Address correspondence to Rainer Haas, Max von Pettenkofer-Institut, Pettenkoferstr. 9a, 80336 Munich, Germany. Phone: 49-89-5160-5255; Fax: 49-89-5260-5223; E-mail: haas{at}m3401.mpk.med.uni-muenchen.de
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Key Words: Helicobacter pylori in vivo essential genes signature tagged mutagenesis gastric adaptation collagenase
Although the current treatments against H. pylori have generally good efficacies, relapses and reinfections do occur, and most significantly, the increasing rate of antibiotic resistance will probably result in rising treatment failures. Therefore, there is a need to search for novel therapies and drug targets. Two complete H. pylori genome sequences are currently available (3, 4). Of the 1590 predicted genes in H. pylori two thirds were assigned biological roles, but about one third did not show any database match. Despite the description of essential and nonessential genes in vitro (5, 6), our understanding of the genes necessary for the specific adaptation of the pathogen to the gastric mucosa is still very limited. There is a need to exploit the information from the H. pylori genome sequences by high throughput functional genomics approaches, as performed for other pathogens (7, 8). Besides the urease enzyme and genes involved in motility of H. pylori, only few further genes have been shown to be essential for colonization in animal models (9). An interesting strategy for this purpose is the identification of specific gene products essential for colonization in the host by signature tagged mutagenesis (STM).* So far only small scale animal-based screening studies for H. pylori colonization factors have been reported (10), which certainly reflects the difficulties in an efficient genome-wide genetic manipulation of these fastidious bacteria.
In the present study, we report about a large scale screen to identify genes in H. pylori essential for gastric colonization using a modified STM procedure. STM was originally established in bacterial pathogens for the detection of genes that are essential for Salmonella typhimurium infection of mice (11). It is a negative selection method allowing large numbers of mutants to be analyzed simultaneously. We improved and adapted the STM method to H. pylori. We generated and analyzed a set of 960 independent H. pylori transposon (Tn) insertion mutants and finally identified 47 genes, which proved to be absolutely essential for gastric colonization of H. pylori in the well-suited gerbil model. In addition, a secreted collagenase activity could be verified as a novel virulence factor of H. pylori for stomach colonization.
General DNA Manipulations and Construction of the pTnHK9 In Vitro Tn.
For cloning of the hp0169 gene, the DNA was PCR-amplified using primers hp0169-start (5'-ggaattcAGTTGAATTACTCTCTCC) and hp0169-stop (ccgctcgagACCTAATCTCTAAACGCC) with H. pylori 26695-DNA as template. The PCR fragment was digested with EcoRI/XhoI and ligated into compatible sites of the vector pGEX4T3 (Amersham Biosciences). Escherichia coli BL21 was transformed and expression of the gst-hp0169-fusion was induced with 1 mM IPTG at 30°C for 2 h.
H. pylori Gene Library Construction and In Vitro Mutagenesis.
Infection and Screening of Tagged Mutant Bank.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Colonization of the gastric mucosa by Helicobacter pylori results in an acute inflammatory response and damage to the gastric epithelium. Inflammation can then progress to several disease states, ranging in severity from superficial gastritis, chronic atrophic gastritis, peptic ulceration, to mucosa-associated lymphoma and gastric cancer (1), and the World Health Organization classified H. pylori as a Group 1 carcinogen (2).
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Bacterial Strains and Growth Conditions.
H. pylori strain G1.1, a human isolate (original named CPY3401), was supplemented with a streptomycin resistance cassette (plasmid pEG21; reference 12) and termed Q1.1 (cagA+, cag-PAI partially deleted). After four animal passages the recovery was optimal and the strain was designated P149. H. pylori cells E. coli Top10 (Invitrogen) and DH5 were grown as described (12), and supplemented with selective antibiotics, as required.
