Gary Sisson

Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen‐insensitive NADPH nitroreductase

Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease. Many H. pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen‐insensitive NADPH nitroreductase activity. The underlying gene (called ‘rdxA’) was identified in several steps: transformation of Mtz‐susceptible (MtzS) H. pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing. We also found that (i) E. coli (normally MtzR) was rendered MtzS by a functional H. pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzRH. pylori rendered it MtzS; and (iii) replacement of rdxA in MtzSH. pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype. The 630 bp rdxA genes of five pairs of H. pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced. In each case, the paired rdxA genes differed from one another by one to three base substitutions. Typical rdxA genes from unrelated isolates differ by ≈ 5% in DNA sequence. Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR. Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles. RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs. 1 or 2 in CNRs) and isoelectric point (pI = 7.99 vs. 5.4–5.6), which might account for its reduction of low redox drugs such as Mtz. We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections. H. pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.

Tumor cell marker pvrl4 (nectin 4) is an epithelial cell receptor for measles virus

Vaccine and laboratory adapted strains of measles virus can use CD46 as a receptor to infect many human cell lines. However, wild type isolates of measles virus cannot use CD46, and they infect activated lymphocytes, dendritic cells, and macrophages via the receptor CD150/SLAM. Wild type virus can also infect epithelial cells of the respiratory tract through an unidentified receptor. We demonstrate that wild type measles virus infects primary airway epithelial cells grown in fetal calf serum and many adenocarcinoma cell lines of the lung, breast, and colon. Transfection of non-infectable adenocarcinoma cell lines with an expression vector encoding CD150/SLAM rendered them susceptible to measles virus, indicating that they were virus replication competent, but lacked a receptor for virus attachment and entry. Microarray analysis of susceptible versus non-susceptible cell lines was performed, and comparison of membrane protein gene transcripts produced a list of 11 candidate receptors. Of these, only the human tumor cell marker PVRL4 (Nectin 4) rendered cells amenable to measles virus infections. Flow cytometry confirmed that PVRL4 is highly expressed on the surfaces of susceptible lung, breast, and colon adenocarcinoma cell lines. Measles virus preferentially infected adenocarcinoma cell lines from the apical surface, although basolateral infection was observed with reduced kinetics. Confocal immune fluorescence microscopy and surface biotinylation experiments revealed that PVRL4 was expressed on both the apical and basolateral surfaces of these cell lines. Antibodies and siRNA directed against PVRL4 were able to block measles virus infections in MCF7 and NCI-H358 cancer cells. A virus binding assay indicated that PVRL4 was a bona fide receptor that supported virus attachment to the host cell. Several strains of measles virus were also shown to use PVRL4 as a receptor. Measles virus infection reduced PVRL4 surface expression in MCF7 cells, a property that is characteristic of receptor-associated viral infections.

Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori, Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

ABSTRACT Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (Ki, 2 to 10 μM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO2) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and 1H nuclear magnetic resonance analyses determined that the spectral shift (A418 to A351) of NTZ was due to protonation of the anion (NTZ−) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pKa = 6.18). We propose that NTZ− intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the “activated cofactor” of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.

The Helicobacter pylori Chemotaxis Receptor TlpB (HP0103) Is Required for pH Taxis and for Colonization of the Gastric Mucosa

The location of Helicobacter pylori in the gastric mucosa of mammals is defined by natural pH gradients within the gastric mucus, which are more alkaline proximal to the mucosal epithelial cells and more acidic toward the lumen. We have used a microscope slide-based pH gradient assay and video data collection system to document pH-tactic behavior. In response to hydrochloric acid (HCl), H. pylori changes its swimming pattern from straight-line random swimming to arcing or circular patterns that move the motile population away from the strong acid. Bacteria in more-alkaline regions did not swim toward the acid, suggesting the pH taxis is a form of negative chemotaxis. To identify the chemoreceptor(s) responsible for the transduction of pH-tactic signals, a vector-free allelic replacement strategy was used to construct mutations in each of the four annotated chemoreceptor genes (tlpA, tlpB, tlpC, and tlpD) in H. pylori strain SS1 and a motile variant of strain KE26695. All deletion mutants were motile and displayed normal chemotaxis in brucella soft agar, but only tlpB mutants were defective for pH taxis. tlpD mutants exhibited more tumbling and arcing swimming, while tlpC mutants were hypermotile and responsive to acid. While tlpA, tlpC, and tlpD mutants colonized mice to near wild-type levels, tlpB mutants were defective for colonization of highly permissive C57BL/6 interleukin-12 (IL-12) (p40-/-)-deficient mice. Complementation of the tlpB mutant (tlpB expressed from the rdxA locus) restored pH taxis and infectivity for mice. pH taxis, like motility and urease activity, is essential for colonization and persistence in the gastric mucosa, and thus TlpB function might represent a novel target in the development of therapeutics that blind tactic behavior.

Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

ABSTRACT Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rifr) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtzr phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.

Molecular diagnosis of Kashmir bee virus infection

SUMMARYMolecular protocols were applied to the diagnosis of Kashmir bee virus (KBV) infection in the honey bee, Apis mellifera. Procedures based on either serology (chemiluminescent Western blotting) or nucleic acid amplification (RT-PCR, reverse transcription-polymerase chain reaction) proved to be very effective, although it was possible in some circumstances to detect viral infection simply by Coomassie blue staining of SDS-PAGE gels. Under laboratory conditions, KBV infections were readily distinguished from those induced by acute paralysis virus, a closely related isolate, by using monospecific antisera raised against the putative picornavirus-like VP4 polypeptide. Since only a very small amount of material was required for either Western blotting or RT-PCR, a single bee could in theory be examined for the presence of a variety of different pathogens. PCR primers based on sequences from the viral RNA polymerase gene amplified a 417 bp amplicon from KBV-infected, but not healthy, pupae. The same prime...

Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori RdxA(+) (Nitroreductase) gene.

Much of the normal high sensitivity of wild-type Helicobacter pylori to metronidazole (Mtz) depends on rdxA (HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance in rdxA(+) (Mtz(s)) and also in rdxA (Mtz(r)) H. pylori strains, and that expression of rdxA in Escherichia coli results in equivalent Mtz-induced mutation. A reversion test using defined lac tester strains of E. coli carrying rdxA(+) indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in H. pylori and in E. coli carrying rdxA(+), suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtz(s) H. pylori with E. coli (highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in E. coli, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected with H. pylori may stimulate mutation and recombination in H. pylori, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.

Replication of Subgenomic Hepatitis C Virus Replicons in Mouse Fibroblasts Is Facilitated by Deletion of Interferon Regulatory Factor 3 and Expression of Liver-Specific MicroRNA 122

ABSTRACT Hepatitis C virus (HCV) infection causes significant morbidity, and efficient mouse models would greatly facilitate virus studies and the development of effective vaccines and new therapeutic agents. Entry factors, innate immunity, and host factors needed for viral replication represent the initial barriers that restrict HCV infection of mouse cells. Experiments in this paper consider early postentry steps of viral infection and investigate the roles of interferon regulatory factors (IRF-3 and IRF-9) and microRNA (miR-122) in promoting HCV replication in mouse embryo fibroblasts (MEFs) that contain viral subgenomic replicons. While wild-type murine fibroblasts are restricted for HCV RNA replication, deletion of IRF-3 alone can facilitate replicon activity in these cells. This effect is thought to be related to the inactivation of the type I interferon synthesis mediated by IRF-3. Additional deletion of IRF-9 to yield IRF-3−/− IRF-9−/− MEFs, which have blocked type I interferon signaling, did not increase HCV replication. Expression of liver-specific miR-122 in MEFs further stimulated the synthesis of HCV replicons in the rodent fibroblasts. The combined effects of miR-122 expression and deletion of IRF-3 produced a cooperative stimulation of HCV subgenome replication. miR-122 and IRF-3 are independent host factors that are capable of influencing HCV replication, and our findings could help to establish mouse models and other cell systems that support HCV growth and particle formation.

A 65-Kilobase Pathogenicity Island Is Unique to Philadelphia-1 Strains of Legionella pneumophila

Nucleotide sequence analysis of an approximately 80-kb genomic region revealed an approximately 65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other Legionella pneumophila strains and serogroups indicated that this approximately 65-kb locus is unique to L. pneumophila serogroup 1 Philadelphia-1 strains.

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we performed genome-wide transcriptional analysis of S. mutans in response to an acidic pH signal. The preliminary results showed that adaptation of S. mutans to pH 5.5 induced differential expression of nearly 14 % of the genes in the genome, including 169 upregulated genes and 108 downregulated genes, largely categorized into nine functional groups. One of the most interesting findings was that the genes encoding multiple two-component systems (TCSs), including CiaHR, LevSR, LiaSR, ScnKR, Hk/Rr1037/1038 and ComDE, were upregulated during acid adaptation. Real-time qRT-PCR confirmed the same trend in the expression profiles of these genes at pH 5.5. To determine the roles of these transduction systems in acid adaptation, mutants with a deletion of the histidine-kinase-encoding genes were constructed and assayed for the acid tolerance response (ATR). The results revealed that inactivation of each of these systems resulted in a mutant that was impaired in ATR, since pre-exposure of these mutants to pH 5.5 did not induce the same level of protection against lethal pH levels as the parent did. A competitive fitness assay showed that all the mutants were unable to compete with the parent strain for persistence in dual-strain mixed cultures at acidic pH, although, with the exception of the mutant in liaS, little effect was observed at neutral pH. The evidence from this study suggests that the multiple TCSs are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.