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Comparative Genomics The Finale Angela Pena, Ambily Sivadas, Amit Rupani Shimantika Sharma, Juliette Zerick Keerti Surapaneni, Artika Nath, Hema Nagrajan Outline Results Goal 1 – PCR Assay Goal 2 – Comparative genome analysis Goal 3 – Haemolysis study Goal 4 – Virulent factors Discussion • • • • • Goal 1 Identification and characterization of target genes for PCR Assay Identification of target genes Fw primer Rv primer A C B A C Hhae NTHi PCR products of different size One copy of B or multiple copies? Is A-C organized the same way in both organisms? Identify candidate clusters/genes for assay development and conserved regions for primer/probe design Cluster Analysis – Genome Set Cluster statistics Protein sequences were clustered using Blastclust Total clusters: 8402 Total common to all genomes : 361 Total unique to Hhae: 82 Total unique to Hinf: 38 Total unique to Pathogenic strains: 0 Common clusters – Functional breakdown Metabolism protein syntheis /post trans transport DNA repair replication chaperone/protein folding proteolysis tRNA processing DNA repair/ replication secretion competence cell division cell cycle regulation Hypothetical ATP/DNA binding unknown transcription stress response secretion antiobiotic resistence Target identification - Protocol 1 1. Take all proteins common to all 25 genomes Common = Most conserved proteins • Target identification - Protocol 1 1. Cluster Analysis: Take all proteins common to all 25 genomes Common = Most conserved proteins • 2. Compute and compare inter-cluster distances for Hhae vs Hinf •Look for species specific patterns •Look for including unique genes If a pistol just isn't working for you . . . Protocol II: BLAST Everything Our method is this: for every unique Hhae gene, we will locate its corresponding contig We checked the flanking regions (on the contig) for conserved genes. We will then locate the conserved genes in the Hinf genome and see if they are adjacent. Since a wide net can be cast with BLASTn searches, this includes homologs. Start with a set of Hhae genes We found a (more) unique gene! Select a unique Hhae gene from the set YES Reject gene and start over Search the set of common Hhae/Hinf (conserved) genes for genes in the flanking regions Is there at least one conserved gene in each flanking region? NO Are the conserved genes adjacent or “close enough” in the Hinf genome? Get the locations of the conserved flanking genes in the Hinf genome NO YES PCR Assay: Results Target 1 50S fatty acid/ ribosomal phospholipid protein synthesis protein 1020 bp Hh NTHi 170 bp Nucleic acid binding protein (hypothetical) 3-oxoacyl-(acyl carrier protein) synthase III PCR product 1250 bp Hh NTHi 380 bp No duplication was found for these genes PCR Assay: Results Target 2 1451 bp Hh NTHi fructosebiphosphate aldolase 1934 bp predicted membrane protein purine nucleoside phosphorylase PCR product 1451 bp Hh NTHi 1934 bp Target validation by Insilico PCR Step 1: Multiple Sequence Alignment by ClustalW2 - Overview 1 870 1775 2749 Non Typable H. influenzae 19 strains + 1 Typeable H. haemolyticus 5 strains Target 1 905 nts Step 2: Phylogenetic analysis Neighbor Joining Tree Percentage of Identity using Jalview Step 3: Finding primers 1 870 Forward 1775 2749 5’-CTCACTTACGCCACCACGTA-3’ Non Typable H. influenzae 20 strains H. haemolyticus 5 strains 3’-TGCAACAATAATCAGTTCAATATCT-5’ Reverse In silico PCR Analysis Non Typable H. influenzae AAZD00000000 Product length: 487 H. haemolyticus M21621 Product length: 1354 In silico PCR Analysis Sequence (5'->3') FORWARD STRAIN PRODUCT LENGTH START STOP M19107 1363 654 673 M19501 1364 654 673 M21127 1363 654 673 M21621 1354 655 673 M21709 487 654 673 CP000671 489 653 672 L42023 487 654 673 MSA – Target 2 1 5372 Non Typable H. influenzae 20 strains H. haemolyticus 5 strains Goal 2 Comparative genomic analysis Horizontal Gene Transfer • Horizontal gene transfer (HGT), also lateral gene transfer (LGT) refers to the transfer of genetic material between organisms Alien Hunter • • • Predicts putative horizontally transferred regions. Standalone software Available at http://www.sanger.ac.uk/Software/analysis/alien_hunter Usage: ./alien-hunter <input_file> <output_file> INPUT: raw genomic sequence PREDICTION: HGT regions based on Interpolated Variable Order Motifs (IVOMs) .sco file Last time, we got many hits with varied scores that covered almost 90% of the genes in each genome. Hence, we decided to place a