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OUTLINE
• Introduction to Comparative Genomics
• Basic biology of Haemophilus spp.
• Specific goals
▫ Unique genes
▫ Virulent Factors
▫ Surface proteins
• Strategy
OUTLINE
• Introduction to Comparative Genomics
• Basic biology of Haemophilus spp.
• Specific goals
▫ Unique genes
▫ Virulent Factors
▫ Surface proteins
• Strategy
TREE OF LIFE
COMPARATIVE GENOMICS: BASIC PRINCIPLES
• DNA sequences encoding proteins and RNA
responsible for function conserved from last ancestor
should be preserved in contemporary genome
sequences.
• DNA sequences controlling expression of genes
regulated similarly in two related species > also be
conserved.
• Sequences that control gene expression, proteins and
RNAs responsible for differences between species
should be divergent.
What is Comparative Genomics
http://www.compsysbio.org
WHY COMPARATIVE GENOMICS?
• To understand the genomic basis of the present
▫ Differences in lifestyle
 pathogen vs. nonpathogenic
 obligate vs. free-living
▫ Host specificity
▫ In the case of emerging pathogens: this understanding
should help us in fighting disease (drug discovery,
vaccines)
• To understand the past
▫ How organisms evolved to be what they are now
What to compare?
What is the common
set of proteins ?
What sequences show a
signature of purifying
selection and are likely
functional ?
What sequence
features are unique to
individual species ?
Genome-wide evolutionary events
• Rearrangements of gene structure
• Gene/region duplication
• Gene/region loss
• Chromosome  plasmid DNA exchange
• Vertical descent (speciation)
• Horizontal gene transfer (HGT)
Horizontal Gene Transfer
• Genetic exchange between different
evolutionary lineages.
- Transformation, Transduction, Conjugation
• Acquire variable number of accessory genes
encoding adaptive traits.
• Most of these accessory genes acquired by HGT
form syntenic blocks recognized as genomic
islands (GEIs)
Genomic Islands
• Large segments of DNA
• Different GC content
• Often inserted at tRNA
genes
• Often flanked by 16-20kb
direct repeats
• Harbour genes encoding
factors involved in mobility
-integrase, transposases and
IS
• Carry genes carrying
seletive advantage
Evolution-Related Concepts
Homologs:
• Genes sharing a common ancestor and
generally retain same function
Orthologs:
• Genes (homologs) in different species
derived from a single ancestral gene in
the last common ancestor (LCA)
(arise from speciation)
Paralogs:
• Homologs in same species related via
duplication
▫ Duplication before speciation
(ancient duplication)
 Out-paralogs; may not have the same
function
▫ Duplication after speciation (recent
duplication)
 In-paralogs; likely to have the same
function
Synteny
Organism A
1a
2a
3a
4a
5a
6a
4b
8b
9b
Block of synteny
7b
2b
3b
• Refers to regions of two
genomes that show
considerable similarity in
terms of
– sequence and
– conservation of the
order of genes
• likely to be related by
common descent
Organism B
OUTLINE
• Introduction to Comparative Genomics
• Basic biology of Haemophilus spp.
• Specific goals
▫ Unique genes
▫ Virulent Factors
▫ Surface proteins
• Strategy
Pasteurellaceae
Comparative phylogeny tree of
16S rRNA gene within the
Pasteurellaceae
Christensen et al. 2004
Characteristics of Haemophilus spp
• Genus of gram negative, coccobaccili bacteria
• Belonging to the Pasteurellaceae family
• Either aerobic or facultative anaerobic
• Of the eight Haemophilus species residing as
commensal organisms in the pharyngeal cavity of
humans.
• H. influenzae is by far the most pathogenic
- Hi Strains possessing a type b capsule are often associated with
invasive diseases such as meningitis, sepsis and pneumonia.
- and strains lacking a capsule (NTHi) are associated with localized
mucosal diseases, such as otitis media, sinusitis, and bronchitis.
• H. haemolyticus emerging pathogen.
Strains of H. haemolyticus
Species
Disease State
State
isolated
Omp2
Hemolysis
Hpd
fucK
M19107
H.haemolyticus
Asymptomatic
Minnesota
Neg
Y
neg
neg
M19501
H.haemolyticus
Asymptomatic
Minnesota
neg
N
pos
neg
M21127
H.haemolyticus
pathogenic
Georgia
ND
Y
neg
neg
M21621
H.haemolyticus
Pathogenic
Texas
ND
Y
neg
neg
M21639
H.haemolyticus
Pathogenic
Illinois
ND
N
neg
neg
M21709
H. influenzae
pathogenic
NY
ND
N
neg
Pos
omp2: encoding the outer membrance protein P2
fucK : ncoding fuculose-kinase. fucK deletion has been observed in some Hi isolates
Hpd: encoding a lipoprotein protein D,
Is H. Haemolyticus opportunistic pathogen?
An organism that can cause infection in
individuals with abnormal host defences.
