Expansion of the Tetracycline-Dependent Regulation Toolbox for Helicobacter pylori

In an effort to gain greater understanding of the biology and infection processes of Helicobacter pylori, we have expanded the functionality of the tetracycline-dependent gene regulation (tet) system to provide more improved and versatile genetic control and facilitate the generation of conditional mutants to study essential genes. Second-generation tetracycline-responsive H. pylori uPtetO5 promoters were based on the mutated core ureA promoter. Single point mutations at either the ribosomal binding site or the start codon were introduced to shift the regulatory range of three uPtetO5 derivatives. All promoters were tested for regulation by TetR and revTetR using dapD, a gene essential to peptidoglycan biosynthesis, as a reporter. All tet promoters were effectively regulated by both TetR and revTetR, and their regulation windows overlapped so as to cover a broad range of expression levels. tet promoters uPtetO5m1 and uPtetO5m2 could be sufficiently silenced by both TetR and revTetR so that the conditional mutants could not grow in the absence of diaminopimelic acid (DAP). Furthermore, through the use of these inducible promoters, we reveal that insufficient DAP biosynthesis results in viable cells with altered morphology. Overall, the development and optimization of tet regulation for H. pylori will not only permit the study of essential genes but also facilitate investigations into gene dosage effects on H. pylori physiology.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.     

Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The −10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.     

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, P_TF, was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked P_TF to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (P_NSE) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.     

Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 μM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.     

Control of gene expression in Helicobacter pylori using the Tet repressor

The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria–host interface. These studies demonstrate the effectiveness of the tetR–tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.     

Integrative elements for Bacillus subtilis yielding tetracycline-dependent growth phenotypes

We describe the construction and application of elements for random insertion of promoter containing DNA into the genome of Bacillus subtilis. The outward-facing promoter of these integrative elements termed InsTet^G+ is inducible by tetracycline so that conditional mutants are generated. We constructed three InsTet^G+ variants using different regulatory windows. In the first, the regulator gene tetR is located within the element, allowing one-step mutagenesis. The second contains tetR in the chromosome and yields the best regulation efficiency. The third exploits xylose-dependent tetR expression from a plasmid, enabling induction of TetR synthesis so that distinct expression levels of an affected gene can be adjusted. We have obtained mutant strains with all three variants. For some of them, growth can be modulated by the presence of effectors. Most growth defects occur in the presence of inducers, presumably due to regulated expression of antisense RNA.     

Motility and Chemotaxis Mediate the Preferential Colonization of Gastric Injury Sites by Helicobacter pylori

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 10⁶ H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10⁶) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10⁶) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.     

Effects of the RNA-binding protein,KSRP, on innate immune response against Helicobacter pylori infection in mice

Helicobacter pylori (H. pylori) contributes to various gastric diseases such as chronic gastritis, gastric ulcer, and gastric carcinoma. Host innate immune response against the pathogen plays a significant role in elimination of pathogen infection. Importantly, pathogen elimination is closely related to numerous inflammatory-related genes that participate in complex biological response of cells to harmful stimuli. Here we studied effects of the KH-type splicing regulatory protein (KSRP), a RNA-binding protein, on innate immune response against H. pylori infection. We found that H. pylori infection downregulated KSRP expression directly, and that KSRP overexpression repressed upregulation of CXCL-2 expression induced by H. pylori and facilitated H. pylori proliferation in vitro. Similarly, KSRP overexpression in H. pylori mice also facilitated H. pylori proliferation and colonization, and induced more severe gastric mucosal damage. Intriguingly, CXCL-2 and HMOX-1 were upregulated in H. pylori infected mice after KSRP overexpression. This difference in expression of these genes implicated that KSRP was closely associated with and directly participated in the innate immune response against H. pylori. These results were beneficial for understanding the in vivo function of KSRP on innate immune response against pathogen infection.     

