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Role of TolC in virulence of salmonella enterica serovar Typhi.

Typhoid fever is a life-threatening febrile illness caused by the bacterium S. Typhi. Typhoid disease is a serious public health problem in developing countries, Typhoid fever remains a significant health burden; the estimated total number of typhoid fever episodes in 2010 was 13.5 million (Buckle et al, 2012). Pathogenic bacteria have evolved various escape mechanisms and strategies, one is efflux pump to survive and propagate in the hostile environment of the host. The gall bladder of the host is inhospitable to most microorganisms due to the presence of bile (Antunes et al, 2011; Hernandez et al, 2012). However, S. Typhi, the causative agent of typhoid fever is able to survive and persist in the gallbladder of typhoid carriers (Crawford et al, 2010; Gonzalez-Escobedo et al, 2011). Bile has been reported to regulate the production of virulence factors, motility, adhesion, and bile resistance, efflux pump expression, and biofilm formation (Crawford et al, 2008). Biofilm formation which prevents the bacteria from being exposed and the efflux systems help the bacteria to grow in adverse conditions by pumping out toxic substances which enable the bacteria to persist even in the macrophage.

TolC is a protein found in all Gram-negative bacteria that forms a channel through the outer membrane while interacting with the AcrAB periplasmic protein (Koronakis et al, 2004). The AcrAB-TolC system is the most prominent of all the multidrug resistance efflux systems. (Koronakis et al, 2004). Apart from its role as an efflux pump, TolC has recently been reported to play multifarious roles ranging from adhesion and invasion of epithelial cells and macrophages to virulence, persistence and stress resistance of Gram-negative bacteria, such as Francisella tularensis, Brucellasuis, Vibrio cholera, Salmonella enteric serovar Typhimurium and Salmonella enteritidis (Bina and Mekalanos, 2001; Buckley et al, 2006; Gil et al, 2006; Posadas et al, 2007; Santos et al, 2010). Piddock and co-workers indicated the requirement of tolC as a general requirement for colonization and persistence of bacteria in the host includes plants, animals, and humans (Piddock, 2006b; Virlogeux-Payant et al., 2008). In addition, tolC also plays an important role to export of chromosomal and plasmid-encoded toxins such as microcins and hemolysin (Delgado et al., 1999; Wandersman and Delepelaire, 1990). Variants of the E. coli tolC have been described in a number of bacterial systems including S. Typhimurium (Cosme et al., 2008; Eaves et al., 2004; Ferhat et al., 2009; Karatzas et al., 2008; Nishino and Yamaguchi, 2001; Posadas et al., 2007) with varying functions. In spite of many reports of the importance of TolC in various systems and its associated with survival in the host. its function has not been studied in detail in S. Typhi.

The aim of this study was to delineate the role of the S. Typhi TolC as an important bacterial virulence mechanism such as the invasion of human macrophages, biofilm formation, and maintenance of the membrane integrity of the bacteria in comparison with that the wild type. Two kanamycin resistant gene knockout tolC mutants ([DELTA]tolC) of S. Typhi were constructed for this purpose. Deletion of tolC gene was observed to have profound effects on the various aspects studied, suggesting that the TolC is an important protein in the maintenance of virulence and pathogenicity of S. Typhi

MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Growth Conditions

The S. Typhi mutant strains were derived from a wild-type strain of a local isolate from an acute fever typhoid patient (acute strain) and from Ty21a vaccine strain (ATCC, USA). Bacterial strains were grown at 37[degrees]C in Luria-Bertani broth (LB) (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl).

Deletion of tolC gene in S. Typhi strains ([DELTA]tolC)

The tolC mutants ([DELTA]tolC) were constructed from two of S. Typhi strains (local clinical isolate from an acute fever typhoid patient also call acute strain and Ty21a vaccine strain) using the method described by Datsenko and Wanner (2000). The target tolC gene was replaced by a kanamycin resistant gene which also serves as a marker for the gene knock-out procedure. Briefly, PCR was used to prepare the replacement gene cassette contained the antibiotic resistance gene, flanked at both ends by 50 bp DNA sequences that were identical to sequences flanking the open reading frame of the target gene, tolC, according to Datsenko and Wanner (2000).

