Although much has been learned about specific H. pylori virulency factors, small is presently understood about why some H. pylori infected persons advancement to develop stomachic malignant neoplastic disease while others remain symptomless. The end of this thesis was to better understand the association between different polymorphisms in CagA and VacA and disease result. Specifically, we showed that East Asian CagA ( EPIYA-ABD ) was linked to patterned advance to stomachic malignant neoplastic disease in a South Korean population ( 39 ) . In fact, all H. pylori strains from malignant neoplastic disease patients expressed and delivered phosphorylatable CagA to host cells, whereas the presence of the cagA cistron did non purely correlate to expression and bringing of CagA with non-cancer strains ( 39 ) . Our survey was the first to statistically associate a specific cagA allelomorph to stomachic malignant neoplastic disease development. We following examined the function of VacA polymorphisms within that population, and found that while the distribution of vacA allelomorphs was non straight associated with disease province, it was associated with the distribution of cagA allelomorphs. Furthermore, the vacA allelomorph was associated with the cagA allelomorph and disease province. Following, we were able to analyse the part of the freshly described one part of VacA to disease development. To this terminal, we identified an amino acid ( 196 ) that was of import for development of stomachic malignant neoplastic disease. We were besides able to place some associations that were CagA-dependent, such as the association of VacA and disease province in the EPIYA-ABD population every bit good as amino acid distribution at place 231 and disease province in the non EPIYA-ABD population. In add-on, in the procedure of finishing this thesis, we were able to optimise techniques that will finally be used to qualify CagA isogenic strains. Those future surveies will assist to clarify the function of the EPIYA motives in H. pylori-induced host cell harm both in vitro and in vivo. En masse, the informations presented herein attention deficit disorder to what we know about the complexness of H. pylori-induced pathogenesis. Overall, it is going progressively more apparent that polymorphisms within CagA and VacA, entirely and in concert, affect H. pylori-induced disease. However, the ground why merely a part of the population develops stomachic malignant neoplastic disease still remains ill-defined. Other bacterial virulency factors, every bit good as multiple host, dietetic, and environmental factors have been indicated as participants in H. pylori-induced disease. Clearly, farther survey is required to find which factors are involved and what function they have in the development of H. pylori-induced stomachic malignant neoplastic disease.
Unanswered Questions Steming from the Epidemiological Surveies
A cardinal inquiry that should be addressed in the hereafter is the difference in the grade of CagA fluctuation between Western and East Asiatic strains. Western isolates vary widely in the figure of EPIYA-C motives that are present ( 7, 8 ) , whereas there is a distinguishable deficiency of fluctuation in East Asiatic strains. In fact, one survey analyzing Gen Bank sequences of 500 East Asiatic strains, found that 441 ( 88.2 % ) contained a canonical EPIYA-ABD motive ( 7 ) . Indeed, extra surveies confirmed this preservation among East Asiatic strains ; greater than 84 % of the examined strains across all three surveies contained an EPIYA-ABD motive ( 7, 12, 39 ) . Furthermore, in our molecular epidemiologic CagA survey, the bulk of strains ( 87.5 % ) contained an EPIYA-D motive. Interestingly, those isolates that contained a nonstandard EPIYA-ABD motive were associated with development of gastritis ( 39 ) . This suggests that fluctuation in East Asiatic cagA is non every bit favourable as in Western isolates and that fluctuation may impact disease patterned advance.
The grounds for rigorous preservation of the EPIYA-ABD motive are unknown, but may be explained by several possible theories. One theory to explicate this preservation could be that there is a difference in the grade of selective force per unit area for fluctuation imposed on Western and East Asiatic strains. Western CagA shows a lower affinity for SHP-2 and is associated with less terrible redness, host cell morphological alterations, and disease every bit compared to East Asiatic strains ( 30, 31 ) . Furthermore, it has besides been demonstrated that there is a dose response in the figure of EPIYA-C motives and the degrees of tyrosine phosphorylation, SHP-2 binding, host cell morphological alterations, and redness induced by Western CagA ( 32, 39, 87 ) . Possibly, increased redness enhances colonisation, and hence may move as a positive selective force per unit area to increase the figure of EPIYA-C motive. This force per unit area would non be experienced by East Asiatic strains since the EPIYA-D motive is already so biologically active. However, if increased redness is of import for colonisation of H. pylori, so there may be a selective force per unit area to maintain a canonical EPIYA-ABD motive. For case, possibly excess EPIYA-A or -B motives in association with an EPIYA-D motive more strongly activate the negative feedback cringle that consequences from EPIYA-A or -B adhering to Csk, thereby diminishing redness ( 7, 82 ) . Additionally, a individual EPIYA-D motive may be optimum for SHP-2 binding, and excess EPIYA-D motive may deform CagA ‘s conformation, thereby destabilising this interaction, once more ensuing in a lessening in redness. The true ground for this preservation among East Asian strains could assist clarify the impact of these motives.
