Speaker
Quick
Reference: Presenter and Title*
*In order of appearance
*In order of appearance
Poster
Session Quick Reference: Presenter and Title**
**In alphabetical order
**In alphabetical order
Speaker Abstracts*
*Presenter in italics
Session 1: Microbes and the Environment
"Dynamics and control of microbial biofilms"
A. Spormann, K. Thormann, R. Saville, S. Shukla and D. Pelletier
*Presenter in italics
Session 1: Microbes and the Environment
"Dynamics and control of microbial biofilms"
A. Spormann, K. Thormann, R. Saville, S. Shukla and D. Pelletier
Shewanella
oneidensis MR1 is a
facultative Fe- and Mn-redudicng
microorganism, and serves as a model system to study microbe-mineral
interactions. Biofilm formation in this microbe leads to direct
metabolic interactions of biofilm cells with the mineral surface, which
may result in direct mineral dissolution or corrosion. In order to
develop a basic understanding of microbe-mineral interactions, we
conducted a detailed molecular and cellular-level analysis of S. oneidensis biofilm formation.
Detailed quantitative studies, using gfp-labelled cells in conjunction
with confocal microscopy, identified key steps and check points in S. oneidensis biofilms. In order to
elucidate the genetic control of early biofilm formation, 15,000 Tn5
mutants were screened for defects in biofilm formation. Transposon
insertions of 80 biofilm-defective mutants were mapped and studied in
detail. Analyses of genes disrupted by Tn5 insertion suggests that
metabolic conditions, including regulation of global and secondary
metabolism, as well as motility are required for biofilm formation of
wild type cells. The results will be discussed with respect to
fundamental processes of biofilm biology as well as to microbe-mineral
interactions.
"Microbial community composition and gaseous nitrogen losses in agroecosystems"
Sasha
Kramer and Brendan J.M. Bohannen
"Microbial community composition and gaseous nitrogen losses in agroecosystems"
Understanding the impacts that
different agricultural management practices have on microbial
communities is critical for the design of sustainable agricultural
systems. Ecologists are just beginning to understand the relationship
between microbial community composition and the regulation of ecosystem
processes. This study focuses on the role of microbial community
composition in mediating gaseous nitrogen losses through nitrification
(the conversion of ammonium to nitrate) and denitrification (the
conversion of nitrate to dinitrogen gas). Agricultural denitrification
is of concern to ecologists because a proportion of the nitrate that is
denitrified will escape from the soil as nitrous oxide (N2O) before
being fully reduced to dinitrogen gas. Nitrous oxide is a potent
greenhouse gas and fertilized agriculture is thought to account for
over 70% of anthropogenic N2O emissions. The objective of this study
was to investigate how organic, integrated, and conventional
agricultural practices in Washington apple orchards influence microbial
community composition and whether differences in microbial communities
have the potential to affect the magnitude and composition of gaseous
nitrogen losses. Microbial communities differ among agricultural
treatments and these differences appear to be functionally relevant to
potential nitrification and denitrification rates and the proportion of
gas that is emitted as N2O. A more thorough understanding of the
explicit role of microbial community structure in agricultural nitrogen
cycling will serve as a useful tool for mitigating greenhouse gas
emissions from agroecosystems.
Vibrio
cholerae O1, the agent of Asiatic cholera, causes a dehydrating
diarrheal illness and sometimes death. Outside the human host, V. cholerae is a member of the
natural aquatic environments, such as lakes, rivers, estuaries, and the
ocean. In the aquatic habitat, V.
cholerae can be found attached to copepods, a subclass of
crustacean zooplankton. A single copepod may carry large numbers of V. cholerae cells. Therefore,
during plankton blooms, ingestion of copepods in drinking water could
result in human infection. Furthermore, copepods and their chitinous
exoskeleton promote V. cholerae
survival by providing a nutrient source and by creating an environment
that protects V. cholerae
from harsh conditions. The main component of the exoskeletons of
copepods is chitin, a polymer of beta-1,4 linked N-acetylglucosamine
residues. Chitin is the most abundant polymer in nature after cellulose
and many Vibrio species that live in aquatic environments are capable
of using chitin as the sole carbon source. Relatively little is known
about the chitin utilization program of V. cholerae. We have performed
expression profiling of V. cholerae
growing on a natural chitin surface. Further expression profiling
experiments with soluble chitin oligosachharides, the
N-acetylglucosamine monomer, and the chitosan glucosamine dimer showed
that each of these three carbohydrate classes regulates different sets
of genes within the transcriptome of chitin surface-attached bacteria.
We also show that most genes within the chitin
oligosaccharide-regulated response are controlled by a newly identified
regulatory protein, ChiS. These results and functional studies with
mutants have led to a model of chitin utilization by V. cholerae.
Session
2: Microbe-Host Interactions
"Assembly
and
disassembly of F-actin on phagosomal membranes"
Patricia T. Yam and Julie A. Theriot
Patricia T. Yam and Julie A. Theriot
Early in infection of the
intracellular pathogen Listeria
monocytogenes in epithelial (MDCK) cells expressing GFP-actin,
we have found that actin rapidly polymerizes and then catastrophically
depolymerizes at the bacterial surface, resulting in a cyclic
appearance and disappearance of a transient actin cloud - a phenomenon
we term “flashing”. Flashing was not dependent on ActA-mediated
nucleation of actin filaments since L.
monocytogenes with a deletion of actA also flashed. A mutant
which was unable to escape from the vacuole ((delta)hly (delta)plcA
(delta)plcB) also flashed. This suggested that flashing was actin
polymerization and depolymerization on the host cell phagosomal
membrane whilst it encapsulated the bacterium. Indirect
immunofluorescence confirmed that the bacteria were completely
intracellular when flashing occurred, indicating that flashing was not
simply abortive phagocytosis. The formation and disappearance of actin
clouds was characterized by a rising phase where net actin
polymerization occurred and a falling phase where net depolymerization
occurred. There was no plateau phase. Both the polymerization and
depolymerization phase showed linear kinetics. The linear rise suggests
that once filament nucleation was initiated, no further nucleation
occurred. Both the rise time and the fall time for a flash were tightly
clustered at 16 - 32 s and 24 - 48 s, respectively. Flashing appeared
to be independent of invasion mechanism. E. coli expressing invA, a gene
product from Yersinia
pseudotuberculosis that binds integrins, also induced flashing.