ALWAYS
PATHOGENIC
POTENTIALLY
PATHOGENIC
COMMENSAL
H. haemolyticus
• As the name of the species implies, is generally
hemolytic on blood agar plates.
• Beta-hemolytic phenotype routinely used in the
clinical setting to distinguish H.h from NTHi.
• Non-hemolytic H. haemolyticus strains are
being isolated > misidentified as NTHI.
Genotyping assays include:
- DNA-DNA hybridization,
- 16S rRNA gene sequencing,
- MLST : internal fragment of seven housing
keeping genes
- others: PCR, DNA blot
Photograph from from
MicrobeLibrary.org
OUTLINE
• Introduction to Comparative Genomics
• Basic biology of Haemophilus spp.
• Specific goals
▫ Unique genes
▫ Virulent Factors
▫ Surface proteins
• Strategy
What are Genes unique to
H. haemolyticus?
Why Unique Genes..?
• They will assist in successful characterization
and distinction of H. Haemolyticus and H.
influenza which is still an open challenge to be
addressed.
• Are there any methods tried or currently
available to address this challenge?
…..Yes!!
Method I: Culture Conditions
• Bacterial culture of
H. influenzae is performed
on agar plates with added
X(hemin) & V(NAD) factors.
• But H.Haemolyticus also
require both X and V factors
for their growth.
Method II: Haemolysis
•
The characterization may be
achieved based on the
H.Haemolyticus’s ability to
lyse Horse red blood cells .
•
But recently some strains of
H.Haemolyticus have been
reported that do not participate
in hemolysis of red blood cells

Method III: Multilocus Sequence Typing
• MLST is highly unambiguous and portable technique
to characterize isolates of bacterial species using
multiple house keeping genes.
• The principle of MLST is simple: the technique involves
PCR amplification followed by DNA sequencing of the
house keeping genes.
• MLST directly measures the DNA sequence variations in
a set of housekeeping genes and characterizes strains by
their unique allelic profiles.
How does MLST work?
• Let us assume there are three strains in certain bacterial
species, say Strain_1, Strain_2 and Strain_3
• The first step in MLST is identification of house
keeping genes. Lets say we have 3 house keeping genes in
this species.
• MLST exploits the possibility of occurring different
(variable) sequences for each house keeping gene.
• All unique sequences for each house keeping genes are
assigned allele numbers
How does MLST work?
Strain
Gene1
Gene2
Gene3
House
Assign
Keeping allele
Gene
Number
s
Total
alleles
Strain_1
1
2
Strain_2
2
Strain_3
1
Strain_1
1
Strain_2
2
Strain_3
3
Strain_1
1
Strain_2
2
Strain_3
2
Allele Profile for
Strain_1
111
Allele Profile for
Strain_2
3
222
Allele Profile for
Strain_3
2
132
Characterize unknown strain
•
Now we have allele profile for all the strains.
Allele Profile for
Strain_1
111
Uncharacterized
Strain in Hand
Allele Profile for
Strain_2
Allele Profile for
Strain_3
222
132
PCR Amplification
and DNA Sequencing
Strain_2

Allele Profile
222
MLST to characterize H.influenzae and
H.haemolyticus
• Seven isolates presumed to be H.influenza were subjected to
multilocus sequence typing by a group of researchers.
• They were consistently unable to amplify fucK from one isolate.
• Failure to amplify the fucK gene fragment from presumptive H.
influenzae isolates has been considered an indicator of a
misidentified strain.
• However, failure to detect the fucK gene cannot be considered
conclusive since some strains of H. influenzae have recently been
shown to lack the fucose operon.
Our Challenge
•
There have been many methods in the past to characterize
and distinguish H. Haemolyticus and H. influenza. None of
those methods saw success due to the associated
disadvantages
•
So now, our challenge is to identify and characterize unique
genes that are specific to H. Haemolyticus.
•
Detecting the presence of these unique genes in unknown
strain using PCR assays will help characterize the strain as
H. Haemolyticus
Which are the virulence factors in
H. haemolyticus ?
VIRULENCE FACTORS
Are molecules expressed and secreted by pathogens (bacteria, virus, fungi
and protozoa) that enable them:
• Colonization of a niche in the host (this includes adhesion to cells)
• Immuno-evasion, evasion of the host's immune response
• Immuno-suppression, inhibition of the host's immune response
• Entry into and exit out of cells (if the pathogen is an intracellular one)
• Obtain nutrition from the host
To understand HOW pathogenic bacteria interact with their
host to produce clinical disease is fundamental
Discovering Virulence Factors is the first step in understanding bacterial
pathogenesis and their interactions with the host, which may also serve as a
novel targets in drugs and vaccine development
Comparative Genomics
& Transcriptomics
Proteomics
Important Tools in discovering VF
in bacterial pathogens
Bacterial VF can be divided into several groups on the
basis of the mechanism of virulence and function:
Membrane Proteins
Adhesion, colonization and invasion
Promote adherence to the host cell surface
Responsible for resistance to antibiotics
Promote intercellular communication
Polysaccharide Capsules
surround the bacterial cell and
have anti-phagocity properties
Secretory Proteins
can be toxins
can modify the host cell environment and
are responsible for some host cell-bacteria
interactions
Major Virulence Factors of Pathogenic Bacteria
Our Main Focus:
1. Genes responsible for Hemolysis
-Hemolysin
2. Genes responsible for colonization and invasion
- LPS biosynthesis
- Adherence and Secretion
pili, Hap, Hia/Hsf , HMW, P2, P5, protein D,
protein E
- IgA protease encoding gene
Hemolysin
• H.ducrey hemolysin is encoded by two genes:
– hhdA encodes the structural protein for hemolysin,
– hhdB which is required for activation and secretion of hhdA
• Serratia marcescens hemolysin which shares homology to H.d
hemolysin are :
– These two genes are transcribed in the order of ShlB ShlA from an
iron regulated promoter upstream of ShlB. Regulated by Fur
protein.
