulence determinants (24, 25, 40).
We recently described a virulence model in which P. aeruginosa PAO1 rapidly paralyzes and kills the nematode C. elegans (17). This killing, termed paralytic
killing, is mediated by a diffusible factor that is under
control of both the LasR and RhlR quorum sensing regulators. This
killing also requires a functional copy of the C. elegans gene egl-9. The EGL-9 protein, which is
strongly expressed in the nematode body wall and pharyngeal muscles,
has homologues in a wide range of organisms, including mammals and Drosophila (3, 22). Paralytic killing of
nematodes by strain PAO1 may be distinct from two modes of nematode
killing reported for strain PA-14 based on differences in gene and
growth condition requirements (17, 41, 62).
In this report we describe experiments designed to identify bacterial
factors that mediate paralytic killing of C. elegans by
strain PAO1. Our results indicate that hydrogen cyanide is the primary
toxic factor responsible for the phenomenon.
Strains, plasmids, growth media, and culture conditions.
The
P. aeruginosa strains used were PAO1 (34) from
the laboratory of B. Iglewski, PAO-R1, a lasR
mutant of PAO1 (26), two
pvd strains carrying transposon insertions in the PA2401 and PA2424 genes (provided by D. D'Argenio), and the mTn5-Tc
(20) insertion mutants listed in Table
1. The Escherichia coli
strains used were DH5
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6207-6214.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Pseudomonas aeruginosa PAO1 Kills
Caenorhabditis elegans by Cyanide
Poisoning
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(52) for plasmid construction and
SM10
pir (55) for conjugal suicide plasmid delivery. The
growth media used were brain heart infusion (BHI) agar (Difco), L agar
(52), skim milk agar (57), King's B medium
(38), and L broth. Plasmids were maintained in P. aeruginosa in media supplemented with 100 µg of carbenicillin
per ml and in E. coli in media supplemented with 100 µg of
ampicillin per ml or 40 µg of tetracycline per ml. To construct
plasmids used for hcn complementation, an 8,968-bp
XhoI fragment carrying the P. aeruginosa hcnABC
operon was gel purified from an
XhoI-BglII-ScaI digest of cosmid 011 (supplied by Matt Wolfgang and S. Lory), whose insert corresponds to
nucleotides 2,396,530 to 2,441,543 in the PAO1 single contig sequence
(www.pseudomonas.com). The XhoI fragment was cloned in both
orientations into the SalI site of pUCP18 (53)
to obtain pLG2 (Fig. 1) and pLG3. pLG3
was then digested with XbaI and religated to obtain pLG4
(Fig. 1). All constructs were confirmed by restriction analysis. For
hcnC complementation assays, MP507 transformed with either
pLG2, pLG4, or pUCP18 was tested in a standard worm killing assay after
growth in individual chambers (see below) on BHI agar supplemented with 40 µg of tetracycline per ml and 100 µg of carbenicillin per ml. Standard molecular biology protocols were used throughout
(52).
TABLE 1.
Mutants defective in paralytic killing
View larger version (13K):
[in a new window]
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
Plac 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.
Nematode paralytic killing assay. Unless indicated otherwise, all paralytic killing assays were carried out by spreading 150 µl of a 2- to 7-day-old P. aeruginosa colony suspended in BHI broth at an optical density at 660 nm (OD660) of ~0.1 onto a 3.5-cm-diameter BHI agar plate containing 4 ml of BHI agar. After the plate was incubated for 24 h at 37°C, N2 nematodes from stock plates were collected in M9 buffer, and a 50-µl aliquot (containing 20 to 200 adult animals) was spotted onto the P. aeruginosa lawn. The plate was then incubated for 4 h at room temperature with the lid on, and paralytic nematode killing was scored with a dissecting microscope. As described previously (17), worms were considered dead if they did not move spontaneously and did not respond detectably to tapping of the assay plate against the microscope stage. For experiments in which individual chambers were used (see below), each 3.5-cm-diameter plate was enclosed in a 10-cm-diameter petri plate, which was then either sealed with Parafilm (sealed chamber) or left unsealed (unsealed chamber).
Transposon mutagenesis of PAO1.