General genetic manipulation was performed following standard procedures. Variable TAG-sequences were generated by oligonucleotide RH171 (5'-cta gggatccAGATCTNNNNNNNNNNNNNNNNNNNNAGATCTATCCACGTT-GAAAATCTCC-3'), which fuses a 20-mer random nucleotide sequence (20 x N; see Fig. 1 B) to a primer binding downstream of the chloramphenicolacetyltransferase gene (cat) in the mini-Tn TnMax5 (13). The variable sequence is flanked by two BglII restriction sites (italics) and carries a 5'-BamHI site (underlined) for cloning. A second primer, RH172, was designed, which binds upstream of the cat gene in pTnMax5. Thus, a PCR amplification using RH171 and RH172 as primers and pTnMax5 as template resulted in a PCR fragment consisting of the cat gene and a set of variable TAG sequences fused to the sequence downstream of cat (13). The variable fragments were digested with HindIII and XbaI and cloned into the corresponding sites of the plasmid pMOD, the EZ::TN Tn vector (Epicentre), which carries the Tn5 inverted repeat sequences. The obtained clone was named pTnHK9 (see Fig. 1 A).
The H. pylori genomic library was constructed by partial digest of chromosomal DNA of H. pylori strain P1 with Sau3A and HpaII, followed by subsequent ligation into cloning vector pSO50 (BglII/ClaI), a derivative of pMin2 (reference 13; see Fig. 2 C). Random in vitro Tn mutagenesis was performed essentially as described by the supplier of the EZ::Tn Tn (Epicentre). After in vitro transposition and transformation into E. coli DH5, plasmid pools from each TAG were extracted (QIAGEN). Transfer of suicide plasmids from E. coli to H. pylori P149 by natural transformation and allelic exchange was performed as described previously (12). After 45 d of growth, 40 individual colonies from each TAG were selected, amplified, and stored at 80°C.
For the gastric infection experiments 12-wk-old specific pathogen free Mongolian gerbils (RCC Ltd.) were used. Animals were maintained under standard laboratory conditions. To ensure that all mutants in the pool would be equally represented, individual mutant clones were grown separately on serum plates and pooled before intragastric infection. Two animals were used for each pool, and they were orally inoculated with 300 µl corresponding to 1.0 x 109 (OD550 = 3.3) H. pylori P149 mutants in broth culture (Brucella medium) at three consecutive days. From the bacterial pool of the last infection, aliquots were taken for the preparation of chromosomal DNA for the input pool (QIAamp® mini kit). Animals were killed 21 d after inoculation. Stomachs were opened along the great curvature, homogenized (glass homogenizer; Wheaton) and aliquots were plated onto selective media for quantitative culture. Colonizing mutants were recovered 45 d after culture, and the chromosomal DNA extracted using the QIAamp® mini kit for generation of the output pool. Detection of specific mutants in the output pool was achieved using a TAG-specific PCR method (Fig. 1
B). One TAG-specific primer was combined with primer SO38 (5'-GAAGATCTCTAAGGAAGCTAAAATGGAG-3') binding at the 5' sequence of the cat gene and the procedure was optimized to use all primer pairs in parallel. In general, thermal cycling was performed at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 52°C for 1 min, and 72°C for 1 min, with a terminal extension at 72°C for 5 min
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Determination of Protease Activity Using the Azocoll Test.
1 mg of insoluble Azocoll was incubated with bacterial crude extract (10100 µl) together with test buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2) (14 h, 37°C shaking at 1,000 rpm). Insoluble Azocoll was removed by centrifugation (18,000 g, 5 min). The protease-digested, solubilized Azocoll in the supernatant was analyzed by measuring with a photometer (442 nm) and comparing it to the control incubation lacking the bacterial supernatant.
Measuring Collagenase Activity.
Bacterial supernatant was concentrated by precipitation with 50% ammonium acetate and the precipitate was dialyzed against test buffer (100 mM Tris-HCl, pH 7.5, 5 mM CaCl2). Type I collagen from calves skin (Sigma-Aldrich) was solubilized in 100 mM acetic acid (1 mg/ml). 10 µl of the solution was mixed with 70 µl test buffer and 20 µl of concentrated bacterial supernatant or purified recombinant HP0169 at 37°C. After different time points aliquots were taken, and prepared for SDS-PAGE. Analysis of collagenase activity was determined by the densitometric evaluation of the intensity of collagen bands in the immunoblot using a collagen type Ispecific antibody (rabbit) (Rockland).