threshold on the scores. • We studied the distribution of scores for each genome by plotting histograms for each genome based on the scores. • We decided to place a threshold of >70 after studying all the histograms. Screenshot of M21621 HGT gene count 6000 5500 5000 4500 4000 3500 before filtering 3000 after filtering 2500 2000 1500 1000 500 0 1 2 3 4 5 6 before filtering after filtering 1699 360 2709 145 3065 225 5601 253 1140 108 2717 185 Insertion elements • An Insertion element is a short DNA sequence that acts as a simple transposable element. • A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". IS Finder FASTA sequences We retrieved FASTA sequences by submitting the accession IDs in NCBI BLAST We blasted these insertion sequences against each of the strains and got the location of the insertion sequences in the strain. A PERL script was written to extract the insertion sequences from their respective contigs in each strain. Feature /Strain Tools M19107 M19501 M21127 M21621 M21639 M21709 Average Genome size - 1774129 1809865 2029793 1959123 2397857 1808157 1963154 GC content % IGIPT 39.39 38.23 39.11 39.11 38.30 38.73 38.81 Total Number of genes - 1973 1785 2086 1923 2669 1840 2046 Operons - 76 (27) 69 (14) 71 (25) 79 (21) 87 (36) 70 (14) 73 124 115 116 115 124 144 115 Comparative Analysis Table Virulence factors HGT gene count Alien Hunter 360 145 225 253 108 185 213 Pathogenic - No No Yes Yes Yes No - Insertion elements IS Finder 6 6 - - 15 - 4.5 Hemolytic activity - Y N Y Y N N - M19107 – Circular alignment using BRIG M19501 – Circular alignment using BRIG M21127– Circular alignment using BRIG M21621– Circular alignment using BRIG M21639 – Circular alignment using BRIG M21709 – Circular alignment using BRIG Goal 3 Identification and Characterization of Haemolysin in Hhae AIM #1 Look for the hemolysin BA operon present in the H.haemolyticus strains and characterize it as present/absent in the hemolytic and non hemolytic strains HEMOLYSIN • Hemophilus ducreyi, requires two adajecent genes, hhdB and hhdA for hemolysis . • hhdB is an outer membrane protein, which is required for secretion and activation of the hemolysin structural protein, hhdA. • Once secreted, hhdA interacts with target cell membranes, oligomerizes, and forms pores 2.5 to 3.0 nm in diameter, which lyse the target cell TWO PROTEIN SECRETION SYESTEM OUR STRATEGY • Downloaded the Fasta files of all hemolysin protein sequence of the Pasteurellaceae family from NCBI protein database. • Blasted the predicted protein sequences of the six strains against these. Cut off threshold: Identity 70% Coverage 80% RESULTS Strain Hemolysis Gene A /contig Gene B /contif Haemophilus haemolyticus M19107 Y 51_11|7343|11596 1417 amino acids ZP_09185204.1| hemolysin [Haemophilus [parainfluenzae] 51_1216|11855|13366 503 amino acids ZP_09185203.1| hemolysin activation/secretion protein [Haemophilus [parainfluenzae] Haemophilus haemolyticus M21127 Y 20_113|106934|111307| 1457 amino acids ZP_09185204.1| hemolysin [Haemophilus [parainfluenzae] Haemophilus haemolyticus M21621 Y 1_361|369207|373580 1457 amino acids ZP_09185204.1| hemolysin [Haemophilus [parainfluenzae] Haemophilus haemolyticus M19501 Haemophilus haemolyticus M21639 Haemophilus influenza M21709 N None 20_112|105150|106760 536 amino acids ZP_09185203.1| hemolysin activation/secretion protein Haemophilus [parainfluenzae 1_362|373754|375349| 531 amino acids |ZP_09185203.1| hemolysin activation/secretion protein [Haemophilus [parainfluenzae] None N None None N None None All hits had 70% and more identity and 95-100 coverage AIM# 2 • Characterize the domains/motifs/residue in hemolysin. • Depict the secondary structures in hemolysin. • Predict the 3D structure of hemolysin. SIGNAL PEPTIDE & HAEMAGGLUTINATION ACTIVITY DOMAIN N’ terminal Haemagglutination activity domain Signal Peptide • A signal peptide (25 aa) to transport the hemolysin to outer membrane or periplasm. LipoP cleavage site Spase I at 25-26. NOT LIPOPROTEIN • Haemagglutination activity domain -suggested that the haemagglutination activity domain is a carbohydrate-dependent haemagglutination activity site which is found in a range of haemagglutinins and haemolysins HAEMAGLUTININ REPEAT • Haemaglutinin repeat is a highly divergent repeat that occurs in number of proteins implicated in cell aggregation TPS DOMAIN All TPS-secreted proteins contain a distinctive N-proximal module essential for secretion, the TPS domain. TpsA proteins display two conserved regions, C1 and C2, and two less-conserved regions, LC region. ANPNL and NPNGIS is found in this region hemolysins/cytolysins ShlA of Serratia marcescens, HpmA of Proteus mirabilis, EthA of Edwardsiella tarda, HhdA of Haemophilus ducreyi, the large supernatant proteins LspA1 and LspA2 of H. ducreyi, and the HecA adhesin of E. chrysanthemi . Clantin et al., 2004. The crystal structure of filamentous hemagglutinin secretion domain and its implications for the twopartner secretion pathway.PNAS. Does the TPS domain exist in H.haemolyticus strains? 