Shaun P. Falk

Phosphorylation of the RitR DNA-binding domain by a Ser–Thr phosphokinase: implications for global gene regulation in the streptococci

We report selective phosphorylation of the DNA‐binding domain of the Streptococcus pneumoniae transcriptional regulator RitR. RitR is annotated as a two‐component response regulator, but lacks a cognate His kinase as a neighbouring locus in the genome. In addition, Asn replaces Asp at the expected acceptor site. By the use of combinatorial phage display, we identified PhpP, a S. pneumoniae Ser–Thr eukaryotic‐like PP2C phosphatase as an interacting partner of RitR. RitR interacts with the phage‐displayed peptide VADGMGGR which forms a part of the active‐site sequence of PhpP. RitR is phosphorylated in vitro by StkP, the presumed cognate kinase of PhpP, and the site on RitR that is phosphorylated has been localized to the RitR DNA‐binding domain. PhpP together with its cognate kinase StkP appear to be necessary for Piu haem transporter expression. In vitro studies suggest that PhpP and StkP interact competitively with RitR in that RitR–PhpP–piu promoter ternary complexes are disrupted by StkP. Our findings indicate a regulatory link between RitR and Ser–Thr kinase–phosphatase‐based bacterial signal transduction.

Nylon-3 Polymers with Selective Antifungal Activity

Host-defense peptides inhibit bacterial growth but show little toxicity toward mammalian cells. A variety of synthetic polymers have been reported to mimic this antibacterial selectivity; however, achieving comparable selectivity for fungi is more difficult because these pathogens are eukaryotes. Here we report nylon-3 polymers based on a novel subunit that display potent antifungal activity (MIC = 3.1 μg/mL for Candida albicans ) and favorable selectivity (IC10 > 400 μg/mL for 3T3 fibroblast toxicity; HC10 > 400 μg/mL for hemolysis).

Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans.

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-β-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure–activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C. albicans that are resistant to the antifungal drugs fluconazole and amphotericin B. These nylon-3 polymers are nonhemolytic. In addition, we have identified cationic–hydrophobic copolymers that are highly active against a second fungal pathogen, Cryptococcus neoformans, and moderately active against a third pathogen, Aspergillus fumigatus.

Nylon-3 polymers active against drug-resistant Candida albicans biofilms.

Candida albicans is the most common fungal pathogen in humans, and most diseases produced by C. albicans are associated with biofilms. We previously developed nylon-3 polymers with potent activity against planktonic C. albicans and excellent C. albicans versus mammalian cell selectivity. Here we show that these nylon-3 polymers have strong and selective activity against drug-resistant C. albicans in biofilms, as manifested by inhibition of biofilm formation and by killing of C. albicans in mature biofilms. The best nylon-3 polymer (poly-βNM) is superior to the antifungal drug fluconazole for all three strains examined. This polymer is slightly less effective than amphotericin B (AmpB) for two strains, but the polymer is superior against an AmpB-resistant strain.

West Nile virus methyltransferase domain interacts with protein kinase G

BackgroundThe flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5.MethodsWe used mass spectrometry to specifically identify NS5 phosphosites. Co-immunoprecipitation assays were used to study protein-protein interactions. Effects on viral replication were measured via replicon system and plaque assay titering.ResultsWe identified multiple sites in West Nile virus (WNV) NS5 that are phosphorylated during a WNV infection, and showed that the N-terminal methyltransferase domain of WNV NS5 can be specifically phosphorylated by PKG in vitro. Expressing PKG in cell culture led to an enhancement of WNV viral production. We hypothesized this effect on replication could be caused by factors beyond the specific phosphorylations of NS5. Here we show for the first time that PKG is also able to stably interact with a viral substrate, WNV NS5, in cell culture and in vitro. While the mosquito-borne WNV NS5 interacted with PKG, tick-borne Langat virus NS5 did not. The methyltransferase domain of NS5 is able to mediate the interaction between NS5 and PKG, and mutating positive residues in the αE region of the methyltransferase interrupts the interaction. These same mutations completely inhibited WNV replication.ConclusionsPKG is not required for WNV replication, but does make a stable interaction with NS5. While the consequence of the NS5:PKG interaction when it occurs is unclear, mutational data demonstrates that this interaction occurs in a region of NS5 that is otherwise necessary for replication. Overall, the results identify an interaction between virus and a cellular kinase and suggest a role for a host kinase in enhancing flaviviral replication.

Screen for Agents that Induce Autolysis in Bacillus subtilis

ABSTRACT The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis. SH-536 distinguishes between the two groups of autolysis-inducing agents by synthesizing and then releasing β-galactosidase (β-Gal) in late stationary phase at a time that cells have nearly stopped growing and are therefore tolerant of cell wall synthesis inhibitors. Four hits, named compound 2, compound 3, compound 5, and compound 24, obtained previously as inducers of autolysis by screening a 10,080-compound discovery library with BAU-102, were probed with SH-536 and found to release β-Gal, indicating that their mode of action was to permeabilize the B. subtilis cell membrane. The four primary hits inhibited growth in Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis, and Bacillus anthracis, with MICs in the 12.5- to 25-μg/ml (20 to 60 μM) range. The four primary hits were further used to probe B. subtilis, and their action was partially characterized with respect to the dependence of induced autolysis on specific autolysins.

