Comparative genomics combined with phenotypical analyses reveal novel acid stress response mechanisms in Listeria monocytogenes
Wu, Jialun
Wu, Jialun
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Publication Date
2023-06-13
Type
Thesis
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Abstract
Listeria monocytogenes is a pathogenic bacterium of significant concern for food safety. Its remarkable ability to withstand food-related stress and survive gastrointestinal passage is important for establishing infections. In this study, the whole genome sequence was determined for a collection of L. monocytogenes strains (n = 168) that were mainly isolated from food and clinical cases in Ireland. Genomic sequence analysis revealed the phylogenetic diversity of this strain collection. To capture the phenotypic diversity across this strain collection, we surveyed the behaviour of these strains under food-related conditions. The ability to grow under salt stress and the ability to survive lethal acid stress is highly diverse across the strain collection, while less pronounced differences were observed in the ability to grow under mild acid stress and utilize difference carbon sources. In order to understand the molecular mechanisms that contribute to the stress response of this pathogen, the genome sequences were carefully interrogated for those strains that exhibited atypical stress response phenotypes comparing to closely related strains. Several genomes were found to carry alleles in sigB operon, which is responsible for the general stress response in L. monocytogenes. Three among these alleles were shown to be loss-of-function mutations and they likely explain the increased salt tolerance and reduced acid resistance in the corresponding strains. Strain 1386 were unable to grow when trehalose was supplied as the sole carbon source due to a single amino acid substitution (N352K) in a putative trehalose transporter. Reversion of this substitution fully restored the ability of this strain to grow on trehalose and thus, confirming the product of this gene transports trehalose. Furthermore, the ability to use trehalose was found to promote biofilm development and acid resistance. Interestingly, strain 1381 was unable to grow at mildly acidic conditions and survived poorly at lethal acidic conditions. We showed that a loss-of-function mutation in a gene encoding a manganese transporter is the cause of the inability to grow at a mildly acidic pH. This manganese transporter was induced under acid conditions and its role in manganese uptake was required for L. monocytogenes to grow at low pH conditions. Further genome sequence analysis identified a mutation in a gene encoding a hitherto unknown RofA-family transcription regulator that explains the poor ability of strain 1381 to survive lethal acidic stress. This transcription regulator, which we name GadR, controls the expression of an important acid survival mechanism glutamate decarboxylase at stationary phase or upon acid exposure. This gadR gene and its regulon is conserved in Listeria species that are frequently isolated from faeces, suggesting this GadR-mediated regulation may be important for gastrointestinal tract colonization. Surprisingly, we observed that premature stop codons (presumably loss-of-function mutations) are prevalent in the genes encoding important stress response regulators sigB and gadR by analysing 44,080 publicly available genome sequence of L. monocytogenes, this suggests that losing the function of these two regulators might confer selective advantages under specific conditions. Further, several strains recommended by European Union Reference Laboratory of L. monocytogenes to be used for determining the growth potential of L. monocytogenes in food products were found to be stress intolerant/sensitive. The findings in this study suggest that caution should be used when selecting the stains for such experiments to avoid underestimating the food safety risks associated with this pathogen. In summary, this study identified multiple acid stress response determinants in L. monocytogenes by combining comparative genomics and phenotypic screening methods, these findings advance the understanding of the physiological response to acid tress in L. monocytogenes and they are of importance from both food safety and functional genomics perspectives.
Publisher
NUI Galway