21127 21621 19107 Fha30 EthA HpmA LspA1 LspA2 ShlA HhdA . Fha30 H.H H.H H.H HhdA EthA ShlA HpmA LSpA1 LSPA2 CONSERVED RESIDUES IN TPS DOMAIN ANPNL NPNLGI NPNL & NPNGI These motifs form type I beta -turns, which might play important stabilizing roles. The conserved residues of the TPS domain serve to drive the folding of the TPS domain into a beta -helix and to stabilize the helix TPS HAD 39-159-Pfam Or TPS 39-270 STRATEGY SECONDARY STRUCTURE AIM #3 • Identify the domains in the hemolysin activator gene • Determine the secondary and 3D structure of hemolysin activator gene HEMOLYSIN ACTIVATOR PROTEIN TRANSMEMBRANE PROTEIN MEMBRANE PROTEINS α-helical β-barrel β-barrel membrane protein class are located in the outer membrane of Gram-negative bacteria. These proteins have membrane spanning segments formed by antiparallel β-strands, creating a channel in the form of a barrel that spans the outer membrane. DOMAIN IS HEMOLYSIN ACTIVATOR SP POTRA _2 Activator Domain SP (LipoP) – SPI cleavage site between pos. 19 and 20. NOT LIPOPROTEIN POTRA_2- polypeptide-transport-associated domain. In ShlB this domain has a chaperone-like function over ShlA. Activator domain in ShlB is shown to interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin. ShlA/B: Serratia marcescens Strategy • Prediction of TransMembrame Beta Barrels (PRED TMBB) Method is powerful when used for discrimination purposes, as it can discriminate with a high accuracy the outer membrane proteins from water soluble in large datasets • The 'TransMembrane protein Re-Presentation in 2 Dimensions' tool, automates the creation of uniform, twodimensional, high analysis graphical images/models of alphahelical or beta-barrel transmembrane proteins. Work Flow Sequence Discrimination score Sequence Step 1: Find Discrimination Score Haemophilus haemolyticus M19107 –hemolystic Contig : 51_1216 Start/End: |11855|13366 Length: 503 amino acids STEP2 Predicted structure 2D of hemolysin activator Haemophilus haemolyticus M19107 –hemolystic CYTOPLASMIC Coloring by Hydrophobic Potential More hydrophobic + - Hydrophilic 3D STRUCTRES STRUCTURE PREDICTION METHODS Homology Modelling Requires a template with a high percentage identity De novo protein structure prediction Models the structure based on general principles that govern protein folding energetics Protein Threadin g Works when homology modelling fails PROTEIN THREADING APPROACH • It follows Protein Threading approach to predict the structure of the target protein sequence. • In practice, when the sequence identity in a sequence alignment is low (i.e. <25%), homology modeling may not produce a significant prediction. In this case, if there is distant homology found for the target, protein threading can generate a good prediction. • Protein threading, also known as fold recognition, is a method of protein modeling (i.e. computational protein structure prediction) which is used to model those proteins which have the same fold as proteins of known structures, but do not have homologous proteins with known structure. • We used RaptorX server which predicts the structure based on protein threading method. 3D STRUCTURES OF HEMOLYSIN FROM RaptorX Segment 1 (37-158) Segment 2 (159-1457) 3D STRUCTURES OF ACTIVATOR FROM RaptorX SP POTRA _2 Activator Domain Segment 3 (1-66) POTRA_2 Domain Segment 2 (67-136) Activator Domain Segment 1 (149-500) POTRA DOMAIN POLYPEPTIDE-TRANSPORT-ASSOCIATED DOMAIN SP POTRA _2 Activator Domain • POTRA domains have similar structure comprising a threestranded b sheet overlaid with a pair of antiparallel helices . E.coli POTRA Domain figure Kim et al., 2007. Structure and Function of an Essential Component of the Outer Membrane Protein Assembly Machine. SCIENCE VOL 317 Goal 4 Identify and characterize the potential virulence factors in Haemophilus haemolyticus Human Immune System An Immune System (IS) is a system of biological structures and processes within an organism that protects