A Genome-Wide Helicobacter pylori Morphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex

Abstract

Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology. IMPORTANCEThe stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen. CM, Leverich CK, Collar AL, Wyckoff TJ, Biboy J, Vollmer W, Salama NR. 2019. A genome-wide Helicobacter pylori morphology screen uncovers a membranespanning helical cell shape complex. J Bacteriol 201:e00724-18. https://doi.org/10.1128/JB .00724-18. FIG 8 Model of the Csd7 protein interaction network. Shown is a schematic diagram of a putative "helical shapesome" which includes the PG hydrolase Csd1, nonenzymatic proteins (CcmA, Csd2, Csd7, and Csd5) that serve to stabilize and/or localize this complex, and the PG precursor synthesis enzyme MurF. See the text for deta...

MicroRNA Modulation of Host Immune Response and Inflammation Triggered by Helicobacter pylori

“…Its helicoidal shape is essential for efficient colonization of the gastric mucosa. An inner membrane protein, i.e., Csd7 plays a key role in both maintaining shape stability and regulating the cellular wall, by interacting with two other proteins: Csd2 protein and Csd1, a peptidoglycan endopeptidase [ 24 ]. This complex helicoidal structure has been considered a virulence factor by some authors [ 25 ].…”

Structural basis of peptidoglycan endopeptidase regulation

“…The gram-positive bacterium Bacillus subtilis (and likely other gram-positive species as well) regulates EP activity via interactions with FtsEX and the PG synthesis elongation machinery (35)(36)(37) and additionally encodes a posttranslational negative regulator, IseA (YoeB) (38), which presumably serves to tone down EP activity during cell wall stress conditions (38,39). Much less is known about EP regulation in gram-negative bacteria, where EPs appear to be kept in check as part of multiprotein complexes (40,41), at the transcriptional level (42,43), or via proteolytic degradation (44,45). Proteolytic turnover appears to be the major mode of regulation of growthpromoting EPs (44,45); however, phenotypes associated with the accumulation of EPs have surprisingly mild phenotypes under normal growth conditions, suggesting an additional unexplored layer of regulation.…”

Review: Pathogenesis of Helicobacter pylori infection

“…This shape is generated and maintained by the action of a combination of peptidoglycan (PG) modifying enzymes and non‐enzymatic proteins that act as scaffolds. With three recent publications, 1‐3 the group of N.R Salama made significant progress in the characterisation of the corresponding actors and underlying mechanisms. Using a genome‐wide screen, they identified Csd7, an integral inner membrane protein, as an essential determinant of H pylori helical shape that is important for efficient mouse colonisation 1 .…”

“…With three recent publications, 1‐3 the group of N.R Salama made significant progress in the characterisation of the corresponding actors and underlying mechanisms. Using a genome‐wide screen, they identified Csd7, an integral inner membrane protein, as an essential determinant of H pylori helical shape that is important for efficient mouse colonisation 1 . Through protein‐protein interaction with two other cell shape determinants, the Csd1 periplasmic PG endopeptidase, the Csd2 protein and interaction with the filament forming bactofilin CcmA, Csd7 was found to stabilise Csd1.…”