Helicobacter pylori gene silencing in vivo demonstrates urease is essential for chronic infection

Helicobacter pylori infection causes chronic active gastritis that after many years of infection can develop into peptic ulceration or gastric adenocarcinoma. The bacterium is highly adapted to surviving in the gastric environment and a key adaptation is the virulence factor urease. Although widely postulated, the requirement of urease expression for persistent infection has not been elucidated experimentally as conventional urease knockout mutants are incapable of colonization. To overcome this constraint, conditional H. pylori urease mutants were constructed by adapting the tetracycline inducible expression system that enabled changing the urease phenotype of the bacteria during established infection. Through tight regulation we demonstrate that urease expression is not only required for establishing initial colonization but also for maintaining chronic infection. Furthermore, successful isolation of tet-escape mutants from a late infection time point revealed the strong selective pressure on this gastric pathogen to continuously express urease in order to maintain chronic infection. In addition to mutations in the conditional gene expression system, escape mutants were found to harbor changes in other genes including the alternative RNA polymerase sigma factor, fliA, highlighting the genetic plasticity of H. pylori to adapt to a changing niche. The tet-system described here opens up opportunities to studying genes involved in the chronic stage of H. pylori infection to gain insight into bacterial mechanisms promoting immune escape and life-long infection. Furthermore, this genetic tool also allows for a new avenue of inquiry into understanding the importance of various virulence determinants in a changing biological environment when the bacterium is put under duress.


Introduction
The human gut pathogen Helicobacter pylori has coevolved with humans over thousands of years to dominate the gastric niche [1][2][3]. The majority of infected individuals (80-90%) carry and transmit H. pylori without any symptoms of disease [4,5]. However, H. pylori infection causes chronic active gastritis that may develop into peptic ulceration (10-20%) or gastric adenocarcinoma (0.5-2%) [6,7] causing a significant burden on public health [8][9][10]. H. pylori infection is persistent and clinical disease usually develops after many years of chronic inflammation and epithelial damage. Furthermore, due to increasing rates of antibiotic treatment failure [11,12] there is a pressing need for further research into the bacterium's mechanisms for persistence and immune evasion strategies. These are of particular importance to understanding H. pylori pathogenesis and to identifying novel targets for the development of new treatment options.
H. pylori is highly adapted to colonizing and surviving in the harsh conditions of the gastric environment. One key adaptation is the virulence factor urease. This multimeric enzyme, consisting of 12 UreA and UreB heterodimers, catalyses the hydrolysis of urea to produce CO 2 and NH 3 , which acts to buffer the acidity of the local environment around the cell [13,14]. Urease is abundantly expressed by H. pylori at levels exceeding that of any other known microbe [15] and is estimated to constitute 10-15% of the bacterium's total protein content [16]. In addition, urease is essential for establishing colonization as H. pylori urease mutants are unable to infect the host [17][18][19][20][21]. Several lines of evidence suggest that urease plays a significantly greater role in infection than simple acid neutralization. Elevating the gastric pH to 7.0 was shown to be insufficient in permitting colonization by a urease negative strain [18]. In several in vitro studies urease and its catalytic products contributed directly to virulence. Ammonia produced by urease activity caused damage to the gastric epithelium by disrupting tight cell junction integrity [22,23] and CO 2 protected against the bactericidal activity of the nitric oxide metabolite, peroxynitrite, produced by phagocytes to kill engulfed bacteria [24]. Furthermore, several studies suggest that urease may directly interact with host epithelial and immune cells. Urease has been shown to bind to major histocompatibility complex (MHC) class II molecules on gastric epithelial cells thereby inducing cell apoptosis [25] and the UreB subunit can stimulate monocytes to release proinflammatory cytokines by binding to cell surface CD74, a MHC class II associated invariant chain [26,27]. In addition, an in vivo study demonstrated that changes to the surface of the urease complex resulted in the eventual clearance of H. pylori infection in mice [28]. Loss of colonization was attributed to the disruption in urease mediated interactions between H. pylori and host cells as urease activity was unaffected by the mutation, ruling out loss of acid resistance or nitrogen assimilation [29] as contributing factors [28]. Clinical isolates maintain high urease activity even after years of chronic infection when the bacterium has established itself in the relatively neutral environment of its gastric niche implicating that ongoing urease expression is required for persistence. However due to the lack of appropriate genetic systems this hypothesis could not be tested experimentally.
The necessity of urease activity in establishing colonization hinders the study of its function during persistence when using conventional knockout mutants. The availability of a conditional urease mutant would overcome this constraint by permitting changes to the urease phenotype during an established infection. To conclusively determine if urease is indeed required after colonization is established, we generated conditional H. pylori urease knockout mutants using a tetracycline repressor (tet) based system [30]. This system controls gene expression by way of a tetracycline repressor (TetR) that binds to specific operator sequences (tetO) in the target promoter and silences transcription of the downstream gene. Expression of the target gene can be turned on by the administration of a potent tetracycline inducer, such as anhydrotetracycline (ATc) or doxycycline (Dox) [31]. This system was recently adapted to H. pylori and gene expression was regulated in vivo during active infection [32]. In the current study, we adapted this system to generate conditional urease mutants. We demonstrate that in an established infection, loss of urease expression is detrimental to the bacterial survival in the host. Strong selective pressure on the bacteria for continuous urease expression is further demonstrated by the emergence of escape mutants that successfully repopulated the mouse stomach six weeks after genetic silencing of urease was initiated.

