Note: Descriptions are shown in the official language in which they were submitted.
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The invention relates to screening methods for identifying pathogen
virulence factors and for identifying drugs that inhibit pathogen infections.
Microbial pathogens use a variety of complex strategies to subvert host
cellular functions to ensure their multiplication and survival. Some pathogens
that have co-evolved or have had a long-standing association with their hosts
utilize finely tuned host-specific strategies to establish a pathogenic
relationslup.
During infection, pathogens encounter different conditions, and respond by
expressing virulence factors that are appropriate for the particular
environment,
host, or both.
Although antibiotics have been effective tools in treating infectious
disease, the emergence of dntg resistant pathogens is becoming problematic in
the
clinical setting. New antibiotic or antipathogenic molecules are therefore
needed
to combat such drug resistant pathogens. Accordingly, there is a need in the
art
for screening methods aimed not only at identifying anal characterizing
potential
antipathogenic agents, but also for identifying and characterizing the
virulence
factors that enable pathogens to infect and debilitate their hosts.
We have discovered that the microbial pathogen, Salmonella typhimurium,
establishes a long-lasting, persistent infection in the nematode,
Caenorhabditis
elegaus. This discovery enables simple screening methods for identifying the
interplay betyveen environmental and host signals (e.g., host-dependent or
host-
independent signals) and physiological pathogenic pathways that control or
regulate genes responsible for establishing a persistent infection, as in the
colonization of the gut of the nematode.
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In one aspect, the invention features an isolated nematode persistently
infected with an isolated pathogen. Preferably, the nematode is C. elegans
(such
as a one-day old adult hermaphrodite worm or an L4 larval stage worm). In
preferred embodiments, the isolated pathogen expresses a detectable marlcer;
or
colonizes the intestine of the nematode. In still other preferred embodiments,
the
pathogen is Salmonella (such as Salmonella typhimuYium strain SL1344 or strain
LT2).
In another aspect, the invention features a method of screening for a
virulence factor that enables a pathogen to develop a persistent infection in
a
nematode. The method generally involves the steps of: (a) exposing a nematode
to a mutagenized pathogen, a pathogen expressing a gene not normally present
in
the pathogen, or a pathogen overexpressing a pathogen gene; (b) determining
whether the mutant or otherwise altered pathogen persistently infects the
nematode, where a reduction or enhancement of disease in the nematode relative
to that caused by the non-mutagenized or otherwise altered pathogen indicates
a
mutation in a virulence factor or a virulence factor gene that enables the
pathogen
to develop a persistent infection in the nematode; and (c) using the mutation
or
virulence factor gene as a marker for identifying the virulence factor.
Preferably, the nematode utilized in the method of screening for a
pathogenic virulence factor is Caefaoy~habclitis elegafZS (such as a one-day
old
adult hermaphrodite worm or an L4 larval stage worm). In preferred
embodiments, the mutated or otherwise altered pathogen used for identifying
the
virulence factor expresses a detectable marker. W other preferred embodiments,
.
colonization of the intestine of the nematode by the mutated or otherwise
altered
pathogen is decreased. In still other preferred embodiments, the pathogen used
is
Salmonella (such as Salmoyaella typhzf~zu~ium strain SL1344 or strain LT2).
In yet other preferred embodiments, the method utilizes a salmonellae/C.
elega~s killing assay. In such methods, the mutated or otherwise altered
pathogen
has reduced or enhanced capacity to develop a persistent infection in C,
elegahs,
causing less or more killing than the non-mutagenized or otherwise altered
pathogen.
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In another aspect, the invention features a method of screening for a
compound that inhibits a persistent pathogenic infection in a nematode. The
method generally involves the steps of: (a) providing a nematode persistently
infected with a pathogen; (b) contacting the persistently infected nematode
with a
test compound; and (c) determining whether the test compound inhibits the
persistent infection in the nematode.
Preferably, the nematode utilized in the compound screening method is
Caeuo~habditis elegans (such as a one-day old adult hermaphrodite worm or an
L4 larval stage worm). In preferred embodiments, the pathogen used in the
compound screening assay expresses a detectable marker. In other preferred
embodiments, colonization of the intestine of the nematode by the pathogen is
reduced. In still other preferred embodiments, the pathogen used is
Salnaofaella
(such as Salmonella typhimurium strain SL1344 or strain LT2).
In yet other preferred embodiments, the test compound is provided in a
compound library; is a small organic compound; or is a peptide,
peptidomimetic,
or an antibody or fragment thereof.
In other preferred embodiments, the compound screening method utilizes
a salmonellae/C. elegans killing assay. In such methods, the pathogen that
persistently infects C. elegayas causes less killing in the presence of the
test
compound than in the absence of the test compound.
In another aspect, the invention features a method of screening for a
virulence factor that enables a pathogen to develop a persistent infection in
a
nematode. The method generally involves the steps of: (a) exposing a nematode
to a pathogen expressing a detectable marker, the pathogen being mutagenized
expressing a gene not normally expressed by the pathogen, or overexpressing a
pathogen gene; (b) determining whether the mutant pathogen persistently
infects
the nematode by measuring the level of detectable marker in the nematode,
where
a decrease or increase of the marker in the nematode relative to that caused
by the
non-mutagenized pathogen indicates a mutation in a virulence factor or a
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virulence factor gene that enables the pathogen to develop a persistent
infection in
the nematode; and (c) using the mutation or virulence factor gene as a marker
for
identifying the virulence factor.
