Helicobacter pylori γ-glutamyltransferase is linked to proteomic adaptions important for colonization

Abstract

Helicobacter pylori γ-glutamyltransferase (gGT) is a virulence factor that promotes bacterial colonization and immune tolerance. Although some studies addressed potential functional mechanisms, the supportive role of gGT for in vivo colonization remains unclear. Additionally, it is unknown how different gGT expression levels may lead to compensatory mechanisms ensuring infection and persistence. Hence, it is crucial to unravel the in vivo function of gGT. We assessed acid survival under conditions mimicking the human gastric fluid and elevated the pH in the murine stomach prior to H. pylori infection to link gGT-mediated acid resistance to colonization. By comparing proteomes of gGT-proficient and -deficient isolates before and after infecting mice, we investigated proteomic adaptations of gGT-deficient bacteria during infection. Our data indicate that gGT is crucial to sustain urease activity in acidic environments, thereby supporting survival and successful colonization. Absence of gGT triggers expression of proteins involved in the nitrogen and iron metabolism and boosts the expression of adhesins and flagellar proteins during infection, resulting in increased motility and adhesion capacity. In summary, gGT-dependent mechanisms confer a growth advantage to the bacterium in the gastric environment, which renders gGT a valuable target for the development of new treatments against H. pylori infection.

Keywords: Gamma-glutamyltransferase; Helicobacter pylori; acid resistance; bacterial metabolism; colonization.

Figure 1.

Establishment and characterization of inducible tet-on gGT H. pylori strains (a) Graphic representation of the tet-on gGT system. The ggt promoter was replaced by the tet inducible promoter tetO1 (P_tetO1). TetR was introduced in the H. pylori genome and is controlled by a flaA promoter (P_FlaA). TetR binds to tetO1 blocking expression of gGT. The inducer anhydrotetracycline (ATc) binds to tetR resulting in disassociation of the repressor and activation of ggt expression. (b) gGT-activity of conditional gGT mutants. Bacteria were cultured in BHI/10% FCS in the presence of 100 ng/ml ATc for 24 h. H. pylori g:O1 t has tetO1 and tetR integrated into its genome. In H. pylori g:O1 C only the ggt promoter was replaced by tetO1, n = 3–6. Bars represent the mean of three to six independent experiments as represented by single dots. Error bars represent the standard deviation. n: number of independent experiments. Mann-Whitney U-test. *p < 0.05.

Figure 2.

H. pylori gGT contributes to initial colonization and persistence colony forming units (CFU) in the stomach (mg) of C57BL/6 mice after infection with PMSS1 wt, ∆ggt or g:O1 C. Mice were inoculated with 2 × 10⁸ bacteria twice and were sacrificed after a defined amount of time (1 week −6 months) after the first infection. Colonization was assessed by plating stomach homogenates. The detection limit is indicated by a dotted line. Each dot represents one individual mouse. PMSS1 wt (n = 15–27), PMSS1 ∆ggt (n = 8–32), PMSS1 g:O1 C (n = 12), n: number of mice. Mann-Whitney U-test. ****p < 0.01.

Figure 3.

H. pylori gGT contributes to acid survival supporting stomach colonization (a) Survival of H. pylori in McIlvaine buffer (pH 3) supplemented with 8 mm urea, n = 3. The detection limit of the assay is indicated by a dotted line. (b) Correlation of survival of clinical isolates after 2.5 h with gGT-activity. Survival was determined in McIlvaine buffer (pH 3) supplemented with 8 mm urea. Each dot represents the mean survival of one clinical isolate, n = 3. (c) Survival of H. pylori in fasted state simulated gastric fluid (FaSSGF) supplemented with 8 mm urea. The detection limit of the assay is indicated by a dotted line., n = 4. (d) Growth of H. pylori in Brucella broth supplemented with 20% FCS at pH 5 normalized to the growth in Brucella broth at pH 7. Growth was quantified spectrophotometrically after 30 h. n = 3. (e) Urease activity after exposure of H. pylori to pH 5. Strains were grown in BHI with 10% FCS, resuspended for 2 h in McIlvaine buffer (pH 5) and lysed for the detection of urease activity. Data was normalized to the mean activity of H. pylori wt or tet-on H. pylori g:O1 t. n = 3. (f) Correlation of urease- and gGT-activity of clinical isolates. Each dot represents the mean activity of an isolate. n = 3. (g, h) Colony forming units (CFU) in the stomach (mg) of C57BL/6 mice after infection with PMSS1 wt or ∆ggt. Mice were inoculated with 2 × 10⁸ bacteria. Colonization was assessed by plating stomach homogenates. The detection limit is indicated by a dotted line. Each dot represents one individual mouse. (g) CFU/mg after 1 week of infection. Two groups of mice received sodium bicarbonate (NaHCO₃) before the infection to elevate the pH in the stomach. (h) CFU/mg in different stomach sections (F: forestomach, S1–S3: stomach sections) after infection for 2 weeks. Colonization was normalized to the mean CFU/mg across sections S1–S3 for each mouse. n: number of independent experiments. Pearson’s correlation (b, f); Mann-Whitney U-test (c, g, h); ANOVA (d, e) *p < 0.05, **p < 0.01.

