Who Killed the Worm and Other
Fungal Pathogenesis Insights
Fungal infections of Caenorhabditis elegans teach us about virulence
factors, antifungal compounds, and host responses
Eleftherios Mylonakis
any pathogens depend on the
same virulence factors to cause
disease in mammals such as mice
and in nonmammalian hosts, thus
sharing a fundamental set of molecular mechanisms across a widely divergent array of hosts. This approach to evaluating virulence
factors by seeking them among divergent model
hosts, referred to as the “multi-host pathogenesis
system” (Fig. 1), offers a number of benefits. For
example, it reduces the number of mammals used
in experiments, and thus minimizes suffering in
rodents and similar species. It also helps investigators who want to run large-scale in vivo screens
because experiments involving nonmammalian
host species are easier and less expensive to do
than studies involving mammals.
In this context, the microscopic nematode
Caenorhabditis elegans is remarkable. It is
genetically tractable, many of its gene functions are identified, and its entire cell lineage
is known in detail and is identical in every
normal adult. Also, because C. elegans is a
hermaphrodite and capable of self-fertilization, genetically identical populations can
be produced by simply allowing an individual animal to produce progeny. Moreover,
because each 1-mm adult worm is transparent, individuals can be examined with a
dissecting microscope or by using Normarski or confocal microscopy. Further, double-stranded RNA molecules can and are
being used to silence nematode genes, determine their functions, and develop an extensive RNA interference (RNAi) library.
M
Eleftherios Mylonakis is an infectious
diseases specialist
at Massachusetts
General Hospital
and assistant professor of medicine
at Harvard Medical
School in Boston,
Mass.
Looking for Fungal Virulence Factors
Frederick M. Ausubel of Massachusetts
General Hospital (MGH) and Harvard
600 Y Microbe / Volume 2, Number 12, 2007
Medical School and his collaborators first used
C. elegans to identify virulence determinants in
several bacterial pathogens that infect humans.
During the past decade, other research groups
contributed similar insights while studying additional pathogenic bacteria, including Burkholderia pseudomallei, Burkholderia cepacia, Enterococcus faecalis, Pseudomonas aeruginosa,
Salmonella enterica, Serratia marcescens, Staphylococcus aureus, Streptococcus pneumoniae,
Streptococcus pyogenes, and Yersinia pestis.
In the 1980s, Hans-Börje Jansson, A. Jeyaprakash, and Bert Zuckerman at the University
of Massachusetts in Amherst studied how C.
elegans interacts with the endoparasitic fungus
Drechmeria coniospora. Spores of this fungus
Summary
• The multi-host pathogenesis system entails using divergent hosts to study microbial pathogenesis.
• Caenorhabditis elegans is being used to study
established and to identify novel virulence determinants among many bacterial and fungal
pathogens.
• Fungi kill nematodes in many different ways—
for example, Candida species adhere to nematode intestine, dissolve nematode tissues, and
break through nematode cuticles by forming
impressive networks of filaments; Drechmeria
coniospora spores cause a localized infection by
attaching to parts of the cuticle, and Cryptococcus neoformans (as well as Candida albicans on
solid media) kill nematodes after being ingested
without forming filaments.
• C. elegans innate immunity pathways are involved in responding to fungi with some degree
of specificity.
attach to parts of the cuticle of C. elegans, particularly the tip of the head
and around the vulva and tail, causing
localized infections. After attaching to a
nematode, fungal cells soon develop filaments that penetrate and eventually
kill the worm.
When we approached this system to
study pathogenic fungi, we first tested
whether C. elegans can use yeasts such
as Cryptococcus laurentii and Cryptococcus kuetzingii as a sole source of
food. We then chose to study Cryptococcus neoformans, which is pathogenic for humans and causes disease via
a number of well-defined virulence
traits. Indeed, several C. neoformans
genes involved in mammalian virulence, including those genes associated
with signal transduction and laccase
production, also partake in nematode
killing. In proof-of-principle studies
that extended these findings, we used
the killing of the nematode C. elegans
by C. neoformans to screen fungal insertion mutants, and several additional
genes identified this way are associated
with virulence in mammals (Fig. 2a).
FIGURE 1
The hypothesis behind the multi-host pathogenesis system: a common, fundamental set
of molecular mechanisms is employed by pathogens against a widely divergent array of
metazoan hosts.