Work on the EPIYA motif part of CagA has chiefly focused on differences in phosphorylation and subsequent transition of phosphorylation-dependent host signaling tracts ( reviewed in ( 40 ) . Recently, nevertheless, a CagA multimerization sphere was described that is located within the EPIYA part, and hence varies as the EPIYA motives vary. Some surveies suggest that this sphere is responsible for the differential transition of some phosphorylation-independent host signaling tracts ( Fig. 2 ; ( 59, 74, 77, 83 ) . However, since the being of this sphere is a really recent find, more work is needed to clearly specify the function of the multimerization sphere on H. pylori-induced alterations in host signaling tracts. Additionally, alterations in this sphere as a consequence of alterations in the EPIYA motive will necessitate to be investigated.
While we have gained much knowledge about the function of the C-terminus of CagA, non much is presently known about the N-terminus of the protein. Furthermore, of the surveies that have been completed, conflicting informations has arisen ; it has been demonstrated that the N-terminus of CagA is responsible for directing CagA to the plasma membrane ( 14 ) , but other informations demonstrated the EPIYA-motifs located in the C-terminus were responsible for proper localisation ( 32, 33 ) . Pelz et Al. late demonstrated that two independent spheres, one in the N-terminus and one in the C-terminus, are responsible for directing CagA to the plasma membrane ( 69 ) . In fact, these writers showed that the first 200 aminic acids of CagA really act as an inhibitory sphere that dampens the host response to the C-terminus of CagA. This sphere reduces activation of the oncogenic written text factor I?-catenin, reduces the length of the “ hummingbird ” bulges, and increases the velocity and strength of new cell to cell contact ( 69 ) . Therefore, an interesting inquiry would be to inquire if the activity of the inhibitory sphere varies in concurrence with changing motives in the C-terminus. In other words, is the consequence of this repressive sphere different based on the fluctuations of the different cagA allelomorphs? This inquiry could be addressed by canceling the inhibitory sphere within the context of isogenic strains that differ merely in the EPIYA part of cagA. Differences in induced host cell morphological alterations could so be assessed when these strains were used to infect AGS cells, and fold alterations in the figure of extended cells could be calculated and compared between the strains and their isogenic strains missing the inhibitory sphere. If the difference in fold alteration was similar across the different EPIYA isogenic strains, this would propose that this inhibitory sphere acts independently of the EPIYA motive. If this inhibitory sphere is influenced by the EPIYA motive that is present, it could besides be interesting to find whether there is besides an interaction with the multermization sphere. This could be addressed by doing phosphorylation immune mutations within the above mentioned strains ; the tyrosine could be changed to an alanine or serine through site specific mutagenesis ( 5, 21, 32 ) . While we know rather a batch about the part of CagA and the EPIYA motives to H. pylori-induced disease, there are clearly more inquiries on the molecular degree that need to be answered.
Many inquiries still remain to be addressed sing the different vacA allelomorphs. Included among these are inquiries refering the signal ( s ) part of the protein. Geographic differences between the s1 part have been reported, and three subtypes have been identified: s1a, s1b, and s1c ( 11 ) . Are these differences of import for how VacA Acts of the Apostless on host cells and/or its interplay with other virulency factors? Although this terminology is now rarely used, it would be interesting to find if the different subtypes display any functional differences in activity, since the s part is responsible for most of the toxicity of the toxin ( 52-54, 72 ) . This could be accomplished by making isogenic strains that vary merely in the s1 subtype or more merely by elating eucaryotic cells with indistinguishable concentrations of purified VacA incorporating one of the three different subtypes. One could measure the ability of the assorted VacA toxins to do vacuoles within host cells every bit good as induce programmed cell death, which could be measured through activated TUNEL checks or by mensurating the sum of activated caspase 3.
Another inquiry that has arisen from our VacA epidemiology work concerns the overall importance of the center ( m ) part of the protein. The first facet of the thousand part that should be addressed is the association between the thousand part and gender. Our work was the first survey to detect such an association ( 37 ) , and begs the inquiry of why this association exists? Females appear more likely to be infected with strains encoding the M2 vacA allelomorph ( 37 ) . Does this association be in populations where the M2 allelomorph is more prevailing, such as in parts of China ( 67 ) and Poland ( 49 ) ? If it does be, so there may be something physiologically different between the stomachic environment of males and females. While what causes this difference may be unknown, there are many possibilities. For case, is at that place a difference in the pH of the tummy acid that may accordingly act upon disease province? Is at that place a difference between the existent stomachic epithelial tissue between males and females? Minute differences in the thickness or composing of the mucous secretion bed, which could in turn impact contact of H. pylori with the stomachic epithelial tissue, could impact the sum of toxin delivered to host cells. Are at that place differences in the sum or type of attachment receptors expressed in the stomachic epithelial tissue of males versus females? Furthermore, what affect does the hormone system, more specifically alterations in endocrine degrees, have on this procedure? Answers to these inquiries may assist to explicate the differences in distribution of the M1 vacA allelomorph between work forces and adult females. Overall, the increased cellular tropism of the M1 vacA allelomorph ( 66 ) , the determination that the presence of the m1 part increases the hazard for stomachic malignant neoplastic disease ( 79 ) , and the fact that patients infected with H. pylori strains encoding for the M2 allelomorph are more likely to be female ( 37 ) may explicate why males are overall more likely to develop stomachic malignant neoplastic disease ( reviewed in 76 ) .