In another aspect, the invention features a method of screening for a
compound that inhibits a persistent pathogenic infection in a nematode. The
method generally features the steps of (a) providing a nematode persistently
infected with a pathogen expressing a detectable marker; (b) contacting the
persistently infected nematode with a test compound; and (c) determining
whether
the pathogen persistently infects the nematode by measuring the level of
detectable marker in the nematode, where a decrease of the detectable marker
in
the nematode indicates that the test compound inhibits a persistent pathogenic
infection in the nematode.
Exemplary pathogenic bacteria useful in the methods of the invention, as
well as for producing persistently infected nematodes, include, without
limitation,
1 S Aerobacte~, Ae~omonas, Acinetobacter, Ag~obacteYium, Bacillus,
Bacte~oides,
Ba~toraella, Bo~detella, Bortella, Bo~~elia, Brucella, BuYkholderia,
Calymmatobacterium, Campylobacter, Citrobacter, Clostridium,
Co~nyebacterium, EnteYObacter, Enterococcus, Escherichia, F~ahcisella,
Gardnerella, HaemoplZilus, Hafnia, Helicobacte~, Klebsiella, Legionella,
Liste~ia, Morganella, Mo~~axella, Mycobacterium, Neisse~ia,
Pasteuf°ella,
P~oteus, P~ovide~zcia, Salmonella, Serr atia, Shigella, Staphylococcus,
Streptococcus, Ste~cto~ophomoyaas, Ti eponema, Xanthomohas, hiby~io, and
YeYSinia.
By "virulence factor" is meant a cellular component (e.g., a protein such
2S as a transcription factor or a molecule) without which a pathogen (e.g., a
bacterium) is incapable of causing disease or infection in a eukaryotic host
organism (e.g., a nematode or mammal). Such components, for example, are
involved in the adaptation of the bacteria to a host (e.g., a nematode host),
establishment of a bacterial infection, maintenance of a bacterial infection,
or
generation of the damaging effects of the infection to the host organism.
Further,
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the phrase includes components that act directly on host tissue, as well as
components which regulate the activity or production of other pathogenesis
factors.
By "inhibits a pathogen" is meant the ability of a test compound to
decrease, suppress, attenuate, diminish, arrest, or stabilize the development
or
progression of a pathogen-mediated disease or infection in a eulcaryotic host
organism. Preferably, such inhibition decreases pathogenicity by at least 5%,
more preferably by at least 25%, and most preferably by at least 50% or more,
as
compared to symptoms in the absence of the test compound in any appropriate
pathogenicity assay (for example, those assays described herein). In one
particular example, inhibition may be measured by monitoring pathogenic
symptoms in a nematode persistently infected with a salmonellae pathogen
exposed to a test compound or extract, a decrease in the level of pathogenic
symptoms relative to the level of symptoms in the host organism not exposed to
the compound indicating compound-mediated inhibition of the salmonellae
pathogen.
By "persistent infection" or "persistently infected" is meant an invasion or
colonization of a host animal (e.g., nematode) by a pathogen (e.g.,
Sal~cohella)
that is damaging to the host, where the size of the persistent pathogenic
population that are associated with the host after the host has been
transferred to a
non-infectious environment remains at least 30%, preferably 50%, more
preferably 80%, a~zd most preferably 90%, or even 95% to 99% of the size of
the
pathogenic population before the transfer of the host to a non-infectious
environment. Such an infection also includes an increase in the numbers of the
pathogenic population that are associated with the host when the host is first
exposed to a relatively small number of the pathogen mixed with an excess of
non-pathogenic bacteria after which the host is transferred to a non-
infectious
environment. A persistent infection is typically measured using a nematode
feeding assay (as described herein) where bacteria are assayed for their
ability to
establish a long-lasting association in the worm intestine.
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By "detectable marlcer" is meant a gene whose expression may be assayed;
such genes include, without limitation, j3-glucuronidase (GUS), luciferase
(LUC),
chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), and
(3-galactosidase.
By "isolated nematode" is meant a nematode that is purified from
contaminating organisms and maintained in a culture. Exemplary nematodes
(wild type or mutant), such as C. elegans, are obtained from publicly
available
sources or purified from the environment according to standard methods known
in
the art.
By "isolated pathogen" is meant a microbial strain that has been cultured,
through the actions of man, and that elicits a disease response in a host.
The present invention provides a number of advantages. For example, the
invention facilitates the identification of novel targets and therapeutic
approaches
for preparing therapeutic agents active on virulence factors and genes that
enable
a pathogen to develop a long-lasting, persistent infection in its host
organism.
The invention also provides long awaited advantages over a wide variety
of standard screening methods used for distinguishing and evaluating the
efficacy
of a compound against salmonellae pathogens. In one particular example, the
screening methods described herein allow for the simultaneous evaluation of
host
toxicity as well as anti-salinonellae potency in a simple iy2 vivo screen.
Moreover,
the methods of the invention allow one to evaluate the ability of a compound
to
inhibit salmonellae pathogenesis, and, at the same time, to evaluate the
ability of
the compound to stimulate and strengthen a host's response to salinonellae
pathogenic attaclc.
Accordingly, the methods of the invention provide a straightforward
means to identify compounds that are both safe for use in eul~aryotic host
organisms (i.e., compounds which do not adversely affect the normal
development and physiology of the organism) and efficacious against pathogenic
microbes that establish persistent infections in their hosts. In addition, the
methods of the invention provide a route for analyzing virtually any number of
compounds for anti-salmonellae pathogenic effect with high-volume throughput,
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high sensitivity, and low complexity. The methods are also relatively
inexpensive
to perform and enable the analysis of small quantities of active substances
found
in either purified or crude extract form. Furthermore, the methods disclosed
herein provide a means for identifying compounds that have the capability of
S crossing eul~aryotic cell membranes and which maintain therapeutic efficacy
in an
ih vivo method of adxninistration. In addition, the above-described methods of
screening are suitable for both lcnown and unl~nown compounds and compound
libraries.
Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments thereof, and from the
claims.
The drawings will first be described.
Dravvin~s
Fig. 1A shows the mechanism C. elegaus billing by S. typhimurium strain
1S SL1344. Between 10 to 20 worms were placed on each plate and each assay
consisted of two replicates. L4 stage (open circle) and 1-day-old adult
hermaphrodite (closed triangle and circles) worms fed either on S.
typhimu~iuyra
SL1344 (closed triangle and open circle) or on E. coli OPSO (closed circle).
The
small bars represent the standard deviations and the results axe
representative of at
least six independent experiments.
Fig. 1B shows the results of a C. elegaris shifting experiment and the
percentages of dead worms after transfer to OPSO-containing plates after
feeding
for S hours on SL1344 (open circle) or on OPSO (closed circle). The small bars
represent the standard deviations and the results are representative of at
least three
2S independent experiments. The insert shows the percentages of dead worms
after
transfer to OP50-containing plates after feeding for 1, 3, or S hours on
SL1344.
The small bars found in the insert represent the standard deviations and the
'results
are representative of at least two independent experiments.
Fig. 2 shows confocal images of bacterial colonization of the C. elega~as
intestine. The worms were fed on E. coli expressing GFP (E. colilGFP) (Panels
A
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and B), S. typhimurium expressing GFP (S. typhimu~iumlGFP) (Panels C and D)
for 72 hours, or on P. ae~uginosa expressing GFP (P. ae~uginosalGFP) (Panels E
and F) for 24 hours. In the transmission images (Panels A, C, and E), the
intestine margins are noted with arrows. Merged images (B, D, and F) show the
S bacterial fluorescence (green channel) and the gut autofluorescence (red
channel).
The bars represent SO ~,m.
Fig. 3 shows confocal images of bacterial colonization of the C. elegaras
intestine in a shifting experiment. The worms were fed on OPSO for 24 hours
after feeding on E. coli expressing GFP (E. coli/GFP)(panel A) or S.
typhimurium
expressing GFP (S. typhimuYiumlGFP)(panels B and C) for 5 hours. The merged
images show the bacterial fluorescence (green channel) and the gut
autofluorescence (red channel). The bars represent 50 ~,m.
Fig. 4A shows that s typlaimu~ium proliferates in the intestine of C.
elegans. Several dilutions of S. typhimurium in E. coli were prepared on NG
1 S plates, and 10 worms were immediately place on the plates. Each assay
consisted
of two replicates. After 24 houa-s, the worms were transferred to new plates
and
the amount of Salmonella in the plates was determined by counting colony
forming units (c.f.u.) on MacConleey agar plates. The small bars represent the
standard deviations and the results are representative of at least two
independent
experiments.
Fig. 4B shows that S. typhimurium proliferates in the intestine of C.
elegans. Seventy to eighty 1-day-old adult hermaphrodite worms were placed on
plates containing S. typhimu~ium expressing GFP (S typhimuriumlGFP) and E.
coli not expressing GFP in a ratio of 1:10,000. In addition, the same number
of
2S worms were placed on E. coli expressing GFP. After S hours the worms were
washed in M9 buffer and transferred to E. coli not expressing GFP plates.
Subsequently, every 24 hours, 10 worms were transferred to M9 buffer
containing
1% Triton X-100, and then the worms were mechanically disrupted to count the
bacteria present in the gut. Bacteria were subsequently diluted in 10 mM
MgSO~,
plated on plates containing ampicillin, and the c.f.u. counted. The data
represent
the means ~ S.D. of two independent experiments.
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Fig. 5A shows that worm billing requires direct interaction between live .S
typhimu~~iuYn and C. elegans. PA14 (open circle) were grown on 0.45 pm filters
placed on PGS plates, and SL1344 were grown on either PGP (closed circle) or
NG plates (square). Following growth of the bacteria, filters were removed,
the
5'plates exposed to UV light for 5 minutes to kill any possible contaminating
bacteria, heat-killed OP50 added as a source of food, and 20 worms were placed
on each plate. Small bars represent the standard deviations and the results
are
representative of at least two independent experiments.
Fig. 5B shows that worm lulling requires direct interaction between live S.
typhisnu~ium and C. elegans. Twenty womns feeding on heat-lulled SL1344
(closed circle) or SL1344 live (open circle) and heat-billed SL14028 (closed
square) or live SL14028 (open square). The small bars represent the standard
deviations and the results are representative of at least two independent
experiments.
Below we describe experimental evidence demonstrating that Salmonella
typhimurium causes disease in the nematode C. elega~as by establishing a
persistent infection, including a long-lasting association in the nematode
intestine,
and that C. elegans feeding on lamzs of S. typhimurium eventually die over the
couxse of a few days as a result of a pathogenic process. The salmonellae/C.
elegans lcilling assay described herein therefore provides a useful system for
identifying novel virulence factors responsible for a pathogen's ability to
develop
a persistent infection, as well as for identifying compounds that either
inhibit
pathogenicity of salmonellae, promote a host's resistance to the pathogen, or
both.
The following experimental examples are intended to illustrate, not limit, the
scope of the claimed invention.
To determine if S. typhimuf°ium kills G elegahs, worms were fed on
a
lawn of bacteria grown on NG agar and the time to bill 50% of the worms (LT50)
was calculated in six independent experiments. The reduced LT50 of worms fed
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with S. typhimu~ium (LTSO = 5.10 X0.7 days) compared with the one obtained
using worms fed with E. coli (LTSO = 9.86 X0.9 days), indicated that the worms
were killed by xS'. typhimu~ium (Fig 1A).