Screening Helps Identify Promising
Antifungal Compounds
This very simple screening approach resembles
what is done with bacterial pathogens, including
gram-negative P. aeruginosa and gram-positive
E. faecalis. However, because this screening approach entails distinguishing live from dead
nematodes, it is time-consuming. An alternative,
more-efficient screening approach depends on
C. neoformans blocking progeny production in
hermaphrodite worms. Although similar to the
“killing assay” screen because random insertional mutants of C. neoformans are used to
identify genes associated with cryptococcal
pathogenesis, the progeny-permissive phenotype is much simpler (Fig. 2b). For example,
fewer worms per microbial lawn are required,
and surveillance need only occur once. A facile
in vivo whole-animal model for evaluating antifungal chemical compounds could overcome
several major obstacles in identifying promising
drug candidates, particularly if it could circum-
vent the problem that so many such compounds
prove toxic to mammalian cells.
C. neoformans cells do not adhere to nematode intestines. Moreover, nematodes exposed
to cryptococcal lawns defecate the cryptococcal
cells upon transfer to liquid media, thereby
clearing the cryptococcal infection. However, in
contrast to C. neoformans, Candida species
readily establish persistent infections of the C.
elegans intestine. Key components of Candida
pathogenesis in mammals, such as biofilm and
filament formation, are also involved in nematode killing. Forming filaments is instrumental
in Candida virulence in mammals, and nonfilamentous mutants of C. albicans are highly attenuated. After the integrity of the nematode cuticle
is breached, the yeast cells remain connected,
forming a “C. elegans ghost,” in which the transition from mostly yeast-like Candida cells to
the filaments outlines where the cuticle used to
be (Fig. 3).
A specific antifungal agent can prolong C.
elegans survival when it is infected with strains
of Candida that are sensitive to that drug. This
approach provides a means for screening large
Volume 2, Number 12, 2007 / Microbe Y 601
FIGURE 2
This liquid-media assay offers advantages over in vitro assays because it
evaluates compounds with toxic, immunomodulatory, or dual effects. Thus,
in this assay, nematode-toxic compounds can be quickly identified and
excluded. Importantly, nematodes in
wells that contain compounds with antifungal activity can be easily monitored using “screening-by-imaging”
technology. Nematodes exposed to
compounds that have significant antifungal efficacy move sinusoidally,
whereas nematodes exposed to compounds without antifungal efficacy do
not move but remain rod-shaped and
develop filaments (Fig. 4).
C. elegans Responds to Fungal
Pathogens through an Innate
Immune System
The innate immune system of C.
elegans includes conserved signaling
pathways such as the PMK-1 MAP
kinase and DAF-2 pathways. Components of these pathways are involved in
defending nematodes against pathogenic fungi with some degree of specificity, depending on which fungal species and strains are infecting the
nematode.
One set of innate responses involves
the tir-1-nsy-1/SARM-mitogen-activated protein kinase (MAPK) signaling
pathway, which includes a conserved
p38 mitogen-activated protein kinase
that functions downstream of TIR-1,
according to Fred Ausubel, Dennis
(a) Outline of a screen utilizing the C. elegans “killing assay”. (A) Random mutants were
Kim, Rhonda Feinbaum, and their colevaluated for attenuated virulence in C. elegans. (B) In most cases, attenuated virulence
persisted after crossing the mutation back into a wild-type strain. (C) The experimental
laborators at MGH. They exposed
phenotype was confirmed in murine models. (b) Outline of the C. elegans “progeny
worms to the mutagen ethyl methane
assay”. Colonies from a library of C. neoformans insertional mutants (A) are streaked for
sulfonate (EMS) and then transferred
isolation (B). A colony from each insertional mutant is grown in YPD liquid media (C).
