Igor N. Olekhnovich

DNA-Binding Activities of the HilC and HilD Virulence Regulatory Proteins of Salmonella enterica Serovar Typhimurium

The HilC and HilD proteins of Salmonella enterica serovar Typhimurium are members of the AraC/XylS family of transcription regulators. They are encoded on Salmonella pathogenicity island 1 (SPI1) and control expression of the hilA gene, which encodes the major transcriptional activator for many genes encoded on SPI1 and elsewhere that contribute to invasion of host cells. Gel electrophoretic shift and DNase footprinting assays revealed that purified HilC and HilD proteins can bind to multiple regions in the hilA and hilC promoters and to a single region in the hilD promoter. Although both HilC and -D proteins can bind to the same DNA regions, they showed different dependencies on the sequence and lengths of their DNA targets. To identify the binding-sequence specificity of HilC and HilD, a series of single base substitutions changing each position in a DNA fragment corresponding to positions -92 to -52 of the hilC promoter was tested for binding to HilC and HilD in a gel shift DNA-binding assay. This mutational analysis in combination with sequence alignments allowed deduction of consensus sequences for binding of both proteins. The consensus sequences overlap but differ so that HilC can bind to both types of sites but HilD only to one. The hilA and hilC promoters contain multiple binding sites of each type, whereas the hilD promoter contains a site that binds HilC but not HilD without additional binding elements. The HilC and HilD proteins had no major effect on transcription from the hilA or hilD promoters using purified proteins in vitro but changed the choice of promoter at hilC. These results are consistent with a model derived from analysis of lacZ fusions stating that HilC and HilD enhance hilA expression by counteracting a repressing activity.

Role of Nucleoid-Associated Proteins Hha and H-NS in Expression of Salmonella enterica Activators HilD, HilC, and RtsA Required for Cell Invasion

The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position -54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hha-mediated repression.

Characterization of the NAD(P)H Oxidase and Metronidazole Reductase Activities of the RdxA Nitroreductase of Helicobacter pylori

Metronidazole (MTZ) is widely used in combination therapies against the human gastric pathogen Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss‐of‐function mutations in rdxA, which encodes an oxygen‐insensitive nitroreductase. The RdxA‐associated MTZ‐reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His6‐tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H‐dependent reductions of nitroaromatic and quinone substrates including nitrofurazone, nitrofurantoin, furazolidone, CB1954 and 1,4‐benzoquinone, but not MTZ. Unlike other nitroreductases, His6–RdxA exhibited potent NAD(P)H‐oxidase activity (kcat = 2.8 s−1) which suggested two possible explanations for the role of oxygen in MTZ reduction: (a) NAD(P)H‐oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ), and (b) molecular oxygen out‐competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, although increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity, even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H‐dependent MTZ‐reductase activity (apparent Km = 122 ± 58 μm, kcat = 0.24 s−1) under strictly anaerobic conditions. The MTZ‐reductase activity of RdxA was 60 times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH‐oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed.

Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide.

Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreductase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route for the synthesis of NTZ-related antibacterial analogues.

The Histidine Kinase Domain of UhpB Inhibits UhpA Action at the Escherichia coli uhpT Promoter

The histidine kinase (HK) component of many two-component regulatory systems exhibits regulated ability to phosphorylate itself and to participate in transfer of phosphate to and from its cognate response regulator. The signaling system that controls expression of the UhpT sugar phosphate transporter in Escherichia coli in response to external glucose 6-phosphate includes the HK protein UhpB and the polytopic membrane protein UhpC, a UhpT homolog which is required for responsiveness to an inducer and activation of UhpB. The existence of a UhpBC signaling complex is suggested by the requirement for UhpC for the activity of certain constitutively active variants of UhpB, the dominance and epistasis relationships of uhp alleles, and the finding that expression of UhpB in excess of UhpC has a strong dominant-negative effect. Expression of a hybrid protein containing the cytoplasmic C-terminal half of UhpB fused to glutathione S-transferase (GST) also interfered with Uhp signaling. This interference phenotype could not result solely from the phosphatase activity of UhpB, because interference affected both overexpressed UhpA and UhpA variants which are active in the absence of phosphorylation. Variant forms of UhpB which were active in the absence of UhpC carried amino acid substitutions near motifs conserved in HK proteins. The GST fusion protein inhibited the ability of UhpA to bind and activate transcription at the uhpT promoter. Unlike the wild-type situation, a GST fusion variant carrying one of the UhpB-activating substitutions, R324C, displayed autokinase activity and phosphate transfer to UhpA but retained the ability to sequester UhpA when it was altered in the conserved residues important for phosphate transfer. Thus, the default state of UhpB is kinase off, and activation of its phosphate transfer activity requires either the action of UhpC or the occurrence of certain mutations in UhpB. The interference phenotype shown by UhpB in excess of UhpC appears to include the binding and sequestration of UhpA.

