Analysis of the Helicobacter pylori proteases HP0087 and HP1037 links HP0087 to growth and cell length defects

Authors:

Miroslaw Jarzab; Saruchi Wadhwa; Christian Daniel Ferner; Sebastian Diechler; Gernot Posselt, Silja Wessler

Keywords:

Helicobacter pylori; protease; bacterial proliferation; hostile niches; host-pathogen interactions

Abstract:

Introduction: 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.

Methods: 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 knockin mutants were studied by confocal laser scanning microscopy, STED microscopy and plate reader assays.

Results: In comparison to HP0087, which was detected as a monomer in a SDS PAGE under non-reducing 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.

Conclusion: 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.

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