A plot of viable bacterial cell count (CFU/ml) against the OD 600 reading based

Transcription

1 RESULTS

2 4.1 H. pylori and MKN28 cells H. pylori Enumeration of H. pylori A plot of viable bacterial cell count (CFU/ml) against the OD 600 reading based on different dilutions of a 3-day-old H. pylori culture was obtained (Figure 4.1). This standard curve was used in subsequent experiments serving as a means to enumerate H. pylori. Figure 4.1 Standard curve for enumeration of H. pylori Viable cell count (CFU/ml) was plotted against OD 600 for H. pylori strain Data shown are representative of three independent experiments. 97

3 Genotyping of H. pylori The study seeks to screen for the presence of the oncoprotein, CagA, and other known virulence factors using H. pylori as the study model. Genomic DNA extracted from H. pylori was amplified by PCR and analyzed by agarose gel electrophoresis. The result shows that H. pylori harbours caga, vaca, icea1, oipa, hopz and hsp20 genes but lacks icea2, baba2 and saba genes (Figure 4.2). Figure 4.2 Genotyping of H. pylori by PCR Lane M: 100 bp DNA ladder marker; Lane 1: caga (400 bp); Lane 2: vaca (1160 bp); Lane 3: icea1 (246 bp); Lane 4: icea2 (229 bp); Lane 5: baba2 (831 bp); Lane 6: saba (643 bp); Lane 7: oipa gene (457 bp); Lane 8: hopz (611 bp); Lane 9: hsp20 (543 bp). 98

4 4.1.2 MKN28: the ideal gastric cell line for studying apical-junctional complexes Non-polarized AGS cells are commonly used to study aspects of H. pylori pathogenesis, such as cytokine secretion. However, these cells are limited for studying the effects of H. pylori on apical-junctional complexes since AGS cells express only low levels of the adherens junction protein E-cadherin (Jawhari et al., 1999) and do not form tight junctions (Amieva et al., 2003). Phase contrast micrograph of AGS cells (Figure 4.3A) shows that the cells do not form a very tight monolayer. Staining of ZO-1 (tight junction protein) and E-cadherin (adherens junction protein) confirm that AGS cells do not form functional tight junctions (Figure 4.3C) and express minimal levels of E-cadherin (Figure 4.3E). In contrast, polarized MKN28 cells are able to form a tight and closely-packed monolayer under phase contrast observation (Figure 4.3B). Furthermore, MKN28 cells form functional tight junctions as the ZO-1 proteins are localized to the sites of cell-cell contact (Figure 4.3D). MKN28 cells also express high levels of E-cadherin as observed in Figure 4.3F. Therefore, polarized gastric MKN28 cells were chosen as the cell line of choice for subsequent experiments on tight junction disruption. 4.2 Oncoprotein CagA Identification of interacting partner of CagA by Co-IP and MS Anti-CagA antibody was used to pull down potential interaction partners of CagA from the total protein extract of WT H. pylori. A specific band of about 18 kda was immunoprecipitated (Figure 4.4A). The band was excised from the gel and identified as HSP20 by MALDI-TOF MS analysis (NCBI accession number: O25253) (Figure 4.4B). 99

5 Figure 4.3 Differences between uninfected AGS and MKN28 host cells Phase contrast micrographs of (A) AGS and (B) MKN28 cells. Magnification 400. ZO-1 staining of (C) AGS and (D) MKN28 cells. ZO-1 was stained red with antimouse Cy3. Scale bar, 10 μm. E-cadherin staining of (E) AGS and (F) MKN28 cells. E-cadherin was stained green with anti-rabbit Alexa Fluor 488. Scale bar, 10 μm. 100