Construction and characterization of urePtetO promoters to drive urease expression
The urease structural genes, ureA and ureB, encoding for the 27 kDa UreA and 62 kDa UreB urease subunits, are transcribed as a single operon under the control of the ureA promoter, P ureA [33]. To regulate the expression of urease in H. pylori, we placed the operon under tet control. Based on previous mutational studies of P ureA [32,34], the promoter was mutated to incorporate one or more tetO sequences to generate a series of different P ureA derivatives, ureP-tetO(I-V) (Fig 1A and 1B). These tet-promoter constructs were made using PCR based techniques and used to replace the native chromosomal P ureA by allelic replacement. This strategy involved first generating a recipient H. pylori strain in which P ureA and ureA has been replaced with a rpsL-cat cassette. The urease negative recipient strain was then naturally transformed with the urePtetO PCR constructs to generate strains with tetO modified P ureA derivatives and a restored ureA gene. H. pylori strains harbouring these constructs were characterized to identify a tet-promoter construct with regulatory properties that would permit the appropriate level of complementation to ensure colonization yet could be sufficiently silenced to prevent infection. The functionality of these tet-promoters was first assessed in the wild-type background for their ability to drive urease expression by measuring urease enzymatic activity, UreB expression and mouse colonization.
The urePtetO constructs were introduced into the wild-type X47 strain, replacing the chromosomal P ureA and generating X47 urePtetO strains (OND2018-OND2022). The urease Urease activity in X47 urePtetO(I-V) strains (OND2018-OND2022) compared to wild-type X47. Fresh cultures of H. pylori grown on CBA plates for less than 24 h were collected and the urease activity for each strain was determined. Urease activity is expressed as a percentage of wild type X47 (WT) urease activity. The urePtetO construct is specified under each bar. All measurements were carried out in triplicate. Data are averages of three independent experiments and error bars represent standard deviations. (E) Two week colonization of C57BL/6J mice by X47 urePtetO(I-V) strains compared to wild-type X47 (WT). Modifications to the ureA promoter did not prevent colonization. Colonization studies were done without prior adaptation of X47 urePtetO strains to mice. Horizontal bars represent median bacterial load per group (n = 3) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null.
https://doi.org/10.1371/journal.ppat.1006464.g001 expression level and the urease enzymatic activity of these strains under standard growing conditions was measured and compared to the parent strain (Fig 1C and 1D). Despite expressing less urease, as determined by immunodetection of UreB in the total cell lysate (Fig 1C), the urease activity measured in strains transformed with urePtetOI, urePtetOII and urePtetOV was found to be comparable to wild-type ( Fig 1D). In comparison, the urease activity in strains transformed with urePtetOIII and urePtetOIV were reduced by 60% and 25% respectively, which was also accompanied by substantially reduced amount of UreB in the total cell lysate ( Fig 1C).
Given that replacement of the wild type P ureA with the urePtetO promoters resulted in reduced urease expression which concomitantly may reduce their ability to establish infection, C57BL/6J mice were challenged with strains X47 urePtetO(I-V) (OND2018-OND2022) to assess if urease expression in these strains was still sufficient to facilitate colonization. All five X47 urePtetO strains were successfully re-isolated from mouse stomachs two weeks after oral challenge ( Fig 1E). The infection rate and bacterial load in mice challenged with strains X47 urePtetOI, X47 urePtetOII and X47 urePtetOV was comparable to the control group challenged with the wild-type strain. However the infection rates were decreased for groups challenged with strains of lower urease activity, with strain X47 urePtetOIII displaying a major defect in colonization. To verify that colonization had not been established due to mutation or reversion of urePtetO, the ureA promoter region of re-isolated strains was sequenced which confirmed that the sequences of the urePtetO constructs remained unaltered after passage through mice.

Tet-regulation of urease expression in H. pylori
After establishing that the tetO modifications to the ureA promoter did not abrogate colonization per se, we evaluated if these promoters could regulate urease expression in a tetracycline dependent manner. All five ureA promoter derivatives were transformed into a X47 recipient strain that expressed TetR under the control of the strong flaA promoter [32]. The resulting strains, X47 mdaB::ptetR4, urePtetO(I-V) (OND1954-OND1958), did not express urease when grown on standard CBA plates however urease expression could be induced when grown on CBA plates containing 50 ng/ml anhydrotetracycline (ATc) (S1 Fig). These results demonstrated that TetR effectively silenced the tet-promoters in these strains.