In addition, the physiology of the worms fed with S. typhinzurium was
different than worms feeding on E. coli OPSO. As reported for P. aenuginosa
infection (Tan et al., Proc. Natl. Acad. Sci. 96:2408-2413, 1999), the
motility of
the worms and the rate of pharyngeal pumping gradually declined, until the
nematodes became immobile and died. In some cases, worms became laden with
eggs, and at early times during the infections embryos hatched internally,
suggesting an egg-laying defect. To study if S. typhimu~ium was capable of
preventing eggs from hatching, eggs were placed in plates containing S.
typhimur~ium. We observed that 9S% of the eggs hatched, indicating that S.
typhimuriuf~z does not prevent hatching under these conditions.
Since S. typlaimu~iuy~a is known for establishing a long-standing
1S association or persistent infection with their hosts (Galan and Bliska,
Anna. Rev.
Cell Dev. Biol. 12:221-2SS, 1996), the bacterium was examined for its ability
to
colonize the worm intestine in an iiTeversible manner. The woz~ns were fed
with
S, typhimu~ium for five hours, and then were transferred to plates containing
E.
coli. The results shown in Fig. 1B indicate that after S hours of infection S.
typhimus°ium colonized the worm intestine, and that the worms died in
the course
of several days. The rate of killing in this shifting experiment was similar
to
those obtained when the C. elegafas was in contact with S. typhimu~~iurn
during the
whole billing process. To study the time course of S. typhimu~ium infection,
adult
worms were allowed to feed on S. typl2imurium for 1, 3, and S hours and then
2S transferred to plates containing E. coli. As shown in the insert presented
in Fig.
1B, the proportion of worms that survived after the transfer to E. coli plates
was
inversely proportional to the time spent feeding on SL1344. At 8 days, only
30%
of the worms that had been transferred to the E. coli plates after feeding on
SL1344 fox 1 hoax died. The proportion of dead worms after 8 days increased to
90 % when they were fed on SL1344 for S hours before being transferred. In
contrast, when C. elegans was fed on Pseudomonas aef°uginosa PA14 for 6
hours
CA 02401067 2002-08-22
WO 01/71343 PCT/USO1/40311
then transferred to E, coli, no killing was observed within the time frame of
the
experiment (60 hours) (or within the normal time frame of 100% killing by
feeding constantly on PA14). This result is interpreted as indicating that
PA14
does not establish a lethal infection within the parameters of the experiments
described fox Salmonella.
Th~I~i .ling._Mediated by~~~>nhimu~iuna Correlates with Proliferation of the
Bacteria in the Gut
To confirm that S. typhimu~ium killing involves an infectious process, we
constructed a S. typhimu~ium strain expressing the Aequo~ea victo~ia GFP.
After
f0 72 hours of feeding on S. typhimu~ium expressing GFP, the intestinal lumen
was
found to be distended and full of intact bacteria, suggesting that S.
typlaimu~ium
may be proliferating in the gut (Fig. 2, Panels B and C). Although Pseudomonas
aeruginosa PA14 is unable to establish a long-lasting infection, similar
results
were obtained when the worms were fed with PA14/GFP for 24 hours (Fig. 2,
Panels E and F). In contrast, no intact bacteria were observed when the worms
were fed with the control E. coli DHSalphalGFP strain and the lumen was not
distended (Fig. 2, Panels A and B). The worms were also fed with S
typhimuriumlGFP or E. coli DHSalpha/GFP for only 5 hours and then transferred
to E. coli plates. Fig. 3 (Panels A, B, and C) shows that no E. coli was found
in
the C. elegans intestine after 24 hours, while S. typhimu~ium survived into
the
intestinal lumen. Intact S. typhimuriumlGFP is shown at higher magnification
(Fig. 3, Panel C).
To evaluate the colonization process of S. typlaimuniuna, the amount of
bacteria used in the feeding assays was reduced. Several dilutions of S
yphimurium in E. coli were prepared on NG plates and the worms were
immediately placed on the plates for feeding. The percentage of .Salmonella
was
then determined by counting the presence of c.fu. on MacConkey agar plates.
The results of these feeding experiments showed that S. typhimurium diluted up
to
1:1,000 was capable of killing G elegans at levels similar to feeding on 100%
S.
typhimuriurn, whereas a 1:10,000 dilution showed reduced virulence (Fig. 4A).
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Since 0.1% of S. typhimurium supported billing, this concentration of
bacteria was used to cant' out a chase experiment to quantify the amount of
bacteria in the lumen of the intestine during the infection. The worms were
exposed for five hours to 0.1% SLI344/GFP in OP50 or to DHSalphalGFP, and
then transferred to OPSO plates. Every 24 hours 10 worms were placed in a 1.5
ml centrifuge tube and disrupted by pressing them with a pellet pestle.
Dilutions
of bacteria were plated and the c.fu. counted. The results showed that after
24
hours DHSalphalGFP practically disappeared from the worm intestine, whereas
SL13441GFP proliferated, colonizing the worm intestine (Fig. 4B).
I O Direct Interaction between ~S'. t~~znh.imurium and r elegans is Reauired
for Killing
P. ae~ugihosa has been shown to lcill G elegans by the secretion of
diffusible toxins referred to as "fast killing" (Tan et al., Proc. Natl. Acad.
Sci.