Furthermore, polystyrene beads coated with E-cadherin were internalized
by MDCK cells and induced flashing, suggesting that signaling events
from the bacterium are not required. We propose that cyclic actin
assembly and disassembly on the phagosomal membrane may be a general
feature of internalization of large objects by epithelial cells.
"Host factors in
mycobacterial pathogenesis"
Marc Dionne and David Schneider
Marc Dionne and David Schneider
Mycobacterium
marinum is a pathogenic mycobacterial species that is closely
related to M. tuberculosis
and causes tuberculosis-like disease in fish and frogs. We have
recently demonstrated that M. marinum
can cause a lethal infection in the fruitfly Drosophila melanogaster. Death is
accompanied by widespread tissue damage. M. marinum initially proliferates
inside the phagocytes of the fly; later in infection, bacteria are
found both inside and outside host cells. M. marinum disrupt lysosome
maturation in Drosophila
phagocytes just as they do in mouse macrophages. Finally, neither M. marinum nor the nonpathogenic
species M. smegmatis induce
the systemic production of circulating antimicrobial peptides; peptide
induction is ordinarily one of the hallmarks of infection in the fly.
We have used the genetic tractability of the fly to perform a genetic
screen for Drosophila mutants
which die more or less rapidly than wild-type flies when infected with M. marinum, or in which M. smegmatis can cause a lethal
infection. We have isolated mutants which alter bacterial growth rates,
and also mutants in which bacterial growth is unaltered but death is
delayed or accelerated. Several mutants from this screen will be
discussed.
Smallpox and Ebola
are two of the deadliest diseases known to humankind. Smallpox is a
calamitous, lethal disease that was endemic in many parts of the world
until its eradication in 1977 through a vigorous vaccination program by
the WHO. Infection with Ebola virus causes a fulminant, often-fatal
hemorrhagic fever (with case fatality rates up to 90%), and
sporadically erupts in Africa. Both viruses remain a potential
bioterror threat. In order to improve our understanding of smallpox and
Ebola pathogenesis and virus-host interactions we have examined the
molecular features of smallpox and Ebola infection in vivo. Using
high-density cDNA microarrays, we analyzed genome-wide host expression
patterns in sequential blood samples from nonhuman primates infected
with smallpox or Ebola. During smallpox infection, several striking
gene expression patterns are prominent: an interferon response, a cell
proliferation/cell cycle response, immunoglobulin gene expression,
lymphocyte-specific signatures, hematologic abnormalities, and possible
viral modulation of the host immune response. Data from Ebola infection
demonstrate cytokine responses, an interferon response, apoptosis
genes, and pre-symptomatic gene expression. Comparison of these two
models of infection illustrates differences relating to the TNFa/NFkB
pathway and a cell proliferation/cell cycle response. Together, these
data may elucidate conserved and specific mechanisms of viral virulence
and host defense, and may suggest new diagnostic, prognostic,
therapeutic and prophylactic approaches.
Session 3: Microbial Cell Cycle
"Functional proteomic analysis of the
cysteine proteases of Plasmodium falciparum"
Doron C. Greenbaum, Amos Baruch, Munira Grainger, Zbynek Bozdech, Katalin F. Medzihradszky, Tony Holder, Joseph DeRisi and Matthew Bogyo
Doron C. Greenbaum, Amos Baruch, Munira Grainger, Zbynek Bozdech, Katalin F. Medzihradszky, Tony Holder, Joseph DeRisi and Matthew Bogyo
Perhaps one of the greatest
challenges resulting from the rapid advances in proteomics and genomics
is finding ways to assign function to the large numbers of genes within
a genome. Furthermore, in biological systems where standard genetic
methods remain difficult, alternative tools must be developed. We have
designed activity based probes that covalently tag cysteine proteases
through selective chemical modification of the active site cysteine
residue. We have used these probes to identify a family of cysteine
proteases in the human malaria parasite, Plasmodium falciparum by virtue of
their reactivity towards our probes. We have also used these reagents
to screen combinatorial libraries of inhibitors in crude parasite
proteomes. Selective inhibitors identified by this method allowed us to
chemically 'knock-out' a specific protease and assess its function
during the parasite blood stage lifecycle. Our results have identified
a protease that is likely to play a role in host cell invasion, thereby
validating it as a potential new anti-malarial drug target. These
results along with other potential applications of small molecule
probes to studies of P. falciparum
will be discussed.
"Regulated intramembrane proteolysis in
Caulobacter crescentus"
Joseph C. Chen, Patrick Viollier and Lucy Shapiro
Joseph C. Chen, Patrick Viollier and Lucy Shapiro
The bacterium Caulobacter crescentus coordinates
cell cycle progression and cellular differentiation to follow a
specific developmental program. Every cell division leads to asymmetric
separation of the predivisional cell into two distinct cell types, a
stalked cell and a swarmer cell. Transition from one stage of the cell
cycle to another involves profound physiological changes. To ensure
faithful execution of the developmental program, the bacterium employs
multiple regulatory strategies, a key one being targeted proteolysis.
Regulatory and structural proteins are degraded at defined times and
locations during the cell cycle. We are investigating how regulated
proteolysis contributes to developmental events. Specifically, we are
identifying membrane-bound proteases, assessing their involvement in
cell cycle progression, and determining their proteolytic activities
and targets. Analysis of the C.
crescentus genome indicates that two ORFs, CC1916 and CC3381,
encode proteins belonging to a family of membrane-embedded
metalloproteases. Characterized members of this family appear to cleave
their respective substrates within or near transmembrane segments. In
Caulobacter, the CC1916 gene product appears to affect the degradation
of PodJ, a localization factor important for the development of polar
organelles. During the cell cycle, full-length PodJ is processed to a
short form, which is subsequently degraded; the long and short forms of
PodJ have distinct developmental functions. Consistent with the
functions of its homologs in other organisms, CC1916 appears to
participate in the second proteolytic process. We are currently
confirming the role of CC1916 in PodJ processing.