Construction of complementation strain ([DELTA]tolC+)

Complementation plasmid pKK-tolC, derived from the low copy number plasmid pKKtolC. For tolC complementation, tolC was amplified from the Ty21a strain with primers using (primers tolC_F, 5'-TTAATGGAATTCTTACGCATTG TGCTGCCC and tolC_R, 5'-GACGGAAGCTTT CAATGCCGGAATGGATT; underlined sequences indicate restriction enzyme recognition sites). These primers were designed to amplify the entire coding sequence of tolC, including the native stop codon from the Ty21a strain. The amplified PCR product was purified, digested with EcoRI and HindIII and ligated into plasmid pKK that was digested with the same enzymes, and the ligation mixture was transformed into DH5a. Plasmid pKKtolC was isolated from the Ampr transformants, and the presence of a tolC was verified by agarose gel electrophoresis, restriction analysis. Plasmid pKK-tolC was transformed into tolC mutant ([DELTA]tolC to create a tolC complemented strain ([DELTA]tolC+). After plasmid pKK-tolC had been introduced into the [DELTA]tolC and selected by growth on ampicillin (50 mg/L) and 0.01% SDS containing agar plates a selective medium on which [DELTA]tolC mutants are not able to grow,

Determination of growth kinetics

The growth kinetics of all S. Typhi strains was determined by bacterial culture by measuring the optical density (OD) at 595 nm as previously described (Sheridan et al., 2013). Growth characteristics of the reference wild-type (WT), complemented ([DELTA]tolC+) and mutant ([DELTA]tolC) strains were assessed by growth kinematics studies at 37[degrees]C. A single colony was picked and placed into a tube containing 10 ml LB broth and incubated for overnight at 37 [degrees]C without shaking. Five microliters from the overnight culture were inoculated into 200 il of LB broth. Samples were placed in a microtiter plate and bacterial growth was assessed by measuring the OD of the microtiter plate wells at scheduled 2 h time intervals at 595 nm (Thermo Scientific, Multiskan Spectrum). All quantitative growth assays were performed in six technical replicates with three independent experiment. They were compared with wild type by one-way analysis of variance (ANOVA).

Cell invasion assay

THP-1 derived macrophages were used for invasion assay (Buckley et al., 2006). The THP1 line is a human monocyte cell line which differentiates into macrophage-like cells when treated with phorbol 12-myristate 13-acetate (PMA), (Sigma). THP-1 derived macrophage cells were grown in RPMI-1640 tissue culture medium containing 10% fetal calf serum (Gibco-BRL).

Invasion assays were performed with gentamycin protection assay originally described by Isberg and Falkow, (1985). THP-1 macrophages (1x[10.sup.6]/ml in 1 ml complete tissue culture media) were allowed to attach to the surface of 6-well tissue culture plates by incubation at 37 [degrees]C in 5.0% C[O.sub.2] for overnight, following which the media was discarded and the plates washed with PBS three times to remove the non-adherent cells. Bacterial cells were grown to mid-log phase (O.D at 595 nm of 0.6), washed with tissue culture medium and 5x[10.sup.7] CFU inoculums in a total of 1 ml volume of tissue culture media was used to infect confluent macrophage monolayers. The bacterial inoculation was followed by incubation of the plates at 37[degrees]C in 5.0% C[O.sub.2] for 2 hours to allow bacterial entry. The monolayers after the incubation were washed with phosphate-buffered saline (PBS; pH 7.0) and tissue culture medium containing 100 [micro]g/ml gentamycin and were subjected to a further incubation for 1 h to kill the extracellular bacteria. After removal of any remaining extracellular bacteria by the washing of the monolayers with PBS 1 ml, ice cold distilled water at 4 [degrees]C was added to each well and incubated further for 20 min for macrophage cell lysis. The intracellular bacteria were quantitated by plating serial dilutions of the macrophage cell lysate. Experiments were performed to ensure that the data obtained were genuine and not experimental artifacts. All quantitative invasion assays were performed in triplicates with three independent experiment. They were compared with wild type by one-way analysis of variance (ANOVA). Crystal violet biofilm assay

