Twists and turns in the study of genomic adaptation in Helicobacter pylori

1. Twists and turns in the study of genomic adaptation in Helicobacter pylori Xuhua Xia University of Ottawa xxia@uottawa.ca http://dambe.bio.uottawa.ca

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

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

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+ Slide 4

5. Positively charged proteins “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.) Slide 5

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. Positively charged membrane: How? 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 Slide 7

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

9. Increased pI mainly due to Lysine usage 12 H. pylori 26695H. pylori J99 11 10 Obs (AAR) 9 8 7 5 6 7 8 9 10 11 Exp (AAR) Slide 9 Xia and Palidwor, 2005. Am Nat

10. 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% Slide 10

11. 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). Slide 11

12. 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). Measured in the exponentially proliferating chick embryo fibroblasts, 2hrs, in moles 10-12 per 106 cells. Slide 12

13. 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 Slide 13

14. Tentative conclusions Our resultis 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 Slide 14

15. 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. hepaticusgenome. In contrast, among the rest of 30 membrane proteins with pI > 7, only one has a homolog in the H. hepaticusgenome. • 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. Slide 15

16. 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 Ancestor H. hepaticus Slide 16

17. 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 Slide 17

18. 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. Slide 18

19. 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. Gene 1 Gene 2 Gene 3 Polycistronic mRNA RNA polymerase Ribosome Protein Slide 19

20. Genomic pI profiling E. coli K12 (intestine pH  8) H. pylori J99 (stomach pH  1.4 and cytoplasmic pH: 5-6) Slide 20

21. pI and CAI in E. coli Slide 21

22. 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) Slide 22

23. 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. Slide 23

24. Testing prediction from PAH Data from H. pylori 26695 Slide 24

25. Testing prediction from AAH Data from H. pylori 26695 Slide 25

26. Testing prediction from AAH Data from H. hepaticus Slide 26

27. H. pylori vs. H. hepaticus Slide 27

28. 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. Slide 28