Session 4:
Manipulations: Microbe vs. Host
"Major Human Cytomegalovirus structural protein pp65 (ppUL83) blocks the IRF-3-dependent interferon response"
Davide A. Abate, Shinya Watanabe and Edward S. Mocarski
"Major Human Cytomegalovirus structural protein pp65 (ppUL83) blocks the IRF-3-dependent interferon response"
Davide A. Abate, Shinya Watanabe and Edward S. Mocarski
The rapid induction of the
interferon response provides a key defense mechanism against many
viruses. Here we use functional genomics to identify the major
cytomegalovirus structural protein, pp65 (ppUL83), as a crucial factor
that counteracts the type I interferon response. This protein prevents
activation of interferon response factor (IRF)-3 but does not affect
NF-kB, STAT1, STAT3 or IRF-7. pp65 appears to act on this central
mediator of the interferon pathway by inhibiting hyperphosphorylation
and nuclear accumulation. When expressed independently, pp65 is
sufficient to prevent IRF-3 translocation to the nucleus in response to
viral or non-viral inducers. This work illustrates how a major virion
protein of a complex DNA virus has evolved to be a immunomodulatory
factor that sabotages the central alarm system devoted to both
immediate antiviral control and regulation of the immune response.
"Rapid generation of siRNA vectors and
libraries from cDNA"
George Sen, Tom Wehrman, Jason Myers and Helen Blau
George Sen, Tom Wehrman, Jason Myers and Helen Blau
Mammalian cell fate and
differentiation are dynamic processes that are continually regulated by
the selective expression and dosage of specific genes. Facilitating
these studies, rapid loss of gene function in mammalian cells has
recently been achieved through small interfering RNA (siRNA)
technology. Application of this technology to cell fate decisions holds
great promise to unraveling the complex network of positive and
negative feedback loops that determine these cellular decisions.
However, the generation of multiple unique siRNA expression vectors is
slow, inefficient and costly. This report describes a novel method for
generating numerous siRNA constructs from any gene of interest or pool
of genes using a combination of restriction enzyme digests and hairpin
loop ligations. To test if Restriction Enzyme Generated siRNAs (REGS)
were functional, a transgene (GFP) and two endogenous genes (Oct 3/4,
and MyoD) were silenced resulting in the expected cell fate decisions:
ES cell to trophoblast differentiation for Oct 3/4, and inhibition of
myotube formation for MyoD. REGS generated approximately 34 unique
siRNAs per kilobase of sequence with greater than 96% of the cloned
inserts containing siRNAs with the appropriate structure. The
efficiency of REGS enabled the creation of an siRNA library from double
stranded cDNA containing approximately 415,000 independent clones. To
our knowledge, the high yield of siRNAs per gene and the creation of
unbiased highly complex siRNA libraries using REGS is unprecedented.
Poster Abstracts*(in alphabetical
order)
*Presenter in italics
"Caenorhabditis elegans as an early infection model for Salmonella enterica serovar typhimurium"
Rosanna Alegado, Stanley Falkow and Man-Wah Tan
*Presenter in italics
"Caenorhabditis elegans as an early infection model for Salmonella enterica serovar typhimurium"
Rosanna Alegado, Stanley Falkow and Man-Wah Tan
We are exploring the utility of Caenorhabditis
elegans as a small
animal model for S. typhimurium-induced gastritis. Currently,
there is no small animal model for gastritis induced by S. typhimurium, since mice infected with S. typhimurium do not develop gastritis.
Moreover, the high cost of the calf model precludes its use for
comprehensive screening of virulence determinants. Previous work has
demonstrated the ability of S. typhimurium to colonize and proliferate in the
gut of C. elegans. Pronounced lumenal distention
observed is a physiological phenomenon that seems to parallel
gastritis. Bacterial regulators such as OmpR, Fur-1, and RpoS have been
implicated in worm mortality. Additionally, programmed cell death,
modulated by interactions between bacterial lipopolysaccharide (LPS)
and host p38 MAP kinase pathway, is also involved in pathogenesis.
However bacterial factors required for initial colonization of the gut
are not known. We performed in vivo observation of colonization within
live animals using wild type Salmonella that constitutively expresses GFP. Salmonella strains deficient in known
virulence factors were assessed for their capacity to colonize and
persist within the worm. Virulence factors important for colonization
and invasion in the murine model, and enteritis in the calf models such
as LPS, Salmonella pathogenicity island 1 (SPI-1),
SPI-2, and the virulence plasmid were also important for colonization
of Salmonella within the gut of the nematode as
these mutants failed to persist after short term exposure. To identify
novel pathogen factors important for initial colonization by S. typhimurium, transposon insertion library of
approximately 900 mutants was screened for Salmonella strains that fail to colonize C. elegans. We reasoned that a Salmonella mutant which is less effective at
colonization should not persist in nematodes. Here, we report on
progress made toward characterization and identification of these
mutants.
"Molecular analysis of gastric
microbial flora"
Elisabeth M. Bik, Paul B. Eckburg, Steven Gill, Karen Nelson, Fritz Francois, Guillermo Perez-Perez, Martin Blaser and David A. Relman
Elisabeth M. Bik, Paul B. Eckburg, Steven Gill, Karen Nelson, Fritz Francois, Guillermo Perez-Perez, Martin Blaser and David A. Relman
Aside from the known role of
Helicobacter pylori (Hp) in colonization of the human stomach, the
composition of the gastric microflora, including unculturable species,
remains largely unknown. In this study, the microbial diversity within
the human stomach was characterized using broad-range PCR on ribosomal
DNA sequences. Gastric biopsy samples from 25 patients (15 Hp-positive
and 10 Hp-negative) were collected during upper endoscopy. Chromosomal
DNA was extracted using a combined QIAamp (QIAgen) / bead-beater
procedure. No fungal or archaeal DNA could be detected using
broad-range ribosomal DNA primers. Broad-range bacterial 16S rDNA
amplification, however, was positive in 24 samples (96%). Bacterial 16S
rRNA sequences from 4 gastric antrum biopsy samples from 4 male
patients (2 Hp-positive and 2 Hp-negative) were cloned and sequenced. A
total of 330 rDNA sequences of 799 bp were analyzed using the
phylogenetic software package ARB. All sequences could be assigned to
the Proteobacteria (7.6% of the combined 157 sequences in the 2
Hp-negative and 77.5% of the combined 173 sequences in the 2
Hp-positive samples, respectively), Firmicutes (70.7 and 17.9%),
Actinobacteria (4.5 and 2.9%) and Bacteroidetes (17.2 and 1.7%) phyla.