Most transposon insertion
mutants were generated by using transposon mTn5-Tc
(20). MP501 and MP551 were generated by using ISphoA/hah-Tc, a transposon Tn5 derivative that
will be described elsewhere (unpublished data), and MP508 was generated
by using Tn5 (18). For transposon mutagenesis,
a 37°C overnight aerated culture of E. coli SM10
pir/pUT-mTn5-Tc (20) or SM10
pir/pUT-IsphoA/hah-Tc grown in L broth
supplemented with 100 µg of ampicillin per ml was diluted 1:10 into
fresh L broth containing ampicillin and grown with aeration for 45 min
at 37°C. A 0.5-ml aliquot of this culture was mixed with 0.5 ml of a
42°C nonaerated overnight L broth culture of PAO1. The mixture was
filtered with a Nalgene analytical test filter (pore size, 0.45 µm)
and washed with 1 ml of 10 mM
Mg2SO4. The filter was then
removed from the apparatus, transferred to an L agar plate, incubated
at 37°C for 1 h to allow conjugation and transposition to occur,
and then transferred to a test tube containing 1 ml of L broth, and the
cells were washed from the filter by vortexing. Cells were plated onto
L agar containing 10 µg of chloramphenicol per ml to counterselect
for E. coli and 60 µg of tetracycline per ml to select for
growth of P. aeruginosa cells carrying transposon
insertions. Individual colonies appeared after 1 to 2 days of
incubation at 37°C.
Mutant screening. To screen for non-nematode-killing mutants, individual transposon insertion mutants were suspended in BHI broth at a density sufficient to make the broth visibly turbid. Then 150 µl of each suspension was plated onto a 3.5-cm-diameter BHI agar plate, and after 24 h of incubation at 37°C worm killing was assayed. Strains which exhibited at least a 10% reduction in killing compared to the wild type were saved and retested. Strains that arose from 37 independent mutagenesis events were screened.
DNA sequencing.
The chromosomal DNA flanking the transposon
insertions was sequenced after semirandom PCR amplification or cloning.
For semirandom PCR, a variation of a protocol described by Chun et al.
(12) was used. One microliter of a 50-µl boiled
single-colony suspension in distilled H2O was
used as the template DNA in a 20-µl PCR mixture containing primer
MTN5I.1 (5'-CGAGGGCTTTACTAAGCTG-3') and either primer CEKG
2A
(5'-GGCCACGCGTCGACTAGTACN10AGAG-3'),
CEKG 2B
(5'-GGCCACGCGTCGACTAGTACN10ACGCC-3'), or CEKG 2C
(5'-GGCCACGCGTCGACTAGTACN10GATAT-3');
1 µl of a 1:5 dilution of this reaction mixture was used as the
template DNA for a second PCR performed with primers MTN5O.1
(5'-ATTCGTCGACAAGCTTCGG-3') and CEKG 4 (5'-GGCCACGCGTCGACTAGTAC-3'). For the first reaction, the
thermocycler conditions were 94°C for 2 min, followed by six cycles
of 94°C for 30 s, 42°C for 30 s (with the temperature
reduced 1°C per cycle), and 72°C for 3 min and then 25 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 3 min; for the second reaction, the thermocycler conditions were 30 cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 3 min. Samples that produced distinct bands on an agarose gel after the
second reaction were cleaned with a PCR purification kit (Qiagen) and
sequenced by using primer MNT5S.1
(5'-GACAAGCTTCGGCCGCCT-3'). For cloning, chromosomal DNA digested with PstI was ligated into PstI-digested
pUC18 (73). The ligation mixture was electroporated into
DH5, and transformants were selected with tetracycline. The
chromosomal locations of the insertions were determined by BLAST
analysis of the transposon-adjacent chromosomal DNA sequences compared
with the complete PAO1 genome (www.pseudomonas.com).
Exoproduct assays. To measure cyanide production, we used a protocol modified from a protocol generously supplied by D. Haas and based on the method of Gewitz et al. (27). Strains were grown on 3.5-cm-diameter BHI agar plates in individual unsealed chambers for 24 h at 37°C and then enclosed without lids in individual sealed chambers which also contained a 1-ml reservoir of 4 M NaOH (in an inverted 3.5-cm-diameter plate lid). After 4 h of incubation at 30°C, the NaOH was collected and diluted to 0.09 M with double-distilled H2O. If necessary, the sample was further diluted with 0.09 M NaOH to bring the cyanide concentration to within the linear range of the detection procedure (0 to 10 µM). The cyanide in the sample was quantified by comparison with standards of KCN dissolved in 0.09 M NaOH: 105-µl aliquots of the samples were mixed with 350-µl aliquots of a fresh 1:1 mixture of 0.1 M o-dinitrobenzene (Sigma) in ethylene-glycol monomethyl ether (Sigma) and 0.2 M p-nitrobenzaldehyde (Sigma) in ethylene-glycol monomethyl ether. After exactly 30 min of incubation at the ambient temperature (22°C), the OD578 was measured.