Cryptococcal lawns are generated on BHI agar (D). L4-stage nematodes are placed on
the F2 population to plates that coneach plate and incubated at 25oC. Plates are examined for progeny production perioditained pathogenic bacteria. They then
cally over the course of 14 days. Various scenarios are depicted (E). Plates producing
collected those mutant worms that died
progeny are indicated (⫹).
soon after exposure to the pathogens,
which do not infect and thus spare C.
libraries of chemical compounds for antifungal
elegans eggs. Candidate mutants were recovered
activity. Automating it involves using robots to
by transferring individual dead worms to plates
dispense and evenly distribute chemical comseeded with nonpathogenic E. coli.
pounds, plate readers, and screening microscopes
These experiments involving bacterial pathoto monitor nematode survival.
gens are relevant to fungal pathogenesis. For
602 Y Microbe / Volume 2, Number 12, 2007
For Mylonakis, Work Is a Hobby, and His Hobby Is His Work
Eleftherios Mylonakis wishes that
everyone would study ancient
Greek literature, which remains a
significant component of education
in his native Greece. He believes it
introduces young minds to important values, including altruism, philanthropy, and a sense of duty.
Mylonakis is strongly committed to his family, including his
wife, who is also a physician, their
two young daughters, and his elderly parents, who live in Athens.
“The care of the elderly is traditionally the responsibility of the
family in Greece,” he says, a responsibility that is not easily
borne from afar. “At times, I just
have to grab my grants and papers, and catch a flight to Greece
and just do the writing from a
hospital room there.”
Mylonakis, 41, moved to Boston
in 1994 after completing an internship and residency at Brown
University’s Miriam Hospital in
Rhode Island. Before that, he attended medical school and completed a doctoral thesis at the National University of Athens in
Greece. “The community at Brown
University made the transition so
easy,” he says, adding: ‘‘The excitement of the New England area with
sports, along with Seinfeld and Car
Talk, also helped.”
Today he is an infectious diseases specialist at Massachusetts
General Hospital and assistant
professor of medicine at Harvard
Medical School in Boston, where
he did postdoctoral research with
Steve Calderwood and Fred Ausubel. His current research focuses
on fungal pathogenesis and drug
discovery. Specifically, he is developing a whole-animal system for
studying fungal pathogenesis and
host responses, and for identifying
antifungal compounds.
“The study of pathogenic fungi
is compromised because the best
way to study the capability of fungi
to cause disease is during infection
and by the complex mammalian
response to fungal infection,” he
says. “This lack of simple model
systems has forced scientists to limit
their studies to an analysis of the
host, the pathogen, or the antimicrobial compound. The separation
of these disciplines is a major hindrance to developing novel antimicrobial agents and groundbreaking
therapies.”
Mylonakis was born in Larisa,
Greece, and lived in Athens during
most of his childhood. His parents
encouraged his interest in science.
“My parents gave me a ‘blue collar’ approach to science, where
hard work is very important, day
in and day out,” he says. “Also,
my mother taught me a way to use
common sense to approach even
complicated problems. She has an
impressive way to simplify the
most complex problem, and make
it look easy. My research is very
important to me, and I feel that if I
do not give my best effort, I cheat
myself. I think this approach was
instilled in me by my parents.”
His parents, growing up during
the 1940s in Greece, lived through
World War II and, later, the country’s civil war. “The generation that
rebuilt Greece after this difficult decade was remarkable,” he says.
“My dad had to move to a city and
live by himself when it was time for
him to go to high school. From
there, he went on to study theology,
graduate from law school, and
study philosophy; all of this while
he was working full time as a Greek
Orthodox priest.” Mylonakis and
his wife, Polyxeni, also from
Greece, have two daughters, ZoeStella, 7, and Sophia-Chrysoula, 6.
He calls parenthood “a most magnificent journey.” “My work is my
hobby and my hobby is my work,”
he says. “My life is between my research and clinical work, my family,
and my little daughters.
“Why do I care about this work?
As a physician, my goal is to help
people fight disease. This can be
done helping one patient at a time
in a clinical setting or trying to
help a number of individuals by
developing new therapies. Simply
put, I find the mortality rate of
fungal infection that, for a simple
candidemia can be over 20 –30%
and for cryptococcal infection can
be even higher, simply unacceptable and I want to do something
about it. Also, I care about it for
the challenge. What can be a bigger challenge than trying to understand nature, trying to make sense
of how and why things happen?”
