outline introduction to comparative genomics basic biology of haemophilus spp. specific goals...
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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 is the common set of proteins ?
What sequences show a signature of purifying selection and are likely functional ?
What sequences show a signature of purifying selection and are likely functional ?
What sequence features are unique to individual species ?
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 ConceptsHomologs:• 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
2a 4a
Organism A
Organism B
1a 3a 5a 6a
2b 4b7b 3b 8b 9b
Block of synteny
Synteny
• 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
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 PasteurellaceaeChristensen 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?
ALWAYS PATHOGENIC
POTENTIALLY PATHOGENIC
COMMENSAL
An organism that can cause infection in individuals with abnormal host defences.
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
•Introduction to Comparative Genomics•Basic biology of Haemophilus spp.•Specific goals
▫Unique genes ▫Virulent Factors▫Surface proteins
•Strategy
OUTLINE
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 performedon agar plates with added X(hemin) & V(NAD) factors.
•But H.Haemolyticus also require both X and V
factorsfor 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 House Keeping Gene
Assign allele Numbers
Total alleles
Gene1 Strain_1 1 2
Strain_2 2
Strain_3 1
Gene2 Strain_1 1 3
Strain_2 2
Strain_3 3
Gene3 Strain_1 1 2
Strain_2 2
Strain_3 2
Allele Profile for Strain_1
111
Allele Profile for Strain_2
222
Allele Profile for Strain_3
132
Characterize unknown strain • Now we have allele profile for all the strains.
PCR Amplification
and DNA Sequencing
Allele Profile for Strain_1
111
Allele Profile for Strain_2
222
Allele Profile for Strain_3
132
Allele Profile
222
Strain_2
Uncharacterized Strain in
Hand
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
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
To understand HOW pathogenic bacteria interact with their host to produce clinical disease is fundamental
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 proteaseMany 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 oligosaccharide3. O-specific antigen
O –unit plays a vital role in bacterial adherence, invasion and immune invasion.
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.
Lipo-polysaccharide (LPS)
Adherence and SecretionViral Factor Description GenesFimbriae Mediate bacterial adherence to mucosal epithelia
Gene cluster pilABCD coding for type IV fimbriae has been identified in a number of Gram-negative pathogens in the genera of Haemophilus
pilApilBpilCpilD
Hap (Haemophilus Adhesion and Penetration )
Promotes adherence and invasionAdhesive activity is localized within the Hap passenger domain. Mediates bacterial
aggregation and microcolony formation.Hap has serine protease activity
hap
Hia/Hsf High-affinity adhesive activity and mediates interaction with a broad array of respiratory epithelial cell typesReceptor unknown
Hia hsf
HMWhigh-molecular-weight
proteins
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
hmw1A hmw1Bhmw1C
hmw2A hmw2Bhmw2C
P2 protein A surface-associated lipoprotein that is responsible for the transparent colony phenotype of H. influenzae
oapA
P5 protein Major outer membrane protein, shares homology with E. coli OmpAAntigenically variable from one train to another
ompA
Protein D A 42-kDa surface-exposed lipoprotein (9) with glycerophosphodieste phosphodiesterase (GlpQ) activity
hpd
Protein E 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.
hpE
•Introduction to Comparative Genomics•Basic biology of Haemophilus spp.•Specific goals
▫Unique genes ▫Virulent Factors▫Surface proteins
•Strategy
OUTLINE
Identify unique genes
Clustering tools: BlastClust, GenomeBlast, PGAP
Characterization (Manual )
Identify virulence factors
Characterization (Manual )
No. of copies, Flanking genes, Gene order
Metabolic pathways, Missing links, SNPs causing LOF, truncated sequences, protein str. predictions
MVirDB, VFDB PHAST (Phage DNA) Plasmid DB Operons
Alien Hunter (HGT) VISTA (Regulatory regions) ACT (Synteny) Transposons / IS elements
Identify surface proteins and
secreted proteins
Characterization (Manual )
LipoP, OCTOPUS, SignalP, Phobius Apply species specific filters
Evaluate specificity / sensitivity
GO
AL
1
GO
AL
2G
OA
L
3PLAN OF ATTACK