Analysis of the Helicobacter pylori Proteases HP0087 and HP1037 Links HP0087 to Growth and Cell Length Defects

Proteases have been identified as important factors in bacterial pathogenesis that assist in hostile niches and to modulate host-pathogen interactions. The human pathogen Helicobacter pylori (H. pylori) expresses a large number of proteases, of which only some have been characterized so far. The aminopeptidase HP1037 has previously been described as an active protease, while HP0087 still represents a hypothetical protease. Here, we investigated the proteases HP0087 and HP1037, providing initial insights into their proteolytic activities and physiological functions.

We cloned and purified HP0087 and HP1037, generated isogenic H. pylori knockout mutants and complementation strains. Recombinant proteins were used to investigate their proteolytic activities and to produce specific anti-HP1037 and anti-HP0087 antibodies. Bacterial morphology and real-time growth of isogenic H. pylori knockout and knock-in mutants were studied by confocal laser scanning microscopy, STED microscopy, and plate reader assays.

In comparison to HP0087, which was detected as a monomer in a SDS-PAGE under nonreducing conditions, HP1037 formed oligomeric structures. We also identified proteolytic activities of HP0087 and HP1037 using the generic substrate FITC-casein. Finally, a generated H. pylori ΔHP0087 mutant displayed significantly impaired growth and a reduced bacterial cell length, whereas the H. pylori ΔHP1037 mutant did not show significant changes in the phenotype.

In summary, we identified novel proteases expressed by H. pylori, one of which has an impact on bacterial proliferation and cell morphology, providing a basis for dissecting their roles in gastric colonization and disease.