The majority (74%) of the combined sequences from the 2 Hp-positive
samples showed over 99% sequence similarity to Hp 16S ribosomal DNA. No
Helicobacter rDNA was found in the 2 gastric samples that were negative
for Hp by conventional methods. Cloning and sequence analysis of
additional ribosomal DNA clones is underway. These results show
differences in the composition of gastric microflora in the presence or
absence of Helicobacter pylori, and will provide insight into the
role of other bacteria in the development of diseases of the upper
gastrointestinal tract.
"A class
of Salmonella
genes important in systemic infection and antimicrobial peptide
resistance"
Igor Brodsky, Denise Monack, Corrie Detweiler, Hanza Matthew and Stanley Falkow
Igor Brodsky, Denise Monack, Corrie Detweiler, Hanza Matthew and Stanley Falkow
Salmonella
enterica serovar Typhimurium is used as a model for studying
systemic salmonellosis in murine models of infection. encodes
a type III secretion system on S.
typhimuriumSalmonella
pathogenicity island 2 (SPI-2)
that is required for replication of S. typhimurium in macrophages, a phenotype that
correlates closely with systemic disease in mice. We used a
microarray-based screen to identify Salmonella genes that are co-regulated with
SPI-2 genes. Several genes were found which, when interupted by a
kanamycin cassette, resulted in mutants that were indeed attenuated in
a systemic mouse infection. Two of these genes, virK, and somA, were
regulated by the PhoPQ two-component system, while the third, rcsC, was
not. virK and somA are homologues of a known virulence gene in Shigella
flexneri, but have not
previously been described as playing a role in S. typhimurium pathogenesis. Other genes of the
PhoPQ regulon are important for replication of Salmonella in tissue culture macrophages. One
PhoP-regulated gene, mig-14, is not required for initial colonization
steps, but is required for S. typhimurium replication in the spleen and
liver. Mutations in mig-14 affect resistance to antimicrobial peptides
polymyxin B and protegrin-1 in vitro by an unknown mechanism. Similar
to mig-14, somA, virK, and rcsC are required for resistance to
polymyxin B. These data suggest that this class of genes may play a
role in resistance to bactericidal concentrations of antimicrobial
peptides and other immune effector molecules encountered systemically
during later stages of infection. Alternatively, these genes may
control changes in the bacterial envelope associated with interactions
with the host adaptive immune system, and may be involved in modulating
host immune responses.
"Ribosomal
proteins L4 and L22 are involved in tryptophan
induction of the tryptophanase operon"
Luis Rogelio Cruz-Vera and Charles Yanofsky
Luis Rogelio Cruz-Vera and Charles Yanofsky
During the translation process the
nascent peptide moves through the ribosomal particle towards its
external surface through a channel, called the tunnel, in the 50S
ribosomal subunit. Current understanding of the translation process and
ribosome structure places the nascent peptide in close contact with the
tunnel components. Translation of the leader transcript derived from
the tryptophanase (tna) operon of E. coli has been useful as a model for
analyzing nascent peptide-ribosome tunnel interactions. Synthesis of
the tna operon leader peptide, TnaC, in the presence of an excess of
free tryptophan, induces expression of this operon by preventing
TnaC-peptidyl-tRNA cleavage and translation termination. The amino acid
sequence of TnaC, specifically one tryptophan residue at position 12,
putatively located in the ribosomal tunnel, is crucial to inhibition of
translation termination. To investigate whether tunnel components are
involved in this process, specifically proteins L4 and L22, we
mutagenized plasmids containing these genes and screened for mutants
affecting tna operon expression. We obtained one mutant altered in each
gene in which expression of a tna reporter gene was reduced
appreciably. The mutated amino acid, in each protein, is normally
located in the ribosomal tunnel. We also obtained one L22 mutant in
which expression was enhanced during induction. The amino acid replaced
in this mutant is normally located in a contact region between protein
L22 and 23S rRNA. The properties of these mutants suggest that proteins
L4 and L22 may play a crucial role in induction of the tna operon, by
potentially interacting with - and recognizing features of - the
nascent TnaC-peptidyl-tRNA.
"The sporulation
inhibitor, Sda,
and its target, KinA"
Katherine Cunningham and William Burkholder
Katherine Cunningham and William Burkholder
Sporulation in Bacillus subtilis results from the
transcription of sporulation specific genes, which are turned on by the
transcription factor Spo0A. Spo0A becomes active after phosphorylation
via a protein phosphorelay that begins with the autophosphorylation of
several histidine kinases. Two of these kinases, KinA and KinB, are
inhibited by a small protein called Sda when there are defects in DNA
replication or DNA damage. My research focuses on how KinA is
specifically recognized and inhibited by Sda. KinA consists of three
domain: an N-terminal sensor domain, a dimerization/histidine
phosphotransfer domain (DHp), and an ATP-binding catalytic domain. It
is active as a dimer and autophosphorylates in trans. Our current model
is that Sda acts as a molecular wedge to prevent communication between
the catalytic domain of one subunit and the histidine residue that is
the target of autophosphorylation on the other. To verify this model, I
am using targeted disulfide crosslinking to see if Sda prevents
crosslinking between the catalytic and DHp domains of KinA. I will also
use site-directed mutagenesis to identify specific KinA residues that
are important for Sda binding.
"Variable cost of
antibiotic
resistance by environment in Escherichia
coli"
Clara L. Davis and Brendan J.M. Bohannan
Clara L. Davis and Brendan J.M. Bohannan
Many studies on antimicrobial
resistance have focused on the cost of antimicrobial resistance in
terms of Darwinian fitness, and the implications of this cost toward
management of antimicrobial resistance. Studies from different labs use
variable media types to measure this fitness cost. We decided to
compare the cost of resistance in multiple environments using Escherichia coli resistant to
rifampin. We isolated a set of 42 rifampin-resistant mutants (all from
the same genetic background) on a rifampin gradient of 0-100 ug/mL. We
measured the fitness cost for each isolate (when compared to the
rifampin-sensitive ancestor) in both minimal media and rich media. The
range of fitnesses was greater in rich media than in minimal media, and
more mutations appeared to carry no fitness cost in minimal media than
in rich media. We are currently correlating the fitness results to
specific mutations conferring rifampin resistance, along with MIC to
rifampin, and planning further experiments to determine the factors
that may cause this difference in fitness costs by media type, such as
population size and type or number of carbon sources. The fact that
measurement of fitness can vary by media type used in a lab may have
important considerations for how we translate in vitro lab results into
management decisions for public health.
