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Establishment
and maintenance of animal/bacterial symbioses
General
research framework
My laboratory studies the relationship between the Hawaiian sepiolid
squid Euprymna scolopes and its luminous bacterial partner
Vibrio fischeri. Thus far, although all animals have beneficial
associations with microbes, this association is the only experimental
model available to biologists. We are studying 4 principal questions
with this model:
› How are the bacteria harvested by the host from the
host's environment during the re-establishment of the association
each generation?
› How are specific bacteria recognized and promoted,
while nonspecific bacteria and potential pathogens discouraged from
colonizing host tissues?
› What are the effects of the bacteria on the development
of the host tissues with which they associate? and,
› How is stability of the symbiosis achieved, such that
neither does the host eliminate the symbionts nor does the symbiont
population overgrow the host and become pathogenic?
Through studies focused at both the morphological/anatomical and
the biochemical/molecular levels, we are beginning to define the
signals transmitted between the animal host and the bacteria. This
series of signals must mediate both the infection process and the
maintenance of the symbiosis throughout the life history of the
host.
Our early work on this association focused on questions 2 and 3,
i.e., recognition/ specificity and the influence of bacteria on
host development. We found that two factors operate to mediate recognition
and specificity--cell-surface recognition molecules on the surfaces
of the host and symbiont, as well as the creation by the host of
an environment in which only the appropriate microbe can survive.
In one set of studies, we have identified a protein in the bacteria-containing
tissue of the squid with high sequence identity to the mammalian
antimicrobial protein myeloperoxidase. The occurrence of a vertebrate
antimicrobial protein in an invertebrate mutualism suggests that
phylogenetically diverse animals use similar mechanisms to control
both pathogenic and cooperative associations with bacteria. We are
continuing to define the expression of the host peroxidase gene
in this symbiosis. In our early studies of bacteria-induced host
development, we described two principal types of changes: irreversibly
signaled changes in tissues remote from the colonizing population
of symbiont and reversible changes in cells directly contacting
the bacterial symbionts. In the former, bacteria induce apoptosis
of the cells of a superficial, ciliated epithelium, which is specific
to the efficient inoculation of host tissues, that results in the
reabsorption of this tissue. In the latter, cells in direct contact
with the bacteria increase the microvillar density of their apical
surfaces and swell 4-fold in volume in response to interaction with
symbionts. If the tissues are cured of symbionts with antibiotics,
whereas the loss of the superficial epithelium will continue, the
microvillar density and the volume of cells adjacent to the bacteria
will return to levels characteristic of newly hatched, unexposed
host animals.
Recent findings
We have recently described a mechanism by which the newly
hatched host harvests the bacteria from the surrounding environment.
Bacteria induce the host to secrete mucus-like materials, most likely
through cytokine release resulting from the interaction of host
cells with bacterial lipopolysaccharides (LPS). This material works
in concert with the ciliated, superficial epithelium of the nascent
symbiotic organ to entrain water into the vicinity of tissues to
be colonized and to trap potential symbionts. This process represents
a newly discovered mechanism by which bacterial symbionts in low
abundance colonize animal tissue.
In collaboration with Dr. M. Apicella, in the Dept of Microbiology
at U of Iowa and an expert in the field of bacterial lipopolysaccharides,
we have shown that the LPS of V. fischeri induces the observed apoptosis
of the host's superficial epithelium. We are also studying LPS receptors.
In both mammals and drosophila, LPS signals through conserved receptors,
that generally lead to the activation of the NF-kappaB pathway,
a pathway that leads to changes in host gene expression. The NF-kappaB
pathway has many effectors and has been implicated in both developmental
processes and responses of the immune system in vertebrates and
invertebrates. Because the squid/vibrio system combines both processes,
i.e., the immune response to bacteria mediated through LPS and the
LPS-triggered developmental responses, the model affords the opportunity
to study the interface of development and 'education' of the animal
immune system during development. In addition, we have shown that
the bacteria-induced host cell swelling does not occur when the
host is inoculated with bacterial mutants (provided by the laboratory
of Dr. E. Ruby, also at UH) in genes of the lux operon. This activity
of these genes not only controls light production, but also affects
(lowers) oxygen tension of colonized host tissues. It is likely
that it is their role in the poising of oxygen that is responsible
for the phenotype of no cell swelling; hypoxia is known to result
in cell swelling in other systems.
We have also recently studied the maintenance of the association.
We discovered that the specific bacterial symbiont, V. fischeri,
turns down the expression of the peroxidase gene in tissues where
it occurs as a beneficial symbiont, but turns up the expression
of this gene in tissues (specifically gills) where V. fischeri is
perceived as a pathogen. This finding provides further evidence
that some of the same genes are involved in the control of both
pathogenic and beneficial associations, and it is their modulation
that defines the nature and outcome of the relationship. Also, we
found that the macrophages sample the environment of symbiotic tissues,
similar to the manner in which macrophages sample the intestine
of vertebrates. We are currently determining whether these phagocytic
cells recognize all bacteria, including V. fischeri, or they only
recognize nonspecific bacteria in host tissues.
Recently, after defining the critical time points during which bacterial
signaling occurs, we have generated cDNA libraries of aposymbiotic
and symbiont juveniles at these time points. We are currently doing
a subtraction of these libraries to determine gene expression that
is unique to symbiotic animals, i.e., gene expression that has been
induced by interaction with V. fischeri. Once candidate genes are
identified, we will study the timing and location of expression
in colonized host tissues.
