National Microbial Pathogen
Data Resource
Connecting Bioinformatics to the Bench
Leslie Klis McNeil
NCSA, University of Illinois, Urbana
NMPDR is a BRC
• NIAID Bioinformatic Resource Centers
 common goals
 different focus organisms
• Provide annotations and tools to develop diagnostics
and therepeutics against Priority Pathogens
• NMPDR core organisms, all category B:
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Campylobacter jejuni
Listeria monocytogenes
Staphylococcus aureus
Strepcococcus pyogenes and pneumoniae
Vibrio cholerae, vulnificus, parahaemolyticus
www.nmpdr.org
Sister BRCs focus on other priority
pathogens
• Unified port of entry at
• Eight BRCs curate viruses, protozoa, and
bacteria, or insect vectors of disease
www.nmpdr.org
Who is NMPDR
• Fellowship for Interpretation of Genomes
 Primary software developers
 Curators who do manual annotation
• Computation Institute at University of Chicago
 Software developers
 Hardware managers
• Argonne National Laboratory
 Software developers
• NCSA University of Illinois at Urbana
 Education, outreach, training
www.nmpdr.org
What is NMPDR
• Genome database with value added
 Manual annotation in context of systems biology
 Comparative analysis tools
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Bidirectional Best Hits—select and align
Functional clusters—genes with conserved proximity
Compare regions—adjust size of region, number of genomes
Pinned regions—phylogenetic comparison with all genomes
Signature genes—find genes in common or that distinguish userselected groups of genomes; groups may contain one or many
 Essential genes page
 Drug target discovery and in silico screening
 Organism pages with phenotype information
www.nmpdr.org
Pathogen-specific gateways to data
www.nmpdr.org
Outreach services in the user interface
• User forum links to iLabs with Inquiry Units for
teaching and training
• PathInfo—VBI’s PIML project, info about
 General info and strain descriptions
 Lab handling and safety
 Epidemiology
• Journals button opens most recent, relevant
ASM articles
• Google news—RSS feed of popular press
• Links to resources such as strain collections
www.nmpdr.org
Annotation Status Table
• Immediate access to genes whose functions
are known with some degree of certainty
 Named genes in subsystems
 Named genes not in subsystems
 Hypothetical genes in subsystems
• Gateway to genes about which nothing is
known
 Hypothetical genes not in subsystems
• List of genes with links to NMPDR analysis tools
• Exploration in comparative framework first step to
formulating working hypotheses about functions
www.nmpdr.org
Pathways to Data
• Start with keyword search for name of gene or protein
• Start with sequence of your gene or protein and blast against
any complete genome
• Start by browsing an organism of interest
 View lists of proteins with/without functional names; included/not in
biological subsystem. Choose one from the list to investigate with
comparative tools.
• Start from subsystems tree to view the phylogenetic
distribution of an interesting biological process
• Start from essential genes page to view essential genes in
model organisms and to project essentiality to closely or
distantly related organisms
• Start from virtual structural proteomes to investigate proteins
about which structural information is available in PDB
www.nmpdr.org
Subsystems approach to genome annotation
• Subsystems annotation provides researchers with corrected
functional annotations in a structured biological context
• Consistency across genomes achieved by vertical annotation
of functions rather than horizontal focus on single genomes
• More than 500 distinct subsystems have been developed
 Metabolic pathways
 Complex structures
 Genotype – phenotype associations
• Subsystems integrate genomic and functional contexts of
genes in metabolic reconstructions or populated subsystem
spreadsheets
• Metabolic reconstructions summarize all subsystems in a
given genome
• Populated subsystems compare all genomes in a given
subsystem
www.nmpdr.org
What is a Subsystem?