J.D. Dunn and John Boothroyd
Upon invasion, the obligate
intracellular protozoan parasite Toxoplasma
gondii forms a parasitophorous vacuole (PV), a novel,
nonfusogenic compartment in which it replicates. The parasite targets
proteins to the PV and the PV membrane from specialized organelles
called rhoptries and dense granules. Some of these secreted proteins
are thought to have a role in nutrient transport while others may
function in the host-pathogen interaction. Recent evidence from studies
on T. gondii cholesterol
acquisition, on attenuation of the host immune response by T. gondii, and on T. gondii egress are consistent
with the hypothesis that the parasite traffics proteins into the host
cell. The overarching goal of this study is to understand the various
host cell modifications potentiated by Toxoplasma gondii during its
intracellular development. Proteins secreted by the parasite appear to
be the mediators of alterations in host cell physiology. Thus, our
immediate goal is to identify proteins secreted by T. gondii to modify its
intracellular residence. To distinguish between proteins of host and
parasite origin, we metabolically label intracellular parasites
subsequently to selectively inhibiting host protein synthesis with
ricin. Using this technique, we have identified radiolabeled proteins,
putatively of parasite origin, on the surface of infected host cells.
Identification of these proteins will elucidate the nuances of the
relationship between Toxoplasma
gondii and its host, will potentially reveal new therapeutic
targets, and will provide new insight into the cell biology of both
host and parasite.
"Temporal and spatial
dynamics of
the bipartite genome of Vibrio
cholera"
Aretha Fiebig and Julie Theriot
Aretha Fiebig and Julie Theriot
Accurate chromosome partitioning is
essential for the health and survival of any cell, ensuring that both
products of a cell division receive a full complement of DNA. Recent
experiments designed to understand DNA partitioning in bacteria have
primarily focused on plasmids or on species with a single chromosome.
However, numerous bacterial species including Vibrio cholera, Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhodobacter spheroides, and Brucella melitensis encode
essential genes on more than one chromosome. In addition, the majority
of studies thus far concern analysis of static images from populations
of cells. The real-time dynamics of chromosome segregation in species
with multiple replicons has not yet been addressed. I am exploring the
dynamics of chromosome behavior in V.
cholera, and plan to correlate this behavior to other markers of
the cell cycle. Moreover, I am investigating the roles of the multiple
partitioning proteins present in the V.
cholera genome.
"Diversity of nitrite
reductase
genes across environmental gradients in estuarine and extreme lake
environments"
Chris Francis and Bess Ward
Laura Hertel and Edward S. Mocarski
Chris Francis and Bess Ward
Denitrification, the dissimilatory
reduction of nitrate and nitrite to gaseous products (NO, N2O, N2)
under suboxic conditions, is the main loss term for fixed nitrogen from
ecosystems. Nitrite reductase, the key denitrification metalloenzyme
responsible for catalyzing the first committed step to a gaseous
product, occurs in two distinct forms: NirS, containing iron
(heme-cd1); and NirK, containing copper. Although nitrite reductase
genes (nirS and nirK) are useful molecular markers for the
environmental detection of denitrifiers, few environments have been
thoroughly explored. My research has exploited nitrite reductase genes
to explore how the distribution and diversity of denitrifiers are
influenced by environmental gradients in two very different aquatic
environments: Chesapeake Bay and Lake Bonney (Antarctica). In
Chesapeake Bay, the largest estuary in North America, my research
investigated the relationship between nitrite reductase diversity and
physical/chemical complexity along the salinity gradient from the
rivers and upper Bay to the sea. nirS genes were PCR-amplified from DNA
extracted from sediment cores collected at five stations spanning
gradients of salinity, NO3, and DOC. Sequencing of >100 nirS clones
from each station revealed extensive and unprecedented diversity, and
detected distinct spatial structure in the nirS sequence "populations".
The vast majority of the nirS sequences fell into phylogenetic clusters
and lineages distinct from known denitrifiers, implying that cultivated
denitrifiers are simply not representative of the natural assemblages.
Lake Bonney is a permanently ice-covered, chemically-stratified
Antarctic lake that is unusual because denitrification occurs in the
deep anoxic waters of the west lobe (WLB) but not the east lobe (ELB).
Environmental factors that usually control denitrification rates (e.g.,
DOC, NO3, O2) do not appear to explain its distribution in this extreme
environment. To help resolve this mystery, nirS genes were
PCR-amplified from DNA extracted from various depths in both lobes.
Although sequencing of >100 nirS clones revealed extremely low
diversity, significant community compositional overlap and greatest
diversity occurred at the two depths where maximal denitrification
rates were recently measured. The detection of nirS genes is consistent
with the immunofluorescence detection of denitrifiers in the lake, but
only deepens the mystery of why there is no denitrification in situ.
"What happens when you are
late?
Statistically significant changes in expression levels of cellular
genes at late times post infection with Cytomegalovirus as determined
by cDNA microarray analysis"Laura Hertel and Edward S. Mocarski
Human cytomegalovirus (CMV)
infection causes profound alterations in the transcriptional levels of
many cellular genes. While previous studies have focused on the
immediate-early and early times post infection, in this study we sought
to determine the global impact of CMV infection on gene transcription
in human fibroblasts (HFs) at late times post infection using the
technology of cDNA microarray analysis. Equivalent amounts of Cy-5
labeled cDNA pools generated from AD169-infected HFs at 50, 72 or 98
hpi and Cy-3 labeled cDNA pools obtained from uninfected HFs and used
as reference for all time points were hybridized to human cDNA
microarrays containing ~ 42,000 spots representing approximately 30,000
unique genes. A set of 12 microarrays was generated by hybridizing four
microarrays per time point. The statistical program SAM (Significance
Analysis of Microarrays) used to select genes significantly induced or
repressed across all 3 time points identified a set of 5658 highly
statistically significant genes. 65% (3675) of these genes are
constantly repressed at all time points and the remaining 35% (1983)
are constantly induced. About half (43%) of the genes are ESTs or
encoding proteins of unknown function. The remaining genes, grouped
into categories according to their function, show that many classes of
genes are prominently affected. Among the most highly repressed is a
set of cytoskeletal genes including microfilament proteins, adhesion
molecules and components of the extracellular matrix and a set of
several members of the small GTPases family. Among the most highly
induced are genes belonging to the cell cycle control, DNA replication,
mitochondrial respiratory chain metabolism and immune function genes.