6 Figure 4.4 HSP20 is an interaction partner of CagA (A) A 18 kda protein was pulled down after WT lysate was subjected to Co-IP using anti-caga antibody. Lane M: prestained protein ladder marker. Lane 1: WT lysate. (B) The 18 kda protein that was pulled down using anti-caga antibody after Co-IP was identified as HSP20 by MALDI-TOF MS analysis. The four matched peptides (11-27 amino acids) with HP0515 are shown in bold red. Data shown are representative of two independent experiments Expression and purification of rcaga The expression of full-length rcaga is problematic because the protein is unstable in recombinant expression systems (Moese et al., 2001). Hence, an immunogenic partial fragment that comprises the first 284 amino acids of the 120 kda CagA protein (Han et al., 2000) was used to construct pet22b(+)-caga852 (Ho, 2006). pet22b(+)-caga852 was expressed in E. coli BL21 cells and confirmed using RE digestion as shown in Figure 4.5A. Digestion of pet22b(+)-caga852 clone 1 101

7 (Lanes 1 and 2) and clone 2 (Lanes 3 and 4) with EcoRI gave a band size of 6.3 kb (Lanes 2 and 4 respectively) while digestion with NdeI and BamHI gave two bands of 5.5 kb and 852 bp (Lanes 1 and 3 respectively). This indicates that the immunogenic caga partial fragment has been successfully inserted into the pet-22b(+) expression vector. The maximum expression level of rcaga was achieved at 3 hours postinduction with 1.0 mm IPTG (Figure 4.5B). rcaga with a molecular weight of ~40 kda existed mainly in the inclusion body after sonication (Figure 4.5C, Lane 2). Purified rcaga was eluted from the His-tag affinity chromatography column using an imidazole concentration gradient range of M in the presence of 6 M urea (Figure 4.5D). Immunoblot analysis with anti-caga antibody was able to detect the purified rcaga band at ~40 kda (Figure 4.5E). The purified rcaga was used to further confirm the interaction between HSP20 and CagA by SPR (section 4.3.4) Detection of T4SS using SEM The presence of the cagpai confers the gastric pathogen the capability to express and translocate the CagA oncoprotein into host cells through the T4SS, which is encoded by genes within the cagpai (Akopyants et al., 1998). SEM was used to check for the presence of T4SS in WT and ΔcagPAI H. pylori. T4SS (represented by white arrows) were observed in WT (Figures 4.6, A and B) but were not present in ΔcagPAI (Figures 4.6, C and D) in the absence of host cells. The absence of T4SS is as expected since the entire cagpai no longer exists in the genome. Similarly, in the presence of host cells, T4SS (represented by white arrows) were observed for WT (Figure 4.7A) but not in ΔcagPAI (Figure 4.7B). This implies that T4SS are already assembled in H. pylori and that its assembly is not induced by the host cells. 102

8 Figure 4.5 Expression and purification of rcaga (A) Restriction enzyme digest of pet22b(+)-caga852. Lane M: 1 kb DNA ladder marker; Lanes 1 and 3: pet22b(+)-caga852 digested with NdeI and BamHI; Lanes 2 and 4: pet22b(+)-caga852 digested with EcoRI; Lane M : 100 bp DNA ladder marker. (B) Expression of rcaga by pet22b(+)-caga852 in E.coli BL21 with 1 mm IPTG induction for 3 hours. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lane 1: uninduced supernatant; Lane 2: uninduced pellet; Lane 3: induced supernatant; Lane 4: induced pellet. (C) Presence of induced rcaga after sonication of E. coli host cells. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lane 1: supernatant after sonication; Lane 2: pellet after sonication. (D) rcaga was purified by His-tag affinity chromatography. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lane 1: induced rcaga pellet; Lanes 2-4: flow-through fractions; Lane 5: wash fraction; Lanes 6-14: eluted fractions. (E) Immunoblot analysis with anti-caga antibody to detect rcaga. Lane M: prestained protein ladder marker; Lane 1: purified rcaga. 103

9 Figure 4.6 Presence of T4SS in H. pylori in the absence of host cells under SEM (A and B) White arrows depict T4SS observed for WT. (C and D) No T4SS were observed in ΔcagPAI. 104