96:2408-2413, 1999 and Mahajan-Miklos et al., Cell 96: 47-56 1999). To
determine if C. elegahs killing by S. typhimm°iun2 is mediated by a
diffusible
toxin, a killing assay was perfonned under fast killing conditions, using
sorbitol
containing plates (PGS plates). It has been shown that L4 worms died much more
rapidly than adults under the P. aeruginosa fast billing conditions (Tan et
al.,
Proc. Natl. Acad. Sci. 96:2408-2413, 1999), therefore, L4 stage worms were
used
in these experiments. S. typhimu~ium and control P, aerugihosa and E. coli
were
grown on 0.45 ~m filters placed on the plates. The filters containing the
bacteria
were removed and heat inactivated E. coli were place as a source of food to
avoid
starvation (Fig. 5A). The results showed that the killing mediated by S.
typhimurium under these conditions is not due to any diffusible toxin. Fig. 5B
shows that C. elegahs survived when fad heat-billed Salmonella. We have also
fed the worms using the heat-killed and live S. typhimurium clinical isolated
strain
14028. The results shown in Fig. 5B indicate that heat-billed SL14028 failed
to
bill C. elegans, and that live SL14028 billed the worms as found for SL1344.
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Materials and Methods
The above-described results were obtained using the following materials
and methods.
Bacterial Strains, la mids, and Growth Conditions
The P. aeruginosa strain PA14 (Rahme et al., Science 268, 1899-1902,
1995), PAl4/GFP strain (Tan et al., Proc Natl Acad Sci U S A 96, 715-720,
1999), ,S typhimunium strain SL1344 (Hoiseth, Nature 291, 238-239, 1981),
SL1344/GFP (this work), S. typhimurium clinical isolated strain 14028
(SL14028)
were kindly provided by E. Hohmann , Eschericlaia coli strain DHSalpha
(Bethesda Research Laboratories), DHSalpha/GFP strain (Tan et al., Proc Natl
Acad Sci U S A 96, 71S-720, 1999), and Esche~ichia coli strain OP50 (Brenner,
Genetics 77, 71-94, 1974) were grown at 37°C in Luria Broth (LB)
media. The
SL1344/GFP strain was made using the construct pSMC21-GFP (Bloernberg et
al., Appl. Environ. Microbiol. 63:4543-51, 1997).
Maintenance of the Nematodes
The nematodes were maintained as hermaphrodites at 20 ° C, grown
on
standard plates and fed with Esche~ichia coli strain OP50 as described by
Sulston
and Hodglcin (Tn: the Nematode Caehorhabditis elegans, ed., W.B. Wood, Cold
Spring Harbor Lab. Press, Plainview, N.Y., pp. 587-606, 1988). Worms were
observed under a dissecting microscope (Leica MZ6).
Bacterial Tnfection avs
The killing assays were conducted by spreading 10 ~,l of bacterial culture
grown overnight in LB on nematode growth (NG; modified from nematode
growtli medium agar described in Sulston and Hodgl~in (Tn: the Nematode
Caeno~°habditis elegans, ed., W.B. Wood, Cold Spring Harbor Lab.
Press,
Plainview, N.Y., pp. 587-606, 1988); using 0.35% instead of 0.25% peptone or .
peptone-glucose-sorbitol (PGS; 1% Bacto-Peptone/1% NaCI/1% glucose/0.15 M
sorbitol/1.7% Bacto-Agar) media for nematode killing assays (3.5 cm diameter
plates). After spreading the bacterial culture, plates were incubated at
37°C for
12 hours and then ten to twenty worms were placed on the assay plate, which
was
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then incubated at 2S °C. When required, the bacteria were killed by
heating at
60°C for 90 minutes. The E. coli strain OPSO was used as a control for
the
assays. Worm mortality was scored over time, and a worm was considered dead
when it failed to respond to touch. Any worms that died as a result of getting
S stacl~ to the wall of the plate were excluded from the analysis. The time to
kill
SO% of the nematodes (LTSO) was calculated using the PRISM (version 2.00)
computer program using the equation: Y=Bottom+(Top-
Bottom)/(1+10~((LogECSO-X)~HilISlope)), where X is the logarithm of days and
Y is the average of Filled worms; Y starts at Bottom and goes to Top with a
sigmoid shape.
~gaas Shifting rim n s
Eighty to one-hundred one-day-old adult hermaphrodite worms were
seeded on bacteria lawns and allowed to feed. After 1, 3, or S hours, the
worms
were transferred to plates containing E. coli OPSO. Before transfer, worms
were
1 S washed two times in M9 buffer, transferred to plates containing OPSO for
two
hours and then transferred to new plates containing OPSO. Every 24 hours the
worms were transferred to new plates and the c.f.u. counted. To count the
c.~u,
10 worms were washed in M9 buffer and transferred to a 1.S ml tube with M9
buffer containing 1% Triton X-100 where they were mechanically disrupted using
a pellet pestle. Dilutions of bacteria were made in 10 mM MgSO4, plated on
ampicillin containing plates or in MacConlcey agar base plates, and the c.f.u.
counted.
C'onfocal MicroscopT
The worms were seeded on DHSalpha/GFP or SL1344/GFP lawns. After S
2S hours, one half the total number of worms was transferred to plates
containing E.
coli OPSO as described above. For each time point, five worms were placed on a
pad of 1% agar in PBS and 30 ~M sodium azide in M9 buffer was used as
anesthetic. The experiments were repeated at least four times and confocal
imaging was performed using a Leica TCS SP confocal microscope. Composite
images were assembled and edited using Adobe PhotoShop 5Ø
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Pe si tent T_n_fections
Based on the results described above showing that Salmonella establishes
a persistent infection in the nematode, G elegaras, we have developed methods
for
identifying virulence determinants important for establishing this type of
infection. The screen, in general, utilizes the above-described
pathogen/nematode
billing assays and exploits the ability to screen thousands of randomly
generated
mutant pathogens. In addition to using wild type host worms in the screening
assays, mutant worms that are constipated or defecation defective, such as aex-
2
and unc-2S, mutants that are grinding defective, such as phm-2 and eat-14, and
specific ABC transporter mutants such as pgp-4 and mrp-1 may be utilized as
well.