• Subsystem is a generalization of pathway
 Collection of functional roles jointly involved in
a biological process or complex
• metabolic, signaling, regulatory, structural
• Functional Role is the abstract biological
function of a gene product
 Atomic or fundamental; examples:
• 6-phosphofructokinase (EC 2.7.1.11)
• LSU ribosomal protein L31p
• cell division protein FtsZ
www.nmpdr.org
Expert-Defined Subsystems
• Curator is researcher with first-hand
knowledge of biological system
• Functional roles defined and grouped into
subsystem and subsets by curator
 universal groups of roles include all organisms
 functional variants are subsets of roles found in
a limited number of organisms
• often represent alternative paths
www.nmpdr.org
Populated Subsystems
• Two-dimensional integration of functional
roles with genomes
 universal groups of roles include all organisms
 functional variants are subsets of roles found in
a limited number of organisms
• Spreadsheet
 Columns of functional roles
 Rows of organisms
 Cells of annotated genes
• Table of functional roles with GO terms
• Diagram
www.nmpdr.org
Simple Example:
Histidine Degradation Subsystem
• Conversion of histidine to glutamate is organizing
principle
• Functional roles defined in table:
Subsystem: Histidine Degradation
1
2
3
4
5
6
7
HutH
HutU
HutI
Glu F
HutG
NfoD
ForI
Histidine ammonia-lyase (EC 4.3.1.3)
Urocanate hydratase (EC 4.2.1.49)
Imidazolonepropionase (EC 3.5.2.7)
Glutamate formiminotransferase (EC 2.1.2.5)
Formiminoglutamase (EC 3.5.3.8)
N-formylglutamate deformylase (EC 3.5.1.68)
Formiminoglutamic iminohydrolase (EC 3.5.3.13)
www.nmpdr.org
Subsystem Diagram
• Three functional variants
• Universal subset has three roles, followed
by three alternative paths from IV to VI
Tetrahy drofolate
S u bsyste m Diagram
Form im inotetrahy drofolate
GluF
NH 3
I
HutH
H2 O
II
H2 O
HutU
III
Form am ide
H2 O
HutI
IV
H2 O
VI
NH 3
ForI
www.nmpdr.org
HutG
V
NfoD
Subsystem Spreadsheet
Subsystem Spreadsheet
Organism
Variant
HutH
HutU
HutI
GluF
HutG
NfoD
Bacteroides thetaiotaomicron
1
Q8A4B3
Q8A4A9
Q8A4B1
Q8A4B0
Desulfotela psychrophila
1
gi51246205
gi51246204
gi51246203
gi51246202
Halobacterium sp.
2
Q9HQD5
Q9HQD8
Q9HQD6
Q9HQD7
Deinococcus radiodurans
2
Q9RZ06
Q9RZ02
Q9RZ05
Q9RZ04
Bacillus subtilis
2
P10944
P25503
P42084
P42068
Caulobacter crescentus
3
P58082
Q9A9MI
P58079
Q9A9M0
Pseudomonas putida
3
Q88CZ7
Q88CZ6
Q88CZ9
Q88D00
Xanthomonas campestris
3
Q8PAA7
P58988
Q8PAA6
Q8PAA8
Listeria monocytogenes
-1
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Column headers taken from table of functional roles
Rows are selected genomes, or organisms
Cells are populated with specific, annotated genes
Shared background color indicates proximity of genes
Functional variants defined by the annotated roles
Variant code -1 indicates subsystem is not functional
www.nmpdr.org
ForI
Missing Genes Noticed by
Subsystems Annotation
• No genes were annotated “ForI (EC 3.5.3.13)
Formiminoglutamic iminohydrolase” when the
Histidine Degradation subsystem was
populated
• Organisms missing ForI convert His to Glu
• Candidate genes that could perform the role
“ForI” must be identified
• Strategy for finding genes is based on
chromosomal clustering and occurrence
profiling
www.nmpdr.org
Finding Genes that Cluster with NfoD
• Green gene is NfoD of Xanthomonas
• Blue genes within 10 kb of NfoD in at least four other species
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finds biggest clusters in other species
• fc-sc shows table of homologous pairs in other genomes
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displays homologous regions in other genomes
www.nmpdr.org
What are Pinned Regions?