10 Figure 4.7 Presence of T4SS in H. pylori in the presence of host cells under SEM (A) White arrows depict T4SS observed for WT upon infection of MKN28 cells. (B) No T4SS were observed in ΔcagPAI upon infection of MKN28 cells. 105

11 4.3 Characterization of HSP Cloning, expression and purification of rhsp Construction of pet16b-hsp20 Full-length hsp20 (543 bp) was cloned into pet-16b expression vector (Figure 3.5) along with a 10 amino acids His-tag at the N-terminal as depicted in Figure 4.8A. The full-length hsp20 gene of H. pylori was first amplified by PCR (Figure 4.8B, Lane 1) and inserted into pet-16b at the BamHI RE site that was fused downstream of the His-tag to obtain pet16b-hsp20. The correct pet16b-hsp20 plasmid was confirmed using RE digestion as shown in Figure 4.8C. Upon digestion with XbaI, a band size of 6.2 kb (Lane 4) was obtained for pet16b-hsp20 compared to a band size of 5.7 kb (Lane 3) for pet-16b alone. To further confirm this, digesting pet16b-hsp20 with BamHI gave two bands of 5.7 kb and 543 bp (Lane 6) as compared to the single 5.7 kb band for BamHI-digested pet-16b vector alone (Lane 5). This implies that the hsp20 gene has been successfully inserted into the pet-16b expression vector. 106

12 Figure 4.8 Construction and identification of pet16b-hsp20 (A) Diagrammatic representation of pet16b-hsp20 (X: XhoI site, B: BamHI site and S:SspI site). (B) PCR amplification of hsp20 gene using H. pylori genomic DNA as template. Lane M: 100 bp DNA ladder marker; Lane 1: hsp20 target gene (543 bp). (C) RE digest of pet-16b and pet16b-hsp20. Lane M: 1 kb DNA ladder marker; Lane 1: uncut pet-16b; Lane 2: uncut pet16b-hsp20; Lane 3: pet-16b digested with XbaI; Lane 4: pet16b-hsp20 digested with XbaI; Lane 5: pet-16b digested with BamHI; Lane 6: pet16b-hsp20 digested with BamHI Expression and purification of rhsp20 Induction of E. coli BL21 cells harbouring pet16b-hsp20 shows that the optimum expression level of rhsp20 was achieved at 4 hours post-induction with 0.6 mm IPTG (Figure 4.9A, Lane 6). As observed from Figure 4.9A, the molecular weight of the expressed rhsp20 protein is ~23 kda. rhsp20 was found mainly in the inclusion body as shown in the pet16b-hsp20 clone after sonication (Figure 4.9B, Lane 2) and the pellet was subsequently dissolved in 6 M urea-binding buffer for His- 107

13 tag affinity purification. Purified rhsp20 was eluted from the His-tag affinity chromatography column using an imidazole concentration gradient range of M in the presence of 6 M urea (Figure 4.9C). Silver staining was also done to check the purity of the purified rhsp20 (Figure 4.9D). Polyclonal anti-hsp20 antibody that was raised in section was then used to detect the purified rhsp20 (Figure 4.9E). Using MALDI-TOF MS analysis, rhsp20 was identified as HSP20 (HP0515) of H. pylori (Figure 4.10). rhsp20 was also passed through an EndoBind-R column to remove LPS and LPS-associated molecules. From Figure 4.11A, rhsp20 was mainly eluted in the second fraction (Lane 2) out of a total of five elute fractions collected. Using PyroGene LAL assay kit, a standard curve (Figure 4.11B) was generated. Based on the standard curve, the amount of endotoxin present in rhsp20 was found to be EU/ml. The concentration of endotoxin showed a reduction of 1900-fold to 0.02 EU/ml after passage through the column (EndoBind-R ). 108

