Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant Staphylococcus aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study, we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan (PGN) synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs PGN biosynthesis at a stage that precedes transpeptidation by penicillin-binding proteins (PBPs). In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal PGN precursors with pentaglycine branches, the correct substrate of S. aureus PBPs. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics.
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 β-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.
The bacterial pathogen Listeria monocytogenes encodes seven homologous small regulatory RNAs, named the LhrC family of sRNAs. The LhrCs are highly induced under infection-relevant conditions and are known to inhibit the expression of multiple target mRNAs encoding virulence-associated surface proteins. In all cases studied so far, the LhrCs use their CU-rich regions for base pairing to complementary AG-rich sequences of the ribosomal binding site (RBS) of specific target mRNAs. Consequently, LhrC-mRNA interaction results in inhibition of translation followed by mRNA degradation, corresponding to the canonical model for sRNA-mediated gene regulation in bacteria. Here, we demonstrate that the LhrC sRNAs employ a different regulatory mechanism when acting to down-regulate the expression of tcsA, encoding a T cell-stimulating antigen. In this case, LhrC base pairs to an AG-rich site located well upstream of the RBS in tcsA mRNA. Using an in vitro translation assay, we found that LhrC could not prevent the ribosome from translating the tcsA messenger. Rather, the LhrC sRNAs act to decrease the half-life of tcsA mRNA in vivo. Importantly, LhrC-mediated destabilization of tcsA mRNA relies on an intact LhrC binding site near the 5´-end of the tcsA mRNA and occurs independently of translation. Based on these findings, we propose an alternative mechanism for LhrC-mediated control in L. monocytogenes that relies solely on sRNA-induced degradation of a target mRNA.
Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to b-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono-and combinationtreated planktonic cultures showed a 1000-to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on b-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to b-lactammediated inhibition of cell division, rapidly appeared upon b-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of b-lactam treatment. Interestingly, the effect of sugar on b-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in b-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.
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