Characterization of urePtetO regulated urease expression and activity in vitro
Regulation of urePtetO promoters by TetR in conditional urease knockout strains X47 mdaB:: ptetR4, urePtetO(I-V) was assessed using the urease enzymatic activity assay. Bacteria were cultured in the absence or presence of 50 ng/ml ATc for two successive passages and then collected for analysis. When strains were cultured in the absence of ATc, urease activity was below the detection limits of the assay (2 U/ml of Type III urease from Jack bean) (Fig 2A). For strains grown in the presence of ATc, urease activity in strains X47 ptetR4; urePtetO-I, -II and -V was induced to wild type levels, while the urease activity for strains X47 ptetR4; ureP-tetO-III and -IV remained below 10% of wild-type activity. These results demonstrated that by using the appropriate tet-promoter urease activity can indeed be regulated by the presence of a small molecule inducer, confirming the generation of conditional H. pylori urease knockout mutants.
The tet-responsive promoters urePtetOI and urePtetOV have different genetic architectures ( Fig 1B) and upon induction also promoted the greatest urease expression levels amongst the tested strains. Based on these results the regulation of these two promoters was further characterized. The kinetics of urePtetO induction and repression was analysed in strain X47 ptetR4; urePtetOI (OND1954) and X47 ptetR4; urePtetOV (OND1958) by immunodetection of the UreB protein (Fig 2B and 2C). After addition of 200 ng/ml ATc to the culture medium, UreB protein expression increased over time and reached maximum levels after 12 h and 8 h for urePtetOI and urePtetOV, respectively ( Fig 2B). Withdrawal of ATc from induced cultures led to a significant decrease in UreB protein levels within 3 h, demonstrating that both urePtetOI and urePtetOV were quickly silenced ( Fig 2C) and that the UreB protein was turned over efficiently, falling to the threshold of detection within 12 h.

Establishing a conditional urease knockout infection model
With the knowledge that tetracycline dependent regulation of urease expression was attainable in vitro we next turned our attention to establishing a mouse model of infection. Based on previous studies involving in vivo tet-systems [35][36][37] the inducer molecule doxycycline (Dox) was first used as a model inducer to identify a maximal dosage of material that could be tolerated by the bacterium in vivo. We found that wild-type X47 could still infect mice when the animals were supplemented with up to 10 mg/l of Dox in their drinking water. Colonization by wild-type X47 was severely attenuated at 100 mg/l of Dox and bacteria could not be reisolated at 1000 mg/l of Dox supplement (S2A Fig). Furthermore, strain X47 ptetR4; urePtetOI (OND1954), which emerged as the prime conditional urease mutant candidate from the in vitro studies, was tested to verify its ability to establish initial colonisation and then used to optimise the dosage of inducer molecule to regulate urease expression in vivo. OND1954 was only capable of establishing infection in C57BL/6J mice when the animals received Dox supplementation in their drinking water, demonstrating that urease is essential for OND1954 to establish colonization in the mouse infection model (S2B Fig). Addition of Dox supplement at 1 mg/l supported colonization of OND1954 and although attenuated, the conditional mutant was also isolated from animals supplemented with Dox at 5 mg/l and 10 mg/l. Having identified a minimal supplement dose of Dox inducer, we then sought to complete the infection model by investigating two more important factors; the use of the less toxic tetracycline derivative ATc, and attempting to improve the robustness of the conditional urease mutant strain. The original wild-type X47 strain underwent four consecutive transformations to generate the conditional urease mutant OND1954 and consequently the strain may have accumulated secondary mutations that would decrease its fitness in vivo. To address this, the output clones of OND1954 isolated from three individual mice were collected and each clone was verified to be a conditional urease mutant. These clones were then pooled, OND3241 (A-E), and used in subsequent infection studies to test if passage though mice led to improved infection rates. Mice were challenged with either the wild-type strain, the original conditional mutant OND1954 or the mouse passaged urease conditional mutant OND3241, and supplemented without or with 5 mg/l Dox or ATc. Colonization of OND3241 remained dependent on inducer supplement and using ATc instead of Dox resulted in an improved infection rate and bacterial load in the infection model (Fig 3).