There are times when Mylonakis
is surprised to be paid “for something that I would be delighted to
do for free,” he says. “At other
times, I just get disconcerted when I
cannot secure enough funding to
move this research forward with the
same sense of urgency that I would
like. Having a project that you really, deeply believe in—and has the
potential to help others—yet being
unable to pursue it because of funding limitations goes against every
fiber of my being.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
Volume 2, Number 12, 2007 / Microbe Y 603
Toll-interleukin 1 receptor (TIR) domain adapter protein. Inactivating tir-1
in wild-type worms leads to their decreased survival after infection with D.
coniospora. However, C. neoformans
infection does not upregulate nlp-31,
indicating specificity of these effects.
Nematodes produce other peptides
that, in some cases, have antifungal activity. For example, an Ascaris suum
antibacterial factor-type antimicrobial
peptide is microbicidal against several
yeasts, including Candida krusei,
Kluyveromyces thermotolerans, Pichia
anomala, and Saccharomyces cerevisiae
as well as many bacterial species, according to Yusuke Kato and colleagues
at the National Institute of Agrobiological Sciences in Tsukuba, Ibaraki,
Japan.
The insulin-like growth factor DAF-2
also appears to play a critical role in the
survival of nematodes, making them
highly resistant to some pathogens.
Stress-response transcription factor
DAF-16 acts downstream of DAF-2.
Resistance to C. neoformans is highly
variable, not only among related nematode species but also between different
sexes of the same species, according to
Filamentation, that is instrumental for Candida virulence in mammals, is also involved in
the killing of C. elegans. C. elegans animals were infected with C. albicans and then
Robin C. May of the University of Birmoved into pathogen-free liquid medium. C. albicans cells persisted within the C.
mingham, United Kingdom.
elegans intestine and formed hyphae (green) that penetrated through the C. elegans
For example, C. briggsae males do
cuticle, leaving a C. elegans “ghost” (dark structure) that outlines where the cuticle used
to be.
not show enhanced resistance to C.
neoformans relative to hermaphrodites,
whereas C. elegans males do. For other
example, we subsequently used a strain with a
species, either the males of C. elegans and C.
mutation involving SEK-1 (that encodes a
japonica or both sexes of C. remanei are resisMAPK kinase that functions directly upstream
tant to this pathogen. In C. elegans, males are
of the C. elegans homologue of the mammalian
more resistant than hermaphrodites to C. neop38 MAPK) and found that it is required for full
formans-mediated killing. DAF-16 may control
resistance to fungi such as C. neoformans and C.
male-specific resistance to C. neoformans. The
albicans. When C. elegans is infected by D.
immune response to C. neoformans also reconiospora, expression of the antimicrobial
quires the function of the intestinal GATA tranpeptides NLP-29 and NLP-31 is increased, acscription factor ELT-2, independently of the
cording to Jonathan Ewbank and collaborators
DAF-2/DAF-16 signaling pathway, according
from the Centre d’Immunologie de Marseilleto Alejandro Aballay at Duke University in
Luminy, France, who followed gene expression
Durham, N.C.
changes using cDNA microarrays. They also
C. elegans is able to avoid infection using
showed that NLP-31 is antifungal towards D.
pathogen recognition and avoidance. In some
coniospora, Neurospora crassa, and A. fumigaway this could be considered an “extension” of
tus. The up-regulation requires the host gene
the nematode responses to pathogens. In C.
tir-1, which encodes an ortholog of SARM, a
elegans, tol-1 mutants are part of a behavioral
FIGURE 3
604 Y Microbe / Volume 2, Number 12, 2007
mechanism that keeps worms away
from potential danger. For example,
tol-1 mutants, defective for the nematode’s single Toll-like receptor, are as
resistant as wild-type worms to D. coniospora, and antimicrobial peptide
gene expression appears to be TLR independent. Work from the Ewbank
group in France demonstrated that
tol-1 mutant nematodes are unable to
avoid bacterial pathogens, something
that we confirmed for C. neoformans.
FIGURE 4
Rich Legacy of Experiments
Involving C. elegans
More than 40 years ago, Sydney Brenner opened a new field when he encouraged biologists to study the relatively
simple nematode C. elegans. His appreciation for this unassuming species
proved prophetic. Thus, experiments
on nematodes continue to expand our
understanding of other specialty areas
Outline of a semi-automated screen for antifungal compounds. C. elegans animals are
of biology, including microbiology and
pre-infected with C. albicans. Nematodes are then pipetted into 96-well plates and
especially mycology.
compounds are added. Survival is monitored and compounds that prolong survival are
identified.