The 2nd facet of the m allele that should be examined is its part to the association between VacA and CagA, every bit good as between VacA, CagA, and disease province. Does the association between the thousand part and the cagA allelomorph occur merely because the vacA parts that are responsible for more terrible disease besides co-vary among themselves? This does non look probably in this population, since we merely found the s1 allelomorph and were still able to observe this association. Furthermore, is the tripartite association between the thousand part, CagA, and disease province due to the fact that the M2 allelomorph has a narrower cell tropism, thereby impacting the types of cells VacA can elate? If this were the instance, one would anticipate to see a direct correlativity between the thousand part and disease province. However, this correlativity is non seen within this South Korean population.
Several inquiries besides still remain sing the intermediate ( I ) part of VacA. The major difference between the i1 and i2 allelomorphs is the add-on of three polar amino acids ( asparagine, histidine, and serine ) in Cluster C ( 37, 38 ) . Since both bunchs B and C have been suggested to impact toxin activity, surveies directed towards understanding the specific function of these aminic acids in vacuolating activity would be interesting. In order to better analyze this, an i1 allelomorph could be genetically engineered through the add-on of merely these three amino acids. Possibly, it would non be surprising if the add-on of these aminic acids decreased toxin activity, since it has been demonstrated that extra amino acids near the cleavage site in the s part decrease the ability of the toxin to incorporate into the cellular membrane, which in bend lessenings toxin activity ( 11, 53 ) . Such a consequence might explicate why the I part has been suggested to be a better forecaster of disease.
Another facet of the one part worth analyzing involves toxin development and the presence of the i3 allelomorph. Is the presence of the i3 part truly a snapshot of the development of the one part from one allelomorph to another ( i1 or i2 ) ? It would be interesting to infect animate beings with H. pylori strains incorporating the i3 allelomorph, and to sequence several recovered isolates after infection to see if the i3 allelomorph has been replaced with either an i1 or i2 allele. This experiment should be performed with an i3 strain where bunch B is from an i1 strain and bunch C is from an i2 strain, every bit good as an i3 strain where bunch B is from an i2 strain and bunch C is from an i1 strain. Consequences from these experiments could bespeak if there is an overall selective force per unit area to germinate in vivo, and if the original bunch sequences influences that development. In this same vena, it would be interesting to find if there is a functional difference between the i3 allelomorph and the i1 and i2 allelomorphs. If so, is at that place a functional difference between i3 strains that contain cluster B from an i1 strain and bunch C from an i2 strain as compared to an i3 strain that contains bunch B from an i2 strain and bunch C from an i1 strain? Alternatively, is toxicity dependant on the sequence of an single bunch?
Finally, it would be interesting to look at a population incorporating an increased per centum of i2 allelomorphs in order to measure the distribution of amino acids at place 196 on the vacA allelomorph every bit good as on disease province. Our work demonstrated that the distribution of amino acids found at this place was linked to more terrible disease, specifically stomachic malignant neoplastic disease ( 38 ) . In the South Korean population analyzed, all of the i2 allelomorphs encoded for a leucine at amino acid 196 ( 38 ) . Therefore, possibly it would be interesting to analyse a population where the i2 allelomorph is more prevailing to see if this tendency persists. If so, the amino acid found at this place may be partly responsible for a old survey that concluded that the one part was the best forecaster of disease ( 75 ) . Further surveies could besides look into fluctuation in the major amino acid differences seen between the i1 and i2 allelomorphs. This might break indicate which amino acids are critical for toxin activity.
Recent surveies have identified an association between the cagA allelomorph and the vacA allelomorph that appears to impact H. pylori toxicity and disease badness ( 37, 84, 89 ) . Infection with H. pylori strains that encode for CagA and s1/m1 VacA consequence in extremely active principal gastritis ( 57 ) , which is linked to the development of stomachic malignant neoplastic disease ( 55-57 ) . Our survey besides found an association between the cagA allelomorph and vacA allele, every bit good as a three manner association between the distribution of the cagA and vacA allelomorphs and disease province ( 37 ) . Indeed, in our South Korean population, the bulk of H. pylori strains carry the most toxic signifier of both VacA and CagA, and this may explicate the high rate of terrible stomachic disease among the South Korean population ( 37 ) .
Conventionally, one might believe that both toxins concomitantly exert drastic effects on the same host cell. However, recent information suggests that the converse is true ; the presence of both CagA and VacA may stifle the consequence of each protein entirely, perchance taking to increased endurance of septic host cells ( 9 ) . In fact, when both toxins are present, there is less VacA-induced programmed cell death so when cells are intoxicated with VacA entirely ( 9 ) . Additionally, eucaryotic cells intoxicated with both toxins demonstrate less CagA-induced morphological alterations as compared to cells intoxicated with CagA entirely ( 9 ) .