Pyocyanin production was assayed by the method of Essar et al. (23): 24-h plate cultures were grown as described above for the nematode killing assay in unsealed individual chambers. The lawn-bearing agar from each plate was diced and extracted for 3 h with 4 ml of chloroform. The chloroform was then extracted with one-seventh volume of 0.2 M HCl, and the pyocyanin in the aqueous phase was quantified by measuring the OD520. Pyoverdine production was assayed by previously described methods (15, 60) by measuring the OD404, relative to that of pvd strains (generously supplied by D. D'Argenio), of cell-free supernatants from saturated overnight 37°C aerated cultures grown in King's B medium and adjusted for culture density (38). Exoprotease production was assessed by spotting 5-µl aliquots of cultures at an OD660 of ~0.1 onto skim milk agar plates, incubating the plates at 37°C overnight, and measuring zones of clearance from the edges of the growth spots.Treatment of nematodes with exogenous cyanide. For direct exposure to exogenous cyanide, nematodes were placed on a 3.5-cm-diameter BHI agar plate without a lid, and this plate was then sealed in a 10-cm-diameter petri plate containing an inverted 3.5-cm-diameter lid. The inverted lid contained separated 0.25-ml aliquots of 0.18 M HCl and a defined amount of KCN dissolved in 0.09 M NaOH. After the 10-cm-diameter plate was sealed, the aliquots were mixed by tipping the plate, thus acidifying the cyanide solution and releasing HCN gas. For experiments in which cyanide exposure in the presence of bacteria was examined (see Fig. 3), worms were placed on a standard 24-h pregrown lawn of bacteria rather than in an empty BHI agar plate.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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P. aeruginosa mutants with impaired ability to
kill C. elegans.
To help identify the substance or
substances produced by P. aeruginosa that are
toxic to C. elegans, we screened chromosomal mTn5-Tc transposon insertion mutants for reduced nematode
killing. Of approximately 3,000 mutants screened, 25 strains with
significant defects were recovered (Table 1). Slowly growing mutants
that formed small colonies on nutrient agar were not included in the analysis. The mutants could be grouped into two classes based on the
strength and reproducibility of their killing defects. Nine of the
mutants (class I) killed 
13% of the nematodes, whereas 16 of the
mutants (class II) killed 27 to 92% of the nematodes. The class
I mutants were quantitatively more reproducible than the class II
mutants in terms of the defects in killing observed in different
trials. Three of the mutant strains listed in Table 1 (MP508,
MP552, and MP553) exhibited abundant papillation of secondary colonies
upon prolonged incubation (several days) on rich media, suggesting that
there was a reduction in the viability of the parent strains with
outgrowth of fitter variants (data not shown).
Cyanide production strongly correlates with nematode killing. To verify that the killing defect in strain MP507 was due to inactivation of the hcnC gene rather than to polar effects of the transposon insertion, we complemented the HCN synthase defect by introducing the hcnABC gene cluster (lacking downstream open reading frames) in trans. The nematode killing phenotype was fully restored in the complemented mutant (Fig. 1), implying that paralytic killing truly depends on the hcn genes.
To determine whether other killing-defective mutants exhibited reduced cyanide production, we measured the level of cyanide generated by each strain under growth conditions mimicking those used to assay nematode killing (see above). As shown in Table 2, there was an excellent correlation between decreased cyanide production and reduced killing for both class I and class II mutants. These data implicate cyanide as a primary component of PAO1 virulence towards C. elegans.
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Cyanide alone is sufficient to kill C. elegans.
We next examined the response of C. elegans to hydrogen
cyanide alone at concentrations comparable to those produced by
bacteria. When exposed to 1 µmol of cyanide gas (HCN) in a sealed
chamber (see above), wild-type worms exhibited a gradual slowing of
movement, and more than 85% of the worms became fully immobile and
unresponsive to touch by 5 h after exposure began (Fig.
2A). By 10 h all of the worms were
immobile and unresponsive. In contrast, although mutant
egl-9 worms exhibited a sluggishness similar to that of wild-type worms soon after cyanide exposure began, they recovered completely within a few hours and remained fully viable. Cyanide gas
thus killed C. elegans with kinetics and genetic dependency similar to the kinetics and genetic dependency of P. aeruginosa-induced paralytic killing, in which complete killing of
wild-type worms but not egl-9 worms occurs after 4 h of
exposure to bacteria (17). The 1 µmol of HCN used in
this protocol approximates the ~300 nmol recovered from wild-type
bacteria grown under standard worm-killing conditions (Table 2).
|
Cyanide as the sole toxic component.
