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Twists and turns in the study of genomic adaptation in Helicobacter pylori Xuhua Xia University of Ottawa

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Presentation on theme: "Twists and turns in the study of genomic adaptation in Helicobacter pylori Xuhua Xia University of Ottawa"— Presentation transcript:

1 Twists and turns in the study of genomic adaptation in Helicobacter pylori Xuhua Xia University of Ottawa

2 Xuhua Xia Slide 2 H. pylori as a gastric pathogen Credit: GastroLab Neutralize the protons leaked into the cell: The urease gene cluster: ureABIEFGH Alleviate the influx of proton by having a positively charged shell (membrane) From Paul S. Hoffman, Univ Virginia

3 Xuhua Xia Slide 3 Acid-resistance mechanisms Ammonia Stomach fluid: Extrinsic source of urea Arginine+arginase: Intrinsic source of urea ureAB-I-EFGH Nickel-inserting Proteins AB I

4 Xuhua Xia Slide 4 The importance of the membrane Protection agaisnt acute exposure to low pH Urease-negative strains of H. pylori can colonize gerbil stomach and induce ulcer (Mine et al. 2005) H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ + + + + + + + + + + + + + + + + + ++ + + + + +

5 Positively charged proteins Xuhua Xia Slide 5 “The basic amino acids, arginine and lysine, occur twice as frequently in H. pylori proteins as in those of H. influenzae and E. coli, perhaps reflecting an adaptation of H. pylori to gastric acidity.” (Tomb, J. F. et al. 1997. Nature 388:539-47.)

6 Conceptual framework Hypothesis: –There is benefit for H. pylori to have positively charged shell –But the lipid bilayer is not charged – need positively charged proteins Prediction: H. pylori should have proteins evolving towards higher pI (isoelectric point)

7 Xuhua Xia Slide 7 Positively charged membrane: How? GenesLengthArgLysHisTyrCysGluAspIE nusB139414131 47.31 ribH1575113431295.1 kdsA277102147315167.43 icfA22281310 42446.19 pyrF22892114417165.24 panC2771622876 118.7 groEL547175389146355.45 groES119214926 66.59 dnaG56021561426639289.48 …… Isoelectric point of the protein (pI): The pH at which the number of negative charges is the same as the number of positive charges of the protein molecule, i.e., the pH at which the protein carries no net charge. Computational method in Xia (2007. Bioinformatics and the cell, Springer) implemented in DAMBE

8 Xuhua Xia Slide 8 Difference in protein pI (all genes) Xia and Palidwor, 2005. Am Nat

9 Xuhua Xia Slide 9 Arguments against AAH The H. pylori genome, like the genomes of other pathogenic eubacteria, is AT-rich, and AT-rich genomes always contain many AAR codons coding for the basic amino acid lysine. The increased Lys usage may simply be a consequence of a high genomic AT% which may result from factors completely unrelated to the acid resistance. At least two factors unrelated to acid resistance have been proposed to change genomic AT%

10 Xuhua Xia Slide 10 I. Factors affecting genomic AT Spontaneous mutation spectrum appears AT-biased, likely mediated by spontaneous deamination –mitochondrial genomes (Marcelino et al. 1998) –prokaryotic genomes (Wang et al. 1996), –pseudogenes in mammalian nuclear genomes (Gojobori et al. 1982; Li et al. 1984). Biased mutations can not only change nucleotide frequencies at the third codon position, but also amino acid usage (Gu et al. 1998; Hickey and Singer 2004; Lobry 2004; Sueoka 1961; Wang et al. 2004). If the high genome AT% and the associated basic proteome result from the biased mutation spectrum, then the basic proteome is an exaptation (Gould and Vrba 1982) instead of an adaptation  The AT-biased mutation hypothesis. Exaptation involves an originally non-selected or neutral trait that has subsequently acquired a beneficial function in a changed environment (Gould and Vrba 1982).