Since these consequences are still reasonably new, there are many inquiries that remain to be answered. For case, is at that place a direct interaction between CagA and VacA, or more likely, is this consequence the consequence of activation of viing tracts by the two toxins ( Fig. 2 and 3 ) ? If there is a direct interaction between these two proteins, this could perchance be detected by executing pull-down checks. Does VacA somehow magnify the map of the freshly identified inhibitory sphere in the N-terminus of CagA ( 69 ) ? Besides, in believing about the chronology of H. pylori infection, does the bacteria use the two toxins to increase the life span of the host cell and therefore, to protract infection? Indeed, it seems plausible that the most terrible signifiers of stomachic disease would ensue from long term infection of cells, and hence, long term H. pylori-induced redness. In footings of CagA and VacA “ interaction, ” does an order of events exist that is of import for the ensuing effects? For illustration, since VacA is secreted while CagA is injected by H. pylori, do cells foremost necessitate to be intoxicated by one or the other toxin to see the protective correlative? This could be straight assessed by elating eucaryotic cells with one toxin and so later with the other toxin at assorted ulterior clip points, followed by analysis of the cells for programmed cell death and for morphological alterations. Additionally, is the muffling consequence of the toxins achieved by making a threshold of both toxins, or is the mere presence of any sum of the two toxins sufficient? This could be assessed by finding if there is a dose response to the toxins. For case, eucaryotic cells could be intoxicated with increasing sums of VacA and a set sum of CagA, or with a set sum of VacA and increasing sums of CagA. Eukaryotic cells would so be analyzed for degrees of programmed cell death every bit good as morphological alterations. Additionally, the order of toxin add-on could be inversed, based on the findings from the above survey measuring the importance of the order of poisoning.
Finally, sing CagA and VacA “ interaction ” in the context of our epidemiological information, what are the physiological effects of poisoning with the different CagA or VacA discrepancies, or combination of these different toxins? For case, if a strain carries EPIYA-ABD CagA and s2/i2/m2 VacA, which typically shows no toxic activity, does it act likewise to a strain that carries an EPIYA-ABD CagA but no VacA? Alternatively, is the combination of Western CagA and s1/i1/m1 VacA more or less deadly than East Asiatic CagA and s2/i2/m2 VacA? These and other allelomorphs based inquiries could be addressed by doing VacA isogenic strains within the context of the CagA EPIYA isogenic strain background as discussed subsequently in this chapter. Since grounds is increasing that the association between the cagA allelomorph and the vacA allele impact disease development, I strongly believe that the hereafter of pathogenesis surveies in H. pylori will hold to concentrate on the consequence of combinations of these virulency factors.
A Hierarchy of Virulence Factors
Another emerging theory is that CagA may be the “ maestro ” virulency factor, and that other virulency factors or polymorphisms are of import merely in the context of which cagA allelomorph is present ( 13, 37, 38 ) . In fact, surveies have found that in footings of stomachic malignant neoplastic disease, the cagA allelomorph carried is the most of import bacterial hazard factor ( 15 ) . Conversely, the one part of VacA is the best forecaster for duodenal ulcers ( 15 ) . Indeed, in our epidemiological surveies, we found that different associations existed in populations transporting peculiar cagA allelomorphs and that these associations were non found in populations encoding for a different cagA allelomorph. For case, when age and gender were taken into history, a two manner association between the distribution of the vacA allelomorph and disease province was found merely within the EPIYA-ABD CagA population. Furthermore, non-s1/i1/m1 vacA allelomorphs were associated with duodenal ulcers within the population transporting the East Asiatic EPIYA-ABD CagA, but with gastritis in the population transporting any other genotype of CagA ( 37 ) . We besides found that an association existed between disease province and amino acid 231 of the VacA one part, but merely within the non EPIYA-ABD population ( 38 ) . These findings once more suggest that the consequence of different virulency factors or polymorphisms within these virulency factors may be masked by which cagA allelomorph is present. Indeed, this fact may assist explicate the huge sum of conflicting literature refering the importance of these different virulency factors. Using the statistical technique of meta-analysis to study the epidemiological informations in different geographic parts might assist to cast visible radiation on some of these reported differences.