In general, a pathogen is assayed (using the method described herein) for
its ability to establish a persistent infection in a nematode. Once
identified, the
pathogen, such as Salmonella typlrimur~ium strain SL1344, is mutated according
to
standard methods brown in the art and then subsequently evaluated for its
ability
to induce disease by establishing a persistent infection in the nematode host
organism. A mutagenized pathogen found to have diminished ability for
establishing a persistent infection is useful in the method of the invention.
Such
mutant pathogens are then used for identifying host-dependent or host-
independent virulence factors responsible for establishing the persistent
infection
according to methods known in the art.
Other screening assays for identifying and characterizing virulence factors
include constitutive or overexpression of putative virulence genes from
Salnaor2ella or other bacterial species. Examples would be constitutively
expressing a putative transcription factor or repressor on a plasmid in the
pathogen, such as Salmonella typhimur~iuna strain SL1344, and testing these
altered pathogens for enhanced or reduced virulence in G elegans.
Alternatively,
a plasmid library of clones covering the entire Salrraorrella typlairnurium
(or other
pathogen) genome, could be screened for enhancement or supression of
CA 02401067 2002-08-22
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Salmonella virulence in C. elegans. The results of these screens identify
virulence factors, and subsequently provided valuable information as to their
downstream targets.
The following is a working example of a virulence factor nematode
screening system which utilizes the human clinical isolate S typlaimu~iufn
strain
SL1344 identified as persistently infecting the gut of C. elegans. The
advantage
of using a nematode as a host for studying microbial pathogens is the relative
simplicity of identifying non-pathogenic mutants in the nematode system.
Tn one preferred working example, in which survival is monitored, four to
eight C. elegans worms (e.g.,one-day old adult hermaphrodites) are placed on a
lawn of a mutagenized strain of a pathogen, and survival is monitored after
approximately five hours according to the methods described herein. For
example, a pathogen, such as Salmonella typhimu~ium strain SL1344, is mutated
according to any standard procedure, e.g., standard in vivo or in vitro
insertional/transposon mutagenesis methods (see, e.g., I~leckner et al., J.
Mol.
Biol. 116: 125, 1977; Simon et al., Gene 80 (1): 161-169, 1989). Other methods
are also available, e.g., chemical mutagenesis, or directed mutagenesis of
DNA.
After very few or no live worms are found on a plate seeded with wild-type,
pathogenic S. typhimurium strain SL1344, whereas on a plate with mutagenized
S.
typhimu~ium strain SL1344, increased survival (e.g., as determined by an
increased LTSO) of the worms is observed. Thus, the ability of worms to grow
in
the presence of mutated S. typhimurium strain SL1344 is an indication that a
gene
responsible for pathogenicity has been inactivated. The positions of the
inactivating mutations are then identified using standard methods, (e.g., by
polymerase chain reaction and sequencing of insertion/transposon junctions or
by
mapping), leading to the cloning and identification of the mutated virulence
factors) (e.g., by nucleotide sequencing).
In another working example, pathogenesis is assayed by monitoring
colonization of the nematode intestine, two to eight C. elegans worms (e.g.,
L4
hermaphrodite laxvae) are placed on a lawn of mutagenized Salmonella
typhimu~ium strain LT2, prepared from a mutagenic library, expressing a
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detectable marker (e.g., GFP) for an appropriate period of time and then
transferred to plates for feeding on non-pathogenic bacteria (e.g., E.
coli/DHSalpha). Strain LT2 is mutated according to standard methods. After
approximately five hours worms are examined for the presence of the pathogen
in
the intestine using confocal microscopy. Worms feeding on wild-type,
pathogenic S. typhimurium strain LT2, will present bacteria growing and
proliferating in the gut. Worms feeding on mutated LT2 that contain a mutation
in a gene that enables the establishment of a persistent infection will
present a
reduced, non-proliferating population in the gut. Thus, the absence or reduced
presence of mutated LT2 in the worm intestine is taken as an indication that a
gene responsible fox pathogenicity has been inactivated. The mutated virulence
factor is then identified using standard methods.
C.ornpound Screenin ssavs
As discussed above, our experimental results demonstrated that virulence
factors are involved in pathogenicity of the nematode, C. elegans. Based on
this
discovery we have also developed a screening procedure for identifying
therapeutic compounds (e.g., anti-pathogenicity pharmaceuticals) which can be
used to inhibit the ability of a pathogen to persistently infection. In
general, the
method involves screening any number of compounds for therapeutically-active
agents by employing the Salinonellae/nematode killing system described herein.
Based on our demonstration that these pathogens infect and bill C elegahs, it
will
be readily understood that a compound which interferes with the pathogenicity
of
a pathogen (e.g., a salmonellae pathogen) in a nematode also provides an
effective therapeutic agent in a mammal (e.g., a human patient). Whereas most
antibiotics currently in medical use are either bactericidal or
bacteriostatic, thus
favoring resistant strains or mutants, the compounds identified in the
screening
procedures described herein do not kill the bacteria or prevent their growth
in
vitro, but instead render them non-pathogenic. Moreover, since the screening
procedures of the invention are performed ifz vivo, it is also unlikely that
the
identified compounds will be highly toxic to the host organism.
