See the article "The identification of drug
targets for a given human disease, whether it is mainly environmental
or genetic in origin, rests on an understanding of the molecular chain
of events that unfold in the disease process. Anatomic pathology,
biochemistry, cellular physiology, and pharmacology constitute the main
traditional approaches towards identifying potential therapeutic
targets. Genetic approaches, by determining the phenotypic consequences
of mutations in genes and ordering these genes into functional
pathways, are uniquely powerful in identifying novel gene products
involved in a disease process. By characterizing mutations that block
or reverse the disease phenotype, genetics can provide a direct route
to target identification. The wild-type versions of these
“suppressor” gene products are potential therapeutic targets,
because chemical compounds that phenocopy suppressor gene mutations
should similarly block the disease phenotype and thus constitute
candidate therapeutic drug leads (1). Such genetic approaches to target
identification are most feasible in well-studied model organisms with
short generation times that are easily maintained in the laboratory,
principally the yeast Saccharomyces cerevisiae, the soil
nematode Caenorhabditis elegans, and the
fruit fly Drosophila melanogaster. Using model organisms to
study homologues of genes causally mutated in human disease is
relatively well established. This approach has not often been applied
to human infectious diseases, however, as most human pathogens have a
highly restricted host range. An exception is the Gram-negative
bacterium Pseudomonas aeruginosa, strains of which are
pathogenic not only to humans but also to C. elegans,
Drosophila, and the genetically tractable model plant,
Arabidopsis thaliana (2, 3). In the December 21 issue of
PNAS, Darby et al. (4) describe a genetic approach toward
the identification of potential therapeutic targets for P.
aeruginosa infection.
P. aeruginosa is a common bacterium in soil and water
worldwide and an opportunistic pathogen in humans, causing acute and
chronic infections in patients with compromised immunity, severe burns,
and cystic fibrosis. A variety of virulence factors, including secreted
enzymes as well as toxic chemicals, contribute to the pathogenesis of
P. aeruginosa infections (for review see ref. 5). The
production and secretion of most known virulence factors is increased
at high versus low bacterial cell density, through a cell-to-cell
signaling mechanism known as “quorum sensing” that links cell
density to gene expression (for review see refs. 6 and 7). In P.
aeruginosa two quorum-sensing systems have been described in some
detail, las and rhl. Each is composed of two components: an inducer
locus, lasI or rhlI, that controls the synthesis
of a diffusible, cell-permeant, pheromone and a responder locus,
lasR or rhlR, that encodes a transcription factor
that binds to and is activated by the pheromone. Activation of LasR
enhances the expression of genes encoding secreted proteases,
phospholipases, ADP-ribosylating enzymes, and genes that control
secretion of virulence factors as well as LasI (thus providing positive
feedback to LasR activation) and RhlR. Activation of RhlR controls some
genes in parallel to LasR and, in addition, activates the expression of
loci responsible for the production of toxic chemicals, including
hydrogen cyanide, rhamnolipids, and phenazines. LasR and RhlR are
further subject to higher-level positive and negative regulators (8,
9).
Two recent PNAS papers extend and expand our knowledge of quorum
sensing. First, Whiteley et al. (10) report the isolation of
47 mutants in 39 different genes that are up-regulated by treatment
with P. aeruginosa pheromones. The authors note that their
screen is far from saturated and speculate that 1–3% of the total
5000–6000 genes of P. aeruginosa are controlled by quorum
sensing. Second, Pesci et al. (11) describe a third
cell-to-cell signaling molecule produced by P. aeruginosa,
2-heptyl-3-hydroxy-4-quinolone (a chemical unrelated to the las and rhl
pheromones, which are acyl homoserine lactones), that regulates the
expression of virulence factors. Synthesis of this new signaling
molecule requires both LasR and RhlR. These studies highlight the
potential value of targeting the quorum-sensing regulatory hierarchy
for new anti-infective therapies (7, 12).
The complexity and long life cycles of mammalian models have limited
the understanding of host factors involved in microbial pathogenesis.
To overcome these limitations, Rahme, Ausubel, and colleagues pioneered
the use of model organisms to identify “universal” virulence
factors for P. aeruginosa pathogenicity. Infection of
Arabidopsis thaliana with P. aeruginosa strain
PA14, a pathogen derived from a human infection, caused soft-rot,
chlorosis, and eventual leaf collapse, and the same strain caused
lethal infections in a mouse full-thickness skin burn model (2, 13).
Three recent papers from this same laboratory describe PA14
pathogenesis in C. elegans (3, 14, 15). In the nematode, two
types of toxicity were observed. Exposure of L4 stage larvae, but not
adults, to PA14 grown on nutrient-rich media at high osmotic strength
killed the nematodes in 4 hr (“fast killing”), whereas exposure
of either L4 or adults to PA14 grown on less rich or minimal media
killed after 1 to 2 days (“slow killing”). Screening for
Pseudomonas mutants with reduced pathogenicity in
Arabidopsis or in slow or fast killing of C.
elegans identified mutations in 23 genes, 19 of which also reduced
virulence in the mouse burn model (refs. 13–15; for review see ref.
16). Because most of these genes were identified by only a single
mutation, this screen also appears to be far from saturated.