IL-22 Negatively Regulates Helicobacter pylori-Induced CCL20 Expression in Gastric Epithelial Cells

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa and causes various gastric diseases. H. pylori infection induces the production of inflammatory chemokine CCL20 in gastric mucosa and leads to gastric inflammation. Given that the IL-22/IL-22R axis plays a critical role in the regulation of homeostasis and inflammation of epithelial cells at barrier surfaces, we investigated the effect of IL-22 on CCL20 expression induced by H. pylori. We demonstrated that H. pylori infection of the gastric epithelia-derived AGS cells significantly induced CCL20 expression and the induction was inhibited by IL-22. Functional analysis of the CCL20 promoter revealed that the H. pylori-induced CCL20 expression required the activation of NF-κB, and that IL-22 inhibited the induction by attenuating NF-κB activation. Knockdown of endogenous STAT3 by either short interfering RNAs or a short hairpin RNA significantly reduced the inhibitory effect of IL-22. Furthermore, STAT3 phosphorylation elicited by IL-22 was crucial for the inhibition of H. pylori-induced CCL20 expression. Consistent with the in vitro data showing that IL-22 negatively regulated H. pylori-induced CCL20 expression in gastric epithelial cells, studies on the tissue sections from patients with H. pylori infection also revealed an inverse association of IL-22 expression and CCL20 expression in vivo. Together, our findings suggest that IL-22 plays a role in the control of overproduction of the inflammatory chemokine and thus may protect the gastric mucosa from inflammation-mediated damage.     

Evaluation and screening of efficient promoters to improve astaxanthin production in Xanthophyllomyces dendrorhous

Astaxanthin is a valuable carotenoid that is widely used in the aquaculture, food, pharmaceutical, and cosmetic industries. Xanthophyllomyces dendrorhous is a carotenoid-synthesizing yeast strain that produces astaxanthin as its main pigment. Although metabolic engineering using gene manipulation is a valuable way to improve astaxanthin production, a gene expression system for X. dendrorhous has been poorly developed. In this study, three known promoters of X. dendrorhous, glycerol-3-phosphate dehydrogenase (gpd) promoter (Pgpd), glucose dehydrogenase (gdh) promoter (Pgdh), and actin (act) promoter (Pact), were evaluated for use in the overexpression of target proteins using green fluorescence protein (GFP) as an expression level indicator protein. The actin promoter, Pact, showed the highest expression level of GFP when compared with Pgpd and Pgdh. Additionally, to obtain new promoters for higher expression of target protein in X. dendrorhous, intracellular GFP intensity was evaluated for 13 candidate promoters. An alcohol dehydrogenase promoter, Padh4, showed more efficient expression of GFP rather than Pact. Overexpression of crtE gene encoding rate-limiting enzyme of carotenoid synthesis under the adh4 promoter yielded an increase in intracellular astaxanthin content of about 1.7-fold compared with the control strain. The promoters identified in this study must be useful for improving carotenoids production in X. dendrorhous.     

N-acetylcysteine prevents the development of gastritis induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection

Helicobacter pylori (H. pylori) is a human gastric pathogen, causing various gastric diseases ranging from gastritis to gastric adenocarcinoma. It has been reported that combining N-acetylcysteine (NAC) with conventional antibiotic therapy increases the success rate of H. pylori eradication. We evaluated the effect of NAC itself on the growth and colonization of H. pylori, and development of gastritis, using in vitro liquid culture system and in vivo animal models. H. pylori growth was evaluated in broth culture containing NAC. The H. pylori load and histopathological scores of stomachs were measured in Mongolian gerbils infected with H. pylori strain 7.13, and fed with NAC-containing diet. In liquid culture, NAC inhibited H. pylori growth in a concentration-dependent manner. In the animal model, 3-day administration of NAC after 1 week from infection reduced the H. pylori load; 6-week administration of NAC after 1 week from infection prevented the development of gastritis and reduced H. pylori colonization. However, no reduction in the bacterial load or degree of gastritis was observed with a 6-week administration of NAC following 6-week infection period. Our results indicate that NAC may exert a beneficial effect on reduction of bacterial colonization, and prevents the development of severe inflammation, in people with initial asymptomatic or mild H. pylori infection.     

Comparative analysis of macrophage associated vectors for use in genetic vaccine

Background

Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

Methods

Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

Results

All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

Conclusions

Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.     

Functional Analysis and Randomization of the Nisin-Inducible Promoter for Tuning Gene Expression in Lactococcus lactis

Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the ?35 and ?10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3–5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside ?35 and ?10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control β-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.