again disease. In order to function properly, IS must detect a wide variety of agents, from viruses to parasitic worm, and distinguish them from the organism’s own healthy tissue. Pathogens can rapidly evolve and adapt to new environments to avoid detection and destruction by the immune system: Phase variation Phase variation Phase variation is defined as the random switching of phenotype at frequencies that are much higher (sometimes >1%) than classical mutation rates. Is a widespread source of intraespecific genotypic and phenotypic variation Several different mechanisms are exploited by bacteria to switch gene and/or protein expression “on” or “off” Combinatorial math: A bacterium with just 20-phase variable loci can exist in 220 different states (more than a million) Multiple defense mechanisms have evolved to recognize and neutralize pathogens: Immune System Innate Adaptive Creates immunological memory after an initial response to a specific pathogen Non-specific response to microorganism or toxins found in the cell Microbes are identified by pattern recognition receptors, eg. LPS Generic response Does not confer longlasting immunity against the pathogen. 1. 2. 3. Inflammatory response Activation of Complement System Antimicrobial peptides Major component of innate IS Immune-Evasion target Complement System Biochemical cascade that attacks the surfaces of foreign cells It can contain over 20 different proteins Complement the killing of pathogens by antibodies: - produces peptides that attract immune cells -opsonize (coat) the surface of a pathogen, marking it for the destruction - increase vascular permeability Host Immune Evasion Microorganisms have developed many ways to evade complement actions: • Trapping endogenous C1 inhibitor • Inactivating antibodies through capture of their FC regions • Mimicking structural regulators • Degradation crucial components of Complement System How we did the analysis ? Search of VF in Haemophilus genus Virulence Factor Data Base 132 VF were retrieved Search in NCBI for all VF in Haemophilus genus RefSeq protein sequences Data Base Blastp against all the 25 genomes of Haemophilus constraints: At least 40% identity At least 70% of query coverage Build a matrix with presence/absence +3 Presence -3 Absence Upload matrix to MeV Heat Map was build HCL was generated Results…. HCL: Hierarchical Cluster Distance Metric: Pearson correlation Linkage method: Average linkage clustering 132 virulence factors analyzed 25 samples: 19 NTHi 5 Hhae 1 THi Cluster A : Uniform/regular pattern Cluster B : heterogeneous/ irregular pattern Cluster A : Uniform/regular pattern • Transferring-binding protein 1 • tad locus • LPS biosynthesis • tad locus • Type IV pili • Adherence • Exopolysaccharide • LPS biosynthesis • Haemophilus iron transport locus • Hemoglobing and hemoglobin-haptoglobin binding proteins • Heme biosynthesis • tad locus • Cytolethal distending toxin Cluster B : heterogeneous/ irregular pattern Btuc vitamin B12 receptor protein, E. coli rfaC 1,5 Heptosyltransferase I ABC_T ATP binding cassette transporter family hptE Lipopolysaccharidae heptosyltransferase I IgA1 hxuA hxuB hxuC FepA Immunoglobulin A protease Heme/hemopexin-binding complex Ferric enterobactin receptor, E. coli Prevalence Ratio Analysis Patho vs Asymptomatic - Adherence HAEMOAGGLUTINATING PILI (hifABCDE operon) Function •Promote adherence to respiratory mucus and human oropharyngeal epithelial cells •Facilitates colonization Mechanism: Binding to the Anton antigen (An-Wj) common to buccal epithelial cells and erythrocytes Prevalence Ratio analysis Patho vs Asymptomatic - Adherence HAEMOAGGLUTINATING PILI (hifABCDE operon) Role in virulence • Expression of pili is a phase-variable phenomenon •Variation in (TA) repeat units within the overlapping promoter region of hifA and hifB regulates the transcription of the gene • 11 repeat units – reduced expression • 10 repeat units – maximal expression • 9 repeat units – transcriptional silencing Found in M19501(5,4),AAZD00000000 (9), AAZE00000000(9),AAZJ00000000(5) Pathogenic – Hi F3047 strain (9) In M19107 – only hifA and hifB – split into two contigs –hence no info on TA repeats Prevalence Ratio Analysis Patho vs Asymptomatic - Adherence High Molecular Weight Protein 1/2 Function •Adhesins that mediate attachment to human epithelial cells Structure features • Autotransport protein • Secretion of these adhesins HMW1A/HMW1B requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C Prevalence