Background. Helicobacter pylori ( Hp) establishes life-long gastric infection in billions of humans, and is often responsible for diseases such as peptic ulcer and gastric cancer. Cumulative actions of genetic drift and natural selection over several millennia sculpted the present Hp population structure, which is characterized by extreme genetic diversity and striking geographic clustering of genotypes. Natural selection is more commonly imprinted in DNA sequences of Hp proteins that interact with host components; however, in most instances biological relevance of selection during Hp infection remains unknown. Here, I attempted to elucidate the consequence of natural selection in two different contexts: (1) on the preservation of duplicated genes in Hp genome; and (2) lineage-specific adaptive evolution in Hp virulence protein HepC.

Principle findings. I characterized the molecular evolutionary dynamics of paralogs, hcpC and hcpG, which belong to the Hp Sel1-like gene family. hcpG genomic analyses identified three distinct states in natural Hp populations, whereby hcpG was either deleted, pseudogenized or encoded highly polymorphic alleles. In contrast, full-length hcpC alleles were conserved in all genomes. Although positive selection was detected in the phylogenies of hcpG and hcpC indicating that both genes had evolved under pressure to diversify, the intensity of selection was much stronger on hcpG than hcpC. The contribution of hcpC to Hp fitness, in the AGS cell culture infection model, was significantly greater than hcpG; however, both genes together demonstrated an additive effect on Hp fitness during infection (24 hrs p.i.: S_ΔhcpC = 0.264 vs. S_ΔhcpG = 0.074, P<0.01; S_ΔhcpC or S _ΔhcpG vs. S_{ΔhcpC::DhcpG} = 0.431, P<0.01, where S=coefficient of median fitness reduction). Furthermore, HcpC was necessary and sufficient for optimal surface expression of Heat-Shock Protein B (HspB), a major contributor to Hp virulence, specifically during infection, and functionally compensated for the lack of HcpG. In contrast, HcpG was only required for optimal HspB expression during early infection, and was unable to compensate for the lack of HcpC during later phases. Thus, a stable, genetically redundant, epistatic and overlapping yet non-reciprocal functional relationship emerged between hcpC and hcpG: natural selection favored retention of the ancestral hcpC function and sub-functionalization by fixation of loss-of-function mutations in hcpG following its origin in the Hp genome.

Earlier studies from my lab showed that the HepC protein, which also belongs to Hp Sel1-like gene family, interacted specifically with human cytoskeletal protein Ezrin, and that lineage-specific positive selection changed HepC-Ezrin interaction affinity. How might alterations in HepC-Ezrin interaction affect progression of Hp infection? As a first step I established that HepC was indeed biologically relevant and contributed significantly to Hp fitness during infection (P=0.05). Furthermore, PCR-array analyses suggested that identical molecules of the human cytoskeletal pathway were differentially up or down-regulated by genetically diverse Hp isolates during infection, and that HepC likely inhibited key components of the human cytoskeletal machinery during infection. Thus HepC (possibly via its interaction with Ezrin) likely contributes a key regulatory role that might determine the pace and trajectory of Hp infection.

Conclusion. Collectively, my thesis proposes a novel mechanism through which natural selection favors the emergence of a stable state of genetic redundancy among duplicated genes, hcpC an hcpG that contribute significantly to Hp infection. This work also establishes a framework within which to further clarify the role of lineage-specific selection in fine-tuning Hp-host interactions.