Hypervariable Genetic Hotspots in Listeria monocytogenes

Listeria (L.) monocytogenes is a facultative intracellular foodborne pathogen responsible for listeriosis, a rare but severe illness in humans. L. monocytogenes can resist extreme environmental stress and survives therefore in multiple habitants e.g. in the host or the food producing environment. Recent genome analyses have revealed that L. monocytogenes comprises multiple hypervariable genetic hotspots harbouring accessory genes which could provide advantage to survive extreme conditions. However the function of most genes located in hypervariable genetic hotspots is unknown. This project focuses on the hypervariable genetic hotspots lmo0443-lmo0449 harbouring at least three different inserts: the stress survival islet SSI-1, lin0464/lin0465 and homologues of the LMOf2364_0481 gene. SSI-1 consists of five genes involved in acidic, salt, bile and gastric stress response. Lin0464, a putative transcriptional regulator and lin0465, a putative intracellular pfpI protease are normally present in non-pathogenic L. innocua; and LMOf2364_0481, harbours a domain of unknown function often associated with a putative nucleic acid binding domain. Preliminary experiments have shown that the pfpI protease Lin0465 is involved in alkaline, oxidative, but not in acidic and salt stress response; and has a role in virulence. Our hypothesis is that the different insert types of the hypervariable genetic hotspot lmo0443-lmo0449 lead to increased survival under distinct stress conditions resulting in adaption to different environments. Therefore we will i) elucidate the role of the pfpI protease Lin0465 in stress survival, biofilm formation, virulence and pathogenicity, ii) characterize the role of Lin0465 in the intracellular recycling of damaged and/or misfolded proteins, and iii) investigate whether Lin0464 regulates transcription of the pfpI protease lin0465. To test the hypothesis that LMOf2365_0481 has a role in the transcriptional regulation of genes involved in specific stress response we will generate a LMOf2365_0481 deletion mutant strain and perform a whole transcriptome sequencing approach, comparing the gene expression of the wildtype and mutant strains under stress conditions. In addition we will perform a large-scale PCR screening of strains to detect novel lmo0443-lmo0449 inserts in L. monocytogenes. The results of this project will give insights in the role of the hypervariable genetic hotspot lmo0443- lmo0449 in the survival of L. monocytogenes under specific stress conditions and in pathogenicity. This will extend our understanding of the influence of hypervariable genetic regions in survival strategies, adaption and persistence of L. monocytogenes and therefore have an impact on food safety.

Listeria monocytogenes is a foodborne pathogen that can colonize the food producing environment and on diverse food products including cheese or meat. The hygiene standards in food production chains, therefore, are quite high. But Listeria is known for its ability to survive in environmental niches in which other microorganisms cannot. The key to the bacteria's survival is its adaptability and persistence in stress situations. Hypervariable, i.e. easily changeable, regions of the genome can contain genetic inserts that help Listeria to survive and adapt. In this project, we analyzed one of these hypervariable genetic hotspots and identified a novel stress islet, which allows L. monocytogenes to react to and block the effects of cleaning solutions and disinfectants. This stress islet, called SSI-2, comprises of two genes, whose expression is increased during alkaline and oxidative stress. The first gene is a transcriptional regulator, which in certain situations regulates the frequency and activity of the second protein. The second is a protease, an enzyme that breaks down other proteins. Proteases help bacteria break down unfunctional proteins that are created during stress situations. If the regulator is not active, there is no protease. Without the protease, L. monocytogenes has a harder time compensating stress caused by cleaning solutions and disinfectants. This stress islet is predominantly found in L. monocytogenes strains that are specialized for food and food processing environments. SSI-2 thus appears to be niche-specific. We were able to identify a specific genome type in which SSI-2 is always present: ST121. This sequence type is found almost exclusively in food and the food processing environment and hardly ever in clinical isolates. Analyzing the genome of ST121 strains showed that this sequence types harbors additional genetic features, supporting the survival under stress conditions. We were able to describe important survival strategies of foodborne Listeria. Knowing the genetic mechanism, allows us to develop new strategies for food safety.