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Accordingly, the methods of the invention simplify the evaluation,
identification, and development of active agents such as drugs for the
treatment of
pathogenic diseases caused by microbes that can establish a long lasting
association or persistent infection with a nematode.
In general, the chemical screening methods of the invention provide a
straightforward means for selecting natural product extracts or compounds of
interest from a large population which are further evaluated and condensed to
a
few active and selective materials. Constituents of this pool are then
purified and
evaluated in the methods of the invention to determine their anti-pathogenic
activity.
Test Extracts and Compo nd
Tn general, novel anti-pathogenic drugs are identified from large libraries
of both natural product or synthetic (or semi-synthetic) extracts or chemical
libraries according to methods pnown in the art. The screening method of the
present invention is appropriate and useful for testing compounds from a
variety
of sources for possible anti-pathogenic activity. The initial screens may be
performed using a diverse library of compounds, but the method is suitable for
a
variety of other compounds and compound libraries. Such compound libraries
can be combinatorial libraries, natural product libraries, or other small
molecule
libraries. In addition, compounds from commercial sources can be tested, as
well
as commercially available analogs of identified inhibitors.
For example, those spilled in the field of drug discovery and development
will understand that the precise source of test extracts or compounds is not
critical
to the screening procedures) of the invention. Accordingly, virtually any
number
of chemical extracts or compounds can be screened using the methods described
herein. Examples of such extracts or compounds include, but are not limited
to,
plant-, fungal-, proparyotic- or animal-based extracts, fermentation broths,
and
synthetic compounds, as well as modification of existing compounds. Numerous
methods are also available for generating random or directed synthesis (e.g.,
semi-
synthesis or total synthesis) of any number of chemical compounds, including,
but
not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based
compounds.
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Synthetic compound libraries are commercially available from Brandon
Associates (Mernmack, NH) and Aldrich Chemical (Milwaukee, WI).
Alternatively, libraries of natural compounds in the form of bacterial,
fungal,
plant, and animal extracts are commercially available from a number of
sources,
including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch
Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge,
MA). In addition, natural and synthetically produced libraries are produced,
if
desired, according to methods known in the art, e.g., by standard extraction
and
fractionation methods. Furthermore, if desired, any library or compound is
readily modified using standard chemical, physical, or biochemical methods.
In addition, those spilled in the art of drug discovery and development
readily understand that methods for dereplication (e.g., taxonomic
dereplication,
biological dereplication, and chemical dereplication, or any combination
thereof]
or the elimination of replicates or repeats of materials already known for
their
anti-pathogenic activity should be employed whenever possible.
When a crude extract is found to have anti-pathogenic activity, further
fractionation of the positive lead extract is necessary to isolate chemical
constituents responsible for the observed effect. Thus, the goal of the
extraction,
fractionation, and purification process is the careful characterization and
identification of a chemical entity within the crude extract having anti-
pathogenic
activity. Methods of fractionation and purification of such heterogenous
extracts
are known in the art. If desired, compounds shown to be useful agents for the
treatment of pathogenicity are chemically modified according to methods known
in the art.
Since many of the compounds in libraries such as combinatorial and
natural products libraries, as well as in natural products preparations, are
not
characterized, the screening methods of this invention provide novel compounds
which are active as inhibitors or inducers in the particular screens, in
addition to
identifying known compounds which are active in the screens. Therefore, this
invention includes such novel compounds, as well as the use of both novel and
known compounds in pharmaceutical compositions and methods of treating.
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FJxemplary Hi T ro ~ n ~t~creening_~, emu
To evaluate the efficacy of a molecule or compound in promoting host
resistance to, or inhibiting pathogenicity of, a number of high throughput
assays
may be utilized.
For example, to enable mass screening of large quantities of natural
products, extracts, or compounds in an efficient and systematic fashion,
Caenorhabditis elegans, (e.g., one-day-old adult hermaphrodite worms, an L4
hermaphrodite larvae or a mutant worm such as aex-2, unc-25, phm-2, eat-14,
pgp-4, or mTp-1), are cultured in wells of a microtiter plate, facilitating
the
semiautomation of manpulations and full automation of data collection. As is
discussed above, salmonellae pathogens establish a persistent infection,
including
a long-lasting association that bills C. elegans. If a salmonellae pathogen
has
diminished pathogenicity, then adult or L4 worms live, develop into adult
hermaphrodites, and produce thousands of live progeny. Accordingly, if C.
elegans is incubated with the pathogen, the worms will die, unless a compound
is
present to reduce pathogenicity. The presence of such live progeny is easily
detected using a variety of methods, including visual screening with standard
microscopes.
To evaluate the ability of a test compound or extract to promote a host's
resistance to a pathogen or to repress pathogenicity of a pathogen, a test
compound or extract is inoculated at an appropriate dosage into an appropriate
agar medium seeded with an appropriate amount of an overnight culture of a
pathogen, e.g., S. typhi~cu~iuyn strain LT2. If desired, various
concentrations of
the test compound or extract can be inoculated to assess dosage effect on both
the
host and the pathogen. Control wells are inoculated with non-pathogenic
bacteria
(negative control) or a pathogen in the absence of a test compound or extract
(positive control). Plates are then incubated 24 hours at 37°C to
facilitate the
growth of the pathogen. Microtiter dishes axe subsequently cooled to 25
°C, and
two C. elegans L4 hermaphrodite larva expressing a detectable marl~er such as
GFP are added to the plate and incubated at 25 ° C, the upper limit for
normal
CA 02401067 2002-08-22
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physiological integrity of C. elegahs. At an appropriate time interval, e.g.,
five
hours, wells are examined for surviving worms, the presence of progeny, or
both,
e.g., by visual screening or monitoring motion of worms using a motion
detector,
or monitoring the fluorescence of the nematodes.