Differential Assay for High-Throughput Screening of Antibacterial Compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/ surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed β-gal in the periplasm, suggesting leakage of β-gal as the means by which this assay detects compound activities. A model is proposed according to which β-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-β-D-galactoside as a single reagent. Cell wall inhibitors release β-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause β-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery. (Journal of Biomolecular Screening 2007:1102-1108)

Screen for Inducers of Autolysis in Bacillus subtilis

ABSTRACT We describe a primary high-throughput screen that uses the reporter strain Bacillus subtilis BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant Escherichia coli β-galactosidase (β-Gal) from the periplasmic space of B. subtilis owing to a loss of integrity of the cell wall. In a model screen, β-Gal release values for 79 members of a library consisting of antibiotics and related compounds were collected, sorted, and plotted as a function of rank. Inducers of autolysis, which included compounds that inhibit cell wall synthesis and those that do not, were readily differentiated from other members of the library on the basis of their elevated β-galactosidase release responses. The results of the BAU-102 model screen called attention to the antibacterial activity of drugs normally used in other applications, describable as “repurposed.” Thus, the screen independently identified the potential antibacterial properties of the antifungal drug miconazole and of the antileishmaniasis drug miltefosine. Daptomycin-induced release of β-Gal was also detected and occurred in a Ca2+-dependent manner.

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5′ end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a Kd = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50 for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5′-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z′ of 0.83 and was successfully used to screen a library of known bioactives.

Inhibition of DNA replication in Staphylococcus aureus by tegaserod

DNA replication is a vital function for cellular growth and proliferation. Enzymes that support this process would serve as useful targets for antibiotic discovery. Here we describe the antibacterial activity of tegaserod (TG) and propose a mechanism of action based on inhibition of Staphylococcus aureus DnaG primase, a DNA-dependent RNA polymerase required for the initiation of replication. TG, whose structure is shown in Figure 1a, was originally developed for clinical use as a 5HT-4 (serotonin) receptor agonist to treat gastrointestinal disorders.1 In the studies described below, TG was, additionally, identified as a hit in a DnaG screen (Profoldin, Hudson, MA, USA) of a library of 450 previously approved drugs (NIH CC1 collection). The screen is based on the measurement of fluorescence enhancement of an intercalating dye into RNA-DNA heteroduplexes synthesized by DnaG. Library compounds were tested at a final concentration of 30 μM in a 20 μl reaction volume. Inhibition was defined as a fluorescence reduction 43 s.d.s compared to untreated control. The identified inhibitors of DnaG, together with their respective percent inhibition in the screen, included: doxorubicin (71%), epirubicin (98%), TG (71%), epigallocathechin gallate (58%), pterostilbene (52%), actinomycin D (52%) and nonyloxyltryptamine (50%). With the exception of TG, the hits that were obtained in the screen had been studied elsewhere as inhibitors of nucleic acid metabolism.2–6 We therefore focused our attention on TG for further characterization of its antimicrobial activity. To confirm these results, a polymerization assay based on the work of Koepsell et al.7 in which the 37-mer single stranded DNA template 5′-CAG(AC)12ACTACACACA-3′ (transcription initiation start site at T, coordinate 30, underlined) was used. The known inhibitor of DnaG primase activity, doxorubicin,4 was included as a positive control for these studies (Figure 1b, Lane 1). The complete in vitro reaction was supplemented with an incremental series of TG concentrations. Polymerization products labeled by inclusion of [α-32P]UTP in the reaction mix were fractionated by PAGE and analyzed by autoradiography.8 The results, Figure 1b, lanes 2 and 3, show the expected 30-mer RNA primer that is formed in the complete uninhibited reaction. Results shown in lanes 4, 5 and 6, indicate that TG inhibits RNA primer formation in a TG-dependent manner with nearly complete inhibition of 30-mer transcript formation at 20 μM, near the MIC. Lanes 6 and 7 show the reappearance of transcript formation and a shift of the transcription product length to 60 nt. The 60-mer product may correspond to RNA formed by extension of the 37-mer template beginning at its 3′OH, and it is similar to the overlong product seen in earlier reports.9 We postulate that the 60-nt product is an RNA-DNA heteroduplex hairpin loop in which 5′-TACACACA-3′ forms the loop portion by association of the 5′T with the 3′A. Lane 8 shows the products formed by DnaG in the presence of 100 μM TG–complete abolition of 30-mer synthesis and marked reduction in 60-mer synthesis. To compare the specificity of DnaG with that of other polymerases, four additional enzymes were tested, namely, Escherichia coli DNA-dependent RNA polymerase and E. coli PolA (NEB, Ipswich, MA, USA); T7 DNA-dependent RNA polymerase (Fermentas, Waltham, MA, USA); and MuLV reverse transcriptase (Thermo Fisher, Fitchburg, WI, USA). The DNA templates used for the four enzymes were, calf thymus DNA, DNAase-treated calf thymus DNA, PCR-amplified T7 polymerase promoter sequence, and Bacillus. subtilis total RNA, respectively. Polymerase reactions were performed based on protocols provided by the vendor. Results, shown in Supplementary Figure 1, indicate that all four polymerases along with DnaG were inhibited by aurintricarboxylic acid, a non-specific inhibitor of nucleic acid-binding proteins.10 Tested at a maximum concentration of 200 μM, doxorubicin selectively inhibited the two RNA polymerases, but not the DNA polymerases PolA or MuLV reverse transcriptase. Importantly, none of the polymerases were inhibited by TG. TG, therefore, appears to have a unique mode of action in its ability to inhibit the polymerase activity of DnaG. To test the susceptibility of S. aureus 1206 cells11 in vivo, we determined the MIC (25 μM) and the MBC (100 μM) for TG. We measured the effect of TG on DNA, RNA and protein synthesis at the MIC using intact cells. Mid-exponential cultures of S. aureus 1206 in LB medium were supplemented with 3H-thymidine, 3H-uracil or 3H-tyrosine followed by a challenge with TG or control antibiotic for 1 h. Results, shown in Figure 2a, indicate that TG at a concentration