– Truncation of the N-terminal region of SHLA no hemolytic activity
• Does H. haemolyticus has fur gene? if so, does it have such
mechanism for regulation?
• hemolysin might enhance invasion into epithelial cells suggestive of
role in invasion and virulence.
IgA protease
Many bacteria which establish infections after invasion at human mucosal
surfaces produce enzymes which cleave immunoglobulin A (IgA)
Secretory immunoglobulin A (IgA) is the primary form of antibody found at
human mucosal surfaces
The IgA proteases cleave within the 16 aa hinge
region which separates the antigen- binding region
(Fab) from the carboxyl (Fc) end of the IgA
molecule
IgA proteases differ in the exact site of
cleavage within the hinge region
Surface Proteins in H. haemolyticus
Lipo-polysaccharide (LPS)
Primary structural and
functional component of the
gram-negative bacterial outer
membrane
Can be recognized and targeted
by the mammalian immune
system
Three biochemical motifs:
1. Lipid A
2. Core oligosaccharide
3. O-specific antigen
O –unit plays a vital role in bacterial adherence, invasion and
immune invasion.
Lipo-polysaccharide (LPS)
Genes essential for the synthesis of lipid A
(lpxC, kdsA, lpxB, kdsB, lpxH, lpxK, lpxD,
lpxA, kdtA, lpxM, kdsC and lpxL) and
core oligosaccharide (rfaE, rfaF, rfaD, lgtF
and gmhA) are present and highly
conserved among the genus Haemophilus.
Adherence and Secretion
Viral Factor
Fimbriae
Description
Mediate bacterial adherence to mucosal epithelia
Gene cluster pilABCD coding for type IV fimbriae has been identified in a number of Gramnegative pathogens in the genera of Haemophilus
Hap
(Haemophilus
Adhesion and
Penetration )
Promotes adherence and invasion
Adhesive activity is localized within the Hap passenger domain. Mediates bacterial
aggregation and microcolony formation.
Hap has serine protease activity
Hia/Hsf
High-affinity adhesive activity and mediates interaction with a broad array of respiratory
epithelial cell types
Receptor unknown
The non-typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that
mediate attachment to human epithelial cells, an essential step in the pathogenesis of
disease.
the hmw genes have only been detected in nontypable strains
HMW
high-molecular-weight
proteins
P2 protein
P5 protein
Protein D
Protein E
A surface-associated lipoprotein that is responsible for the transparent colony phenotype
of H. influenzae
Major outer membrane protein, shares homology with E. coli OmpA
Antigenically variable from one train to another
A 42-kDa surface-exposed lipoprotein (9) with glycerophosphodieste phosphodiesterase
(GlpQ) activity
protein E (PE) is a low-molecular-mass (16 kDa) outer membrane lipoprotein with
adhesive properties
Induces a pro-inflammatory immune response in lung epithelial cells.
Genes
pilA
pilB
pilC
pilD
hap
Hia
hsf
hmw1A
hmw1B
hmw1C
hmw2A
hmw2B
hmw2C
oapA
ompA
hpd
hpE
OUTLINE
• Introduction to Comparative Genomics
• Basic biology of Haemophilus spp.
• Specific goals
▫ Unique genes
▫ Virulent Factors
▫ Surface proteins
• Strategy
GOAL 3
GOAL 2
GOAL 1
PLAN OF ATTACK
Identify unique genes
 Clustering tools: BlastClust, GenomeBlast, PGAP
Characterization
(Manual )
 No. of copies, Flanking genes, Gene order
Identify virulence
factors
 MVirDB, VFDB
 PHAST (Phage DNA)
 Plasmid DB
 Operons
Characterization
(Manual )
 Metabolic pathways, Missing links, SNPs causing
LOF, truncated sequences, protein str. predictions
Identify surface
proteins and secreted
proteins
Characterization
(Manual )
 Alien Hunter (HGT)
 VISTA (Regulatory regions)
 ACT (Synteny)
 Transposons / IS elements
 LipoP, OCTOPUS, SignalP, Phobius
 Apply species specific filters
 Evaluate specificity / sensitivity