Taken together these data provide useful insight into an as yet poorly
understood period of CMV life cycle.
"Predator resistance
versus
competitive ability: effects of trade-off magnitude on community
structure and dynamics"
Christine Jessup and Brendan Bohannan
Christine Jessup and Brendan Bohannan
The trade-off between predator
resistance and competitive ability is one of several trade-offs
believed to be central to the maintenance of diversity. In laboratory
communities of bacteriophage and E.
coli, mutant bacteria that are resistant to infection by
co-occurring phage frequently arise and coexist with sensitive
bacteria. Because mutations conferring resistance also affect important
cellular processes, resistant bacteria often incur a competitive cost
in their ability to obtain resources. Theoretical predictions show that
the magnitude of the cost of bacteriophage resistance determines the
equilibrium resource concentration, thereby affecting both the
phage-sensitive population and the equilibrium density of phage. We
assembled communities containing bacteriophage T4, T4-sensitive E. coli and either a high-cost or
low-cost T4-resistant strain to explore how the magnitude of the
trade-off between predator resistance and competitive ability affects
community structure and dynamics. Cost of resistance of the
T4-resistant strain had no effect on the equilibrium density of
bacteriophage, and we observed the extinction of the T4-resistant E. coli strain in several
communities. Therefore, we revisited the theoretical predictions for
these communities by running numerical simulations parameterized with
measured strain characteristics. The results of these simulations
showed that our empirical results were, in fact, consistent with
theoretical predictions for the strains used. The model also revealed
that lower cost phage-resistant E.
coli strains are required to explore the effects of trade-off
magnitude on community structure. We are currently conducting
experiments with such lower cost T4-resistant strains to achieve
coexistence of the two competitors, and to further explore the effects
of the cost of predator resistance.
"Fluorescence bleaching
reveals
asymmetric compartment formation prior to cell division in Caulobacter"
Ellen M. Judd, Kathleen R. Ryan, W. E. Moerner, Lucy Shapiro and Harley H. McAdams
Ellen M. Judd, Kathleen R. Ryan, W. E. Moerner, Lucy Shapiro and Harley H. McAdams
Asymmetric cell division in Caulobacter
crescentus yields
daughter cells that have different cell fates. Compartmentalization of
the predivisional cell is a critical event in the establishment of the
differential distribution of regulatory factors that specify cell fate.
To determine when during the cell cycle the cytoplasm is
compartmentalized so that cytoplasmic proteins can no longer diffuse
between the two nascent progeny cell compartments, we designed a
fluorescence loss in photobleaching (FLIP) assay. Individual cells
containing green fluorescent protein (EGFP) were exposed to a bleaching
laser pulse tightly focused at one cell pole at different times in the
cell cycle. In compartmentalized cells, fluorescence disappears only in
the compartment receiving the bleaching beam; in non-compartmentalized
cells, fluorescence disappears from the entire cell. In a 135-minute
cell cycle, the cells were compartmentalized 18+5 minutes before the
progeny cells separated. Clearance of the 22000 CtrA master
transcriptional regulator molecules from the stalked portion of the
predivisional cell is a controlling element of Caulobacter asymmetry. Monitoring of a
fluorescent marker for CtrA showed that the differential degradation of
CtrA in the nascent stalk cell compartment occurs only after the
cytoplasm is compartmentalized.
"Dissecting malaria
vector-pathogen
interactions using Drosophila-Plasmodium genetic system"
Hirotaka Kanuka, Stephanie M. Brandt and David S. Schneider
Hirotaka Kanuka, Stephanie M. Brandt and David S. Schneider
Malaria is a devastating public
health menace, killing over one million people every year and infecting
about half a billion. The malarial parasite, Plasmodium, has an intricate
lifecycle in both its vertebrate and insect hosts. We previously showed
that the protozoan Plasmodium
gallinaceum, a close relative of the human malaria parasite Plasmodium falciparum, can develop
in the fruit fly Drosophila
melanogaster. For the purpose of understanding the factors in an
insect-parasite relationship that control the ability of an insect to
act as a disease vector, we identify and analyze Drosophila mutants affecting these
processes. To identify genes involved in the insect response to Plasmodium, we performed a genetic
screen for genes that affect a fly's ability to support the growth of Plasmodium gallinaceum by using
real time reverse transcription PCR to determine the parasite load in
each strain. Approximately 700 fly mutant strains have been screened
and we identified one mutant in which we can observe more Plasmodium growth than in the wild
type strain. We determined tht mutated gene encodes a putative
cell-adhesion/recognition receptor protein. We will discuss about some
predicted functions of this gene product in relation to Plasmodium growth in insect vector.
"Nitrogenase gene diversity
in
subgingival plaque"
Paul W. Lepp, Megan Dennis, Cleber Ouverney, Gary Armitage and David Relman
Paul W. Lepp, Megan Dennis, Cleber Ouverney, Gary Armitage and David Relman
Phylogenetic analysis of 16S rDNA
is currently being used by many investigators to characterize the
bacterial community found in subgingival plaque. Recent advances
permits us to investigate the functional role of specific members of
that community. Using broad-range PCR primers for the dinitrogenase
reductase gene (nifH) involved in nitrogen fixation we have identified
four distinct phylotypes that appear to be widely distributed in
specimens from periodontitis patients. All four phylotypes fall within
nifH Cluster IV which is comprised primarily of nifH genes from
Treponema and methanogens. The translated amino acid sequence of the
predominant oral phylotype shared 64% identity and 77% similarity to
the most closely related phylotype within the public database. None of
the phylotypes shared more than 69% identity and 85% similarity to
phylotypes from public databases. Two of the phylotypes were most
closely related to sequences retrieved from the gut of termites. The
remaining two phylotypes were most closely related to phylotypes
associated with deep-sea hydrothermal vents. We developed a specific
oligonucleotide probe targeting mRNA for the predominant dominant nifH
phylotype. Fluorescent in situ hybridization revealed an approximately
5 mm x 1 mm bacillus that co-localizes with a bacterial specific probe.