Biofilm assay was established on 96-well polystyrene microtiter plates from a previously described method with some modifications (Sheridan et al, 2013). A single colony was selected and inoculated into 5 ml of LB media and incubated at 37 [degrees]C for 18 h. Two [micro]l aliquots from the overnight culture were diluted in 1 ml of PBS. 1: 100 diluted bacteria in fresh media antibiotic-free LB broth containing 3.0% bile (Begley et al., 2009). The ninety-six-well polystyrene microtiter plate each well was inoculated with 200 [micro]l of this suspension and incubated at 37 [degrees]C for 48 h with gentle agitation. After 48-hour incubation the medium was removed from the wells and the microtiter plate was washed five times with sterile distilled water to remove unbound bacteria and media. The plate was air dried for 45 min and each well was stained with 1.0% (w/v) crystal violet solution in distilled water for 45 min. After staining, the wells of the plate were washed twice with sterile distilled water. The amount of bacterial biofilm produced was estimated by determining the amount of dye restrained in the microtiter plate wells by the addition of 95% ethanol to the wells, and the dye solution was measured using a microtiter plate reader at 595 nm. All biofilm assays were performed three times with three biological and twelve technical replicates per study. One-way analysis of variance (ANOVA) was used to compare the statistical significance of results each mutant to wild-type and complemented strains.

Scanning electron microscopy of bacterial cells

Cell from all three strains, i.e. [DELTA]tolC, wild-type, and [DELTA]tolC+ were processed and observed via scanning electron microscopy (SEM) (Yuen et al., 2012). Briefly, bacteria were grown to the exponential phase in LB broth and centrifuged at 2,000 xg for 15 min. The pellet was fixed overnight with fixing solution and centrifuged at 2,000 xg for 15 min, dehydrated through a graded ethanol series (20%, 40%, 60 %, 80%, 95%, 100% ethanol) and subjected to gold coating. The cells were viewed using a Leo Supra 50 VP field emission scanning electron microscope (Carl-Zeiss SMT, Oberkochen, Germany) equipped with an Oxford NCA 400 energy dispersion X-ray microanalysis system (Oxford Instruments, Bucks, UK).

RESULTS

Verification of tolC gene deletion in S. Typhi

Deletion of the tolC gene was confirmed by colony PCR. Based on the results of these assays and sequencing (sequencing result is not shown), we concluded that tolC was successfully deleted from the wild-type strains, resulting is strain [DELTA]tolC strains were unable to grow on 0.01% SD S Luria agar (LA) plate but wild-type and [DELTA]tolC+ strains could grow on 0.01% SDS LA (result is not shown).

Growth Curve

Growth characteristics of [DELTA]tolC strain derived from both vaccine and acute strains, acute wild-type reference strains were examined to measure bacterial fitness (Fig. 1A). The ability of the [DELTA]tolC strain to growth was compared with that of the wild-type and [DELTA]tolC+. The results showed that [DELTA]tolC strain displayed similar growth kinetics to the wild-type parent strain over the before 8 h duration of the experiment. After 8h, the growth of [DELTA]tolC significantly lower than wild-type and [DELTA]tolC+ (p<0.05). The result has shown here acute strains because the [DELTA]tolC of vaccine strain Ty21a was given similar results like [DELTA]tolC of acute strain.

In vitro invasion assay

The invasion ability of the [DELTA]tolC in THP1 derived macrophages was significantly reduced when compared to the wild-type strain and [DELTA]tolC+ strain of acute strains (p<0.0001, Fig. 1B). The defect was, however, restored in the [DELTA]tolC+ strain (Fig. 1B), even to a higher degree than with the wild type strains. [DELTA]tolC+ corresponds to [DELTA]tolC in which a TolC-positive phenotype was reintroduced. Results correspond to the mean of three independent experiments carried out in three replicates. Statistical significance: p <0.0001 wildtype vs. [DELTA]tolC/[DELTA]tolC+. An approximate similar result was observed for [DELTA]tolC of vaccine strain result is not shown here.