14 Figure 4.9 Expression and purification of rhsp20 (A) Optimization for rhsp20 expression with various concentrations of IPTG. SDS- PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lane 1: E. coli BL21; Lane 2: BL21 + pet-16b; Lane 3: uninduced pet16b-hsp20 clone; pet16b-hsp20 clone induced with IPTG at various concentrations for 4 hours - Lane 4: 0.2 mm, Lane 5: 0.4 mm; Lane 6: 0.6 mm; Lane 7: 0.8 mm, Lane 8: 1.0 mm. (B) Presence of induced rhsp20 after sonication of E. coli host cells. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lane 1: supernatant after sonication; Lane 2: pellet after sonication. (C) rhsp20 was purified by His-tag affinity chromatography. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lanes 1-4: flow-through fractions; Lanes 5-6: wash fractions; Lanes 7-10: eluted fractions. (D) Silver-stained SDS-PAGE gel confirming eluted rhsp20 (Lane 1) is free from contaminating proteins. Lane M: prestained protein ladder marker. (E) Immunoblot analysis with anti-hsp20 antibody to detect rhsp20. Lane M: prestained protein ladder marker; Lane 1: purified rhsp

15 Figure 4.10 Identification of purified rhsp20 using MALDI-TOF analysis The purified recombinant protein was identified as HSP20 of H. pylori The three matched peptides (14-26 amino acids) with HP0515 are shown in bold red. Figure 4.11 Removal of endotoxin from rhsp20 (A) rhsp20 was eluted in the second fraction after passing the sample through the EndoBind-R column. SDS-PAGE gel stained with Coomassie blue. Lane M: prestained protein ladder marker; Lanes 1-5: fractions of rhsp20 eluted. (B) A standard curve was generated using PyroGene LAL assay kit and the level of endotoxin present in rhsp20 was reduced by 1900-fold. Data shown are representative of two independent experiments. 110

16 4.3.2 Raising and purification of antibody against rhsp20 The antibody titre against rhsp20 raised in a female New Zealand rabbit immunized with 150 g of antigen over a 13 week period was screened using indirect ELISA as shown in Figure 4.12A. The antibody titer increased 28 days postimmunization and reached a peak at day 71. Immunoblot analysis of the polyclonal HSP20 antibody raised showed that the pre-immune serum did not have antibody against rhsp20 (Figure 4.12B) whereas the final bleed serum was able to detect the HSP20 band in the WT lysate (Lane 1) but not the Δhsp20 lysate (Lane 2), which acts as the negative control (Figure 4.12C). After purification, the serum shows the presence of heavy chain (55 kda) and light chain (25 kda) of the rabbit IgG only as confirmed by the Coomassie blue-stained SDS-PAGE gel (Figure 4.12D) Construction of Δhsp Construction of the gene-targeting construct The construction of Δhsp20 involves constructing the gene-targeting construct as well as natural transformation of H. pylori with the correct construct. The gene-targeting construct was obtained as described in section by which the kanamycin resistance cassette replaced the entire hsp20 gene. This was done by inserting the kanamycin resistance gene in between the upstream and downstream genes of hsp20 (Figure 3.1). Using genomic DNA from H. pylori as a template, two different PCR products were obtained with two different sets of primers (Table 3.3). The kanamycin resistance cassette was obtained by performing a PCR reaction (Figure 4.13, Lane 1) with the pill600 plasmid as template (Ferrero et al., 1992). The presence of the three PCR products was confirmed by agarose gel electrophoresis (Figure 4.13). 111

17 Figure 4.12 Raising and purification of antibody against rhsp20 (A) Antibody production profile. Antibody titer against rhsp20 collected at different time-points. The graph was plotted based on values of 1:800 diluted sera. (B and C) Specificity of the polyclonal antibody against rhsp20 checked using immunoblot analysis. B: Pre-immune serum. C: Final bleed serum. Lane M: prestained protein ladder marker; Lane 1: WT lysate; Lane 2: Δhsp20 lysate. (D) Coomassie blue-stained SDS-PAGE gel showing purified IgG from immunized rabbit. Lane M: prestained protein ladder marker; Lane 1: purified IgG against rhsp

