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International audience; Background: Small regulatory RNAs (sRNAs) can influence cellular processes by targeting mRNAs, leading to translation inhibition and mRNA degradation. Previous studies have shown that the σB-dependent sRNA Rli47 is induced in L. monocytogenes stationary phase cells, in the intestinal lumen and macrophages, but its function has remained elusive. Objectives: This study aimed to comprehensively characterize Rli47 and determine its biological function in L. monocytogenes. Methods: Besides in silico predictions, several molecular approaches such as DMS-MaPseq, northern blotting, EMSAs, RTqPCR, RNA-seq and phenotypic experiments were applied. Results: Rli47 was shown to basepair through a single-stranded CU-rich loop with the ribosome binding site of ilvA mRNA, which encodes for threonine deaminase (TD), the first enzyme of the isoleucine biosynthetic pathway. Mutational analysis of the predicted binding regions on either Rli47 or ilvA mRNA abolished the basepairing. Rli47 was shown to reduce the expression of ilvA. A significantly shorter growth lag was found on Drli47 and DsigB than the wild-type in isoleucine-depleted medium. Moreover, TD activity was higher in both mutant strains under this condition. Complementary transcriptomic analysis identified complex changes in gene expression in the Drli47 mutant strain and highlighted a significant role for Rli47 in modulating amino acid metabolism. A potential role for Rli47 in the regulation of isoleucine biosynthesis emerged. Data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis even in conditions where it is absent, suggesting that isoleucine might act as a critical signal for L. monocytogenes at certain times during its life cycle.

Listeria monocytogenes, a foodborne pathogenic bacterium, remains a serious public health concern due to its frequent occurrence in food products coupled with a high mortality rate. Bacterial pathogenicity depends greatly on the ability to secrete virulence factors to or beyond the bacterial cell surface. The Tat pathway, one of the secretion systems present in L. monocytogenes, was until now only investigated in silico. In L. monocytogenes strain EGDe two genes constitute this pathway, tatC(lmo0361) and tatA(lmo0362). Here we show that tatC and tatA are cotranscribed in a bicistronic- and growth-phase-dependent manner, being downregulated in the stationary phase. An EGDe tatAC mutant strain (EGDe ΔtatAC) was constructed, confirming that the Tat pathway is not essential for L.monocytogenes survival or biofilm-forming ability. When compared to the wild-type EGDe, deletion of tatAC did not decrease the virulence potential of EGDe ΔtatAC in HT-29 human epithelial cell line and even increased (p < 0.05) the virulence potential for mice. Moreover, we show that tat genes are prevalent in L. monocytogenes strains belonging to genetic lineage II and are generally absent from lineage I, which is more associated with human cases, thus excluding the possibility of using the Tat system as a target for novel antimicrobial compounds targeting L.monocytogenes.

In this study we employ a biophysical and molecular approach for the investigation of qualitative and quantitative changes in both food surface and bacteria upon surface decontamination by SonoSteam®. SonoSteam® is a recently developed method of food surface decontamination, which employs steam and ultrasound for effective heat transfer and short treatment times, resulting in significant reduction in surface bacteria. An efficient decontamination method should be cheap and fast, while eliminating harmful microorganism without decreasing the quality of the food. However, all known methods represent compromises between the abovementioned facts. Therefore it is important to develop new tools of analysis, which allow for an optimization of food decontamination. In this study we developed biophysical evaluation methods employing differential scanning calorimetry, second harmonic generation microscopy, two photon fluorescence microscopy and green fluorescence protein expressing Escherichia coli and Campylobacter jejuni. We employ these methods in the investigation of the effects of SonoSteam on both bacteria and broiler skin and compare our results with those obtained by traditional methods of food quality and safety evaluations. Our results show that there are no contradictions between data obtained by either approach. However, the biophysical methods draw a much more nuanced picture of the effects and efficiency of the investigated decontamination method, revealing e.g. an exponential dose/response relationship between SonoSteam treatment time and changes in collagen I, and a depth dependency in bacterial reduction, which points toward CFU counts overestimating total bacterial reduction. In conclusion the biophysical methods provide a less biased, reproducible and highly detailed system description, allowing for focused optimization and method validation.

The antipsychotic drug thioridazine is a candidate drug for an alternative treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in combination with the β-lactam antibiotic oxacillin. The drug has been shown to have the capability to resensitize MRSA to oxacillin. We have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall and affect the ability of the bacteria to sustain oxacillin treatment. Furthermore, we found that thioridazine itself reduces the expression level of selected virulence genes and that selected toxin genes are not induced by thioridazine. In the present study, we find indications that the mechanism underlying reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis.