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

OUTLINE•Introduction to Comparative Genomics•Basic biology of Haemophilus spp.•Specific goals


•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


•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



•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


222


132

Characterize unknown strain • Now we have allele profile for all the strains.

PCR Amplification

and DNA Sequencing


111


222


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



•Strategy

OUTLINE

Identify unique genes

Clustering tools: BlastClust, GenomeBlast, PGAP

Characterization (Manual )

Identify virulence factors


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


LipoP, OCTOPUS, SignalP, Phobius Apply species specific filters

Evaluate specificity / sensitivity

GO

AL

1

GO

AL

2G

OA

L

3PLAN OF ATTACK

Documents

OUTLINE Introduction to Comparative Genomics Basic biology of Haemophilus spp. Specific goals ▫Unique genes ▫Virulent Factors ▫Surface proteins Strategy