Purification of the GST-HP0169 Fusion Protein.
For heterologous overproduction of HP0169, the glutathion S-transferase system was used (Amersham Biosciences). E. coli BL21 carrying the fusion gene was grown in 100 ml LB to an OD550 of 0.8 and the tac-promoter of the plasmid was induced by the addition of 1 mM isopropylthiogalactosid (IPTG). The bacterial culture was chilled on ice (15 min) and pelleted (6,000 g, 4°C, 10 min). The pellet was suspended in 4 ml ice-cold buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2) and lysed by sonification. The lysate was cleared by centrifugation (15,000 g, 4°C, 15 min) and the supernatant was mixed with 0.4 ml of glutathion S-sepharose 4B solution. After 30 min incubation (4°C) the sepharose was removed by centrifugation (500 g) and washed three times in 2 ml buffer (50 mM Tris-HCl, pH 7.5, 5 mM CaCl2). Finally, the bound fusion protein was eluted by adding 10 mM glutathion. The eluate was dialyzed against 3 liter ice-cold buffer.
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Construction of an H. pylori Signature Tagged Mutant Library.
The original STM approach was modified by selecting 24 defined 20-mer sequence TAGs, based on their efficiency of amplification and lack of crosshybridization with each other. The TAGs were incorporated close to the left inverted repeat (IR) of TnHK9 (see Materials and Methods, and Fig. 1, A and B). A plasmid gene library was constructed, statistically covering the H. pylori genome 20-fold (Figs. 1 C and 2 C). In separate in vitro reactions a random mutagenesis of the plasmid library was performed, using the respective pTnHK9-TAG124 constructs and the hyperactive mutant transposase (EZ::Tn, Epicentre; Fig. 1 C). After transposition and subsequent transformation of individual TAG groups into E. coli strain DH5
(amplification step), total plasmids were extracted from the resulting transformants (1005,000 transformants per in vitro mutagenesis reaction) and analyzed by restriction for random Tn insertion (unpublished data). Transfer of plasmid pools containing defined TAGs into H. pylori P149 by natural transformation resulted in the final library of H. pylori mutants (Fig. 1 C). For a single TAG we obtained at least 40 independent H. pylori mutants, resulting in 960 (24 x 40) H. pylori mutants in total, which were stored at 80°C. To confirm that transformants harbored random insertions of a single Tn, 12 mutant clones were selected from TAG groups 2 and 12 each and the chromosomal DNA was subjected to restriction enzyme (PvuII) digestion. Southern analysis using TnHK9 DNA as probe revealed that all mutants tested had single Tn insertions in apparently different loci (Fig. 2, A and B)
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The Essential ORF hp0169 Encodes a Secreted Collagenase.
Interestingly, the gene product of hp0169 was annotated as a putative protease of the U32 protein family, which is characterized by the consensus sequence E-x-F-x(2)-G-[SA]-[LIVM]-C-x(4)-G-x-C-x-[LIVM]-S (14). Only one member of this family of proteases, PrtC of Porphyromonas gingivalis, has been characterized biochemically as a Ca2+-dependent metallo-protease with collagenolytic activity. No functional biochemical data were, however, present for HP0169, which reveals 29.4%/49.5% sequence identity/similarity with PrtC. Furthermore, the database search using the HP0169 sequence identified several other putative bacterial collagenases, such as PA5440 of Pseudomonas aeruginosa (41.0%/60.8%) and YdcP of E. coli O157:H7 (33.8%/55.8%) with significant sequence identity/similarity.