Crystal violet biofilm assay

[DELTA]tolC strain is unable to produce biofilm on biofilm inducing media

The role of tolC in biofilm formation was investigated using a standard microtiter plate assay. We compared biofilm formation by the wildtype, [DELTA]tolC and [DELTA]tolC+ of both, Ty21a and acute strains of S. Typhi on LB media containing bile. The ability of wild-type, [DELTA]tolC and [DELTA]tolC+ strains to attach to the wells of polystyrene microtiter plates was assessed in biofilm inducing media. Results of the assays revealed that wild-type and [DELTA]tolC+ strains were able to attach to polystyrene and showed strong biofilm formation. However, the [DELTA]tolC strain was unable to attach to polystyrene wells and lost ability to form biofilm in the crystal violet assays (Fig. 2D,3D). The formations of biofilm by the different strains were compared by one-way analysis of variance (ANOVA). The results show a significant difference (p < 0.0001) in the degree of biofilm formation in the [DELTA]tolC strains. Restored expression of the deleted tolC by using complementation vectors contain tolC, resulted in the strain returning to biofilm phenotype.

Cell membrane integrity

The scanning electron microscopy comparison of cell surfaces of the wild-type, [DELTA]tolC and [DELTA]tolC+ S. Typhi strains of both strains, Ty21a and acute of S. Typhi showed observable differences affecting cell membrane integrity in the [DELTA]tolC strains (Fig. 2A, 2B, 2C and 3A, 3B, 3C).

DISCUSSION

The study of functions of tolC in S. Typhi, two mutants was generated by deleting tolC from different strains of S. Typhi. In this study, these [DELTA]tolC showed significant reduction in: 1) SDS tolerance, 2) biofilm formation ability, 3) macrophage invasion ability, and 4) cell membrane integrity, as compared with their parent (wild-type) strains.

Demonstrating that, tolC, contributed to in vitro fitness and that deletion of tolC gene resulted in additive effects during the stationary phase of growth. Proving that growth delays were directly due to lack of tolC, growth during the stationary phase of the genetically complemented mutant ([DELTA]tolC+) were approximately similar to that of the wild-type reference proving that growth delays were directly due to absence of tolC. The ability of the mutant lacking tolC to grow anaerobically was not affected in log phase of growth (Webber et al., 2009) according to our result. Also our result similar to the previous report on the study on Sinorhizobium meliloti (Santos et al., 2010). Here, growth rates of wild-type and the [DELTA]tolC of Sinorhizobium meliloti growth were determined. During the first 8 hours the growth rate was similar to both strains; afterwards the [DELTA]tolC presented a lower growth rate and reduced biomass formation. It is probable that under the cytoplasmic and extra cytoplasmic stress conditions suffered by the [DELTA]tolC, many proteins and cofactors become defective and essential to be synthesized de novo or defended from denaturation. (Santos et al., 2010). if. Extra ATP would be spent to maintain cell homeostasis and not to produce biomass. It is also a formal possibility that uncertainties to the cell envelope may decrease the proton electrochemical gradient, negatively affecting ATP synthesis and, therefore, generating the need to increase the expression of genes related to energy metabolism not related to growth (Santos et al., 2010; Zgurskaya et al., 2011).

The study of the "tolC showed that these mutant exhibit reduced invasion ability compared to their wild-type parental strains. Complementation of this [DELTA]tolC restored to ability to invade human macrophage cell similar to their wild-type parents' strains this result conformed that tolC play role in during invasion of S. Typhi to human host. It can therefore be consider having impact on host-bacteria interaction. This result is in line with previous research on other bacteria which demonstrated that these [DELTA]tolC were loss ability to invade host cell and reduction in adherence to host epithelial cells when compared to parental wild type bacteria was a feature obseved in studies with S. Typhimurium. This study was further supported by similar observations with reduction in ability to adhere, invade and survive in human embryonic intestine cells (INT-407, RAW 264.7 cell lines) and mouse monocyte macrophages virulence using [DELTA]tolC strains of S. Typhimurium and L. pneumophila (Buckley et al., 2006; Ferhat et al., 2009). In this context, the work of Stone and Miller, 1995 who observed that S. Typhimurium strain that was generated using a transposon mutagenesis system was less virulent than wildtype holds interest. Not much is known on the role of the tolC in S. Typhi infection. Since epithelial adherence and penetration are the most important pathogenecity factors in S. Typhi infection, this aspect was investigated with the help of the invasion assay using the THP-1 derived macrophge cell line. The results of this study was in agreement with the earlier observations in other systems supporting that tolC is an important player in the pathogenecity of S. Typhi. In addtion Intracellular yield of wild-type S. Typhi in invasion assays were in the range of 3.5 x [10.sup.5]"5.1 x [10.sup.5] cfu/ml. similar to the observations of Buckley et al., 2006; DibbFuller et al., 1999 and Virlogeux-Payant et al., 2008, with wild-type S. Typhimurium. There is no clear mechanism for function of TolC in invasion but report from Virlogeux-Payant et al. (2008); Webber et al. (2009) have shown tolC deletion decrease expression of SPI-1 genes, which involved in invasion of bacteria to host cell. Other possibility, TolC have role in secretion of virulent factor which may be control invasion toward host cell (Song et al., 2015). Complementation with the tolC-bearing plasmid had significant effect on increase invasion of [DELTA]tolC+, this is because may be multiple copy of tolC present at cytoplasmic level as compared to single tolC present in wild-type strain.