18 The two primers, hsp20kanr2 and hsp20kanf, consist of a 5 leader that are complementary to kanf and aphar and a gene specific sequence of 22 bp and 25 bp respectively. Thus, the resulting PCR products (Figure 4.13, Lanes 2 and 3) have sequences complementary to that of the kanamycin resistance cassette. In this way, the three purified PCR products (Figure 4.13, Lanes 1-3) obtained were ligated to obtain the final gene-targeting construct of 2522 bp by performing another PCR reaction (Table 3.4). The final gene-targeting construct was confirmed by agarose gel electrophoresis as shown in Figure 4.13 (Lane 4). Figure 4.13 Generation of hsp20 gene-targeting construct Lane M: 1 kb DNA ladder marker; Lane 1: kanamycin resistance cassette (1402 bp); Lane 2: fragment of hsp20 upstream region (564 bp); Lane 3: fragment of hsp20 downstream region (556 bp); Lane 4: final hsp20 knockout construct (2522 bp); Lane M : 100 bp DNA ladder marker. 113

19 Identification and confirmation of various isogenic mutants of H. pylori The hsp20 knockout construct (obtained in Section ) was transformed into WT H. pylori to obtain Δhsp20. ΔcagA, ΔcagPAI and Δhsp20/ΔcagA H. pylori were constructed earlier by Dr. S.Y. Lui of our lab. Genomic DNA of each respective isogenic mutant was extracted and analyzed by PCR to check for the presence of hsp20 or caga. Figure 4.14A shows that WT possesses both hsp20 (Lane 1) and caga (Lane 2) while Δhsp20 only harbours caga (Lane 4). Both ΔcagA (Lane 5) and ΔcagPAI (Lane 7) possess hsp20 only. As expected, both hsp20 and caga were absent in Δhsp20/ΔcagA (Lanes 9 and 10). To further confirm the expression of HSP20 and CagA, total cell lysates of the WT as well as the various H. pylori mutants were obtained and probed with anti-hsp20 and anti-caga antibodies. Figure 4.14B shows that WT expresses both HSP20 and CagA (Lane 1) while Δhsp20 only expresses CagA (Lane 2) but not HSP20. Both ΔcagA (Lane 3) and ΔcagPAI (Lane 4) expressed HSP20 only. As expected, Δhsp20/ΔcagA expresses neither HSP20 nor CagA (Lane 5). 114

20 Figure 4.14 Identification and confirmation of various H. pylori isogenic mutants (A) Detecting the presence of hsp20 (543 bp) and caga (400 bp) by PCR. Genomic DNAs of various H. pylori strains were used as templates for PCR using the respective primers for hsp20 and caga. Lane M: 100 bp DNA ladder marker; Lanes 1 and 2: WT; Lanes 3 and 4: Δhsp20; Lanes 5 and 6: ΔcagA; Lanes 7 and 8: ΔcagPAI; Lanes 9 and 10: Δhsp20/ΔcagA. (B) Immunoblot analysis for CagA (120 kda) and HSP20 (20 kda) from total cell lysates of H. pylori WT and various isogenic mutants. Lane 1: WT; Lane 2: Δhsp20; Lane 3: ΔcagA; Lane 4: ΔcagPAI; Lane 5: Δhsp20/ΔcagA Confirmation of the interaction between HSP20 and CagA using immunoblot analysis and SPR HSP20 was only detected in the WT lysate but not in lysates of Δhsp20 and ΔcagA obtained from Co-IP (section 4.2.1) using the anti-hsp20 antibody that was raised previously (section 4.3.2). This further confirms the interaction between HSP20 and CagA. The absence of HSP20 in Δhsp20 and ΔcagA lysates acts as negative controls (Figure 4.15A). SPR analysis between rhsp20 and rcaga (purified in sections and respectively) gave a K D value of M (Figure 4.15B) indicating a reasonable interaction despite using an immunogenic partial 115