To obtain a first insight about a potential proteolytic activity of HP0169, total lysates of H. pylori P149 wild-type and the corresponding hp0169 mutant strain were compared using an Azocoll assay (15), which determines the release of an azo-dye due to proteolytic activity by a photometrical quantification. The wild-type strain revealed twice as much proteolytic activity as the mutant strain in this assay, but addition of EDTA reduced the proteolytic activity in the lysate to 25% of the wild type activity (Fig. 4 A), indicating that a proteolytic activity might be associated with HP0169.
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Finally, the hp0169 gene was fused to the glutathione-S-transferase gene (gst) and expressed in E. coli BL21 to produce the recombinant protein. The purified GST-HP0169 fusion protein, but not the purified GST protein alone, was active in degrading type 1 collagen (Fig. 4 D). Thus, we could show that HP0169, which was found to be essential for H. pylori to colonize in the gerbil stomach, is a secreted collagenase.
The In Vivo Essential Function of ComB4 Is Distinct from Its Role in Natural Transformation Competence.
The comB4 gene, which encodes a putative ATPase involved in natural transformation competence of H. pylori (16), was identified as essential for gastric colonization in the gerbil model (C.I. <0.0010, Table II). As the general screen resulted only in a single comB4 mutant, the mutation was reconstructed de novo in the wt strain background by transferring the mutated gene and was verified to be colonization defective. To prove experimentally whether or not natural transformation competence is the biological function determining the essential phenotype for in vivo colonization, we generated a deletion mutant in the comB810 genes of H. pylori P149 (see Materials and Methods). The ComB810 proteins are structural components of the type IV transport system involved in DNA transformation of H. pylori, probably by building a channel between inner and outer membrane (16, 17). Interestingly, the comB810 mutant strain, which was deficient in natural transformation competence, was still able to colonize the gerbil very efficiently in single infection experiments (Fig. 5)
. From these data we would conclude that the ComB4 protein, besides its role in natural transformation competence, might have an additional essential function for H. pylori, which has not been identified yet.
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Discussion |
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Salama et al. defined recently a minimal functional core of 1281 H. pylori genes by comparing the gene content of 15 clinical isolates using a whole genome DNA microarray (20). Interestingly, 46/47 genes that we identified as essential for gastric colonization belong to this core group of H. pylori genes. The only exception was ureH, which was found in 93.3% of the strains (20). It is considered to be essential in vivo. This indicates that the genes we identified are in the pool of common H. pylori genes, which supports their essentiality.
A fairly large number of mutants in the essential group consists of genes affecting bacterial chemotaxis and motility. In addition to the major flagellin flaA, we identified mutants in genes, such as fliD, fliF, fliI, fliP, fliS, and flgE, which validated our method, as they have already been described to be involved in the assembly of a functional flagellar apparatus (2125). In addition, we added several new genes, which are annotated as being involved in flagellar biosynthesis from their sequence homology, but which have not been verified experimentally.
The outer membrane proteins (OMP8, HP0788) might have a function as adhesins, as described for the AlpAB, the BabA, the HopZ (26), and other outer membrane proteins from in vitro studies. Alternatively, they might fulfill an essential role as porins, or in stabilizing the integrity of the outer membrane to resist the harsh conditions in the gastric mucus. The LPS of H. pylori is known to be essential for colonization in the mouse model (27). This explains the galE mutant to be colonization defective, supporting the essential role of the O-side chain and/or Lewisx/y mimicry of LPS as an important feature for survival of H. pylori within the host (28).
Several genes in the urease gene cluster were found to be essential for gastric colonization, thus validating again our screening model (29, 30). Only relatively few genes encoding enzymes of the central intermediary metabolism and for amino acid biosynthesis were disrupted by the Tn, indicating that many of these genes might be essential in vitro and therefore not present in our collection of mutants.
Another group of essential functions relates to the putative membrane transport systems. Surprisingly, two putative transport ATPases usually associated with type IV secretion systems, the VirB4 homologous ATPase ComB4 (hp0017; reference 16), and the product of hp1421, a VirB11-homologous ATPase, were essential for colonization. ComB4 was recently identified as an essential component of the natural transformation associated type IV transport system (16). The comB810 deletion mutant of H. pylori P149 was only weakly attenuated for gastric colonization (Fig. 5). In contrast, ComB4 seems to play a major role in the colonization process (see C.I., Table II) and might therefore have a further function in addition to mediating DNA uptake. This function has to be identified in future experiments.