Ratio Analysis Patho vs Asymptomatic-LOS rfaC lic3A Characteristics: Biosynthesis pathway of LOS is producing a branched oligosaccharide attached to a lipid A via two 3-deoxy-Dmanno-2-octulosonic acid (KDO) molecules. rfaC gene product adds the first heptose (Hep I) to KDO lex2B siaA rfaC mutants are shown to produce truncated LPS rfaC is absent in all H.influenzae, but present in all H. haemolyticus rfaC mutants also shown to decrease haemolytic activity and expression in E Coli So, may be, retaining rfaC helps H. hae retain its haemolytic activity lgtA Prevalence Ratio Analysis Patho vs Asymptomatic-LOS rfaC lic3A Sialic Acid Transporter Lic3A,SiaA or LsgB Characteristics: Sialic acid is added as terminal nonreducing sugar to LOS – important for bacterial virulence. lex2B siaA These genes code for sialyltransferases which incorporates sialic acid into LOS In the absence of this transporter, Hinf cannot survive when exposed to serum Found to be absent in all H.haemolyticus lgtA Prevalence Ratio Analysis Patho vs Asymptomatic-LOS rfaC lic3A Phase variable glycosyl transferases Lex2B, LgtB lex2B Characteristics: Contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae siaA And phase variable expression Found to be absent in all H. haemolyticus lgtA Prevalence Ratio Analysis Patho vs Asymptomatic- Immuno-evasion • No difference in pattern observed in this case! Prevalence Ratio Analysis Patho vs Asymptomatic- Iron acquisition HxuABC HxuA binds to hemehemopexin HxuB releases HxuA from the cell surface into the medium HxuC is involved in the transport of heme within the cell Function • Using host heme-hemopexin as the source of heme iron for growth Absent in all H.haemolyticus YadA is a potent serum resistance factor as it inhibits the classical pathway of complement, Yersinia adhesin A Fba - Fibronectin-binding protein, streptococcus spp IgA1 – serine protease, cleaves IgA1 Protein E – adhesine protein, captures vitonecting (Vn), which prevents the formation of MAC IgA1 MAC (Membrane Attack Complex) it forms transmembrane channels, disrupting the phospholipid bilayer of target cells, leading to cell lysis and death. IgA1 mrsA, glmM, galU, galE, manA, manB Protein E Is H. haemolyticus an opportunistic or pathogenic bacterium ? Innate IS Adaptive IS Ambiental Opportunistic Pathogenic IgA1 ompP2 Protein E Exopolysaccharides Btuc YadA HuxABC rfaC/hptE FepA fba DISCUSSION Hydrogenase-4 10-gene operon • First identified in E. Coli in 1997 • hyfABCDEFGHIJ - hyfR (transcriptional activator) • The proteins encoded by the hyf operon are proposed to constitute a proton-translocating formate hydrogenlyase • Hyf catalyzes dihydrogen production and ion transport when the cells are grown at a starting pH of 7.5 • This operon is silent in E. Coli – Hyd-3 is the active H2 evolving operon. Hydrogenase and Virulence • As per recent studies, Hyd-4 in Yersinia enterocolitica helps in gut colonization ▫ Using H2 produced during fermentation by intestinal microflora • Also, hydrogenases facilitated respiratory hydrogen use by Salmonella enterica and is considered essential for virulence • So, understanding the expression and role of this hydrogenase operon could provide critical information for ▫ Characterization of Hhae ▫ Understanding a new mode of virulence in Hhae (May be!) ABC transporter system • ATP-binding cassette (ABC) transporter system • One of the largest protein families. • Found in all species and are evolutionarily related. • Functionally diverse and have roles in a wide range of important cellular functions. Structural schema • Bacterial genomes encode different numbers of ABC transporters, which correlate with their lifestyles, suggests that bacterial ABC transporters are likely to be necessary for growth and/or survival of the bacteria in their ecological niches ABC Transporters and Virulence • Virulence associated with uptake of nutrients ▫ Polyamine, glutamine, sugar • Virulence associated with uptake of metal ions ▫ such as iron, zinc, and manganese • Virulence associated with cell attachment • ABC transporter (outer membrane) proteins are sometimes immunogenic too. • Based on the role of the ABC system in virulence, certain components could be potential targets for developing vaccines too • In our case, one of the closest homologs is the SalX gene from Pasteurella multocida– which is an ABC-type antimicrobial peptide transport system, ATPase component. • Hence, characterizing this ABC transporter system could be insightful.