We demonstrate the in vitro biofilm formation assay it was observed significantly reduced biofilm production in [DELTA]tolC as compared to their wild-type parental strains. Complementation of this [DELTA]tolC restored ability to form biofilm similar to their wild-type parents strains this result conformed that tolC of S. Typhi have role in biofilm formation in human host. This biofilm defect was might be due to decrease in growth rates after 8h of growth. In our study [DELTA]tolC+ of acute strain unable to produce biofilm similar to [DELTA]tolC+ of Ty21a strain, possible reasons for this, might be over expression of AcrAB-TolC in [DELTA]tolC+ acute strain, previous report shown that MexEF-OprM over expressing strains of P. aeruginosa exhibit compromised biofilm formation ability (Fernando and Kumar, 2013), OprM which is a homologous of TolC. We expecting some other possible reasons for this may be for this difference are (1) SEM picture of [DELTA]tolC+ of Ty21a strain has shown membrane similar to wild type Ty21 a strain (Fig.2A, 2C) but [DELTA]tolC+ acute strain unable to repair membrane as in acute wild type (Fig.3A, 3C). (2) An intact, functional AcrAB-TolC efflux system is crucial for S. Typhi to form a biofilm. Defects in this system blocks biofilm formation. The role of AcrAB-TolC in biofilm formation appears to be related to regulation of matrix formation by activation of transcription of biofilm matrix components (Baugh et al., 2014). [DELTA]tolC, AacrB, S. Typhimurium are unable to form biofilm, unlike AacrA, which was able to compromise to form biofilm (Baugh et al., 2014). Chemical inhibition and genetic inactivation of efflux pumps results in transcriptional repression of biofilm matrix components and a lack of biofilm formation (Baugh et al., 2014), therefore we used tolC from Ty21a strain to develop [DELTA]tolC+ of acute strain. This may be possible as tolC from Ty21a strains unable to dock with AcrAB in acute strain. Since this defect in acute strain unable to form biofilm, this might be attributed to transcriptional repression of biofilm matrix components. Our results are in line with previous research on S. Typhimurium and other bacterial pathogens play role in many persistent human infections, are observed to be associated with biofilm growth (Crawford et al., 2010). It has been known for decades that bacterial biofilm can tolerate antibacterial agents like biocides, detergents, and antibiotics far better than planktonic cells (Anderl et al., 2000) and possible roles of AcrAB -TolC channel in biofilm formation has been stressed upon. Studies in E. coli, and other bacteria observed tolC to be active in promoting cell aggregation between bacteria, biofilm formation, adhesion to cells and AcrAB-TolC efflux pumps functioning (Imuta et al., 2008; Kvist et al., 2008; Costerton et al., 1999; Roberts, 1996). Bacterial biofilm, in turn, aids the bacteria to colonize and develop resistance towards antimicrobial agents and host immune responses (Costerton et al., 1999).

