From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma

Abstract

Background: Helicobacter pylori infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.

Results: A set of 254 H. pylori genes was used to perform array-based comparative genomic hybridization among 120 French H. pylori strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with cagPAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released H. pylori genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest H. pylori genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the vacAs2m2 allele and lacks the genes encoding the major virulence factors (absence of cagPAI, babB, babC, sabB, and homB). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 H. pylori sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.

Conclusion: These isolates are deprived of the main H. pylori virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.

Figure 1

Hybridization reactions on a DNA macroarray membrane containing 254 PCR products that are representative of H. pylori strain 26695 (41 ubiquitous genes + 213 non-ubiquitous or strain-specific genes). Bacterial DNAs from 120 isolates involved in various diseases, including chronic gastritis (yellow), intestinal metaplasia (pink), duodenal ulcer (blue) and gastric MZBL (green), were tested by hybridization. Isolates are listed on the horizontal axis, and the genes tested, on the vertical axis. Clustering (genesis software) was carried out using the continuous values from 120 heterologous hybridization experiments, where each value corresponds to the (log₂₆₆₉₅-log_{heterol.strain}) value for each tested gene (see materials & methods). Colors of the line range from blue, if the gene is present, to red, if absent. The range of intermediate colors reflects the degree of hybridization and thus homology, but also the redundancy of the tested genes. This figure represents the clustering based on the complete set of 254 genes.

Figure 2

Hybridization reactions on a DNA macroarray membrane: clustering based on the 48 most discriminatory genes identified as key combinations of variables (genes/axes) from Principal Component Analysis. These 48 genes are labeled in Addional file 1.

Figure 3

Genome map of Helicobacter pylori strain B38. From outside to inside: - GC skew (window 2500, step 500) in blue. - Total CDSs (green) with pseudogenes/partial genes (purple). - CDSs coding for hypothetical restriction/modification systems (purple), phage proteins (orange), or insertion sequences (ISHp609) (green). - Total CDSs according to the matrix defined for gene identification (matrix n°1 in red, matrix N°2 in black, matrix n°3 in green). - RNA (rRNA in green, tRNA in purple and misc_RNA in red). - Rule. - GC% (window 5000, step 2000) in yellow. Red arrow indicates the position of the origin of replication.

Figure 4

Synteny lineplot pair-wise analyses between B38 and the H. pylori strain 26695, J99, HPAG1, Shi470, P12, G27, Helicobacter hepaticus, or Helicobacter acinonychis.