Now, Darby et al. (4) describe a third model of
Pseudomonas pathogenesis in C. elegans. Exposure
of adult C. elegans to P. aeruginosa strain PAO1
(a standard laboratory strain isolated some 50 years ago from a human
infection) inhibited feeding of the nematodes within seconds to
minutes, more gradually slowed and disorganized locomotory behavior and
caused paralysis and death after several hours. These effects were not
observed when C. elegans was exposed to PAO1 derivatives
bearing a mutation in the quorum-sensing gene lasR or
rhlR. Mutations in genes encoding secreted enzymes
controlled by quorum sensing, however, had no effect in this model,
suggesting that one or more of the toxic chemicals regulated by quorum
sensing is responsible for neuromuscular toxicity. Several outstanding
questions remain regarding the mechanism of PAO1-induced paralysis:
First, does toxicity result from the action of a single toxin or is
more than one component required? And what is the chemical nature of
the toxin(s)? Second, what genes are responsible for production of the
toxin(s)? And what are their functions? Given that the genome of
P. aeruginosa (PAO1) is completely sequenced and that
powerful screens for virulence factors and for genes controlled by
quorum sensing are available, the identity of the genes responsible for
toxicity in this model is likely to be forthcoming soon. The time and
effort required to determine the chemical nature of the toxin(s), if
there are no breakthrough insights from the genetic studies, are more
difficult to predict.
Darby et al. (4) further describe nematode mutants that
resist the lethal effects of exposure to PAO1. Two strongly resistant
strains were identified from among 8,000 mutagenized genomes. Genetic
analysis revealed that both strains carry recessive mutations in a
known gene, egl-9, previously identified in screens for
egg-laying-defective mutants (17). Two additional egl-9
alleles isolated on the basis of their egg-laying phenotype are also
resistant to P. aeruginosa-induced paralysis. Darby et
al. (4) cloned the egl-9 gene and showed that the
molecular lesions in the four mutations most likely generate
nonfunctional EGL-9 proteins. These results are consistent with a model
in which exposure to P. aeruginosa PAO1 induces paralysis
and death by excessive or inappropriate activation of EGL-9. Because
egl-9 function appears to be nonessential for C.
elegans (in the comfortable confines of the laboratory), strains
that lack egl-9 activity are both viable and resistant to
PAO1-induced paralysis. It would be interesting to know whether
egl-9 is the only gene that can mutate to give resistance to
Pseudomonas-induced toxicity. The experiments performed so
far do not survey a sufficiently large number of genomes to reliably
answer this question.
EGL-9 is a novel 723 amino acid protein with no obvious signal sequence
or other readily identified functional motifs. EGL-9 is homologous to
SM-20, a 355 amino acid protein identified as an “immediate early
gene” induced upon treatment of rat aortic smooth muscle cells with
a mixture of growth factors (18). SM-20 appears to be a cytoplasmic
protein localized to multiple types of muscle cells, scattered
epithelial cells, and neurons (19), which resembles the expression
pattern of EGL-9 in C. elegans (4). The function of SM-20,
however, remains unknown. Furthermore, the amino acid similarity is
limited to a domain in the C-terminal half of the predicted EGL-9
protein. Whether alternative splicing of egl-9 could produce
an SM-20-sized protein product, or whether SM-20 or another mammalian
homologue contains sequences homologous to the N-terminal half of
EGL-9, remain to be seen. Of note, a preliminary search (L.X.L) of the
emerging but unannotated Drosophila genome reveals fly genes
with homology to EGL-9 and SM-20. Clearly, the completion of genomic
sequencing in metazoans besides C. elegans will ease the
identification and assignment of homologous and orthologous genes.
The genetic approach to selection of human therapeutic targets
involves, as a start, the identification of mutations that suppress a
surrogate disease phenotype in a genetically tractable organism.
Loss-of-function mutations of egl-9 block P.
aeruginosa-induced paralysis of C. elegans. Might
inhibition of an EGL-9 homologue, possibly SM-20, similarly block
Pseudomonas-induced pathology in infected humans?
Accumulated knowledge suggests that P. aeruginosa
pathogenesis in human disease involves multiple virulence factors and
pathogenic mechanisms. Accordingly, a therapeutic strategy focused on a
single mechanism is not likely to provide a completely satisfactory
treatment. Nonetheless, the possibility that it might provide valuable
supportive or ancillary therapeutic effects remains to be tested.
Traditionally, antimicrobial drug discovery has entailed
screening candidate compounds directly on target microorganisms. The
clear scientific advance represented by the dual model organism
infection systems described above is the capacity to dissect specific
interactions by using both pathogen and host genetics. These studies
can be expected to yield both novel and “broad host spectrum”
bacterial virulence factors and toxins, and to help elucidate
mechanisms of toxicity and identify targets of toxin action. On the
basis of these studies, one can envision microtiter plate-based
high-throughput screens using a pathogenic Pseudomonas
strain and susceptible C. elegans nematodes to identify
chemical compounds that “cure” host pathology regardless of
whether the compound blocks bacterial virulence or a deleterious
host response. Of more direct utility, however, may be an enhanced
focus on identifying chemical suppressors of P. aeruginosa
virulence factor expression or secretion as candidate novel
antibiotics, with Arabidopsis, C. elegans, and the mouse as
confirmatory models. Finally, numerous microbial toxins have been
invaluable as selective probes of biological function, such as tetanus,
botulinum, pertussis, cholera, and diphtheria toxins. Because
egl-9 appears to regulate neuromuscular activity, dissection
of the Pseudomonas PAO1–C. elegans egl-9
interaction may reveal as much, if not more, about the control of
muscle contraction or neuronal signaling as it reveals about microbial
virulence.