Fig 3. Infection of conditional urease mutant is dependent on tetracycline inducers.
Optimization of in vivo model for tet-regulated urease expression. Mice groups were supplemented without (C) or with 5 mg/l of either Dox or ATc in their drinking water. Animals were orally challenged with wild-type strain X47, with pre-induced OND1954 or a pool of pre-induced clones of OND1954 re-isolated from mice (OND3241A-E). Animals were sacrificed two weeks after oral challenge. Bars represent median bacterial load per group (n = 5) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null. Statistical analysis was performed to test if Dox or ATc treatment influenced the infection rate of any of the given bacterial strains. Treatment did not significantly impact on the infection rate of the wild-type strain. However ATc supplementation significantly increased the infection rate for OND1954 and OND3241 (* p < 0.05). Unpaired two sided Fisher's exact test followed by Bonferroni correction was used for pairwise testing of the infection status. https://doi.org/10.1371/journal.ppat.1006464.g003

Silencing urease during active infection
Having established an infection model in which the H. pylori urease phenotype could be regulated in vivo, we proceeded to investigate what effect tet-mediated silencing of urease expression had on established H. pylori infections. Mice were challenged with the conditional strain OND3241 and provided with 5 mg/l ATc supplement to establish infection. After two weeks, the supplement was withdrawn and the animals were sacrificed at indicated time points. The conditional H. pylori urease mutant could still be isolated on days 1 and 3 after supplement withdrawal, however on days 5 and 7 the bacterial load had decreased to below our detection limit ( Fig 4A). This data demonstrated for the first time that continuous urease expression is required by H. pylori to maintain colonization even after the bacteria have become established in the gastric niche.

Isolation of escape mutants demonstrates strong selective pressure for urease expression
To test if H. pylori were under selective pressure to overcome tet-regulation the suppression experiment was repeated and the animals were sacrificed at a much later time point. Mice were challenged with either wild-type X47 or OND3241 and provided with 5 mg/l ATc supplement to establish infection. After two weeks, the supplement was withdrawn from half the groups (both OND3241 and wild-type) while the remaining groups were maintained on ATc supplement and the animals were sacrificed at different time points (Fig 4).
No differences in bacterial load or infection rate was observed for animals infected with wild-type X47 over the course of the experiment demonstrating that long-term ATc supplement (5 mg/l for 8 wks) does not interfere with H. pylori infection (Fig 4B). Animals challenged with OND3241 and maintained continuously on ATc supplement had a consistent infection rate of 60% (Fig 4B). In vitro tests confirmed that bacteria re-isolated from these groups remained conditional urease mutants, even after a total infection time of 8 weeks. Withdrawal of ATc supplement from the animal groups challenged with OND3241 resulted in reduced infection load on days 3 and 5 and, although not completely cleared to below our detection limit in all animals, the infection rate had decreased to 20% on day 5. However, when mice challenged with the OND3241 were left in the absence of ATc for 42 days, the bacterial load and the infection rate had increased resulting in 80% of the animals bearing bacteria in the stomach above our detection limit. Importantly, unlike the bacteria re-isolated at the earlier time points (day 3 and day 5), H. pylori re-isolated from this last group of mice were all urease positive and they were no longer conditional urease mutants as tested qualitatively in vitro. This result revealed that the strain was under selective pressure to restore urease expression.
In an effort to identify possible genetic mutations to overcome tet-regulation of the urease operon, whole genome sequencing of the original conditional urease mutant strain OND1954, the individual clones of input strain OND3241(A-E), and 34 output strains recovered at day 42 (5 conditional urease-negative strains and 29 urease-positive tet-escape mutants) was undertaken. Sequence data were mapped against reference strain OND1954 and variants specific to tet-escape mutants were identified (S1 Table). Sequence analysis of the ureAB locus, including the upstream regulatory region, revealed no changes between OND1954, OND3241 and the tet-escape mutants. Interestingly, the tet-escape mutants harboured non-synonymous substitutions or frameshift mutations in at least one of the following genes, tetR and the flagellar biosynthesis genes fliA, fliE and flgE. These data reveal that tet-regulation was overcome in the tet-escape mutants not by altering the tetO binding sites but through affecting the repressor protein.