11 Xuhua Xia Slide 11 II. Factors affecting genomic AT% The CTP concentration is generally the lowest among the four NTPs, and dCTP the lowest among the four dNTPs (Colby and Edlin 1970). rATP1890 rCTP53 rGTP190 rUTP130 Measured in the exponentially proliferating chick embryo fibroblasts, 2hrs, in moles  10 -12 per 10 6 cells.

12 Xuhua Xia Slide 12 C-Minimization CTP and dCTP are limited  selection against C-usage  increased AT%: The C-minimization hypothesis. –The protozoan parasite, Trypanosoma brucei, that causes the African sleeping sickness maintains its de novo synthesis pathway for CTP and inhibiting its CTP synthetase effectively eradicates the parasite population in the host (Hofer et al. 2001). In contrast, the parasite does not have de novo synthesis pathways for purines, suggesting that the parasite can obtain the purines by its salvage pathway. This suggests that little CTP can be salvaged from the host. –H. pylori maintains an active biosynthesis pathway, and a much less active salvage pathway, for pyrimidine nucleotides (Mendz et al. 1994). –It might be evolutionarily beneficial for a mammalian parasite or symbiont to minimize the use of CTP in its DNA (and consequently GTP because of the complementary nature of the DNA double helix) in building its genomes and in transcription (Rocha and Danchin 2002; Xia 1996). Preadaptation involves a trait originally selected for one function but subsequently gained a different function beneficial to the carrier of the trait. Three hypotheses resolved in Xia and Palidwor, 2005. Am Nat

13 Xuhua Xia Slide 13 Increased pI mainly due to Lysine usage 7 8 9 10 11 12 567891011 Exp (AAR) Obs (AAR) H. pylori 26695 H. pylori J99 Buchnera aphidicola str. APS Buchnera aphidicola str. Bp Buchnera aphidicola str. Sg Campylobacter jejuni RM1221 Campylobacter jejuni NCTC 11168 Helicobacter hepaticus ATCC 51449 Helicobacter pylori 26695 Helicobacter pylori J99 Wigglesworthia glossinidia Xia and Palidwor, 2005. Am Nat


15 Xuhua Xia Slide 15 Tentative conclusions Our result is consistent with the acid-adaptation hypothesis but inconsistent with the AT-biased mutation hypothesis and the C-minimization hypothesis. The H. pylori proteins, especially membrane proteins, have evolved to have more basic amino acids. Xia and Palidwor, 2005. Am Nat

16 Xuhua Xia Slide 16 Membrane proteins A recent characterization of membrane proteins of H. pylori STR 26695 found membrane proteins to be mainly basic (Baik et al. 2004). The protein pI for the 34 identified membrane proteins. –The average pI is very high (=8.9221) relative to the genomic average  Membrane proteins are significantly more basic than the rest of the proteins in H. pylori. Four proteins (HP0243, HP0072, HP0512 and HP1563) have low pI values ranging from 5.86 to 6.25, all have homologs in the H. hepaticus genome. In contrast, among the rest of 30 membrane proteins with pI > 7, only one has a homolog in the H. hepaticus genome. Thus, nearly all those positively charged membrane proteins in H. pylori are unique, and most likely result from the evolution along the H. pylori lineage.

17 Xuhua Xia Slide 17 The evolutionary mechanism? Natural selection modifying existing genes to increase positively charged amino acids: –Large population size –High mutation rate (Bjorkholm et al. 2001; Wang et al. 1999). –High recombination rate (Bjorkholm et al. 2001; Kersulyte et al. 1999; Suerbaum et al. 1998). –Extract homologous proteins from H. pylori and the non-acid-resistant H. hepaticus and test the prediction that mean pI for H. pylori proteins are higher than that for H. hepaticus. Evolution of acquired characters by assimilating environmental DNA –Strains of H. pylori are naturally competent for uptake of chromosomal DNA (Wang et al. 1993), leading to the horizontal gene transfer (Alm and Trust 1999; Axon 1999; Censini et al. 1996; Covacci et al. 1997). –Extract proteins unique in H. pylori and H. hepaticus, and test the prediction that mean pI for H. pylori proteins are higher than that for H. hepaticus. H. pylori H. hepaticus Ancestor