The ability to visualize this organism using a mRNA targeted probe
provides preliminary evidence for active dinitrogenase reductase
expression. We are currently developing a quantitative RT-PCR assay to
determine the relative expression of each of the four phylotypes. These
data suggest that a previously unrecognized guild of nitrogen fixing
microorganisms may influence the community composition of subgingival
plaque.
Entamoeba
histolytica is a
protozoan parasite and the causative agent of amebiasis, a disease
resulting in ~ 100,000 deaths per year. In order to identify genes
involved in the pathogenesis of amebiasis we have constructed an E. histolytica microarray. The array consists of
11,500 sequenced genomic clones and we have demonstrated that it
accurately detects transcript abundance. Using Caco-2 cells to model
the interaction of E. histolytica with colonic epithelial cells, we
have demonstrated that there is a limited transcriptional response by
the parasite during enterocyte interaction. Preliminary results
identify a small number of upregulated and down-regulated transcripts.
Interestingly, genes previously characterized as virulence determinants
(cysteine proteinases, lectin, amoebapore) were not specifically
regulated in our model. Additional studies will focus on expression
differences
between different strains of E. histolytica (NIH200, HK9 and Rahman) as
well as the avirulent Entamoeba dispar.
"Translational
regulation of the
DNA checkpoint protein, Sda"
Emily Nahas and William Burkholder
"The role of human
CMV UL37X1 gene product vMIA"
A. Louise McCormick, Geoff Smith, Vanessa Smith and Edward S. Mocarski
A. Louise McCormick, Geoff Smith, Vanessa Smith and Edward S. Mocarski
Infection with human
cytomegalovirus altered the reticular mitochondrial network
characteristic of uninfected fibroblasts. A kinetic association between
mitochondrial alterations and expression of the immediate early (a)
anti-apoptotic UL37x1 gene product, viral mitochondria-localized
inhibitor of apoptosis (vMIA) was evident in virus-infected and
vMIA-transfected cells. A 68 aa fully func-tional derivative of vMIA
also induced disruption. vMIA-function may be tied to the
fission/fusion process that normally controls mitochondrial networks.
Importantly, mutations that prevent anti-apoptotic activity also
prevent mitochondrial disruption. GFP-tagged vMIA has been used to
further clarify the relationship between cell death suppression and
disruption of mitochondrial networks.
"Gene expression
during form
transition in Paracoccidioides
brasiliensis"
J. P. Monteiro, K. V. Clemons, L. F. Mirels, C. R. Lopes and D. A. Stevens
J. P. Monteiro, K. V. Clemons, L. F. Mirels, C. R. Lopes and D. A. Stevens
This project intends to apply
high-density DNA microarray analysis to
Paracoccidioides brasiliensis to identify and characterize
differentially expressed genes during the mycelium-to-yeast transition,
a key event during the initiation of infection. Total DNA was extracted
from isolate Pb 01 (ATCC MYA-826), and randomly sheared into 1-2 kb
fragments. The fragments were cloned into ë ZAP II phage to
generate a random shear genomic library. We have isolated approximately
12,000 clones from this library. Inserts have been amplified by PCR
using a single primer-pair. After purification, the inserts will be
used for the construction of arrays of about 12,000 elements. Over 100
clones were sequenced and blasted against nucleotide and protein
sequences present in the GenBank databases. Forty-six sequences showed
strong similarity to known fungal genes or proteins, ESTs from P. brasiliensis and/or transposable
elements. These sequences confirmed our presumption that most of the
inserts would contain only one gene, and be most similar to fungal
genomic DNA, cDNA or protein sequences even if its function was
unknown. Interestingly, no clones proved to contain rDNA sequences. We
have also designed primers for 26 known genes (e.g., rDNA, chitin
synthases, ribosomal proteins and heat shock proteins) for use as
controls on the array. We will isolate total RNA from P. brasiliensis at various time
points before, during and after the thermal dimorphic transformation.
We expect gene expression changes to occur early during this process.
Polyadenylated RNA will be converted to cDNA containing
fluorescent-modified (Cy3 and Cy5) nucleotides, which will be
hybridized to the microarray and scanned to generate a dynamic picture
of gene expression during morphogenesis. The spots showing changes in
expression will have their corresponding DNA fragment sequenced and
analyzed. This information will be important for the understanding of
the biology of P. brasiliensis.
Emily Nahas and William Burkholder
Sporulation in Bacillus subtilis is regulated in
response to DNA damage or replication blocks by an inhibitor of
sporulation, Sda. Sda acts by inhibiting two key histadine kinases in
the sporulation phosphorelay, KinA and KinB. Our research focuses on
what regions of Sda are responsible for recognition and inhibition of
KinA and KinB. While characterizing truncations of Sda, we have found
that regions of the sda mRNA, including parts of the open reading
frame, may regulate translation of Sda. Preliminary results indicate
that the 5’ mRNA leader and the “spacer” region between the ribosome
binding site and the start codon are necessary for normal expression of
Sda, while the 3’ untranslated region of the mRNA appears to play an
inhibitory role. Furthermore, deletion of the first six codons of the
gene results in over-expression of Sda. The truncated Sda is largely
inactive in vivo despite being fully active in vitro, suggesting that
the first six residues may play a role in targeting or specificity. We
will use both genetic and biochemical approaches to further
characterize the role of the sda mRNA in regulating translation and the
role of the peptide leader of Sda in regulating in vivo function.
"MCK-2, a murine
cytomegalovirus
encoded CC chemokine homolog, recruits immature myeloid cells to the
initial sites of infection"
Satoshi Noda, 4, Shirley S. Aguirre, Andrew BitMansour, Thomas Schall and and Edward S. Mocarski
Satoshi Noda, 4, Shirley S. Aguirre, Andrew BitMansour, Thomas Schall and and Edward S. Mocarski
MCK-2, a murine cytomegalovirus
encoded CC-chemokine homolog, is responsible for recruiting leukocytes
to the sites of infection. We sought to identify the phenotype of these
leukocytes, by comparing infections with mck-deficient virus RM461 and
RQ461, a virus in which this mutation was rescued. When the
inflammatory infiltrates were compared, a population of Mac-1+Gr-1int
myeloid lineage cells was strikingly greater during wild type virus
infection. A recombinant form of MCK-2 (N-Met-MCK-2) was administered
to mice and this treatment was found to recruit a similar cell type.