21 fragment that comprises the first 284 amino acids of the 120 kda CagA protein (Han et al., 2000; Ho, 2006). Figure 4.15 Confirmation of the interaction between HSP20 and CagA (A) Co-IP: Anti-CagA antibody was used to pull down potential interaction partners of CagA from the total protein lysates of indicated H. pylori strains. Total protein lysates of indicated H. pylori strains were then probed with antibodies against HSP20 (20 kda) or CagA (120 kda). Top panel: HSP20 was detected when using WT lysate but not when using Δhsp20 and ΔcagA lysates. Bottom panel: CagA was detected when using both the WT and Δhsp20 lysates but not when using ΔcagA lysate. (B) Sensogram showing kinetic analysis of rcaga and rhsp20. The partial fragment (first 284 amino acids) of rcaga was used as the analyte at a range of indicated concentrations to show the association and dissociation from immobilized rhsp20 on CM5 sensor chip surface. SPR was performed as described to give a K D value of 9.11x10 5 M. RU, response units. Data shown are representative of two independent experiments. 116

22 4.4 Localization of HSP20 and CagA within H. pylori using immuno-gold labeling TEM In the absence of host cells Since HSP20 was identified to be an interacting partner of CagA using Co-IP and SPR analysis (Figure 4.15), immuno-gold labeling TEM was employed to examine the interaction between these two proteins of interest. In the absence of host cells, HSP20 (represented by 10 nm gold particles) preferentially localized on the cell surface of the bacterium while CagA (represented by 20 nm gold particles) was localized in the cytoplasm (Figure 4.16A) for the WT H. pylori strain. Similarly, only CagA was found in the cytoplasm of Δhsp20 (Figure 4.16B) while only HSP20 was mainly localized at the cell surface of ΔcagA (Figure 4.16C). As Δhsp20/ΔcagA does not possess hsp20 and caga genes, both HSP20 and CagA were absent in the ultrathin sections (Figure 4.16D). T4SS (represented by green arrows) were also observed under TEM and HSP20 (represented by 10 nm gold particles) was found to be localized near the proximal end of the T4SS (Figure 4.17). 117

23 118

24 Figure 4.16 Localization of HSP20 and CagA within H. pylori in the absence of host cells under TEM Ultrathin sections of H. pylori were double-labeled with immuno-gold particles of size 10 nm (HSP20 ) or 20 nm (CagA ). (A) CagA is found in the cytoplasm while HSP20 is on the surface of WT. (B) Only CagA is localized in the cytoplasm in Δhsp20. (C) Only HSP20 is found on the cell surface in ΔcagA. (D) Both HSP20 and CagA are absent in Δhsp20/ΔcagA. 119

25 Figure 4.17 Presence of T4SS in H. pylori under TEM Ultrathin sections of WT H. pylori were labeled with immuno-gold particles of size 10 nm (HSP20 ). Green arrows ( ) are pointing to T4SS observed under TEM In the presence of host cells It has been reported that CagA needs to be first membrane-presented before its translocation into the host cells (Covacci et al., 1993). As HSP20 was found to interact with CagA, it was postulated that HSP20 could play a role in facilitating the membrane-presentation of CagA prior to its translocation since majority of HSPs are chaperonic in nature. Interestingly, HSP20 and CagA were observed to co-localize near the surface of WT H. pylori in the presence of host cells (Figures 4.18, A and B), lending support to its chaperonic role in the membrane-presentation of CagA. 120

26 Figure 4.18 Localization of HSP20 and CagA in WT H. pylori in the presence of host cells under TEM Ultrathin sections of WT H. pylori were double-labeled with immuno-gold particles of size 10 nm (HSP20 ) or 20 nm (CagA ). (A and B) HSP20 and CagA colocalize near the surface of H. pylori. 121