Discussion
A set of conditional urease mutants were generated to demonstrate for the first time that urease expression is essential in the persistence stage of H. pylori infection, which broadens our understanding of the role of this enzyme during chronic infection.
Genetic manipulation of P ureA to place urease under tet-control led to a decrease in the basal levels of urease expression for all urePtetO constructs tested. However, under the conditions tested, strains transformed with urePtetOI, urePtetOII and urePtetOV were found to have comparable urease activity to that of wild-type. This data can be reconciled as it has been reported that under neutral in vitro growth conditions without added nickel, such as the growth conditions used in this study, a significant amount of urease in wild-type strains is in the inactive apoenzyme form and only a minor fraction of urease is active [38][39][40]. Urease activity in H. pylori is highly controlled and is modulated through several different mechanisms in response to various environmental cues [39]. Since our goal was to establish a working in vivo model, we decided to directly test if the decrease in urease expression could be tolerated by the bacteria by assessing if the urePtetO strains were capable of colonizing the murine stomach.
Interestingly, when analyzing the in vivo colonization data from the X47 urePtetO strains (OND2018-OND2022) a positive correlation between infection rate and in vitro urease expression and activity but not to bacterial load in colonized animals was observed (Fig 1E). This finding suggests that for initial colonization of the murine stomach the amount of urease activity is an important factor likely due to the fact that the bacteria need to withstand the acidity of the gastric lumen until they reach their gastric niche, deep into the gastric mucus near the epithelial surface. However, once the bacteria are established within their environmental niche, although urease is still required for growth, the level of urease expression may be less important for maintaining colonization as mice colonized with strains transformed with ureP-tetOIII and urePtetOIV had a similar bacterial burden compared to mice infected with strains expressing more urease.
Infection of the mouse host by the conditional urease mutant was strictly dependant on supplementation with a tetracycline inducer, confirming that genetic regulation of urease expression was stringent enough to prevent colonization. Furthermore, in the induced state, tet-mediated expression of urease was sufficient to allow and maintain infection by the conditional urease mutant. Withdrawal of the supplement resulted in clearance of the bacterium within 5 days. This is in line with the slow shut-off observed in other mouse models using tetbased regulation systems which has been attributed to the persistence of doxycycline in tissues [41,42].
Notably the longer time of clearance of the bacterium in vivo provided the opportunity for the emergence and selection of tet-escape mutants. H. pylori possess several mechanisms, such as an error prone PolA [43] and efficient DNA homologous recombination and transformation systems [44,45], that permit the bacterium to undergo rapid microevolution to adapt to changing environments in its specific host [46,47]. The emergence of tet-escape mutants in this study suggests that there is strong selective pressure on the bacterium for continuous represent median bacterial load per group (n = 10) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null. Statistical analysis using the unpaired two-sided Mann-Whitney test are indicated above the compared groups (* indicates that the geometric value of the (+/-, □) group is significantly different (p 0.05) from the (+, ■) group of the same time point) and unpaired two-sided Fisher's exact test analysis of the infection rate by the conditional mutant is presented below the compared groups (* p< 0.05; *** p < 0.001). For the day 42 time point in panel B, as all the analysed colonies isolated from the (+/-, □) group were found to be escape mutants, none of the animals were considered infected with the conditional mutant resulting in a 0/10 infection rate. urease expression to maintain chronic infection. Whole genome sequence analysis of the tetescape mutants identified several different mutations that likely explain how tet-regulation of the ureAB operon was overcome.
One group of escape mutants had missense or nonsense mutation within the tetR gene. TetR is a finely tuned transcriptional regulator [31] and therefore most amino acid changes are likely to have deleterious effects to the function of TetR by inhibiting repressor dimerization or DNA binding [48][49][50]. Interestingly, another group of tet-escape mutants harbored either amino acid substitutions within the DNA binding domains of the alternative RNA polymerase sigma factor FliA (σ 28 ) or a truncated FliA due to nonsense mutation and frameshift alteration. FliA controls the transcription of some late flagellar genes (class 3) including the flagellin subunit, flaA [51,52]. Previous studies have reported that H. pylori fliA mutants have no detectable flaA transcript and have truncated flagella [51]. In the conditional H. pylori urease mutants generated in this study, the transcription of tetR is driven by a flaA promoter. Therefore, it is reasonable to suggest that the fliA mutations identified in the tet-escape mutants likely affect the transcription of tetR from P fla and consequently release the ureAB operon from tet-regulation. Additionally, some tet-escape mutants acquired non-synonymous mutations in fliE and flgE, genes that encode for components of the flagellum hook-basal body complex. Flagellar biosynthesis is a highly ordered and regulated process and transcription of late flagellar genes by fliA proceeds only once the hook-basal body complex is complete [53,54]. Mutation of fliE has been shown in other studies to affect the regulation of fliA dependent genes, leading to a two-fold reduction in flaA transcript levels [54]. Thus mutation of fliE and flgE may indirectly impact on the transcription of tetR from P flaA through the negative control of FliA [51,53]. Motility is essential for H. pylori colonization [19, 55,56], and therefore the isolation of tetescape mutants with mutations in genes involved in flagellar biosynthesis (fliA, fliE and flgE) raises interesting questions. During the chronic stage of infection, once the bacteria are established in their environmental niche and have adhered to gastric epithelial cells [57], can motility be compromised in preference for improved urease expression? Further investigations with the appropriate conditional mutants are necessary to better understand the escape and in particular to test whether motility is still required once colonization is established. Another question is whether there are other mutations acquired by the tet-escape mutants that could compensate for mutations in fliA, fliE and flgE? In-depth analysis of the whole genome sequencing data together with mutational studies needs to be undertaken to discern how these tet-escape mutants are able to survive in the host.
The sequencing results underline the importance of urease for H. pylori to maintain persistence infection and reveal the high selective pressure for continuous expression of urease even after colonisation is successfully established. The mutations identified in the tet-escape mutants add support to the hypothesis that the genomic plasticity of H. pylori is an important mechanism for adaptation to new and changing environments [58]. Furthermore these findings also highlight the potential of using tet-based genetic tools together with whole genome sequencing to study H. pylori genetic plasticity and adaptation in a changing biological environment when the bacterium is put under duress.
Using the conditional mutant, we have demonstrated that urease is essential for chronic infection and that repression of urease expression results in the loss of bacterial load within 5 to 7 days. The availability of these tools now allows for new questions to be asked regarding the reason behind the relatively rapid loss in colonization. One reason may be that the loss of urease activity results in the bacteria being more susceptible to clearance by phagocytic cells [24]. Another possibility is that the loss of urease activity negatively affects H. pylori ability to swim through gastric mucus, as the bacteria would no longer have the ability to decrease the viscosity of mucus through the elevation of local pH [59], and consequently are cleared due to turnover of the mucus lining. The conditional urease mutant will serve as a valuable tool in further studies that pursue this line of investigation.
In this study, H. pylori conditional urease mutants were generated by placing the expression of the urease subunits, UreA and UreB, under tet-control and have permitted the first direct testing of the hypothesis that urease is required by H. pylori for chronic infection. Furthermore, eventual escape from tet-regulated urease expression by H. pylori demonstrates that there is a very strong selective pressure on the bacterium to maintain urease expression during infection. Our data validates urease as a good target for therapeutic intervention. The conditional urease mutants generated here can also be used to gain more detailed insight into the role of urease in the persistence stage of infection including its interactions with MHC class II molecules [25], induction of proinflammatory cytokine [27] and its potential role in motility [59]. Furthermore, this study demonstrates the need for conditional mutants, generated by using genetic tools such as the tet-system, to study H. pylori virulence factors, persistence and the bacterium's influence on the host microbiota.

