The Virulence and Infectivity of Listeria monocytogenes Are Not Substantially Altered by Elevated SigB Activity.

Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed" stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

A Highly Substituted Ring-Fused 2-Pyridone Compound Targeting PrfA and the Efflux Regulator BrtA in Listeria monocytogenes.

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.

Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Complete Genome Sequence of the Uropathogenic Methicillin-Resistant Staphylococcus aureus Strain MRSA-1369.

MRSA-1369 is a uropathogenic methicillin-resistant Staphylococcus aureus (MRSA) strain. Here, we present the complete genome sequence of MRSA-1369, which consists of one chromosome (2.87 Mb) and two plasmids (16.68 kb and 3.13 kb). This will serve as a reference genome for future Staphylococcus aureus pathogenesis and multiomic studies.

Listeria monocytogenes Requires the RsbX Protein To Prevent SigB Activation under Nonstressed Conditions.

The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multiprotein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins poststress. We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation levels under nonstress conditions and that it is required for appropriate SigB-mediated stress adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation and also an increased survival at low pH. Our results could suggest that absence of RsbX alters the multiprotein composition of the stressosome without dramatically affecting its phosphorylation status. Overall, the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under nonstressed conditions. IMPORTANCE Pathogenic bacteria need to sense and respond to stresses to survive harsh environments and also to turn off the response when no longer facing stress. Activity of the stress sigma factor SigB in the human pathogen Listeria monocytogenes is controlled by a hierarchic system having a large stress-sensing multiprotein complex known as the stressosome at the top. Following stress exposure, proteins in the stressosome become phosphorylated, leading to SigB activation. We have studied the role of a putative phosphatase, RsbX, which is hypothesized to dephosphorylate stressosome proteins. RsbX is critical not only to switch off the stress response poststress but also to keep the activity of SigB low at nonstressed conditions to prevent unnecessary gene expression and save energy.

Mycobacterium tuberculosis Rv3160c is a TetR-like transcriptional repressor that regulates expression of the putative oxygenase Rv3161c.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is a major health threat listed among the top 10 causes of death worldwide. Treatment of multidrug-resistant Mtb requires use of additional second-line drugs that prolong the treatment process and result in higher death rates. Our team previously identified a 2-pyridone molecule (C10) that blocks tolerance to the first-line drug isoniazid at C10 concentrations that do not inhibit bacterial growth. Here, we discovered that the genes rv3160c and rv3161c are highly induced by C10, which led us to investigate them as potential targets. We show that Rv3160c acts as a TetR-like transcriptional repressor binding to a palindromic sequence located in the rv3161c promoter. We also demonstrate that C10 interacts with Rv3160c, inhibiting its binding to DNA. We deleted the rv3161c gene, coding for a putative oxygenase, to investigate its role in drug and stress sensitivity as well as C10 activity. This Δrv3161c strain was more tolerant to isoniazid and lysozyme than wild type Mtb. However, this tolerance could still be blocked by C10, suggesting that C10 functions independently of Rv3161c to influence isoniazid and lysozyme sensitivity.

Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Corticosteroids protect infected cells against mycobacterial killing in vitro.

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 µM. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling.

Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening.

High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents.

Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile.

INTRODUCTION: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified [Formula: see text], which in wild-type strains are modified with mcm5s2U, partially suppress phenotypes of an elp3Δ strain. OBJECTIVES: To identify metabolic alterations in an elp3Δ strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified [Formula: see text]. METHOD: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3Δ strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing [Formula: see text]. The temperature sensitive elp3Δ strain derivatives were cultivated at permissive (30 °C) or semi-permissive (34 °C) growth conditions. RESULTS: Culturing an elp3Δ strain at 30 or 34 °C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3Δ strain carrying the wild-type ELP3 gene. Overexpression of hypomodified [Formula: see text] suppressed a subset of the metabolic alterations observed in the elp3Δ strain. CONCLUSION: Our results suggest that the presence of ncm5- and mcm5-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis.

Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies.

The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes.

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or 5-carbamoylmethyl-2'-O-methyluridine (ncm(5)Um) nucleosides in the anticodon at the wobble position (U34). Earlier we showed that mutants unable to form the side chain at position 5 (ncm(5) or mcm(5)) or lacking sulphur at position 2 (s(2)) of U34 result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm(5) and s(2) side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.

Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor.

The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA.

Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcm(5)s(2)U in tRNA.

Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides.

Elongator, a conserved complex required for wobble uridine modifications in eukaryotes.

Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U34 and their influence on Elongator activity.