Figure 4.

H. pylori gGT promotes survival in iron-restricted environments (a) Log₂ transformed fold change of proteins potentially involved in metal acquisition comparing the proteome of PMSS1 wt and ∆ggt. The proteomes of three single clones isolated after 1 week, 1 month, and 3 months of infection from different mice were determined. Significant differences (-log₁₀(p-value) > 1.3 and log₂(fold change) ≥ I1I) are marked with gray boxes. Missing data points are colored white and are marked with a cross. (b) Schematic representation of the poly(a)tract in the fecA3 upstream region between positions − 181 and − 163 before the translation start. The number of adenine bases in the tract was quantified for all re-isolates (RI) and for the aliquot used for infection (BI) (c) Growth of H. pylori in iron-restricted medium. Deferoxamine mesylate (DFO) was added to the culture in different concentrations (20 µm, 50 µm, and 75 µm). Growth was monitored spectrophotometrically and was normalized to the growth without supplementation. Bars represent the mean of three to four independent experiments as indicated by dots. Error bars represent the standard deviation. (d) Correlation of growth in iron-limited conditions with gGT-activity. Each dot represents the mean growth of one clinical isolate as measured in three independent experiments. Clinical isolates were grown in the presence of 50 µm DFO and growth data were normalized to the growth without supplementation. (e) Reduction of iron(iii)citrate to Fe²⁺ in the presence of varying concentrations of Cys-Gly or GSH. As a positive control (+) 1 M ascorbic acid was added to fully reduce the available iron. As a negative control (-) Fe²⁺ concentrations were measured in a Fe(III) citrate solution. (f) Uptake of iron(iii)citrate into H. pylori in the presence and absence of 2 mm GSH or Cys-Gly. Students t-test (a, c, e, f). ANOVA (c); Pearson’s correlation (d). *p < 0.05.

Figure 5.

H. pylori’s motility and adhesion capacity is increased in the absence of gGT during infection (a) Log₂ transformed fold change of flagellar proteins and adhesion factors comparing the proteome of PMSS1 wt and ∆ggt. The proteomes of three single clones isolated after 1 week, 1 month, and 3 months of infection from different mice were determined. Significant differences (-log₁₀(p-value) > 1.3 and log₂(fold change) ≥ I1I) are marked with gray boxes. Missing data points are colored white and are marked with a cross. Values out of the range of the heat map are colored gray. (b) Schematic representation of the genomic organization of the hpaA locus as determined by whole genome sequencing. ins: insertion, del: deletion. ORF: open reading frame, BI: before infection, RI: re-isolate (c,d) Flagellar motility of H. pylori strains in the presence and absence of gGT. Soft agar assays on Brucella/10% FCS plates were performed. 100 ng/ml ATc was added to the plates to switch on gGT-activity in inducible strains. gGT-activity was not induced in the pre-culture prepared for the experiment. Motility of deletion strains and inducible strains was determined (c) as well as motility before (BI) and after infection (AI) (D). Bars represent the mean of 8 to 14 independent experiments as indicated by single dots (C) or the mean motility of 4 to 5 isolates as measured in three independent experiments (d). (e) Correlation of flagellar motility of clinical isolates with gGT-activity. Each dot represents the mean motility of an isolate as determined by 3 independent experiments (f) Adhesion capacity of H. pylori before infection (BI) and after infection (AI) to AGS cells. Bars represent the mean of three independent experiments. MFI is the median fluorescence intensity on the PE channel. Error bars represent the standard deviation. Student’s t-test (a, c, f). ANOVA (d), Pearson’s correlation (e). **p < 0.01, ****p < 0.0001.