Whole-genome plasticity among Mycobacterium avium subspecies: insights from comparative genomic hybridizations

Abstract

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is also implicated in cases of Crohn's disease in humans. Another closely related strain, M. avium subsp. avium, is a health problem for immunocompromised patients. To understand the molecular pathogenesis of M. avium subspecies, we analyzed the genome contents of isolates collected from humans and domesticated or wildlife animals. Comparative genomic hybridizations indicated distinct lineages for each subspecies where the closest genomic relatedness existed between M. avium subsp. paratuberculosis isolates collected from human and clinical cow samples. Genomic islands (n = 24) comprising 846 kb were present in the reference M. avium subsp. avium strain but absent from 95% of M. avium subsp. paratuberculosis isolates. Additional analysis identified a group of 18 M. avium subsp. paratuberculosis-associated islands comprising 240 kb that were absent from most of the M. avium subsp. avium isolates. Sequence analysis of DNA regions flanking the genomic islands identified three large inversions in addition to several small inversions that could play a role in regulation of gene expression. Analysis of genes encoded in the genomic islands reveals factors that are probably important for various mechanisms of virulence. Overall, M. avium subsp. avium isolates displayed a higher level of genomic diversity than M. avium subsp. paratuberculosis isolates. Among M. avium subsp. paratuberculosis isolates, those from wildlife animals displayed the highest level of genomic rearrangements that were not observed in other isolates. The presented findings will affect the future design of diagnostics and vaccines for Johne's and Crohn's diseases and provide a model for genomic analysis of closely related bacteria.

FIG. 1.

Comparative genomic hybridization of M. avium subspecies by use of DNA microarrays. A) PCR confirmation of the identity of the examined genomes. An ethidium bromide-stained agarose gel (2%) shows the PstI digestion pattern (lane 2 of each set) of the PCR amplicons (lane 1 of each set) seen when different templates (name on top of each set) were used to amplify a 500-bp fragment of the hsp65 gene. A 100-bp marker (Promega) is included. B) Reproducibility of the genomic microarray hybridizations. The overall Pearson's correlation values are plotted for biological replicates (denoted as a or b) of all mycobacterial genomic DNA used for microarray analysis. The black-to-red scale bar represents low to high correlation levels. Note that all replicates have r values close to 1. M. avium, M. avium subsp. avium; M. para, M. avium subsp. paratuberculosis. C) A dendrogram displaying the overall genomic hybridization signals generated from biological replicates of different mycobacterial isolates from animal or human (HU) sources. The name of each sample is indicated in the dendrogram tree. An example of the hierarchical cluster analysis of the hybridization signals from a region encompassing M. avium subsp. avium GIs 16 and 17 is chosen. The green-to-red color bar represents low to high log ratios of the hybridization signals generated from each genome relative to that of M. avium subsp. avium 104.

FIG. 2.

Identification of genomic islands in the M. avium subsp. avium genome. A) A genome map based on M. avium subsp. avium sequences, displaying GIs deleted from the examined strains as predicted by DNA microarrays. Inner circles denote the microarray hybridization signals for each examined genome (see legend in center of panel). The outermost red boxes denote the locations of all GIs associated with M. avium subsp. avium. M. av., M. avium subsp. avium; M. ap., M. avium subsp. paratuberculosis. B) A diagram illustrating the PCR and sequence-based strategy implemented to verify genomic deletions. Three primers for each island were designed, including a forward (F) and two reverse primers. C) PCR confirmation of genomic deletions. An ethidium bromide-stained agarose gel (1%) displays amplicons from two GIs by use of DNA templates extracted from five different isolates of M. avium subsp. avium. The first and middle lanes are occupied by the 100 bp and 1 kb DNA markers (Promega). Note that GI 8 was only 3 kb, i.e., within the amplification range for M. avium subsp. avium as well as M. avium subsp. paratuberculosis isolates.

FIG. 3.

Synteny of M. avium subsp. avium and M. avium subsp. paratuberculosis genomes. The locations of genomic islands present in M. avium subsp. avium (red boxes) or in M. avium subsp. paratuberculosis (green boxes) genomes are drawn to scale on the circular map of M. avium subsp. avium (outer circle) as well as the map of M. avium subsp. paratuberculosis (inner circle). The sequences of M. avium subsp. paratuberculosis k10 (query sequence) were compared with the whole genome sequence of the M. avium subsp. avium 104 ORFs (target sequence) by use of the BLAST algorithm; cutoff values of E > 0.001 and alignment percentages of <25% for the whole gene were accepted as indications for gene deletion. Blue short bars represent predicted ORFs in forward (outermost) or reverse (innermost) orientations. Large gray arrows indicate sites of genomic inversions.