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Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

ABSTRACT: In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces "detergent-like" LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes.

SUBMITTER: Kutsch M

PROVIDER: S-EPMC7327485 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

Kutsch Miriam M Sistemich Linda L Lesser Cammie F CF Goldberg Marcia B MB Herrmann Christian C Coers Jörn J

The EMBO journal 20200608 13

In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1' ...[more]

PMID: 32510692

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Project description:The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the ?M-dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the ?M regulon to cell envelope homeostasis pathways.The emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

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Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

Publications

Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions.

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