Although the results
described above indicated that the amount of cyanide normally produced
by PAO1 should be enough to kill C. elegans (Table 2 and
Fig. 2A), we wondered whether additional factors produced by the
bacteria contribute to the killing. To address this possibility, we
compared the kinetics of killing by cyanide gas when the worms were
placed on a lawn of hcnC mutant bacteria and on agar lacking
bacteria. As shown in Fig. 3, the effect
of the hcnC mutant bacteria on the kinetics of the response to cyanide was negligible for both wild-type and egl-9
nematodes. The bacteria thus did not augment the toxicity of the
cyanide added, suggesting that hydrogen cyanide alone can kill
nematodes.
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Phenazine production does not correlate with nematode killing. Recent studies of a form of killing (fast killing) of C. elegans by a different strain of P. aeruginosa (PA-14) showed that production of the blue phenazine pigment pyocyanin occurred in a subset of mutants defective in killing (41). Phenazines are redox-active compounds secreted by pseudomonads, and pyocyanin is the characteristic phenazine produced by P. aeruginosa (23, 31, 66). To examine the potential involvement of phenazines in nematode killing by strain PAO1, we measured the amount of pyocyanin produced by our mutants under the growth conditions used to assay killing. As shown in Table 2, only about one-half of the mutants were defective in production of pyocyanin. Curiously, the hcnC mutant (MP507) produced more than twice as much pyocyanin as its parent. These data show that there was not a strong correlation between reduced pyocyanin production and loss of virulence towards nematodes in the assay described here.
Defects in pyocyanin production in some of the non-worm-killing mutants may simply reflect pleiotropic effects of the mutations (51, 70). To further assess pleiotropy in the killing-defective strains, we assayed production of total secreted protease (7) and the secreted siderophore pyoverdine (13, 43). Of the 14 mutants examined, 7 showed reduced pyoverdine production and 2 showed reduced secreted protease production (Table 2). One of the nine class I mutants, the hcnC mutant, was exceptional in that reduced pyocyanin, pyoverdine, or secreted protease production was not evident.| |
DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this report we describe experiments in which we investigated the mechanism by which P. aeruginosa PAO1 rapidly paralyzes and kills C. elegans (17). Our results imply that the poison hydrogen cyanide is the sole or primary bacterial factor responsible for killing of the nematode. That cyanide is necessary for the virulence is implied by the finding that 17 transposon insertion mutants impaired in worm killing, including 1 mutant in which inactivated hydrogen cyanide synthase itself was inactivated, all exhibited reduced cyanide production. That cyanide is sufficient for nematode killing is implied by the finding that exposure to exogenous cyanide at levels comparable to that produced by the bacteria kills nematodes with kinetics similar to those observed with bacteria. Furthermore, a nematode mutant (egl-9) resistant to P. aeruginosa killing was also resistant to killing by exogenous HCN.
Hydrogen cyanide is a typical pseudomonad secondary metabolite, a compound which is not required for growth, energy storage, or primary metabolism but which may provide some ecological advantage to the organism (67). In addition to cyanide, the pseudomonad secondary metabolites include siderophores, such as pyoverdine; redox-active compounds, such as phenazines; and polyketide antibiotics (10). Cyanide is produced in Pseudomonas strains by oxidative decarboxylation of glycine by the three-subunit membrane-bound flavoenzyme encoded by hcnABC (4). P. aeruginosa produces HCN maximally in the late exponential and early stationary phases under microaerophilic conditions (4), and transcription of the hcn genes appears to depend directly on the quorum sensor regulators LasR and RhlR, as well as the anaerobic regulator Anr (7, 47, 70). Additional components of the complex regulatory circuitry controlling the production of cyanide and other secondary metabolites have been identified (2, 11, 42, 46, 71).
The mutations that we identified which reduced cyanide production and virulence towards C. elegans affect a variety of regulatory and metabolic functions (Table 1). Two of the regulatory mutations affect quorum sensing indirectly; one is in gacS, which encodes a two-component sensor that influences autoinducer levels (51), and the other is in a locus (PA2587) needed for synthesis of a quinolone signal required for RhlI-RhlR function (E. Pesci, personal communication). Mutations in three additional putative regulators were also identified; one of these regulators (PA3946) is homologous to the Bordetella pertussis virulence regulator BvgS (1), another (PA1003) belongs to the LysR family (32), and the third (PA4725) resembles a two-component sensor fused to a membrane permease. A mutation affecting PA1003 was previously identified in a study to screen for mutations that reduce virulence towards Arabidopsis (50). The mutations affecting metabolic functions inactivate enzymes that participate in central carbon metabolism, fatty acid breakdown, and proline biosynthesis. With the notable exception of the mutation of the HCN synthase mutant, all of the strongest (class I) non-worm-killing mutations reduced the production of pyocyanin, pyoverdine, or secreted protease. This pleiotropy would have made identification of cyanide as the worm-killing poison difficult if the hcnC mutant had not been isolated. An unanticipated benefit of the genetic approach taken in this study is that it appears to have identified several new regulatory and metabolic components of the circuitry controlling the production of secondary metabolites.
