Shedding Light on the African Enigma: In Vitro Testing of Homo sapiens-Helicobacter pylori Coevolution

Abstract

The continuous characterization of genome-wide diversity in population and case-cohort samples, allied to the development of new algorithms, are shedding light on host ancestry impact and selection events on various infectious diseases. Especially interesting are the long-standing associations between humans and certain bacteria, such as the case of Helicobacter pylori, which could have been strong drivers of adaptation leading to coevolution. Some evidence on admixed gastric cancer cohorts have been suggested as supporting Homo-Helicobacter coevolution, but reliable experimental data that control both the bacterium and the host ancestries are lacking. Here, we conducted the first in vitro coinfection assays with dual human- and bacterium-matched and -mismatched ancestries, in African and European backgrounds, to evaluate the genome wide gene expression host response to H. pylori. Our results showed that: (1) the host response to H. pylori infection was greatly shaped by the human ancestry, with variability on innate immune system and metabolism; (2) African human ancestry showed signs of coevolution with H. pylori while European ancestry appeared to be maladapted; and (3) mismatched ancestry did not seem to be an important differentiator of gene expression at the initial stages of infection as assayed here.

Keywords: Helicobacter pylori; Homo sapiens; ancestry background; coevolution; genome-wide gene expression; innate immune response.

Figure 1

Ancestry profiles of human gastric cancer cell lines and genome-wide expression profiles. (A)—Population structure inferred by ADMIXTURE analysis of 1000 Genomes populations and CCLE panel, in which each individual is represented by a vertical (100%) stacked column of genetic components proportions shown in color for K = 3 (AFR—African ancestry in red, EUR—European in blue and EAS—Asian in green; AMR and SAS are, respectively, American and South Asian populations). Lower plot is a zoom of the CCLE panel. (B)—PCA plot (PC1 explaining 54% of variation vs. PC2 explaining 31% of variation) of the human transcriptomic profile of the three gastric cell lines (triplicates for each condition; blue colors for the European; green for the Asian and pink for the African) without and after infection with HpAFR and HpEUR H. pylori strains. (C)—Venn diagrams for up-regulated and (D)—down-regulated genes in all experimental H. pylori infected settings compared with the uninfected status (indicated by Ø). (E)—Top significantly enriched pathways in pairwise comparisons between infected (positive side) and uninfected (negative side) conditions in the three cell lines. The different color represents H. pylori strains (pink for African and blue for European ancestries). The scale reports the normalized enrichment score (NES) values.

Figure 2

Expression profiles for innate immune system genes based on InnateDB database and IL8 variability. (A)—Clustering and heat map of the most significant differences in expression (in any pairwise comparison of infection versus uninfected) in at least one of the three cell lines. (B)—Log2 changes in expression of gene represented in A when infections were conducted with matched (for HsEUR and HsAFR) or close-mismatch (for HsEAS) bacteria in blue, and between mismatched (for HsEUR and HsAFR) or distant-mismatch (for HsEAS) bacteria in red. (C)—IL8 gene expression profiles in African (Yoruban population; in pink) and European (Great Britain population; in blue) in the RNAseq data available in the 1000 Genomes consortium website. (D)—IL8 gene expression profiles depending on the genotypes at rs4073 SNP (AA in pink; AT in green; TT in blue). (E)—Genotype frequency distributions (same colors as in B) across the globe as inferred from 1000 Genomes populations.

Figure 3

Metabolism of L-Lactate. (A)—Statistically significant fold changes (up-regulation in pink and down-regulation in blue) in gene expression for the HsEUR × HpEUR setting versus uninfected in the glycolysis, Krebs cycle and fatty acids synthesis. (B)—Statistically significant fold changes (up-regulation in pink and down-regulation in blue) in gene expression for the HsAFR × HpAFR setting versus uninfected in the glycolysis, Krebs cycle and fatty acids synthesis. (C)—Extracellular lactate concentration in uninfected and infected settings in the three cell lines (mean and standard deviations of triplicates). Statistically significant comparisons are indicated by *.

Figure 4

Reactive oxygen species (ROS). (A–C)—MitoSOX fluorescence values for the various settings in each cell line. (D)—Mean values and standard deviations of fluorescence values for the various settings in each cell line. (E)—Statistically significant fold changes (up-regulation in pink and down-regulation in blue) in gene expression for the HsEUR × HpEUR setting versus uninfected in the response to ROS pathway. (F)—Statistically significant fold changes (up-regulation in pink and down-regulation in blue) in gene expression for the HsAFR × HpAFR setting versus uninfected in the response to ROS pathway. Statistically significant comparisons are indicated by *. * means: p < 0.05, ** means: p < 0.01, *** means: p < 0.001.

Figure 5

Mean and standard deviation values for cellular cytotoxicity (A), viability (B) and apoptosis (C) in each cell line infected for 24 h. Statistically significant comparisons are indicated by *. * means: p < 0.05, *** means: p < 0.001.