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Listeria monocytogenes is a facultative Gram-positive bacterium that causes listeriosis, a severe foodborne disease. We previously discovered that ring-fused 2-pyridone compounds can decrease virulence factor expression in Listeria by binding and inactivating the PrfA virulence activator. In this study, we tested PS900, a highly substituted 2-pyridone that was recently discovered to be bactericidal to other Gram-positive pathogenic bacteria, such as Staphylococcus aureus and Enterococcus faecalis. We show that PS900 can interact with PrfA and reduce the expression of virulence factors. Unlike previous ring-fused 2-pyridones shown to inactivate PrfA, PS900 had an additional antibacterial activity and was found to potentiate sensitivity toward cholic acid. Two PS900-tolerant mutants able to grow in the presence of PS900 carried mutations in the brtA gene, encoding the BrtA repressor. In wild-type (WT) bacteria, cholic acid binds and inactivates BrtA, thereby alleviating the expression of the multidrug transporter MdrT. Interestingly, we found that PS900 also binds to BrtA and that this interaction causes BrtA to dissociate from its binding site in front of the mdrT gene. In addition, we observed that PS900 potentiated the effect of different osmolytes. We suggest that the increased potency of cholic acid and osmolytes to kill bacteria in the presence of PS900 is due to the ability of the latter to inhibit general efflux, through a yet-unknown mechanism. Our data indicate that thiazolino 2-pyridones constitute an attractive scaffold when designing new types of antibacterial agents.

IMPORTANCE: Bacteria resistant to one or several antibiotics are a very large problem, threatening not only treatment of infections but also surgery and cancer treatments. Thus, new types of antibacterial drugs are desperately needed. In this work, we show that a new generation of substituted ring-fused 2-pyridones not only inhibit Listeria monocytogenes virulence gene expression, presumably by inactivating the PrfA virulence regulator, but also potentiate the bactericidal effects of cholic acid and different osmolytes. We identified a multidrug repressor as a second target of 2-pyridones. The repressor–2-pyridone interaction displaces the repressor from DNA, thus increasing the expression of a multidrug transporter. In addition, our data suggest that the new class of ring-fused 2-pyridones are efficient efflux inhibitors, possibly explaining why the simultaneous addition of 2-pyridones together with cholic acid or osmolytes is detrimental for the bacterium. This work proves conclusively that 2-pyridones constitute a promising scaffold to build on for future antibacterial drug design.

HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.

Bacterial pathogens often employ RNA regulatory elements located in the 5' untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5' UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5' UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions. We demonstrate that the 5' UTR of the prsA2 mRNA base pairs with the 3' UTR of the full-length hly mRNA encoding listeriolysin O, thus preventing RNase J1-mediated degradation of the prsA2 transcript. Mutants lacking the hly-prsA2 interaction exhibit reduced virulence properties. This work highlights an additional level of RNA regulation, where the mRNA encoding a chaperone is stabilized by the mRNA encoding its substrate.

The structure of 5′ untranslated regions (5′ UTRs) of bacterial mRNAs often determines the fate of the transcripts. Using a dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) approach, we developed a protocol to generate sequence libraries to determine the base-pairing status of adenines and cytosines in the 5′ UTRs of bacterial mRNAs. Our method increases the sequencing depth of the 5′ UTRs and allows detection of changes in their structures by sequencing libraries of moderate sizes. For complete details on the use and execution of this protocol, please refer to Ignatov et al. (2020).

The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.

RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain.

RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37 degrees C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26 degrees C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20 degrees C, whereas the effect was marginal at 37 degrees C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food-industry. In this work, we have investigated the physiological role of one DExD-box RNA-helicase in Listeria monocytogenes. The RNA-helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to a down-regulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low temperature growth, motility, 23S rRNA processing and appropriate ribosomal maturation. However, Lmo1722 lacking the C-terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C-terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, ribosomal RNA processing and is important for ribosomal maturation being associated mainly with the 50S subunit of the ribosome.

Expression of virulence factors in the human bacterial pathogen Listeria monocytogenes is almost exclusively regulated by the transcriptional activator PrfA. The translation of prfA is controlled by a thermosensor located in the 5'-untranslated RNA (UTR), and is high at 37 degrees C and low at temperatures < 30 degrees C. In order to develop a thermoregulated translational expression system, the 5'-UTR and different lengths of the prfA-coding sequences were placed in front of lacZ. When expressed in Escherichia coli, the beta-galactosidase expression was directly correlated to the length of the prfA-coding mRNA lying in front of lacZ. A similar effect was detected with gfp as a reporter gene in both L. monocytogenes and E. coli, emphasizing the requirement of the prfA-coding RNA for maximal expression. In vitro transcription/translation and mutational analysis suggests a role for the first 20 codons of the native prfA-mRNA for maximal expression. By toe-print and RNA-probing analysis, a flexible hairpin-loop located immediately downstream of the start-codon was shown to be important for ribosomal binding. The present work determines the importance of an unstructured part of the 5'-coding region of the prfA-mRNA for efficient translation.