Lessons Learned from the Current Undertaking
Unfortunately, the largest molecular constituent of my thesis undertaking encountered jobs since it was finally discovered that the isogenic cagA strains that were constructed really contained secondary mutants. While we do non understand precisely how these mutants arose, some hints may come from believing about the built-in familial variableness of the bacteria. For case, H. pylori strains are of course competent, which allows for the changeless exchange of DNA. In fact, the bacteria has the ability to uptake new DNA in vivo, which creates a changeless opportunity for familial exchange since a host can be infected with multiple H. pylori strains ( 24, 25, 36, 43 ) . This phenomenon has been good documented with one of the H. pylori mention strains ( J99 ) . When an archived isolate of J99 was compared to isolates from the same patient taken 6 old ages apart, there was a high degree of familial diverseness ( 36 ) . Jointly, the new isolates had lost up to 2.3 % of the unfastened reading frames compared to the archived J99 strain. Additionally these strains had gained DNA that was non found in the original J99 strain ( 36 ) . Overall, natural competency has been proposed to lend to the huge allelomorphic diverseness of the being, and to assist history for the considerable familial variableness ( 6-7 % ) that is seen between strains ( 4, 26, 50, 80 ) . Furthermore, carnal transition of strains has been shown to bring on formation of big Numberss of disconnected cistrons and repeated parts ( 22 ) . H. pylori besides has an increased rate of self-generated mutants as compared to E. coli, with initial surveies showing that the self-generated mutant rate in H. pylori was 10-7-10-8 ( 28, 35, 85 ) . Again, this rate varies among strains and a rate every bit high as 3 ten 10-4 has been observed ( 46 ) . In fact, familial polymorphisms seem to be normal between strains. In a survey that examined the familial sequence of a house-keeping cistron ( glmM ) it was found that the sequence was alone in all the strains examined ( 44 ) . Furthermore, this microdiversity has been observed in a figure of other cistrons ( 1, 3, 4, 34, 65, 78 ) , every bit good as within strains taken from the same patient ( 36 ) . Additionally, H. pylori ‘s genome contains multiple cistrons that are phase variable. Indeed, when a individual mention strain was sequenced, up to 27 cistrons were identified that contained nucleotide repetitions that could ease stage fluctuation ( 51, 81 ) ; two illustrations of these cistrons that have been examined in more item include somersault ( 41 ) and oipA ( 88 ) .
In order to cut down the potency for familial fluctuation that could impact our experiments, the Merrell group has adapted certain lab protocols. For case, when we create a mutant strain, we select a individual settlement of the mutant strain and so ne’er use individual settlements once more. Bacterias are expanded as spots of cells from the deep-freeze stock ( -80A°C ) on antibiotic-supplemented Equus caballus blood agar home bases for 36-42 hours, which is the minimum sum of clip for growing. Bacterias are so expanded as lawns from these spots for approximately 20 hours on home bases. The lawn is so used to inoculate 18 hr liquid starting motor civilizations that are finally used to inoculate OD controlled experimental liquid civilizations. All of these protocols are performed in an effort to minimise the figure of lab transitions of the strain, and to do certain that if familial variableness occurs, it does so in the context of a population of cells. Furthermore, when executable, we besides create an independent biological isolate of all mutant strains.
In inventing our isogenic strain survey, we foremost created a I”EPIYA strain, which was used as the strain background for all subsequent strains. Furthermore, we followed the aforesaid lab protocols for spread outing bacteriums from deep-freeze stocks, transmutation, choice, and growing, which were designed to minimise the possibility of fluctuation. Therefore, there was no ground to believe that these strains would incorporate secondary mutants. However, upon contemplation on the undertaking, there were several different points throughout the procedure when the information suggested that there might be a secondary mutant that would perplex our survey. Sequence analysis every bit good as growing kineticss suggested that the strains were in fact isogenic. However, the first indicant that the strains were non acting as expected came with the quantification of CagA look. Theoretically, all of the isogenic strains, with the exclusions of the I”cagA and I”EPIYA strains, should hold expressed CagA at similar degrees. However, up to a double difference could be seen between the EPIYA-ABtCCCC and wild type strains ( Fig. 16 ) . Conversely, there were no major differences when CagA look was compared to the restorant strain, which I believed to be a sensible comparing ; the restorant strain should be genetically indistinguishable to the 7.13 wild type strain, and had undergone the same familial use as the other isogenic strains. Minor differences in the restorant suggested that possibly the familial use of the strain somewhat altered the overall look degree of CagA. At the clip, we considered this as no surprise ; nevertheless, in hindsight, this consequence may hold been the first indicant that there were jobs with the strains.
While CagA has non been shown to impact the attachment of H. pylori ( 5 ) , any difference in attachment of strains to host cells would potentially change the sum of CagA that could be translocated and phosphorylated, thereby altering the deregulating of host cell signaling tracts and potentially impacting development of stomachic disease. In our surveies, the attachment check was the first check that showed marked differences between the isogenic strains. Indeed, the restorant, EPIYA-ABtC, and wild type strains, which should hold all adhered at similar degrees, did non act as expected ; the restorant strain was 10-fold less adherent than the wild type or EPIYA-ABtC strains. When these preliminary consequences showed the deficiency of consistence between the restorant, EPIYA-ABtC, and wild type strains, we sought to govern out the possible confounding consequence of little differences in the growing stage of the different isogenic strains ( Fig. 15 ) . Re-examination of the growing curves suggested that at 18 hours some of the isogenic strains may hold entered stationary stage, which could adversely impact the attachment of the bacteriums to the AGS cells. We hence repeated the check utilizing 12 hr liquid civilizations to infect the AGS cells. We found that while the figure of adherent bacteriums was increased, the tendencies stayed the same, proposing that there were secondary mutants within the strains ( Fig. 18 ) .