• Focus gene is number 1, colored red
• Most frequently co-localized homolog
numbered 2, colored green
• Homologous genes presented in the same
color with the same numerical label
• Numerical labels correspond to rank
ordered frequency of co-localization with
the focus gene
 Focus gene labeled 1
 Gene 17 is homolog 16th most frequently colocalized with focus gene
www.nmpdr.org
Candidate ForI in Context with NfoD
• Homologous
regions around
NfoD, red,
center
• Same color
indicates
homology
BLAST cutoff
1e-20
• HutH, the first
functional role
in the
subsystem, is
green, 2
• Candidate ForI
is pink, 4,
“conserved
hypothetical”
www.nmpdr.org
Annotation of ForI EC 3.5.3.13
• Metabolic context proves need for role
 Organisms missing annotated ForI degrade His to Glu
• Chromosomal context points to candidate
 Clusters with NfoD and other genes in subsystem
• Occurrence context supports candidate
 Organisms containing NfoD lack GluF and HutG,
required for functional variants 1 and 2, respectively
 Organisms containing candidate ForI also contain
NfoD, indicating functional variant 3
• Phylogenetic trees of candidate ForI genes are
coherent
www.nmpdr.org
Conjectures archived in HOPS
• Hypotheses and Open Problems identified by
Subsystems
 HOPS linked from NMPDR’s FAQ
• Subsystems point to missing or alternative
genes
• Bioinformatic predictions need to be tested at
the bench
• ForI candidate now verified experimentally
• Connections forged between bench and
bioinformatics
www.nmpdr.org
Bioinformatics to Bench
• Essential genes page at NMPDR
 Click bar to search for essential genes
 Follow NMPDR link to compare with other genomes
www.nmpdr.org
Candidate Drug Targets
• First-draft table (manually derived) links to
biochemical data in BRENDA or TCDB
• Candidate proteins
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essential in at least one of the NMPDR pathogens
included in subsystems by our curators
orthologs in the Protein Data Bank
orthologs in a substantial number of bacterial priority
pathogens curated in the BRC system
• Second-draft table to be automatically generated
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annotations include essential for growth or virulence
PDB and pathogen orthologs
No good hit in host
targets without crystallized orthologs suggested to HTS
project at Argonne National Laboratory
www.nmpdr.org
NMPDR efforts feed into high-throughput
structure project at Argonne
www.nmpdr.org
In Silico Screening
• Targets docked with 10 K random compounds
as training set
• Neural network program tracks 9 properties of
compounds to learn characteristics of those
that bind and those that do not
• ZINC compound db screened to find 10K
likely binders predicted to be ligands
• Targets docked against 10K predicted ligands
on BlueGene with Dock5
• Top 1000 docked compounds soon to be
linked to NMPDR
www.nmpdr.org
IBM BlueGene Supercomputer
World’s fastest
Supercomputer
280 TeraFLOPS
www.nmpdr.org
Live Demo of NMPDR
• From essential genes, click H.pylori, then click NMPDR for first protein
• Show compare regions
 Possible to increase/decrease size of region
 Possible to “walk” chromosome
 Possible to include more genomes--type in 10 and click resubmit
• Click on the homologous gene 1 in the second genome, Campylobacter
• Ask, is this function also essential in Campy,is this a good drug target?
• Investigate the campy homolog by using Pins, Compare Regions, find
best clusters (CL)
• What is the pathway or biological system that this protein is essential for?
 IF not included in a subsystem by NMPDR curators, follow alias link to KEGG
• Pathway is lysine biosynthesis—Ask:
 Does this protein catalyze the rate-limiting step?
 Is this the best function in this pathway to target for inhibition by a drug?
 Does this protein have a close structural/functional homolog in human or
PDB? Use BLAST to find homologs.
 Is this a broad or narrow spectrum target? Show all homologs using
Bidirectional Best Hits button.
www.nmpdr.org