Materials and methods
Bacterial strains and culture conditions H. pylori X47 strains used in this study are listed in S2 Table. Bacteria were grown at 37˚C under microaerobic conditions on Columbia blood agar (CBA) plates containing 5% horse blood and Dent's antibiotic supplement (Oxoid). When appropriate, antibiotic selection was carried out by supplementing media with chloramphenicol or streptomycin at a final concentration of 10 μg/ml. Microaerobic conditions were established in sealed jars using the Anoxomat MarkII system (Mart Microbiology B.V., the Netherlands) after one atmosphere replacement with the following gas composition N 2 :H 2 :CO 2 , 85:5:10.

Genetic manipulation and H. pylori strain construction
All genetic manipulation of H. pylori strains was done using genomic insertion and replacement of a counter-selectable rpsL-cat cassette [60]. The use of the counterselectable streptomycin susceptibility (rpsL-based) system requires a host strain that possesses a streptomycinresistant phenotype [61]. The H. pylori X47 host strain is naturally streptomycin-resistant and no modifications to this strain were required. The genotype of all mutants was confirmed by PCR and/or DNA sequencing.

Oligonucleotides
Oligonucleotides used in this study are listed in S3 Table. Construction of the H. pylori tetracycline responsive urePtetO promoters To place ureA and ureB under tet control, wild-type nucleotide sequences flanking the -35 and -10 promoter regions of the ureA promoter, P ureA , were replaced with tetO sequences to generate five derivatives of P ureA , urePtetO(-I through -V) (Fig 1A and 1B). These promoter constructs were used to replace the native urease promoter, using the two-step rpsL-cat based transformation approach.

Construction of ureA::rpsL-cat recipient strain
A construct composed of the counterselection cassette flanked by DNAs homologous to regions of the ureA locus, ureA::rpsL-cat, was made by SOE PCR [62,63] (S3 Fig) and used to generate recipient strains in which P ureA and ureA were replaced with rpsL-cat. Two 1 kb regions flanking P ureA and ureA (HP0073), were amplified from 26695 genomic DNA using primers ureArcat1 and ureArcat2, and ureArcat3 and ureArcat4 respectively. The rpsL-cat selection cassette was amplified using primers ureArcat5 and ureArcat6. Nested primers ureArcat7 and ureArcat8, were used to generate and amplify a final 3.4 kb PCR product, ureA:: rpsL-cat. Natural transformation of the H. pylori strains with the ureA::rpsL-cat PCR construct was performed to obtain the recipient strain OND2017. Transformants isolated on chloramphenicol plates were urease negative.
Reconstruction of ureA promoter to incorporate tetO sites and generation of conditional urease mutants Five tetO modified ureA promoter constructs urePtetO(I-V), containing up to three tetO sites, were constructed by SOE PCR (S4 Fig). The primer pairs used to make each urePtetO construct are listed in S4 Table. Briefly, a 1 kb fragment upstream, arm I, and a 1.5 kb fragment downstream, arm II, of P ureA were amplified using 26695 genomic DNA as a template. Long primer tails were used to reconstruct the ureA promoter region upon fusion of arms I and II by SOE PCR. Primers ureArcat7 and ureArcat8 were used to amplify the final 2.5 kb products, urePtetO(I-V), and sequencing confirmed that the modified ureA promoters were reconstructed correctly. Natural transformation of the recipient strain OND2017 with urePtetO PCR constructs resulted in replacement of the rpsL-cat with urePtetO and restoration of ureA, generating strains X47 urePtetOI through X47 urePtetOV (OND2018-OND2022). Correct allelic replacement of the resulting Str r transformants was confirmed by colony PCR using primers ureAP1 and ureArcat8 and by sequencing using primer urePseq.
Conditional urease strains were generated by transforming the TetR expressing H. pylori strain, X47 mdaB::ptetR4 (OND1987) [32], with the ureA::rpsL-cat PCR construct to generate the recipient strain OND2026. This urease negative strain was then transformed with each of the five urePtetO constructs to generate conditional urease mutant strains X47 mdaB::ptetR4; urePtetO(I-V) (OND1954-OND1958). Transformants were first screened for tetracycline dependent urease expression using the urease phenotype assay and additional characterization was done using the urease activity assay and immunoblot analysis.