27 4.5 Host-pathogen interactions Intercellular leakage upon H. pylori infection Optimum infection period Solute leakage may be attributed to cells dying and the disintegration of the cell monolayer instead of disruption of the tight junction complex. In barrier function test, solute leakage is captured in the apical chamber of the Transwell filter apparatus (Figure 3.8). In order to reduce the possibility of leaked biotinylated albumin into the apical chamber due to spontaneous cell death, the viability of H. pylori-infected MKN28 cells was determined using MTT assay. Figure 4.19 shows that there was a gradual decrease of cell viability with increasing length of infection time. Close to 70% of MKN28 cells were still viable 48 post-infection by WT H. pylori. Hence, 48 hours post-infection was chosen as the maximum period of infection for MKN28 cells in barrier function test. Figure 4.19 Cell viability reflecting optimum infection time for barrier function test MKN28 cells were infected with WT for the indicated number of hours. Data shown are representative of two independent experiments. 122

28 Detection of barrier function disruption by conducting barrier function test Figure 4.20 shows that in uninfected MKN28 monolayers, tight junctions were deemed functional as judged by the exclusion of basolaterally-applied biotinylated albumin in the apical chamber. The findings show that MKN28 cells infected with WT, ΔcagA or ΔcagPAI H. pylori strains resulted in 3.6-fold more leakage of biotinylated albumin into the apical chamber as compared to Δhsp20 and Δhsp20/ΔcagA mutants as analyzed using ImageJ software version 1.44i. The amount of biotinylated albumin diffusing from the basolateral chamber to the apical chamber also increased for all H. pylori strains tested from 4 hours to 48 hours post-infection (Figure 4.20). Interestingly, when 6 μg/ml of rhsp20 was supplemented exogenously to Δhsp20-infected and Δhsp20/ΔcagA-infected MKN28 monolayer, the extent of albumin leakage was restored to a level similar to that of WT-infected cells within 4 hours (Figure 4.21). It was therefore of no surprise that co-incubation of exogenous rhsp20 with WT, ΔcagA or ΔcagPAI caused an exacerbated leakage of albumin as compared to that induced by the respective bacterial strains alone (Figure 4.21) Visualization of barrier function disruption using SEM SEM micrograph reveals that cell-cell junctions of WT-infected MKN28 monolayer were disrupted 4 hours post-infection (Figure 4.22A). Cracks were observed as cell-cell contacts were disrupted and this could account for the leakage of biotinylated albumin from the basolateral to the apical chamber as shown in Figures 4.20 and In contrast, cell-cell contacts were still functional after 4 hours of infection by Δhsp20 (Figure 4.22B) and barrier function of the monolayer was mainly intact. This could be the reason why the amount of albumin leakage for the WT- 123

29 infected monolayer was increased 3.6-fold compared to the Δhsp20-infected MKN28 monolayer as analyzed using ImageJ software version 1.44i (Figure 4.20). Figure 4.20 Barrier function test of H. pylori-infected MKN28 cells Immunoblot analysis of diffused biotinylated albumin in the apical chambers of confluent MKN28 monolayer cultures grown on Transwell filters infected with either WT or respective isogenic H. pylori mutants. The amount of diffused biotinylated albumin was detected with a streptavidin-hrp probe and densitometric quantification of the corresponding bands was performed using ImageJ version 1.44i analysis software. Data shown are representative of two independent experiments. 124

30 Figure 4.21 Barrier function test of H. pylori-infected MKN28 cells supplemented with 6 μg/ml of rhsp20 Immunoblot analysis of diffused biotinylated albumin in the apical chambers of confluent MKN28 monolayer cultures grown on Transwell filters. These co-cultures were exogenously supplemented with 6 μg/ml of rhsp20 in the apical chamber upon infection with either WT or respective isogenic H. pylori mutants. The amount of diffused biotinylated albumin was detected with a streptavidin-hrp probe and densitometric quantification of the corresponding bands was performed using ImageJ version 1.44i analysis software. Data shown are representative of two independent experiments. 125

31 Figure 4.22 SEM of MKN28 monolayer infected with H. pylori Micrographs of (A) WT-infected and (B) Δhsp20-infected MKN28 monolayer at 4 hours post-infection. 126