The results of the present study highlight the effect of tolC on cell membrane integrity in both Ty21a and acute S. Typhi strains. [DELTA]tolC have some phenotypic difference on cell membranes from wild type strain and [DELTA]tolC+ strain (Fig.2B 3B). No previous reports have documented this morphological difference in [DELTA]tolC of S. Typhi before. This difference suggests that disruption of tolC, which encodes TolC outer membrane channel protein, an integral part of AcrAB-TolC channel, may affect the structural integrity of the outer membrane of S. Typhi cells. Our results on structural integrity of the outer membrane have some degree of relationship on previous research on role of tolC deletion on integrity of the outer membrane defects such as depletion of essential metabolites, cytoplasmic membrane stress and alteration in NAD+/NADH ratios have been reported in literature (Dhamdhere and Zgurskaya, 2010). Studies available on [DELTA]tolC of E. coli have observed that the loss of tolC leads to metabolic shut down of E. coli grown in a minimal medium with glucose. [DELTA]tolC is reported to compromise the integrity of the bacterial inner membrane leading to inactivation of NADH oxidases and overconsumption of proton motive Force (Dhamdhere and Zgurskaya, 2010). Rosner and Martin, 2009 suggested that the up regulation of MarA, SoxS and Rob activity in [DELTA]tolC could be due to the response of membrane stress and also metabolic imbalance, this may be also possible reason for growth defect after 8h (fig 1A). These observations point towards the role of TolC in the maintenance of membrane integrity in survival process of the bacteria. However, the role of tolC in relation to infection process remains obscure.

CONCLUSION

In conclusion, the [DELTA]tolC strain exhibited differences in phenotype and morphology compared to the wild type. tolC may operate as part of the multiple regulatory mechanisms governing external stress response and is involved in membrane integrity in S. Typhi, although tolC was found in our study to be involved in biofilm formation and involved in invasion into macrophage cell. It would also be interesting to study the effect of tolC deletion on other functions such as signal transduction and quorum sensing.

ACKNOWLEDGEMENTS

The authors acknowledge Universiti Sains Malaysia, Malaysia for providing world class infrastructure and financial support (RUC 101.PSK. BP.8630011, RU-PRGS 1001/CIPPM/846046 and USM post graduate fellowship) to continue the research work.

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Ashraf Hussain [1], Eugene Boon Beng Ong [1], Priscilla Saw Cheah Phei [1], Kaizar Hossain [2], Prabha Balaram [1], Asma Ismail [1], Phua Kia Kien [1] *

[1] Institute for Research in Molecular Medicine (INFORMM), Main Campus, Universiti Sains Malaysia, Penang, 11800, USM, Malaysia.

[2] School of Industrial Technology, Universiti Sains Malaysia, Penang, 11800 USM, Malaysia.

(Received: 11 March 2016; accepted: 19 April 2016)

* To whom all correspondence should be addressed. Tel: +604 653 4801; Fax: +604 653 4803;

E-mail: kkphua7@gmail.com

Caption: Fig.1. Growth plot and histogram of invasion assay of the acute S. Typhi wild-type reference strain and its +tolC and +tolC+

Caption: Fig. 2. Ty21a S. Typhi recombinant and wild strains--Scanning electron micrographs and biofilm formation Scanning electron micrographs of three stains: 2A - Ty21a S. Typhi wild strain, 2B - [DELTA]tolC and 2C - [DELTA]tolC+ strain. Arrows indicate altered surfaces of [DELTA]tolC bacterial cells. The cells were observed at 100,000X magnification. 2D -Biofilm formation by S. Typhi Ty21a wild-type reference strain, [DELTA]tolC and [DELTA]tolC+ on polystyrene in LB containing bile. Each bar represents the mean optical density measured at 595 nm of 12 replicates from a single sample taken from each of three independent trials for all strains.

Caption: Fig. 3. Acute S. Typhi recombinant and wild strains - Scanning electron micrographs and biofilm formation Scanning electron micrographs of three stains: 3A - Acute S. Typhi wild strain, 3B - "tolC, and 3 C - "tolC+ strain. Arrows indicate change surfaces of "tolC bacterial cells. The cells were observed at 100,000X magnification. 3D - Biofilm formation of S. Typhi acute reference strain, "tolC and "tolC+ on polystyrene in LB containing bile. Each bar represents the mean optical density measured at 595 nm of 12 replicates from a single sample taken from each of three independent trials for all strains.
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Author:Hussain, Ashraf; Ong, Eugene Boon Beng; Phei, Priscilla Saw Cheah; Hossain, Kaizar; Balaram, Prabha;
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Jun 1, 2016
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