Urease phenotype assay
Urea culture plates (Brucella broth, 7% NCS, 1 mM urea, phenol red 100 mg/l, vancomycin 6 mg/l, pH 6) were used to assay the urease phenotype of H. pylori clones. The pH of the media was adjusted with 1 M HCl before the addition of NCS and vancomycin. The pH was low enough to observe the colourimetric change of phenol red, from yellow to red, due to the catalytic activity of urease on urea, but not acidic enough to inhibit the growth of urease negative strains. To screen for tet-regulated urease activity, transformants and colonies re-isolated from infected animals were replica plated onto CBA plates with or without 50 ng/ml of ATc and cultured for 48 h. Bacteria were then patched onto urea plates and incubated under microaerobic conditions. Urea plates were examined after 16 h of incubation to identify clones that had switched urease phenotype upon exposure to ATc. Localized changes in colour around each growing colony identified urease positive clones. Conditional urease mutant strains grown on CBA plates without ATc remained urease negative, while strains grown on CBA plates with ATc became urease positive (Example S5 Fig).

Measurement of urease enzymatic activity
The urease activity assay used in this study was adapted from the protocol previously described [28]. Strains were grown on CBA plates without or with 50 ng/ml ATc for two successive passages. Bacteria from 24 h plate cultures were collected and resuspended in cold buffer A (25 mM phosphate buffer, pH 6.8) and standardized to an OD 600 = 4.0. A 50 μl aliquot of the standardized bacterial suspension was then diluted with 50 μl of buffer B (25 mM phosphate buffer, pH 6.8, 0.2% Tween-20). A 25 μl aliquot of this diluted bacterial suspension was transferred into one well of a 96 well plate, diluted with 150 μl of buffer C (25 mM phosphate buffer, pH 6.8, 250 μM phenol red) and incubated for 5 min at 37˚C. A 75 μl aliquot of urea solution (0.5 M) was then added to the well and the absorbance at 560 nm was measured every 72 s for 75 cycles using a POLARstar Omega (BGM Labtech) plate reader. Activity was measured as the rate of change in absorbance over time and expressed as percent of urease activity of the wildtype X47 strain. All urease activity measurements were carried out in triplicate and experiments were repeated at least three times.

Time course experiments of tet-regulation
Bacteria were grown in Heart Infusion (HI) medium supplemented with 10% Newborn Calf Serum (NCS) and vancomycin (6 μg/ml). Cultures were inoculated with bacteria suspended in PBS to give a starting OD 600 = 0.05, and grown under microaerobic conditions at 37˚C and 120 rpm. For induction, H. pylori cultures were grown to mid-log phase in 10 ml of media. Cultures were induced with 200 ng/ml ATc and bacteria were incubated for another 12 h, with aliquots were taken at indicated time points. For gene silencing, conditional strains were cultured in the presence of 200 ng/ml ATc to mid-log phase. Fresh HI media, with or without 200 ng/ml ATc, was inoculated with pre-induced bacteria (OD 600 = 0.5) and grown for 12 h, with aliquots taken at indicated time points. Bacterial cells were collected by centrifugation and washed twice with PBS before processing for immunoblot analysis.