One putative ATP-binding cassette transporter, as well as three permeases for glutamate, proline and -ketoglutarate were found to be essential for H. pylori to colonize in vivo, indicating that H. pylori apparently relies on the exogenous uptake of these amino acids and precursors in the stomach mucosa, as shown previously for growth of H. pylori in vitro (31). Such an amino acid exchange between bacteria and host is frequently observed in primary and secondary symbionts of plants and animals. The squid Euprymna scolopes, for instance, provides at least 9 amino acids to support the growth of its auxotroph symbiont Vibrio fischeri, present in its light-emitting organ (32). The amino acid dependence demonstrated here might be in support of the view that H. pylori originated as a symbiont of humans (33).
The RpoN sigma factor and a stationary phase survival protein (SurE) are supposed to fulfill essential regulatory functions (Table II). Among others, RpoN is known to be involved in flagellin gene transcription, which might be one reason for its essential function. SurE is a member of a novel family of metal ion-dependent phosphatases responsible for survival of bacteria under certain stress conditions (34), as they may be present in the gastric mucosa. The VacA-paralogous protein HP0289 is an OMP expected to be exported by a putative autotransporter mechanism to the bacterial surface, analogous to the VacA cytotoxin, but its function or role in colonization is completely unknown. Another group of identified genes corresponds to those encoding hypothetical proteins. This group consists of 6 mutants (Table II; hp) encoding hypothetical genes, which are, according to their original annotation (3), only present in H. pylori. The corresponding proteins might be involved in specific functions to adapt H. pylori to its special niche, the gastric mucosa. Two genes encoded conserved hypothetical integral membrane proteins (chimp).
Interestingly, a putative collagenase (HP0169) was identified by sequence homology to other bacterial collagenases, especially of the U32 protein family. Using a biochemical approach, we could verify that (a) the gene hp0169 actually encodes an active bacterial collagenase and that (b) HP0169 is actively transported to the cell surface or secreted. As HP0169 does not carry a typical hydrophobic signal sequence to enter the general secretory pathway, we assume that the protein is secreted via a specific secretion mechanism.
What might be the benefit for H. pylori to secrete a collagenase? Collagen type I and III are important components of the extracellular matrix of the stomach epithelium. The synthesis of type I collagen is induced in regions around gastric ulcers and collagens are of outmost importance for the process of ulcer healing (35). Thus, secretion of a collagen degrading enzyme by H. pylori could be responsible for the chronicity of gastric or duodenal ulcerogenesis and the delayed healing process. For H. pylori the degradation of collagen might be used as a source for the uptake of certain amino acids or short peptides. Alternatively, the enzyme might be important for the proteolytic degradation of components of the innate and acquired immune system, such as IgA antibodies or components of the complement system, to evade the immune response, as described for other mucosal pathogens (36). Both could explain the essential nature of the enzyme in vivo.
In conclusion, our approach allowed the identification of genes required for survival in the host, which has provided physiologically significant information about genes required for the adaptation of H. pylori to its specific niche, the gastric mucosa. Of particular significance, a more complete understanding of the role that the hypothetical proteins play in the infection process of gastric colonization of H. pylori may lead to a more rational understanding of the Helicobacter infection process and finally allow a specific intervention.
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Acknowledgments |
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This work was funded by grants from the Deutsche Forschungsgemeinschaft (HA 2697/1-4) and by Byk Gulden Konstanz, Germany. B.P. Burns was funded by a fellowship from the Alexander von Humboldt Foundation.
Submitted: August 30, 2002
Revised: December 20, 2002
Accepted: December 30, 2002
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Footnotes |
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* Abbreviations used in this paper: STM, signature tagged mutagenesis; Tn, transposon.
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References |
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