Amino-Terminal Processing of Helicobacter pylori Serine Protease HtrA: Role in Oligomerization and Activity Regulation

Abstract

The HtrA family of serine proteases is found in most bacteria, and plays an essential role in the virulence of the gastric pathogen Helicobacter pylori. Secreted H. pylori HtrA (HtrAHp) cleaves various junctional proteins such as E-cadherin disrupting the epithelial barrier, which is crucial for bacterial transmigration across the polarized epithelium. Recent studies indicated the presence of two characteristic HtrAHp forms of 55 and 52 kDa (termed p55 and p52, respectively), in worldwide strains. In addition, p55 and p52 were produced by recombinant HtrAHp, indicating auto-cleavage. However, the cleavage sites and their functional importance are yet unclear. Here, we determined the amino-terminal ends of p55 and p52 by Edman sequencing. Two proteolytic cleavage sites were identified (H46/D47 and K50/D51). Remarkably, the cleavage site sequences are conserved in HtrAHp from worldwide isolates, but not in other Gram-negative pathogens, suggesting a highly specific assignment in H. pylori. We analyzed the role of the amino-terminal cleavage sites on activity, secretion and function of HtrAHp. Three-dimensional modeling suggested a trimeric structure and a role of amino-terminal processing in oligomerization and regulation of proteolytic activity of HtrAHp. Furthermore, point and deletion mutants of these processing sites were generated in the recently reported Campylobacter jejuni ΔhtrA/htrAHp genetic complementation system and the minimal sequence requirements for processing were determined. Polarized Caco-2 epithelial cells were infected with these strains and analyzed by immunofluorescence microscopy. The results indicated that HtrAHp processing strongly affected the ability of the protease to disrupt the E-cadherin-based cell-to-cell junctions. Casein zymography confirmed that the amino-terminal region is required for maintaining the proteolytic activity of HtrAHp. Furthermore, we demonstrated that this cleavage influences the secretion of HtrAHp in the extracellular space as an important prerequisite for its virulence activity. Taken together, our data demonstrate that amino-terminal cleavage of HtrAHp is conserved in this pathogen and affects oligomerization and thus, secretion and regulatory activities, suggesting an important role in the pathogenesis of H. pylori.

How many protein molecules are secreted by single Helicobacter pylori cells: Quantification of serine protease HtrA

“…Western blotting was performed onto polyvinylidene difluoride membranes (Carl Roth) by standard procedures, after the membranes were blocked with 5% milk powder in 0.2 M of Tris, 1.4 M of NaCl, 1% Tween at 4°C overnight. HtrA was detected by incubation with primary rabbit antibody α‐HtrA as previously described (Albrecht et al, ), incubated for 2 hr at room temperature, followed by incubation with horseradish peroxidase‐conjugated secondary antibody for 1 hr. The membrane was also incubated with α‐VacA and α‐FlaA antibodies as controls (Austral Biologicals, San Ramon/USA).…”

“…pylori G27 bacteria were collected from agar plates and resuspended in prewarmed brain heart infusion medium (Oxoid) using sterile cotton swabs (Carl Roth, Karlsruhe/Germany) to OD 600 = 0.3 to start the HtrA secretion assay. primary rabbit antibody α-HtrA as previously described (Albrecht et al, 2018), incubated for 2 hr at room temperature, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hr. The membrane was also incubated with α-VacA and α-FlaA antibodies as controls (Austral Biologicals, San Ramon/USA).…”

Pathogenesis of Helicobacter pylori infection

“…The amino-terminal variations may affect the oligomerization, secretion, and regulatory activities. 23 The overexpression of HtrA did not affect the expression of other virulence proteins, such as VacA and γ-glutamyl-transpeptidase (GGT). 22 A structural similarity analysis using a DALI server, a protein structure comparison server, 24 showed that Lpp20 is very similar to the TNFα inducing protein (Tipα), with an additional short α-helix at the N-terminal.…”

“…In addition, the HtrA activity was affected by the variation on the amino‐terminal between H46/D47 and K50/D51. The amino‐terminal variations may affect the oligomerization, secretion, and regulatory activities . The overexpression of HtrA did not affect the expression of other virulence proteins, such as VacA and γ‐glutamyl‐transpeptidase (GGT) …”

Function of serine protease HtrA in the lifecycle of the foodborne pathogen Campylobacter jejuni

“…HtrA undergoes oligomerization and conformational changes when encountering denatured proteins, which switches on its proteolytic activity. These oligomerization and configuration processes have been studied in detail for HtrA homologs from a number of organisms, such as Thermotoga maritima [ 57 ], E. coli [ 58 , 59 ], Legionella pneumophila [ 60 ] and Helicobacter pylori [ 61 ]. Bacterial HtrAs are predominantly localized in the periplasm and de novo protein typically contains an N-terminal signal peptide that must be removed during transport across the inner membrane before the protein becomes active.…”