C. elegans assays are helping to
bridge a gap between in vitro assays and
those done in mammals. However, the
valuable only so long as those findings prove
homo-mensura statement of Protagoras, the
relevant to how those pathogens behave in huancient Greek philosopher, applies. He said,
mans.Within this context, studying fungal
“⌸␣´ ␷␶␻␷ ␮⑀´ ␶␳␱␷ ␣´ ␷␪␳␻␲␱␵.” That is, “Man is
pathogens in disparate hosts can help us to learn
the measure of all things.” Put another way,
more about virulence factors, antifungal comwhat we learn from studying infectious diseases
in C. elegans or other invertebrate species will be
pounds, and host responses.
ACKNOWLEDGMENTS
Support was provided by NIH K08 award AI63084 and a New Scholar Award in Global Infectious Diseases from the Ellison
Medical Foundation. I would like to acknowledge the enthusiastic and tireless work of the members of my group, mentors, and
collaborators. I extend my appreciation for the great work from a number of exceptional colleagues that, because of format
restrictions, I was unable to highlight in detail. I acknowledge the help of Roanna London in Figure 2b and Julia Breger and
Beth Burgwyn Fuchs in Fig. 3.
SUGGESTED READING
Breger, J., B. B. Fuchs, G. Aperis, T. I. Moy, F. M. Ausubel, and E. Mylonakis. 2007. Antifungal chemical compounds
identified using a C. elegans pathogenicity assay. PLoS Pathog. 3:e18.
Chamilos, G., M. S. Lionakis, R. E. Lewis, J. L. Lopez-Ribot, S. P. Saville, N. D. Albert, G. Halder, and D. P. Kontoyiannis.
2006. Drosophila melanogaster as a facile model for large-scale studies of virulence mechanisms and antifungal drug efficacy
in Candida species. J. Infect. Dis. 193:1014 –1022.
Couillault, C., N. Pujol, J. Reboul, L. Sabatier, J. F. Guichou, Y. ., Kohara, and J. J. Ewbank. 2004. TLR-independent control
of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM.
Nature Immunol. 5:488 – 494.
Kim, D. H., R. Feinbaum, G. Alloing, F. E. Emerson, D. A. Garsin, H. Inoue, M. Tanaka-Hino, N. Hisamoto, K. Matsumoto,
M. W. Tan, and F. M. Ausubel. 2002. A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity.
Science 297:623– 626.
Volume 2, Number 12, 2007 / Microbe Y 605
Mylonakis, E., F. M. Ausubel, J. R. Perfect, J. Heitman, and S. B. Calderwood. 2002. Killing of Caenorhabditis elegans by
Cryptococcus neoformans as a model of yeast pathogenesis. Proc. Natl. Acad. Sci. USA 99:15675–15680.
Mylonakis, E., A. Casadevall, and F. M. Ausubel. 2007. Exploiting amoeboid and non-vertebrate animal model systems to
study the virulence of human pathogenic fungi. PLoS Pathog. 3:e101.
Pujol, N., E. M. Link, L. X. Liu, C. L. Kurz, G. Alloing, M. W. Tan, K. P. Ray, R. Solari, C. D. Johnson, and J. J. Ewbank.
2001. A reverse genetic analysis of components of the Toll signaling pathway in Caenorhabditis elegans. Curr. Biol.
11:809 – 821.
Tang, R. J., J. Breger, A. Idnurm, K. J. Gerik, J. K. Lodge, J. Heitman, S. B. Calderwood, and E. Mylonakis. 2005.
Cryptococcus neoformans gene involved in mammalian pathogenesis identified by a Caenorhabditis elegans progeny-based
approach. Infect. Immun. 73:8219 – 8225.
Troemel, E. R., S. W. Chu, V. Reinke, S. S. Lee, F. M. Ausubel, and D. H. Kim. 2006. p38 MAPK regulates expression of
immune response genes and contributes to longevity in C. elegans. PLoS Genet. 10:e183.
van den Berg, M. C., J. Z. Woerlee, H. Ma, and R. C. May. 2006. Sex-dependent resistance to the pathogenic fungus
Cryptococcus neoformans. Genetics 173:677– 6
83.
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