The menaquinone pathway is important for susceptibility of Staphylococcus aureus to the antibiotic adjuvant, cannabidiol.

Emergence of antibiotic resistant bacteria is evolving at an alarming pace; therefore, we must start turning to alternative approaches. One of these, could be the use of antibiotic adjuvants that enhances the effect of antibiotics towards resistant bacteria. A novel antibiotic adjuvant is cannabidiol (CBD), which we have previously shown can enhance the effect of bacitracin (BAC). BAC targets cell wall synthesis by inhibiting dephosphorylation of the lipid carrier undecaprenyl pyrophosphate prior to recycling across the membrane. However, the mechanism underlying this CBD mediated potentiation of BAC has remained unknown. To explore this, we examined resistance to CBD in Staphylococcus aureus through daily exposures to CBD. By subsequent whole genome sequencing, we observed multiple genes to be mutated, including the farE/farR system encoding a fatty acid efflux pump (FarE) and its regulator (FarR). Importantly, recreation of mutations in these genes showed decreased susceptibility towards the combination of CBD and BAC. Furthermore, we searched the Nebraska Transposon Mutant Library for CBD susceptible strains and identified menH encoding a protein participating in menaquinone biosynthesis. Strains containing deletions in this and other menaquinone related genes showed increased susceptibility towards CBD, while addition of exogenous menaquinone reversed the effect and reduced susceptible towards CBD. These results suggest that CBD potentiates BAC by redirecting the isoprenoid precursor isopentenyl pyrophosphate towards production of menaquinone rather than the lipid carrier undecaprenyl pyrophosphate, which dephosphorylation is inhibited by BAC. This in turn might decrease the level of undecaprenyl pyrophosphate thus enhancing the effect of BAC. Our study illustrates how antibiotic adjuvants may apply to enhance efficacy of antimicrobial compounds.

Bacteria-host transcriptional response during endothelial invasion by Staphylococcus aureus.

Staphylococcus aureus is the cause of serious vascular infections such as sepsis and endocarditis. These infections are notoriously difficult to treat, and it is believed that the ability of S. aureus to invade endothelial cells and persist intracellularly is a key mechanism for persistence despite ongoing antibiotic treatment. Here, we used dual RNA sequencing to study the simultaneous transcriptional response of S. aureus and human endothelial cells during in vitro infections. We revealed discrete and shared differentially expressed genes for both host and pathogen at the different stages of infection. While the endothelial cells upregulated genes involved in interferon signalling and antigen presentation during late infection, S. aureus downregulated toxin expression while upregulating genes related to iron scavenging. In conclusion, the presented data provide an important resource to facilitate functional investigations into host-pathogen interaction during S. aureus invasive infection and a basis for identifying novel drug target sites.

Cannabidiol is an effective helper compound in combination with bacitracin to kill Gram-positive bacteria.

The cannabinoid cannabidiol (CBD) is characterised in this study as a helper compound against resistant bacteria. CBD potentiates the effect of bacitracin (BAC) against Gram-positive bacteria (Staphylococcus species, Listeria monocytogenes, and Enterococcus faecalis) but appears ineffective against Gram-negative bacteria. CBD reduced the MIC value of BAC by at least 64-fold and the combination yielded an FIC index of 0.5 or below in most Gram-positive bacteria tested. Morphological changes in S. aureus as a result of the combination of CBD and BAC included several septa formations during cell division along with membrane irregularities. Analysis of the muropeptide composition of treated S. aureus indicated no changes in the cell wall composition. However, CBD and BAC treated bacteria did show a decreased rate of autolysis. The bacteria further showed a decreased membrane potential upon treatment with CBD; yet, they did not show any further decrease upon combination treatment. Noticeably, expression of a major cell division regulator gene, ezrA, was reduced two-fold upon combination treatment emphasising the impact of the combination on cell division. Based on these observations, the combination of CBD and BAC is suggested to be a putative novel treatment in clinical settings for treatment of infections with antibiotic resistant Gram-positive bacteria.

Studies of Impending Oligonucleotide Therapeutics in Simulated Biofluids.

Synthetic oligonucleotides, their complexes and conjugates with other biomolecules represent valuable research tools and therapeutic agents. In spite of growing applications in basic research and clinical science, only few studies have addressed the issue of such compounds' stability in biological media. Herein, we studied the stability of two therapeutically relevant oligonucleotide probes in simulated biofluids; the 21 nucleotide-long DNA/locked nucleic acid oligonucleotide ON targeted toward cancer-associated BRAF V600E mutation, and a longer DNA analog (TTC) originating from BRAF gene. We found that stability of peptide-oligonucleotide conjugates (POCs) in human serum (HS) was superior compared with the naked or complexed 21mer oligonucleotide, whereas stability of POCs in simulated gastric juice (GJ) was dependent on the peptide sequence. Addition of pepstatin A in general increased the stability of oligonucleotides after 24 h digestion in HS and simulated GJ. Similarly, complexation with optimal amounts of histone proteins was found to rescue oligonucleotide stability after 24 h digestion in hydrochloric acid.

Molecular mechanisms of thioridazine resistance in Staphylococcus aureus.

Staphylococcus aureus has developed resistance towards the most commonly used anti-staphylococcal antibiotics. Therefore, there is an urgent need to find new treatment opportunities. A new approach relies on the use of helper compounds, which are able to potentiate the effect of antibiotics. A well-studied helper compound is thioridazine, which potentiates the effect of the ß-lactam antibiotic dicloxacillin against Methicillin-resistant Staphylococcus aureus (MRSA). In order to identify thioridazine's mechanism of action and how it potentiates the effect of dicloxacillin, we generated thioridazine resistant strains of MRSA USA300 by serial passage experiments. Selected strains were whole-genome sequenced to find mutations causing thioridazine resistance. Genes observed to be mutated were attempted deleted in MRSA USA300. The cls gene encoding a cardiolipin synthase important for synthesis of the membrane lipid cardiolipin was found to be mutated in thioridazine resistant strains. Deletion of this gene resulted in a two-fold increased Minimum inhibitory concentrations (MIC) value for thioridazine compared to the wild type and decreased susceptibility similar to the thioridazine resistant strains. Since cardiolipin likely plays a role in resistance towards thioridazine, it might also be important for the mechanism of action behind the potentiating effect of thioridazine. TDZ is known to intercalate into the membrane and we show here that TDZ can depolarize the plasma membrane. However, our results indicate that the membrane potential reducing effect of TDZ is independent of the resistance mechanism.

Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes.

Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species.