We next assessed the ability of the strains to present CagA to host cells, where it is phosphorylated by host cell kinases, thereby doing morphological alterations within the cells. While there were little differences in the peak phosphorylation clip of CagA between the strains, the tendencies across the strains were the same. However, appraisal of the five hr clip point, which was a clip point shown in a old survey to let sensing of high degrees of phosphorylation ( 39 ) , presented more grounds that there were secondary mutants in our strains. While increasing Numberss of EPIYA-C motives corresponded to increasing sums of phosphorylated CagA, the three isogenic strains that contained merely one EPIYA motive ( EPIYA-ABtC, EPIYA-ABtD, and the restorant ) showed similar degrees of phosphorylation ( Fig. 19 ) . Unfortunately, these degrees of phosphorylation were 2.5 times less than the sum of phosphorylated CagA from the wildtype strain ( Fig. 19 ) . This fact combined with the fact that the isogenic strains expressed about 70 % of the sum of CagA that the wild type strain expressed, instantly suggested that there was something different among the isogenic strains. Additionally, the degrees of phosphorylation did non interpret into the expected alterations in host cell morphology. Once once more, the the wild type, EPIYA-ABtC, and restorant strains all behaved otherwise ( Fig. 20 ) proposing that there was a 2nd site mutant within at least some of the strains.
Based on the long length of the experiments, the large-scale animate being survey was regretably started prior to the completion of the in vitro word picture of the isogenic strains. The animate being survey showed that there was a complete deficiency of differences in disease patterned advance among Mongolian gerbils infected with the different isogenic strains. Furthermore, there was a drastic difference in the pathology induced in animate beings infected with the wild type strain as compared to the EPIYA-ABtC strain. This fact entirely suggests that there is something basically different between these two strains that is non CagA-related. Knowing what we know now, ideally a little pilot experiment of eight to ten hebdomads continuance with fewer Numberss of animate beings should hold been conducted before continuing with the big graduated table animate being survey.
Additionally, all H. pylori infection groups progressed to gastritis and finally stomachic malignant neoplastic disease at the same rate as gerbils infected with the I”cagA strain ( Fig. 22 ) , which has been shown antecedently to do no stomachic malignant neoplastic disease in this theoretical account ( 23 ) . This led to the re-creation of the I”EPIYA strain followed by the creative activity of a new restorant strain. This new restorant strain induced pathology similar to the wild type strain in gerbils six hebdomads after infection ; these animate beings displayed dysplasia and were come oning to stomachic malignant neoplastic disease ( Fig. 23 ) . This consequence combined with all the incompatibilities in the in vitro work suggests that there was a 2nd site mutant within the original I”EPIYA strain ; hence, no correlativities could be made and there was no ground to further qualify these strains.
Since that clip, we have learned that strain 7.13 loses in vivo virulency after low figure of lab transitions ( R. Peek, personal communicating ) . Additionally, other work in our lab has shown drastic in vivo differences for strains that have been idenitically manipulated to cancel the same cistron. Knowing this now, it might hold been prudent to mensurate the mutant rate of 7.13 anterior to get downing our surveies in order to find whether this strain has a higher mutant rate than other strains of H. pylori.
Since H. pylori strain 7.13 was excessively genetically unstable to utilize for these surveies, is at that place a hereafter for this undertaking? A I believe that there is and would suggest the undermentioned possibilities: 1 ) usage strain G27 for in vitro word picture and 2 ) usage strain PMSS1 for both in vitro and in vivo word picture. G27 is our lab ‘s normally used mention strain and has been shown to be reasonably genetically stable. Presently, all of the CagA isogenic strains have been created in the G27 background, and these strains can now be used to find the consequence of the EPIYA motives on host cell signaling. To this terminal, if I was finishing these surveies, I would once more get down by measuring growing dynamicss of these strains, every bit good as analysing look of CagA via Western smudge analysis. The Western smudge analysis should place any differences in CagA look that could perplex future experiments. Following, I would look at interaction of the strains with host cells by mensurating bacterial localisation, attachment, internalisation, CagA phosphorylation, and induced host cell morphological alterations. A Though I would originate these surveies utilizing AGS cells, it might besides be interesting to measure other cell lines: MDCK, T84, and HGE, as they are frequently used to analyze H. pylori. Using the protocols in chapter five as a starting point, each check would necessitate to be optimized for the alteration in H. pylori strain background.
After completion of the basic word picture, transition of host cell signalling tracts could be assessed. This should ab initio be assessed through analyzing of alterations in host cell morphology, as described in chapter five. However, there are some alterations that might be considered. One concern is that we possibly did non of all time achieve the maximum per centum of extended cells in our check ; subsequent smaller experiments showed that the highest per centum of extended cells was observed between 12-18 hours station infection. Additionally, while the per centum of extended cells tapered off, in a individual experiment I conducted, the length of the bulges in extended cells infected with the EPIYA-ABtD strain still continued to increase even at 18 hours. These consequences could bespeak that the EPIYA-ABtD strain may arouse reactions more easy, yet its overall effects may be more drastic. Therefore, it would be wise to research extra clip points, such as 24 and 36 hours post infection, to measure this possiblity. Of class, if these strains take longer to adhere or if they adhere at much lower degrees, so the MOI or clip points studied station infection would hold to be adjusted consequently.
Once the basic charaterization of these strains is complete, transition of specific tracts could be assessed. For illustration, Erk activation could be assessed via Western smudge analysis, as could activation of NF-I?B. Localization of I?-catenin to the karyon could be assessed via microscopy or Western smudge analysis of atomic infusions of septic cell lysates. An alternate option to measure the function of the EPIYA motives in vitro could besides be to make these strains in other mention strains, such as J99, 26695, or HPAG-1.
An extra option for traveling this undertaking frontward, could be the usage of the parental strain ( PMSS1 ) of the mouse derivative Sydney strain 1 ( SS1 ) . PMSS1 has late been characterized ( 10, 47 ) , and colonizes mice, but at a lower degree than SS1 ( 10 ) . PMSS1 encodes for a functional CagA protein, whereas SS1 does non show or present functional CagA ( 10, 71 ) . Not merely does PMSS1 bring forth a functional CagA, but this strain has been shown to do terrible pathology in mice, including wasting, hyperplasia, and metaplasia ( 10 ) . Since our cognition about this strain is limited, and it is known to lose the ability to shoot CagA into host cells after 1 month in vivo ( 10 ) , it might be wise to mensurate the self-generated mutant rate of PMSS1 before once and for all make up one’s minding to utilize it for isogenic strain building. Provided preliminary consequences are satisfactory, new primers to magnify the upstream ( 5 ‘ ) cagA part and the downstream ( 3 ‘ ) cagA part would necessitate to be designed in order to make the concepts needed to bring forth the isogenic strains. Then, in vitro word picture of the freshly constructed strains could continue as described above. Once these checks are completed, a little pilot animate being survey to measure colonisation burden, histology, and the timeline for disease patterned advance should be completed. Overall the use of this strain could supply another option to research the function of the EPIYA motifs non merely in vitro but besides in in vivo checks.
Importance and the Impact of Future Studies
Gastric malignant neoplastic disease is still the 2nd most common cause of malignant neoplastic disease morbidity and mortality, and this could be brooding of the high incidence of H. pylori infection ( 20, 60, 68, 86 ) . It could besides be a consequence of the high prevalence of cagA in many H. pylori strains, or due to the presence of certain CagA polymorphisms that predominate in geographic countries that have high rates of stomachic malignant neoplastic disease ( 2, 18, 20, 27, 29, 45, 86 ) . Due to the fact that we do non yet exhaustively understand the procedure of H. pylori induced pathogenesis, including development of stomachic malignant neoplastic disease, elucidation of virulency factors or virulency factor polymorphisms that impact disease is imperative. Unfortunately, H. pylori is an being that shows a high rate of familial variableness, which makes traditional epidemiological surveies merely good indexs of tendencies. Therefore, in order to clarify the exact function of virulency factors or polymorphisms, it is best to measure differences by making isogenic strains.
The successful creative activity of EPIYA isogenic strains will non merely reply the inquiry as to what function the EPIYA motifs play in disease manifestations, but will open the door to the appraisal of multiple virulency factors. For case, it has already been demonstrated that CagA is a “ maestro ” virulency factor ( 13, 37, 38 ) , and that there is an association between other virulency factors and disease among the different cagA allelomorphs. This is particularly true for different vacA allelomorphs ( 38, 42 ) . Thus, one time the EPIYA isogenic strains are created and characterized, they could so be used as the parental strain background to make isogenic strains incorporating different polymorphous signifiers of other virulency factors. This would let us to non merely measure the function of different virulency factors in disease, but besides their function in disease development within the context of a peculiar cagA allelomorph.
If I were to set about these checks personally, I would concentrate foremost on VacA. As discussed earlier, VacA is polymorphous and different vacA allelomorphs impact disease otherwise ( 15, 37, 48, 58 ) . Therefore, we could make vacA isogenic strains within certain cagA isogenic backgrounds to more conclusively expression at CagA and VacA interaction. Specifically, I would change the vacA allelomorphs within the context of the EPIYA-ABtC, EPIYA-ABtCCC, EPIYA-ABtD and the restorant strains. Experiments with these strains would place any differences between East Asian ( EPIYA-ABtD ) and Western ( EPIYA-ABtC and EPIYA-ABtCCC ) strains. They would besides bespeak if the figure of EPIYA-C motives influences the effects of the different vacA allelomorphs. The restorant strain would supply an of import control for familial use of our strains. These types of surveies would let our lab to non merely analyze the function of the different vacA allelomorphs, but to measure the impact of these allelomorphs within the context of the cagA allelomorph. Furthermore, these types of surveies may besides clear up some of the disagreements in the literature as to the impact of different vacA parts ( 37, 75 ) .
