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

Xuhua XiaUniversity of Ottawa

xxia@uottawa.cahttp://dambe.bio.uottawa.ca

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

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

AB AB

I

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+

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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.)

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)

Xuhua Xia Slide 7

Positively charged membrane: How?Genes Length Arg Lys His Tyr Cys Glu Asp IE

nusB 139 4 14 1 3 1 14 4 7.31

ribH 157 5 11 3 4 3 12 9 5.1

kdsA 277 10 21 4 7 3 15 16 7.43

icfA 222 8 13 10 10 4 24 4 6.19

pyrF 228 9 21 1 4 4 17 16 5.24

panC 277 16 22 8 7 6 22 11 8.7

groEL 547 17 53 8 9 1 46 35 5.45

groES 119 2 14 9 2 6 14 6 6.59

dnaG 560 21 56 14 26 6 39 28 9.48

……

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

Xuhua Xia Slide 8

Difference in protein pI (all genes)

H. pylori J99 H. hepaticusMean 8.18150 7.60268Variance 3.66710 3.95961N 1491 1875df 3364t Stat 8.52359p (t-tail) 0.00000

Xia and Palidwor, 2005. Am Nat

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%

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).

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).

rATP 1890rCTP 53rGTP 190rUTP 130

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

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

Xuhua Xia Slide 13

Increased pI mainly due to Lysine usage

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ylori 2

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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

Xia and Palidwor, 2005. Am Nat

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

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.

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. hepaticusAncestor

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

H. hepaticus H. pylori J99Mean 7.35306 7.82751Variance 3.91172 3.67699Observations 503 503Pearson r 0.79837df 502t Stat -8.59411p (2-tailed) 0.00000

Hh pI HpJ99 pIaccA 5.4 accA 5.39accB 5.05 accB 5.65accC 6.52 accC 7.15accD 6.77 accD 9.92ackA 6.91 ackA 6.95… … … …

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.

UniqHh UniqHpJ99Mean 7.04456 8.25034Variance 2.79449 3.06298N 182 175df 355T -6.65803p (2-tailed) 0.00000

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

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)

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pI and CAI in E. coli

Xuhua Xia Slide 22

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)

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.

Testing prediction from PAH

Xuhua Xia Slide 25Data from H. pylori 26695

Testing prediction from AAH

Xuhua Xia Slide 26

Groups Count Sum Average VarianceCytoplasmic Membrane 296 2698.5400 9.1167 2.4680Outer Membrane 43 401.1100 9.3281 1.8902Periplasmic 16 146.4000 9.1500 2.8344Cytoplasmic 807 6167.6100 7.6426 3.4820

ANOVASource of Variation SS df MS F P-value

Between Groups 557.2659 3 185.7553 58.8286 0.0000Within Groups 3656.4625 1158 3.1576

Total 4213.7284 1161

Data from H. pylori 26695

Testing prediction from AAH

Xuhua Xia Slide 27

Groups Count Sum Average VarianceCytoplasmic Membrane 356 2859.2300 8.0315 3.2819Outer Membrane 26 218.1900 8.3919 2.2434Periplasmic 25 202.6100 8.1044 4.1021Cytoplasmic 896 6462.3400 7.2124 3.4181

ANOVASource of Variation SS df MS F P-value

Between Groups 203.7982 3 67.9327 20.1526 0.0000Within Groups 4378.8106 1299 3.3709

Total 4582.6088 1302

Data from H. hepaticus

H. pylori vs. H. hepaticus

Xuhua Xia Slide 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.

Xuhua Xia Slide 29