Studies of a different strain of P. aeruginosa (PA-14)
showed that about one-half of a collection of transposon insertion mutations that eliminated a fast-killing form of virulence towards C. elegans also reduced production of pyocyanin, as did a
constructed deletion mutation (
phnAB) that decreased
phenazine biosynthesis (41). The results were interpreted
in terms of a model in which phenazines are one component of a
multifactorial killing process. Phenazines are toxic to a
variety of cell types and are thought to act by generating reactive
oxygen species by redox cycling (59). For strain PAO1, we
found no convincing indication that pyocyanin or any other phenazine
plays a direct role in killing C. elegans. Although five of
nine strongly avirulent mutants produced significantly less pyocyanin
than the parent, the reduction in production is readily explained by
the pleiotropy of the mutations (Table 2). Indeed, two of the mutations
affect regulators (LasR and GacS) already known to be required for
expression of multiple genes, and a third affects an enzyme of central
carbon catabolism in Pseudomonas (Entner-Douderoff aldolase)
whose loss might also be expected to be highly pleiotropic (51,
63, 70). Furthermore, since exposure of nematodes to HCN in the
absence of bacteria reproduced the nematode paralytic killing
phenomenon, no additional bacterial substances are required.
The classic cellular target of cyanide inhibition is cytochrome oxidase, although other metalloenzymes are also sensitive to the poison (58). Inhibition of mitochondrial respiration can easily account for the rapid and dramatic paralytic killing of nematodes by P. aeruginosa PAO1. Pseudomonads appear to protect themselves from cyanide poisoning by expressing an unusual cyanide-resistant cytochrome oxidase (16). Studies of human and animal cyanide poisoning indicate that the poison strongly affects neurological tissue (69), and it is possible that nematode killing also reflects hypersensitivity of neuromuscular tissues to the poison.
It is striking that loss-of-function mutations in a single nematode gene (egl-9) confer strong resistance to cyanide poisoning. The mechanism underlying this resistance is mysterious. Since HCN is predominantly uncharged at physiological pH (pK 9.3) and is expected to diffuse freely through membranes, it appears unlikely that a cyanide transporter is eliminated by the mutations. One possibility is that the egl-9 mutations constitutively activate an adaptive response to hypoxia (54), thus conferring some resistance to cytochrome oxidase inhibition by cyanide. Another possibility is that reactive oxygen species generated by cyanide inhibition activate an Egl-9-dependent pathway, such as a stress-dependent MAP (mitogen-activated protein) kinase pathway (65), leading to paralysis and death. Homologues of egl-9 exist in humans (3, 22) and may represent potential therapeutic targets for countering the toxic effects of cyanide.
Cyanide is a potent poison expected to be active against most eukaryotic species (4, 58). This compound thus could contribute profoundly to the broad pathogenic host range of P. aeruginosa (5, 9, 36, 40, 45). It is thought that cyanide inhibition of fungal growth helps account for the suppression of several plant root and leaf fungal diseases (30, 68). The activities of cyanide and other small-molecule poisons may also contribute to the pathogenesis accompanying the variety of opportunistic infections caused by P. aeruginosa (39). Although the role of cyanide in Pseudomonas pathogenesis in humans is largely unexplored, an early study of burn infections detected this poison (28). The recent finding that sputa of cystic fibrosis patients contain P. aeruginosa in the appropriate quorum-sensing physiological state to produce cyanide (56) suggests that the poison could also contribute to the tissue destruction that accompanies lung infections in this disease.
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ACKNOWLEDGMENTS |
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We thank Jeannie Bailey, Chris Cosma, Dave D'Argenio, Creg Darby, Steve Lory, Maynard Olson, James Thomas, and Mike Vasil for helpful discussions; Christina Buchanan, Brian Buchwitz, Denise Gaunt, and Allyson McCormick for assisting with the mutant screening; Everett Pesci for providing unpublished data; and Dieter Haas for sharing the cyanide quantification protocol.
This research was supported by a grant from the Cystic Fibrosis Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Genetics, University of Washington, Box 357360, Seattle, WA 98195-7360. Phone: (206) 543-7800. Fax: (206) 543-0754. E-mail: manoil{at}u.washington.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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) 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.
) were also present in the chamber but were not in contact with the
nematodes.