Pathogenic Differences of Type 1 Restriction-Modification Allele Variants in Experimental Listeria monocytogenes Meningitis

Abstract

Background: L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis.Methods: L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis.Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion:We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and Zbinden et al. Phenotypic Switching in Experimental MeningitisD worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

Different Shades of Listeria monocytogenes: Strain, Serotype, and Lineage-Based Variability in Virulence and Stress Tolerance Profiles

“…Such shifts between phenotypic forms are also observed in other bacteria such as Streptococcus pneumoniae ( Manso et al, 2014 ). A previous study using a neonatal rat-based Listeria meningitis model and an L. monocytogenes CC4 strain carrying a similar type I RMS as N12-0794 showed that phase variations linked to these systems result in altered disease severity outcomes ( Zbinden et al, 2020 ). These phase variable epigenetic modifications might allow some strains to finely tune their virulence gene expression, only expressing them when specific host conditions are encountered.…”

Editorial: Host-Pathogen Interaction in the Central Nervous System

“…On the other hand, the expression of hsdSC and D worsened the disease onset in L. monocytogenes meningitis (Zbinden et al, 2020). This observation shows important phenotypical switching that has crucial role in regulating the virulence of CNS infections by L. monocytogenes (Zbinden et al, 2020). Nevertheless, further studies are necessary to investigate how this therapy can also be effective in reducing brain injury.…”

“…L. monocytogenes meningoencephalitis has a mortality rate of up to 50%, and severe neurofunctional sequelae are prevalent. Zbinden et al have identified that L. monocytogenes expressing multiple sequence recognition (hsdS) A causes less damage than when other hsdS genes (B, C or D) are present (Zbinden et al, 2020). On the other hand, the expression of hsdSC and D worsened the disease onset in L. monocytogenes meningitis (Zbinden et al, 2020).…”

“…Zbinden et al have identified that L. monocytogenes expressing multiple sequence recognition (hsdS) A causes less damage than when other hsdS genes (B, C or D) are present (Zbinden et al, 2020). On the other hand, the expression of hsdSC and D worsened the disease onset in L. monocytogenes meningitis (Zbinden et al, 2020). This observation shows important phenotypical switching that has crucial role in regulating the virulence of CNS infections by L. monocytogenes (Zbinden et al, 2020).…”

Table_4.xlsx

“…Such shifts between phenotypic forms are also observed in other bacteria such as Streptococcus pneumoniae (Manso et al, 2014). A previous study using a neonatal ratbased Listeria meningitis model and an L. monocytogenes CC4 strain carrying a similar type I RMS as N12-0794 showed that phase variations linked to these systems result in altered disease severity outcomes (Zbinden et al, 2020). These phase variable epigenetic modifications might allow some strains to finely tune their virulence gene expression, only expressing them when specific host conditions are encountered.…”