Morphological transformation in Helicobacter pylori is a dynamic process leading to two types of coccoid

ABSTRACT The helical shape of Helicobacter pylori is crucial for successful colonization of the human stomach. However, this pathogen can shapeshift into another form termed the “coccoid form” with a spherical shape, through a mechanism that remains elusive. Here, by a combination of fluorescence microscopy using fluorescent D-aminoacids, cryoelectron and atom force microscopy, we explored the dynamics of coccoid formation in H. pylori through interrogation of the peptidoglycan layer. Contrary to the widely held hypothesis, we showed that helical-rod H. pylori transformed into a coccoid without transiting through a U-form. We show that U-forms, characterized by a U-shaped peptidoglycan with enlarged periplasmic space, altered genetic material, and red autofluorescence, are the output of a parallel pathway, which, unlike the coccoid pathway, is independent of the HdpA/Csd3 peptidoglycan endopeptidase. Coccoid formation occurred along a rigid timeline, by bulging of the cytoplasmic membrane through a peptidoglycan crack, resulting in a spheroplast-like structure with the peptidoglycan stacked into a thick layer near the original cell poles. Resistance of that structure against lysis likely involves a switch in metabolic profile reminiscent of bacteria in dormancy, with a notable accumulation of lysophospholipids, demonstrated in this work. Altogether, the ultrastructure and properties of H. pylori coccoids evidenced here are compatible with a role of this form in relapse after antibiotic treatment. SIGNIFICANCE STATEMENT Upon prolonged growth, stomach pathogen Helicobacter pylori undergoes a morphological change from a helical to a spherical form called coccoid, which may be involved in bacterial persistence and immune evasion. The pathway leading to this form, as well as its precise architecture, were unclear. In this work, we show by different microscopy techniques that the transition to coccoid does not involve a U-shaped intermediate as proposed before, but is triggered by progressive thinning, due to the activity of endopeptidase HdpA/Csd3, of the peptidoglycan meshwork that normally protects the cell, eventually leading to rupture. Because of the hole thus created, peptidoglycan can no longer contain the osmotic pressure in the cytoplasm, which leaks out within a membrane bulge, to eventually give rise to a kind of sphaeroplast, expected to be insensitive to cell wall-targeted antibiotics.