Further study indicated that this Mac-1+Gr-1int population was granular
and expressed CD31 (PECAM-1), a marker of immature myeloid cells when
found in conjunction with myeloid cell markers. These cells also
expressed CD45, a marker for leukocyte, but lacked markers of dendritic
cells (CD11c, MHC class II), macrophages (Mac-3, F4/80),
monocytes/glanurocytes (CD13, CD14) and stem cells (CD34). This cell
population was the predominant MCMV permissive cell population in
infiltrates, thus implying the Mac-1+Gr-1intCD31low cells could carry
virus. Interestingly, a higher number of Mac-1+Gr-1int cells was
recruited into the peripheral blood in an MCK-2-dependent fashion.
These results suggest that MCK-2 is responsible for recruiting
CD31lowMac-1+Gr-1int immature myeloid progenitors to sites of
infection. These cells may facilitate viral dissemination to salivary
glands via the blood stream, while also contributing in other ways to
viral pathogenesis.
"What type III effector proteins are
Xanthomonas injecting into a
plant cell during pathogenesis?"
Julie Roden and Mary Beth Mudgett
Julie Roden and Mary Beth Mudgett
We are interested in understanding
the mechanism used by the bacterial pathogen Xanthomonas
campestris pv. vesicatoria (Xcv), causal agent of black spot,
to alter plant physiology during infection of pepper and tomato plants.
It is unknown why this extracellular pathogen infects plant cells. We
do know this microbe uses a specialized type III secretion system
(TTSS) to deliver TTSS effectors (ie: virulence proteins) directly into
the plant cell. The function of these type III effectors in planta
remains elusive. Furthermore, the number and diversity of these
translocated proteins in Xanthomonas
has not been studied, though studies of other plant pathogens suggest
that Xcv could contain as many as 50 – 100 of these proteins. This work
is focused on identifying and characterizing novel Xanthomonas proteins translocated
into the plant cell by using a sensitive bacterial transposon-mediated
AvrBs2 reporter system in Xcv. Using this method, we have identified
six Xanthomonas translocated
proteins (Xtps). Several of these proteins appear to have structural
homology to eukaryotic proteins. One protein also appears to play a
role in the virulence of the pathogen on a susceptible plant host. By
identifying Xtp proteins and their plant host targets, we expect to
reveal the mechanisms used by Xcv to manipulate its plant host during
pathogenesis.
"Post-translation
regulation of
Sda: a developmental checkpoint protein in Bacillus subtilis"
Michael Ruvolo and William Burkholder
Michael Ruvolo and William Burkholder
Sda is a small protein that
establishes a developmental checkpoint inhibiting sporulation in
Bacillus subtilis under conditions of DNA damage or blocks in
replication. Cells can proceed into sporulation when these blocks are
lifted and the chromosome has been replicated. For this to occur, the
levels of Sda must be reduced. We are investigating the role of
post-translational regulation in controlling Sda levels. Using
pulse-chase analysis we have shown that Sda is an unstable protein with
a half-life of five minutes, consistent with regulation by proteolysis.
To understand the mechanism of Sda regulation we introduced mutations
to a putative protease recognition site at the C-terminus of Sda. We
have shown that variants of Sda with C-terminal mutations accumulate to
higher levels in vivo. We are in the process of determining if this is
due to increased stability of Sda. Bacillus
subtilis contains a number of proteases that degrade substrates
in a sequence specific manner. To identify which protease targets Sda,
we are measuring Sda’s stability in Bacillus
strains deleted for the genes encoded these candidate proteases.
Preliminary results show that Sda levels are increased in strains
lacking the ClpP protease. We are determining the effect of deleting
clpP, and the other proteases, on Sda stability. We are also interested
in establishing if Sda stability is regulated, and if so, how its
stability is controlled.
Outside of the laboratory, bacteria
predominately favor the biofilm mode of growth rather than the
free-swimming planktonic mode. While most of classical microbiology has
focused on studying planktonic pure cultures, in recent years there has
been growing interest in various aspects of biofilm growth. Stages in
the development of a biofilm include initial attachment of cells to a
surface, growth of microcolonies on that surface and secretion of
extracellular polymeric substances, further growth of microcolonies
resulting in maturation of biofilm architecture, and eventual sloughing
of biomass from the biofilm once it reaches a critical size. Cell
shedding into the aqueous environment occurs throughout this process,
and it has been observed that widespread shedding of cells—termed
detachment—can be induced under certain conditions. Such detachments
are critical for many environmental proceeses but also for the spread
of infectious diseases in natural environments, such as for cholera
outbreaks induced by dispersal of Vibrio
cholerae, and from vascular catheter biofilms. Shewanella oneidensis MR1 is a
metabolically diverse environmental organism that readily forms
biofilms in laboratory-built flow cells. In order to characterize the
phenomenon of detachment in S.
oneidensis biofilms, we developed a detachment assay whereby we
were able to measure the relative induced detachment in biofilms at
varying stages of development. Knockout mutants were made that targeted
genes putatively involved in detachment and these mutants were screened
with the assay. These experiments will help to gain an understanding of
the physical and molecular aspects of biofilm detachment.
"Protein-protein
interactions in
poliovirus"
Andres Tellez and Karla Kirkegaard
Andres Tellez and Karla Kirkegaard
Poliovirus transcription occurs via
an RNA dependent RNA polymerase (called 3D) and a peptide primer (3B).
In the presence of a poly-A template strand and UTP, 3B is uridylylated
by the polymerase 3D. The uridylylated peptide then primes
transcription of the polyadenylated RNA, producing 3B-linked poly U.
The structure of 3B and mechanism of the peptide polymerase interaction
is unknown; my work uses computational methods to determine putative
folds for 3B and then docks these folds onto 3B. Specific predictions
about residue-residue contacts in the ligand-enzyme complex will be
tested with a gel shift binding assay. Using a combination of
computation and traditional bench techniques, we hope to move toward a
model of primer-polymerase interactions in Poliovirus and its homologs.
Stephanie Brandt: sbrandt@stanford.edu
Julie Wong: juwong@stanford.edu
Matt Taylor: mptaylor@stanford.edu