We have also studied the role of nitric oxide (NO) production in
the control of the symbiotic association. We have found that the
ducts leading to the internal crypts that are colonized by bacteria
have very high activity for nitric oxide synthase, the enzyme that
catalyzes the production of NO. Because other bacterial species
in the water column do not seem to be able to negotiate their way
through the ducts into the crypts, we are investigating whether
NO production is, at least in part, responsible for the specificity
of the tissues for colonization by V. fischeri.
Recently, my laboratory began a collaboration with Dr. Mary Montgomery
(Asst. Prof., Macalaster College, MN), a former student in the McFall-Ngai
laboratory. We are using the new technique of RNAi to attempt to
block specific gene expression in E. scolopes. Dr. Montgomery and
I have begun a collaboration to determine whether this new method
will block gene expression in the host Euprymna scolopes. If this
method is effective, we will be afforded a mechanism by which to
manipulate the host, a model animal in which classical genetics
has not been developed.
Recent publications:
Journal articles
Lemus, JD and MJ McFall-Ngai* (2000) Alterations in the proteome
of the Euprymna scolopes light organ in response to symbiotic Vibrio
fischeri. Appl Environ Microbiol 66:4091-4097.
Visick, KL, JS Foster, J Doino, MJ McFall-Ngai, and EG Ruby* (2000)
Vibrio fischeri lux genes play an important role in colonization
and development of the host light organ. J Bacteriol 182:4578-4586.
Foster, JS, MA Apicella, and MJ McFall-Ngai* (2000) Vibrio fischeri
lipopolysaccharide induces developmental apoptosis, but not complete
morphogenesis, of the Euprymna scolopes symbiotic light organ. Dev
Biol 226:242-254.
Nyholm, SV, EV Stabb, EG Ruby and MJ McFall-Ngai* (2000) Harvesting
symbiotic vibrios: Imposing a magnet on the environmental haystack.
Proc Natl Acad Sci USA 97:10231-10294.
Small, AL and MJ McFall-Ngai* (1999) A halide peroxidase in tissues
that interact with bacteria in the host squid Euprymna scolopes.
J Cellul Biochem 72:445-457.
Montgomery, MK and MJ McFall-Ngai* (1998) Late postembryonic development
of the symbiotic light organ of Euprymna scolopes (Cephalopoda:Sepiolidae).
Biol Bull 195:326-336.
Nyholm, SV and MJ McFall-Ngai* (1998) Sampling the light organ microenvironment
of Euprymna scolopes: Description of a population of host cells
in association with the bacterial symbiont Vibrio fischeri. Biol
Bull 195:89-97.
Nishiguchi, MK, EG Ruby, and MJ McFall-Ngai* (1998) Competitive
dominance among strains of luminous bacteria provides an unusual
form of evidence for parallel evolution in the sepiolid squid-vibrio
symbioses. Appl Environ Microbiol 64:3209-3213.
Foster, JS and MJ McFall-Ngai*(1998) Induction of apoptosis by cooperative
bacteria in the morphogenesis of host epithelial tissues. Dev Genes
Evol 208:295-303.
Lamarcq, L, and MJ McFall-Ngai* (1998) Induction of a gradual, reversible
morphogenesis of its host's epithelial brush border by Vibrio fischeri.
Infect Immun 66:777-785.
Reviews
McFall-Ngai, MJ (Solicited review) Unseen forces: The influence
of bacteria on animal development, (In press, Dev Biol)
McFall-Ngai (Solicited review) Unseen forces: symbiosis and the
evolution of multicellularity. [Invited contribution, Proceedings
of the Society for Experimental Biology Mtg., Cambridge, Eng] Comp
Biochem Physiol 129:711-723.
Hirsch, A* and MJ McFall-Ngai (Solicited review) Fundamental concepts
in symbiotic interactions. J Plant Growth Regul 19:113-130.
McFall-Ngai*, MJ and EG Ruby (Solicited review) Forming partnerships
in the marine environment: the squid-vibrio developmental model.
Curr Opinion Microbiol 3:603-607.
McFall-Ngai, MJ (2000) Negotiations between animals and bacteria:
The diplomacy' of the squid-vibrio symbiosis. Comp Biochem Physiol
126:471-480.
Visick*, KL and MJ McFall-Ngai (2000) Minireview. An exclusive contract:
Specificity in the iVibrio fischeri-Euprymna scolopes partnership.
J Bacteriol 182:1779-1787.
Ruby, EG* and MJ McFall-Ngai (1999) The many roles of oxygen in
the symbiotic bacterial colonization of an animal epithelium. Trends
Microbiol 7:414-419.
McFall-Ngai, MJ (1999) Consequences of evolving with bacterial symbionts:
Lessons from the squid-vibrio association. Annu Rev Ecol Syst 30:235-256.
McFall-Ngai, MJ (1998) Pioneering the squid-vibrio model. ASM News
64:639-645.
McFall-Ngai, MJ*, and EG Ruby (1998) Squids and vibrios: when first
they meet. BioScience 48:257-265.
McFall-Ngai, MJ (1998) The development of cooperative associations
between animals and bacteria: establishing détente among
Domains. Amer Zool 38:593-608.
* Communicating author
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