In another working example, the presence of a persistent infection in gut
of C. elegahs is carried out as follows. Media is prepared as described herein
and
a test compound or compound library is also added. On the tissue culture
plate,
approximately eight worms, at the one-day old adult stage, are placed on the
lawn
of pathogenic bacteria expressing a detectable markers such as GFP from a
plate
of OP50 E, coli. The plates are incubated at 25 °C for 5 hours and then
the worms
are transferred to a plate of OP50 E. coli not expressing GFP. After 24 hours,
the
worms are examined by fluorsecent microcopy for the presence of the pathogen
in
the worm intestine. Each experimental condition is done in triplicate and
repeated
at least twice. Test compounds that reduce the presence the pathogen in the
worm
intestine are taken as being useful for treating microbial infection.
Comparative studies between treated and control worms (or larvae) are
used to determine the relative efficacy of the test molecule or compound in
promoting the host's resistance to the pathogen or inhibiting the
establishment of
a persistent infection. A test compound which effectively stimulates, boosts,
enhances, increases, or promotes the host's resistance to the pathogen or
which
inhibits, inactivates, suppresses, represses, or controls pathogenicity of the
pathogen, and does not significantly adversely affect the normal physiology,
reproduction, or development of the worms is considered useful in the
invention.
The methods of the invention provide a simple means for identifying
virulence factors that enable a pathogen to establish a persistent infection
in a
nematode and compounds capable of either inhibiting pathogeucity or enhancing
an organism's resistance capabilities to such pathogens. Accordingly, a
chemical
entity discovered to have medicinal value using the methods described herein
are
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useful as either drugs, or as information for structural modification of
existing
anti-pathogenic compounds, e.g., by rational drug design.
For therapeutic uses, the compositions or agents identified using the
methods disclosed herein may be administered systemically, for example,
formulated in a pharmaceutically-acceptable buffer such as physiological
saline.
Preferable routes of administration include, for example, subcutaneous,
intravenous, interperitoneally, intramuscular, or intradermal injections which
provide continuous, sustained levels of the drug in the patient. Treatment of
human patients or other animals will be carried out using a therapeutically
effective amount of an anti-pathogenic agent in a physiologically-acceptable
Garner. In the context of treating a bacterial infection a "therapeutically
effective
amount" or "pharmaceutically effective amount" indicates an amount of an
antibacterial agent, e.g., as disclosed for this invention, which has a
therapeutic
effect. This generally refers to the inhibition, to some extent, of the normal
cellular functioning of bacterial cells (e.g., salmonellae cells) causing or
contributing to a bacterial infection. The dose of antibacterial agent which
is
useful as a treatment is a "therapeutically effective amount." Thus, as used
herein,
a therapeutically effective amount means an amount of an antibacterial agent
which produces the desired therapeutic effect as judged by clinical trial
results,
standard animal models of infection, or both. This amount can be routinely
determined by one spilled in the art and will vary depending upon several
factors,
such as the particular bacterial strain involved and the particular
antibacterial
agent used. This amount can further depend on the patient's height, weight,
sex,
age, and renal and liver function or other medical history. For these
purposes, a
therapeutic effect is one which relieves to some extent one or more of the
symptoms of the infection and includes curing an infection.
The compositions containing antibacterial agents of virulence factors or
genes can be administered for prophylactic or therapeutic treatments, or both.
In
therapeutic applications, the compositions are administered to a patient
already
suffering from an infection from bacteria (similarly for infections by other
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microbes), in an amount sufficient to cure or at least partially arrest the
symptoms
of the infection. An amount adequate to accomplish this is defined as
"therapeutically effective amount." Amounts effective for this use will depend
on
the severity and course of the infection, previous therapy, the patient's
health
status and response to the drugs, and the judgment of the treating physician.
In
prophylactic applications, compositions containing the compounds of the
invention are administered to a patient susceptible to, or otherwise at risk
of, a
particular infection. Such an amount is defined to be a "prophylactically
effective
amount." In this use, the precise amounts again depend on the patient's state
of
I O health, weight, and the like. However, generally, a suitable effective
dose will be
in the range of 0:1 to 10000 milligrams (mg) per recipient per day, preferably
in
the range of I O-5000 mg per day. The desired dosage is preferably presented
in
one, two, three, four, or more subdoses administered at appropriate intervals
throughout the day. These subdoses can be administered as unit dosage forms,
for
example, containing 5 to 1000 mg, preferably 10 to 100 mg of active ingredient
per unit dosage form. Preferably, the compounds of the invention will be
administered in amounts of between about 2.0 mg/kg to 25 mg/kg of patient body
weight, between about one to four times per day.
Suitable carriers and their formulation are described, for example, in
Remington's Pharmaceutical Sciences by E.W. Martin. The amount of the anti-
pathogenic agent to be administered varies depending upon the manner of
administration, the age and body weight of the patient, and with the type of
disease and extensiveness of the disease. Generally, amounts will be in the
range
of those used for other agents used in the treatment of other microbial
diseases,
although in certain instances lower amounts will be needed because of the
increased specificity of the compound. A compound is administered at a dosage
that inhibits microbial proliferation.
All publications and patents mentioned in this specification are herein
incorporated by reference to the same extent as if each individual publication
or
patent was specifically and individually indicated to be incorporated by
reference.
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From the foregoing description, one skilled in the art can easily ascertain
the essential characteristics of this invention, can make various changes and
modifications of the invention to adapt it to various usages and conditions.
Thus,
other embodiments are also within the claims.
What is claimed is:
24
