Development of inducible systems to engineer conditional mutants of essential genes of Helicobacter pylori

The Escherichia coli-Helicobacter pylori shuttle vector pHeL2 was modified to introduce the inducible LacI(q)-pTac system of E. coli, in which the promoters were engineered to be under the control of H. pylori RNA polymerase. The amiE gene promoter of H. pylori was taken to constitutively express the LacI(q) repressor. Expression of the reporter gene lacZ was driven by either pTac (pILL2150) or a modified version of the ureI gene promoter in which one or two LacI-binding sites and/or mutated nucleotides between the ribosomal binding site and the ATG start codon (pILL2153 and pILL2157) were introduced. Promoter activity was evaluated by measuring beta-galactosidase activity. pILL2150 is a tightly regulated expression system suitable for the analysis of genes with low-level expression, while pILL2157 is well adapted for the controlled expression of genes encoding recombinant proteins in H. pylori. To exemplify the usefulness of these tools, we constructed conditional mutants of the putative essential pbp1 and ftsI genes encoding penicillin-binding proteins 1 and 3 of H. pylori, respectively. Both genes were cloned into pILL2150 and introduced in the parental H. pylori strain N6. The chromosomally harbored pbp1 and ftsI genes were then inactivated by replacing them with a nonpolar kanamycin cassette. Inactivation was strictly dependent upon addition of isopropyl-beta-d-thiogalactopyranoside. Hence, we were able to construct the first conditional mutants of H. pylori. Finally, we demonstrated that following in vitro methylation of the recombinant plasmids, these could be introduced into a large variety of H. pylori isolates with different genetic backgrounds.     

The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola, allowing single cell and microcolony detection

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola -specific phage (φ11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola φ11P, 19H and P. aeruginosa φPLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux ⁺ bacteria were inoculated onto French bean leaf ( Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux ⁺ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.     

Lipopolysaccharides from Helicobacter pylori can act as antagonists for Toll-like receptor 4

Infection with Helicobacter pylori, a Gram-negative bacterium, is strongly associated with gastric ulcers and adenocarcinoma. The mechanisms by which the innate immune system recognizes H. pylori lipopolysaccharide (LPS) remain unclear. Contradictory reports exist that suggest that Toll-like receptors are involved. In this study we evaluated the interactions of Toll-like receptors with LPS from different strains of H. pylori. Using reporter cell lines, as well as HEK293 cells transfected with either CD14 and TLR4, or CD14 and TLR2, we show that H. pylori LPS-induced cell activation is mediated through TLR2. In addition, for the first time, we report that LPS from some H. pylori strains are able to antagonize TLR4. The antagonistic activity of H. pylori LPS from certain strains, as well as the activation via TLR2, might give H. pylori an advantage over the host that may be associated with the clinical outcome of H. pylori infection.     

Recombinase-based reporter system and antisense technology to study gene expression and essentiality in hypoxic nonreplicating mycobacteria

The study of the mechanisms used by Mycobacterium tuberculosis to survive in the absence of growth is hampered by the absence of appropriate genetic tools. Here, we report two strategies, a recombinase-based reporter system and an antisense technology, to study gene expression and essentiality in hypoxic nonreplicating mycobacteria. The recombinase-based reporter system relies on the resolution of an antibiotic marker flanked by the gammadelta-res sites. This system was developed to identify M. tuberculosis promoters, which are specifically expressed under anaerobic conditions. The antisense strategy was designed to study the role of a gene candidate during anaerobic survival. To validate this approach, the dosR, narK2 and rv2466c promoters were selected to drive dosR antisense mRNA expression in quiescent mycobacteria. The conditional knockout strains were found to be attenuated to adapt and survive under anaerobic conditions, as observed for the dosR knockout strain. Together, our work demonstrates that the recombinase-based reporter system and antisense technology represent two genetic tools useful for the identification and characterization of genes essential for the survival of hypoxic nonreplicating M. tuberculosis.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

Pharmacological and genetic modulation of Wnt-targeted Cre-Lox-mediated gene expression in colorectal cancer cells

Wnt-targeted gene therapy has been proposed as a treatment for human colorectal cancer (CRC). The Cre-Lox system consists of methodology for enhancing targeted expression from tissue-specific or cancer-specific promoters. We analyzed the efficiency of Wnt-specific promoters as drivers of the Cre-mediated activity of a luciferase reporter gene or cell death effector gene in CRC cell lines in the presence and absence of two modulators of Wnt activity, sodium butyrate and lithium chloride. Butyrate is present in the colonic lumen after digestion of fiber-rich foods, whereas the colonic lumen is readily accessible to lithium chloride. In both SW620 and HCT-116 CRC cells, a physiologically relevant concentration of butyrate upregulated reporter and effector activity and altered the Wnt-specific expression pattern. Lithium chloride markedly enhanced Cre-Lox-mediated Wnt-specific reporter expression only in APC wild-type CRC cells. Possibilities for genetic modulation of the proposed CRC therapy included Wnt-specific expression of a floxed Lef1-VP16 fusion that enhanced Wnt-specific cell death and of a floxed dominant-negative Tcf4 that specifically downregulated endogenous Wnt activity. These findings demonstrated that the Cre-Lox system, in combination with pharmacological and genetic modulators, represents effective methodology for enhancing Wnt-targeted gene therapy.