18 Xuhua Xia Slide 18 Difference in Protein pI (shared genes) 503 pairs of genes between H. pylori and H. hepaticus, with the same gene name and clear homology H. pylori proteins have higher pI Mean pI is low for shared genes between the two species The difference in mean pI is highly significant between the two species: homologous genes have been modified to increase protein pI along the H. pylori lineage

19 Xuhua Xia Slide 19 Uniquely named genes The difference is larger in unique genes between H. hepaticus and H. pylori H. pylori must have either lost genes with low pI and gained genes with high pI, or both.

20 Xuhua Xia Slide 20 A new twist The electrostatic repulsion between proteins is smallest when pH = pI, leading to protein aggregation and precipitation. Prediction: A species should generally evolve to avoid having many proteins with a pI that is the same or very close to its physiological pH. Polycistronic mRNA Ribosome Gene 1 Gene 2 Gene 3 RNA polymerase Protein

21 Xuhua Xia Slide 21 Genomic pI profiling E. coli K12 (intestine pH  8) H. pylori J99 (stomach pH  1.4 and cytoplasmic pH: 5-6)

22 pI and CAI in E. coli Xuhua Xia Slide 22

23 Xuhua Xia Slide 23 An alternative hypothesis H. pylori has a basic proteome not because it needs those positively charged proteins on the membrane to restrict the influx of protons, but because it should avoid having many proteins with a pI at or near its physiological cytoplasmic pH  5. This is still an acid-adaptation hypothesis, but invokes a different selection factor. Which one is correct? –Positively charged proteins against proton influx (Acid- adaptation hypothesis or AAH) –Positively charged proteins against aggregation and precipitation (Precipitation-avoidance hypothesis or PAH)

24 Xuhua Xia Slide 24 Predictions PAH: highly expressed proteins in H. pylori should be more positively charged than lowly expressed proteins. AAH: Membrane proteins should be under stronger selection to gain positive charge than cytoplasmic proteins.

25 Testing prediction from PAH Xuhua Xia Slide 25 Data from H. pylori 26695

26 Testing prediction from AAH Xuhua Xia Slide 26 GroupsCountSumAverageVariance Cytoplasmic Membrane2962698.54009.11672.4680 Outer Membrane43401.11009.32811.8902 Periplasmic16146.40009.15002.8344 Cytoplasmic8076167.61007.64263.4820 ANOVA Source of VariationSSdfMSFP-value Between Groups557.26593185.755358.82860.0000 Within Groups3656.462511583.1576 Total4213.72841161 Data from H. pylori 26695

27 Testing prediction from AAH Xuhua Xia Slide 27 GroupsCountSumAverageVariance Cytoplasmic Membrane3562859.23008.03153.2819 Outer Membrane26218.19008.39192.2434 Periplasmic25202.61008.10444.1021 Cytoplasmic8966462.34007.21243.4181 ANOVA Source of VariationSSdfMSFP-value Between Groups203.7982367.932720.15260.0000 Within Groups4378.810612993.3709 Total4582.60881302 Data from H. hepaticus

28 H. pylori vs. H. hepaticus Xuhua Xia Slide 28

29 Conclusion The positively charged proteins in H. pylori were gained both by acquiring horizontally transferred genes and by modifying existing genes. The relationship between protein expression and protein isoelectric point is not consistent with prediction from precipitation-resistance hypothesis. Membrane proteins in H. pylori exhibit the most dramatic increase in protein isoelectric point, which is consistent with the acid-resistance hypothesis. Xuhua Xia Slide 29

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