Additionally, I would probably concentrate on two other polymorphous virulency factors that have been implicated in disease development, HomB ( 19, 42, 64 ) and OipA ( 16, 61 ) . The Helicobacter outer membrane ( Hom ) proteins are complex because H. pylori has two venue that can encode for a Hom protein ; strains can encode for either homA, homB, homA/homB, homA/homA, homB/homB, or be negative for both homA and homB ( 19, 42, 64 ) . The presence of homB has been linked to the development of more terrible disease, as compared to the presence of homA ( 19, 42, 64 ) . Additionally, a dose response has been identified for strains encoding for homB/homB ( 62, 63 ) . OipA is an outer membrane protein whose look has been shown to be capable to phase fluctuation due to the figure of CT repetitions found in the oipA signal sequence ( 6 ) . OipA “ on ” is used to denominate a strain that expresses a functional protein ( 16, 61 ) . Again, strains that encode for an OipA protein are associated with more terrible disease results ( 16, 61 ) . Furthermore, the OipA “ on ” phenotype is frequently found in cagA positive strains ( 6 ) . The impact of homB and oipA could be assessed separately or in the context of different virulency factors. In other words, besides making isogenic derived function of these factors within the EPIYA-ABtC, EPIYA-ABtCCC, EPIYA-ABtD and restorant strains to place difference among the different cagA allelomorphs, they could besides be assessed within the EPIYA isogenic strains that carried different vacA allelomorphs. For case, I would probably first measure the EPIYA-ABtC, EPIYA-ABtCCC, EPIYA-ABtD and the restorant strains that were s1/i1/m1 and s2/i2/m2. This would assist restrict the figure of strains, particularly since there are six different hom combinations that could be assessed. This would besides look to be the most likely topographic point to detect differences, since the s1/i1/m1 is the most deadly and the s2/i2/m2 is the least deadly vacA allelomorph. If a difference between these populations was observed, isogenic strains within the different vacA allelomorphs could so be created and assessed. This system would besides let us to consistently analyze these virulency factors in the context of the whole bacteria, and to measure their function in disease patterned advance, including patterned advance to stomachic malignant neoplastic disease. This would besides be a better system to analyse the function of these different virulency factors in the context of geographical differences, which could truly assist spread out the field of H. pylori pathogenesis.
H. pylori is a medically of import pathogen that has successfully challenged the preconceived thought that bacteriums can non do stomachic disease. However, more than a few inquiries remain about this procedure every bit good as what host, environmental, and bacterial factors are of import for the patterned advance to severe disease. This thesis focused on the bacterial toxins, CagA and VacA, and their function in act uponing patterned advance to more terrible disease. To that terminal, we were the first to statistically associate a specific cagA allelomorph ( EPIYA-ABD ) to stomachic malignant neoplastic disease development. We were besides able to analyse this big population of clinical isolates for the function of VacA polymorphisms, and found that while the distribution of vacA allelomorphs was non straight associated with disease province, it was associated with the distribution of cagA allelomorphs and in a tripartite association that included the vacA allelomorph, the cagA allelomorph and disease province. During the class of this work, we identified an amino acid ( 196 ) of import for the development of stomachic malignant neoplastic disease within the intermediate part of VacA. Additionally, we identified some associations that were CagA-dependent, such as the association of VacA and disease province in the EPIYA-ABD population and amino acerb distribution at place 231 and disease province in the non EPIYA-ABD population. Finally, we were able to optimise techniques that will be used in future surveies aimed at qualifying CagA isogenic strains.
While small is presently understood about why some H. pylori infected persons develop stomachic malignant neoplastic disease while others remain symptomless, the informations gathered during the class of this thesis will assist cast some visible radiation on the pathogensis of H. pyloriA-induced disease. Indeed, the elucidation of bacterial factors that are involved in the pathogensis of H. pyloriA-induced disease, such as EPIYA-ABD CagA, is of import, because they can function as a diagnostic marker of infection with a more deadly strain. Understanding any hierarchy of virulency factors is imperative, because more grounds has accumulated that underscores the fact that colonisation with H. pylori is protective against other unwellnesss, including asthma ( 73 ) , TB ( 70 ) and esophageal malignant neoplastic disease ( reviewed in ( 17 ) . Therefore, proposing that intervention should merely be used for persons infected with extremely deadly strains. Such interventions that merely mark patients infected with strains that could do terrible disease would be kindred to geographically individualized intervention, which I believe is the hereafter for handling H. pylori-induced disease. Implementing such location-specific interventions could help in extinguishing a bulk of stomachic malignant neoplastic disease mortality and morbidity worldwide without giving the protective effects provided by infection with H. pylori.