Synthesis and antimicrobial evaluation of nitazoxanide-based analogues: identification of selective and broad spectrum activity.

A library composed of nitazoxanide‐based analogues was synthesized and assayed for increased antibacterial efficacy against the pyruvate–ferredoxin oxidoreductase (PFOR) using microorganisms Helicobacter pylori, Campylobacter jejuni and Clostridium difficile. Derivatives were found to recapitulate and improve activity against these organisms and select analogues were tested for their ability to disrupt the PFOR enzyme directly. The library was also screened for activity against staphylococci and resulted in the identification of analogues capable of inhibiting both staphylococci and all PFOR organisms at low micromolar minimum inhibitory concentrations with low toxicity to human foreskin cells.

Response to Metronidazole and Oxidative Stress is Mediated Through Homeostatic Regulator HsrA (HP1043) in Helicobacter pylori

Metronidazole (MTZ) is often used in combination therapies to treat infections caused by the gastric pathogen Helicobacter pylori. Resistance to MTZ results from loss-of-function mutations in genes encoding RdxA and FrxA nitroreductases. MTZ-resistant strains, when cultured at sub-MICs of MTZ (5 to 20 μg/ml), show dose-dependent defects in bacterial growth; depressed activities of many Krebs cycle enzymes, including pyruvate:ferredoxin oxidoreductase (PFOR); and low transcript levels of porGDAB (primer extension), phenotypes consistent with an involvement of a transcriptional regulator. Using a combination of protein purification steps, electrophoretic mobility shift assays (EMSAs), and mass spectrometry analyses of proteins bound to porG promoter sequences, we identified HP1043, an essential homeostatic global regulator (HsrA [for homeostatic stress regulator]). Competition EMSAs and supershift analyses with HsrA-enriched protein fractions confirmed specific binding to porGDAB and hsrA promoter sequences. Exposure to MTZ resulted in >10-fold decreases in levels of HsrA and in levels of the HsrA-regulated gene products PFOR and TlpB. Exposure to paraquat (PQ), hydrogen peroxide, or organic peroxides showed near equivalence with MTZ, revealing a common oxidative stress response pathway. Finally, direct superoxide dismutase (SOD) assays showed an inverse relationship between HsrA levels and SOD activity, suggesting that HsrA may serve as a repressor of sodB. As a homeostatic sentinel, HsrA appears to be ideally positioned to enable rapid shutdown of genes associated with metabolism and growth while activating (directly or indirectly) oxidative defense genes in response to low levels of toxic metabolites (MTZ or oxygen) before they reach DNA-damaging levels.

Contribution of the RpoA C-Terminal Domain to Stimulation of the Salmonella enterica hilA Promoter by HilC and HilD

Expression of invasion genes in Salmonella pathogenicity island 1 (SPI-1) is mainly driven by the transcriptional activator HilA. Transcription of hilA is subject to complex control and is stimulated by the SPI-1-encoded HilC and HilD proteins. The C-terminal domain of RpoA contributes to hilA activation by HilC/D under certain inducing conditions.

Preclinical Studies of Amixicile, a Systemic Therapeutic Developed for Treatment of Clostridium difficile Infections That Also Shows Efficacy against Helicobacter pylori

ABSTRACT Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 μM (NTZ was toxic above 10 μM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · μg/ml (30 mg/kg dose) to 328 h · μg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 μg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

Effects of Mutations in the Pseudomonas putida miaA Gene: Regulation of the trpE and trpGDC Operons in P. putida by Attenuation

Tn5 insertion mutants defective in regulation of the Pseudomonas putida trpE and trpGDC operons by tryptophan were found to contain insertions in the P. putida miaA gene, whose product (in Escherichia coli) modifies tRNA(Trp) and is required for attenuation. Nucleotide sequences upstream of trpE and trpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.