Characterization of <i>Helicobacter pylori</i> aggregation reveals a requirement for both AlpA and AlpB

ABSTRACT Helicobacter pylori is a gram-negative human pathogen that colonizes the stomach and is a major risk factor for gastric ulcers and cancer. A common but poorly understood characteristic of H. pylori is its propensity to aggregate in liquid culture. To investigate this phenomenon, we developed protocols to form, measure, and disperse aggregates. Using these tools, we determined that H. pylori aggregation is protein-dependent and can occur non-clonally, from the binding of distinct cells. Motility, flagella, quorum sensing, and exogenous host proteins were not necessary for aggregation, but the outer membrane proteins AlpA and AlpB were. H. pylori lacking either alpA , alpB , or both were unable to form aggregates. While bacterial aggregation often confers tolerance to antibiotics, H. pylori aggregates were no more tolerant than dispersed cells to several clinically used antibiotics and human serum. Instead, we found that alpA mutants were highly deficient in biofilm formation, suggesting that aggregation may be a step along the H. pylori biofilm formation pathway. IMPORTANCE Helicobacter pylori is a common human pathogen. Infection by this bacterium can lead to gastric cancers and ulcers. H. pylori infections present major global health challenges due to rising antibiotic resistance that complicates treatment. While bacterial aggregation is a recognized driver of antibiotic tolerance and persistence in other pathogens, its role in H. pylori remained unexplored. This work provides the first comprehensive characterization of H. pylori aggregation, demonstrating that it is a protein-mediated but flagella-independent process. We find that, unlike in many other bacteria, aggregation did not confer tolerance to tested antibiotics or serum antimicrobials, but instead may be an initial step on the pathway to forming biofilms. Characterizing H. pylori aggregation is a crucial step toward understanding the microbe’s life cycle and may inform novel strategies to disrupt its colonization and persistence.