SDS-PAGE and immunoblot analysis
Bacterial whole cell lysates were prepared as previously described [32]. The protein concentration of bacterial cell whole cell lysate samples was determined using the Micro BCA protein assay reagent kit (Pierce) with bovine serum albumin as the standard. Equal amounts of protein for each sample were mixed with 3x SDS-PAGE sample loading buffer, incubated at 95˚C for 10 min, and proteins were separated by 10% SDS-PAGE and electrotransferred to a PVDF membrane. For detection of the UreB subunit of urease, mouse anti-UreB (Austral biologicals) was used at a dilution of 1:8000. Secondary antibody rabbit anti-mouse-HRP (Jackson Immu-noResearch Laboratories) was used at a dilution of 1:10,000 and detection of the secondary HRP conjugate was accomplished by chemiluminescence (Sigma) using LAS 3000 (Fujifilm) (software Image reader LAS 3000 V2.2). For loading controls, duplicate gels were run in parallel and stained with Coomaise Brilliant Blue R-250 (S1B Fig, S6 Fig and S7 Fig).

Animal experiments
Mouse procedures were reviewed and approved by the Institutional Animal Care and the Animal Ethics Committee of the University of Western Australia. 6-7 week old C57BL/6J female mice were challenged once by oral gavage with 200 μl of 1 x 10 9 CFU/ml of bacteria suspended in HI broth. Groups of infected mice received doxycycline (Dox), anhydrotetracycline (ATc) or no supplement in drinking water containing 5% sucrose. Water was kept in light-protected bottles and changed every three days. Mice were sacrificed at indicated time points and stomachs were removed and homogenized in 1 ml HI using a tissue lyser (Retch). Homogenates were serially diluted and plated out on H. pylori selective plates (CBA containing 5% Horse blood, Dent, nalidixic acid 10 mg/l and Bacitracin 100 mg/l) to determine the bacterial burden. Were appropriate, re-isolated clones were assayed for tet-responsive gene expression.

Illumina library preparation and sequencing
Preparation of MiSeq library was performed using Illumina Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) as previously described with minor modifications [64]. In brief, 1 ng of genomic DNA was fragmented in 5 μl of Amplicon Tagment Mix and 10 μl of Tagment DNA buffer. Tagmentation reaction was performed by incubation at 55˚C for 10 min followed by neutralisation with 5 μl of Neutralise Tagment Buffer for 5 min. Tagmented DNA (25 μl) was indexed in a 50 μl limited-cycle PCR (12 cycles) as outlined in the Nextera XT protocol and subsequently purified using 25 μl of AMPure XP beads (Beckman Coulter Inc, Australia). The fragment size distribution of the purified DNA was analysed utilising a LabChip GXII 2100 Bioanalyser. DNA libraries were adjusted to 2 nM, pooled in equal volumes and then denatured with 0.2 N NaOH according to the Nextera protocol. The libraries were sequenced using 2 × 300 paired-end protocols on an Illumina MiSeq instrument (MiSeq Reagent Kit v3 for 600 cycles). The draft genome sequence of H. pylori OND1954 has been deposited at DDBJ/ENA/GenBank under the accession MVFB00000000. The version described in this paper is version MVFB01000000. All raw sequence data generated in this study have been submitted to Sequence Read Archives (SRA) database with accession numbers listed in S5 Table. Sequence data de novo assembly, annotation, reference mapping and variant calling The generated MiSeq reads of H. pylori strain OND1954 was assembled using SPAdes genome assembler (version 3.8.2) with careful option [65]. The draft genome sequence was subsequently annotated using Prokka (version 1.11) with Swiss-Prot, Pfam (release 30.0), TIGRFAMs (release 15.0) and Superfamily (version 1.75) databases [66][67][68][69][70]. The annotation features are available in S1 File. The raw reads of OND1954-derivative strains were trimmed and mapped against the annotated draft genome using Bowtie2 on Geneious R7 platform [71,72]. Variants were called using the following parameters: minimum coverage = 10 and minimum variant frequency = 0.7.

Statistical analysis
For mouse colonization assays (where n ! 5) the Mann-Whitney unpaired two-tailed test was used to compare colonization loads and the two sided Fisher's exact test was used to compare infection rates. Bonferroni correction was used for multiple pairwise testing. Statistical analysis was performed using GraphPad Prism version 7 for Windows, (GraphPad Software) and Stata Statistical Software (StataCorp. 2015. Release 14. College Station, TX: StataCorp LP.). . Mice were orally challenged with wildtype X47 strain and supplemented with a range of Dox concentrations (1, 10, 100 and 1000 mg/l) in their drinking water. Bars represent median bacterial load per group and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null. (B) Urease expression in conditional urease mutant strain OND1954 was induced with 50 ng/ml ATc for 48 h prior to oral challenge. Mice were orally challenged with wild-type X47 strain or pre-induced OND1954 and supplemented with a range of Dox concentrations (1-20 mg/l) in their drinking water. Animals were sacrificed one week after oral challenge. Bars represent median bacterial load per group (n = 3) and points plotted represent colonization density for each individual animal. Detection limit was < 50 CFU per stomach (dotted horizontal line). Gastric specimens without H. pylori re-isolation are shown as null.