Whole Genome Sequencing of Listeria monocytogenes Strains Carrying Loss of Function Mutations in inlA Supports These Strains are Evolving Away from a Pathogenic Lifestyle

Introduction: Listeria monocytogenes is capable of thriving as a saprotroph and causing invasive disease as an intracellular pathogen. Bacterial adaptation to a lifestyle can occur quickly by loss-of-function (LOF) mutations that increase fitness in a given niche. Previous studies support that L. monocytogenes strains carrying LOF mutations in inlA have adapted to thrive in non-host environments as a tradeoff for loss of full virulence. If these strains are indeed adapted to non-host environments, we assume additional genes involved in host-pathogen interactions carry similar LOF mutations.

Purpose: This study aimed to use Whole Genome Sequencing (WGS) to characterize L. monocytogenes strains with naturally occurring premature stop codons (PMSCs) in inlA and identify genome-wide mutations that may contribute to evolution towards a non-pathogenic lifestyle.

Methods: 43 L. monocytogenes isolates carrying naturally occurring PMSCs in inlA were characterized by WGS. The resulting genome sequences were analyzed by constructing a phylogenetic tree followed by comparing each generated sequence to the closest available clonal complex (CC) sequence without a PMSC in inlA. Variant nucleotides were annotated to determine if each mutation resulted in a potential LOF.

Results: The 43 strains fell into eight previously identified CCs. Analyses of each CC independently resulted in the identification of several LOF mutations across the CC level, but not at the lineage level. LOF mutations potentially associated with decreased fitness in a host were identified in genes for sugar hydrolase enzymes (lmo2733), genes for the production of menaquinone (lmo1201 and lmo1932), and genes for DNA repair and replication (mutS and dnaG).

Significance: The LOF mutations identified in this study suggest that L. monocytogenes carrying a PMSC in inlA are independently losing genes necessary for survival and replication intestinally and intracellularly. L. monocytogenes carrying a PMSC mutation in inlA are likely accumulating mutations that result in loss of the ability to lead a pathogenic lifestyle.