Whole-genome analysis of Mycobacterium avium subsp. paratuberculosis IS900 insertions reveals strain type-specific modalities

Authors: CONDE, CYRIL - Universite De Tours; PRICE-CARTER, MARIAN - Agresearch; COCHARD, THIERRY - Universite De Tours; BRANGER, MAXIME - Universite De Tours; STEVENSON, KAREN - The Moredun Group; WHITTINGTON, RICHARD - University Of Sydney; Bannantine, John; BIET, FRANCK - Universite De Tours

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/2021
Publication Date: 5/10/2021
Citation: Conde, C., Price-Carter, M.L., Cochard, T., Branger, M., Stevenson, K., Whittington, R., Bannantine, J.P., Biet, F. 2021. Whole-genome analysis of Mycobacterium avium subsp. paratuberculosis IS900 insertions reveals strain type-specific modalities. Frontiers in Microbiology. Vol. 12, Article 660002. https://doi.org/10.3389/fmicb.2021.660002.
DOI: https://doi.org/10.3389/fmicb.2021.660002

Interpretive Summary: The IS900 insertion sequence is specific to Mycobacterium avium subsp. paratuberculosis (Map), the etiological of Johne’s disease, and has been used widely as an epidemiological marker and for PCR diagnosis. In this study, available Map complete genomes were used to analyze the distribution of this important nsertion sequence. We used bioinformatic approaches to study the distribution and the polymorphism of IS900 in the complete genomes very recently available for the three major phylogenetic lineages of Map. Between 16 to 22 copies of the IS900 sequence were detected in the studied genomes. An in silico IS900 RFLP analysis method was developed. The alignment of all copies within the three genomes revealed new sequence polymorphisms that define the three sequevars distinguishing the three Map types. Only nine IS900 insertion site locations were conserved across all genomes. IS900 distribution analysis in Map genomes enrich our knowledge on the dynamics of distribution of this IS for epidemiological purposes, for understanding the evolution within the Map species and studying the biological implication of the presence of IS900.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis or Johne’s disease that causes chronic intestinal inflammation in ruminants. The IS900 insertion sequence, specific for Map, has been used widely as an epidemiological marker and target for qPCR diagnosis. The improvement of sequencing technologies has led to a rapid increase in the number of available Map genomes, which makes it possible to analyze the distribution of IS900 in this very slow-growing bacterium. The objective of this study was to use bioinformatic approaches to study the polymorphism of IS900 in Map and to develop automated in silico IS900 restriction fragment length polymorphism (IS900-RFLP) analysis. The complete genomes of strains from the three major phylogenetic lineages known as C-type II and Subtypes I and III, were chosen to represent the genetic diversity of Map. IS900 elements were located in these genomes using BLAST software and the relevant fragments extracted from complete genomes. An in silico RFLP analysis using the BstEII restriction site was performed to obtain the exact size of each of the DNA fragments carrying a copy of the IS900 and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The program developed for this study allowed automated localization of IS900 sequences to identify their positions and their exact number. Between 16 to 22 copies of the IS900 sequence were detected in the studied genomes. A loci-by-loci sequence alignment of all IS900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing between the three subtypes. Nine IS900 insertion site locations were conserved across all genomes studied while a smaller subset were unique to a particular lineage. This study provided a program making it possible to automate IS900 distribution analysis in Map genomes to enrich our knowledge on the dynamics of distribution of this IS for epidemiological purposes, for understanding the evolution within the Map species and studying the biological implication of the presence of IS900.