<i>The Helicobacter pylori</i> cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton

Blair, Kris M.; Mears, Kevin S.; Taylor, Jennifer A.; Fero, Jutta; Jones, Lisa; Gafken, Philip R.; Whitney, John C.; Salama, Nina R.

doi:10.1111/mmi.14087

Cited by 26 publications

(39 citation statements)

References 75 publications

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“…The specific morphological and antibiotic susceptibility changes in elsL, elsS, and dacC mutants matching those caused by Rod-system block could be explained in at least two ways: the mutations could cause defects that indirectly affect Rod system function, or the encoded proteins could themselves be important components of the Rod complex. We considered ElsS a candidate for the latter, based on analogy with Helicobacter pylori, in which an SH3 domain protein serves to scaffold a cell wall synthesis complex 67 . To determine protein interactions with ElsS, we fused its predicted soluble C-terminus ( Fig.…”

Section: Coordination Of Chromosome Segregation and Cell Divisionmentioning

confidence: 99%

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

et al. 2020

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A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

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Section: Coordination Of Chromosome Segregation and Cell Divisionmentioning

confidence: 99%

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

et al. 2020

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“…We also provide evidence to suggest that CcmA may indirectly interact with Csd1 through Csd7, which would support a multiprotein transmembrane complex that provides a link from the cytoplasm to the periplasm via Csd7. In a separate study, we showed that Csd5 interacts with CcmA and the cytoplasmic PG precursor enzyme MurF and directly binds PG in the periplasm (39). Interestingly, among the top 20 hits in the Csd7 IP was MurF.…”

Section: Discussionmentioning

confidence: 94%

“…For mass spectrometry, samples were analyzed as previously reported (39). Briefly, the immunoprecipitated fractions were run for approximately 5 min into the wells of an SDS-PAGE gel (Bio-Rad TGX), excised as a gel slice, and submitted for analysis to the Proteomics Shared Resource of the Fred Hutchinson Cancer Research Center.…”

Section: Mutant Library Preparation and Flow Cytometry Screeningmentioning

confidence: 99%

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

et al. 2019

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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...

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“…Some biochemical evidence has suggested that BacAB are peripheral membrane proteins 3 . forward in which CcmA bactofilin filaments position lytic endopeptidases Csd1-3 in the periplasm, to remodel the shape-giving cell wall 12,13 . Bactofilins have also been described in Leptospira biflexa, with one of its bactofilin paralogues controlling the helical pitch of cells 14 .…”

Section: Introductionmentioning

confidence: 99%

The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity

et al. 2019

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Bactofilins are small β-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions from cell stalk formation in C. crescentus to chromosome segregation and motility in M. xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here we revealed by sequence analysis and electron microscopy that in addition to wide distribution across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Cryo-EM analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerise through end-to-end association of the β-helical domains. With the aid of a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerisation-impairing mutation enabled crystallisation and structure determination, while reaffirming the lack of polarity and the strength of the beta-stacking interface. To confirm the generality of the lack of polarity, we performed co-evolutionary analysis of a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes both on liposomes and in E. coli cells. Membrane binding is likely a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.

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The Helicobacter pylori cell shape promoting protein Csd5 interacts with the cell wall, MurF, and the bacterial cytoskeleton

Cited by 26 publications

References 75 publications

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope

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

The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity

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