Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

Sherman, David J.; Okuda, Suguru; Denny, William A.; Kahne, Daniel

doi:10.1016/j.bmc.2013.04.020

Cited by 42 publications

(30 citation statements)

References 45 publications

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“…Nonetheless, these proteins might represent interesting candidates for drug targets, given their association with the accessible outer membrane or cell periplasm. Consistent with this idea, orthologs of these proteins have been validated as drug targets, given their essential roles in either lipopolysaccharide or outer membrane protein transport (69)(70)(71)(72)(73)(74).…”

Section: Resultsmentioning

confidence: 69%

Proteomic analysis of Vibrio cholerae outer membrane vesicles

Altındiş

Mekalanos

2014

Proc. Natl. Acad. Sci. U.S.A.

139

110

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Outer membrane vesicles (OMVs) produced by Gram-negative bacteria provide an interesting research material for defining cellenvelope proteins without experimental cell disruption. OMVs are also promising immunogenic platforms and may play important roles in bacterial survival and pathogenesis. We used in-solution trypsin digestion coupled to mass spectrometry to identify 90 proteins present in OMVs of Vibrio cholerae when grown under conditions that activate the TCP pilus virulence regulatory protein (ToxT) virulence regulon. The ToxT expression profile and potential contribution to virulence of these proteins were assessed using ToxT and in vivo RNA-seq, Tn-seq, and cholera stool proteomic and other genome-wide data sets. Thirteen OMV-associated proteins appear to be essential for cell growth, and therefore may represent antibacterial drug targets. Another 12 nonessential OMV proteins, including DegP protease, were required for intestinal colonization in rabbits. Comparative proteomics of a degP mutant revealed the importance of DegP in the incorporation of nine proteins into OMVs, including ones involved in biofilm matrix formation and various substrates of the type II secretion system. Taken together, these results suggest that DegP plays an important role in determining the content of OMVs and also affects phenotypes such as intestinal colonization, proper function of the type II secretion system, and formation of biofilm matrix.HtrA family | in-solution digestion | biofilm formation | CTXϕ phage

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Section: Resultsmentioning

confidence: 69%

Proteomic analysis of Vibrio cholerae outer membrane vesicles

Altındiş

Mekalanos

2014

Proc. Natl. Acad. Sci. U.S.A.

139

110

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“…Modifications to affinity purifications (15) are explained in SI Materials and Methods. ATPase assays were conducted as described with 5 mM ATP/MgCl 2 for both LptB and LptB 2 FGC variants (21).…”

Section: Methodsmentioning

confidence: 99%

“…LptB, both alone and in a complex with LptF and LptG, has ATPase activity in vitro (19)(20)(21). In addition, the LptB 2 FG complex is closely associated with the bitopic IM protein LptC, which binds LPS (20)(21)(22)(23) and is part of the transenvelope bridge (15,16). Based on these findings, the current model is that LptB 2 FG extracts LPS from the outer leaflet of the IM and is the sole energy input responsible for the entire process of transport and assembly of LPS on the cell surface against a concentration gradient (22).…”

Section: Abc Transporter | Antibiotic Target | Membrane Biogenesismentioning

confidence: 99%

“…3B) reported inaccurately on the activity of the full complex (21). Therefore, we overexpressed and purified LptB 2 FGC in detergent and measured the ATPase activity of the wild-type complex and complexes containing LptB-E163Q and LptB-H195A (Fig.…”

Section: Atp Hydrolysis Induces Conformational Changesmentioning

confidence: 99%

“…Three of these Lpt proteins form an ABC system composed of a heteromeric TMD complex (LptF and LptG) and a homodimeric NBD complex (LptB). LptB, both alone and in a complex with LptF and LptG, has ATPase activity in vitro (19)(20)(21). In addition, the LptB 2 FG complex is closely associated with the bitopic IM protein LptC, which binds LPS (20-23) and is part of the transenvelope bridge (15,16).…”

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confidence: 99%

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Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport

Sherman

Lazarus

Murphy

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

127

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The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre-and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.ABC transporter | antibiotic target | membrane biogenesis A TP-binding cassette (ABC) systems represent one of the largest protein superfamilies across all domains of life (1, 2). Many of these systems are transporters that share a common architecture of two transmembrane domains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs), which bind and hydrolyze ATP. They couple the energy of ATP binding and hydrolysis to the transport of a variety of substrates against a concentration gradient. Although eukaryotic ABC transporters involved in human diseases have received much attention (3), the canonical ABC systems that have been most extensively studied are Gram-negative bacterial importers (2).Gram-negative bacteria, such as Escherichia coli, have a unique double-membrane architecture that allows them to colonize harsh environments. The inner membrane (IM) contains phospholipids, whereas the outer membrane (OM) is an asymmetric lipid bilayer composed of phospholipids in the inner leaflet and LPS in the outer leaflet (4). These two membranes are separated by an aqueous periplasmic compartment. LPS is a complex glycolipid composed of hundreds of sugars attached to a core containing fatty acyl chains (Fig. 1). Millions of LPS molecules must be properly assembled during each division cycle (4) on the cell surface to establish a permeability barrier that prevents the entry of hydrophobic molecules, including antibiotics (5). Therefore, understanding how LPS is assembled at the OM could lead to the development of better strategies to target Gram-negative infections. In 1972, it was established that the biosynthesis of LPS is completed on the outer leaflet of the IM. Because LPS cannot move passively across the aqueous periplasm and through the OM, it was recognized that there must be machinery to transport LPS across the cell envelope (6, 7).In the past decade, all of the components essential for the transport and assembly of...

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Fat Matters for Bugs: How Lipids and Lipid Modifications Make the Difference in Bacterial Life

Sperandeo

Polissi

Fabiani

2019

Euro J Lipid Sci & Tech

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Lipids are building blocks of biological membranes in the three domains of life and are endowed with several important biological functions. Lipopolysaccharide (LPS) is a peculiar glycolipid that represents the hallmark of the cell envelope of Gram‐negative bacteria, a group comprising several important human pathogens. The presence of LPS in the outer membrane (OM) of Gram‐negative bacteria correlates not only with the intrinsic resistance of this group of bacteria toward several antibiotics currently in use, but also with the worrisome increase in antibiotic resistance that is depleting the arsenal of efficacious molecules to cure bacterial infections. LPS consists of a conserved internal moiety decorated by a more variable distal unit. Several modifications occur at the canonical LPS structure during bacterial infection which are exploited by the microorganism to co‐evolve with the host thus evading its immune system. This viewpoint article discusses the current knowledge on LPS biogenesis and modification systems and their consequences on recognition by immune cells and antimicrobial resistance. It also discusses how knowledge on LPS biogenesis can be exploited to develop molecules able to dismantle the Gram‐negative protective barrier and to interfere with the interaction with the mammalian host. Practical Applications: Antibiotic resistance is a major threat for human health leading to inefficacy of many antibiotics to treat infectious diseases. Resistance to antibiotics causes around 25 000 deaths per year in the European Union alone and 700 000 deaths per year globally. This results in an increase of 1.5 billion euros per year in healthcare costs and productivity losses for illness. Gram‐negative bacteria are intrinsically resistant to many antibiotics, due to their LPS‐coated cell surface that protects them from the environment. LPS is sensed by the host immune system as a signal of bacterial infection, moreover bacteria exploit the complexity of LPS modifications to modulate the host immune response and eventually escape it. A deep understanding of the molecular mechanisms underlying LPS biogenesis, including the physico‐chemical and biological consequences of its lipid modifications, will be instrumental to develop novel antibiotic treatments aimed at dismantling the Gram‐negative bacteria barrier and restoring drug sensitivity. The hallmark of Gram‐negative bacteria is the presence of an asymmetric outer membrane (OM) surrounding the cytoplasmic membrane (inner membrane, IM), whose outer leaflet is made by lipopolysaccharide (LPS). A layer of peptidoglycan (PG) is sandwiched in between. LPS endows the bacteria with increased resistance to many antibiotics and is the first line of interaction with the host cell immune system. Chemical modifications of the canonical LPS structure determine different stimulatory effects of the immune response and allow the bacteria to escape from the killing action of effector molecules such as antimicrobial peptides.

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Validation of inhibitors of an ABC transporter required to transport lipopolysaccharide to the cell surface in Escherichia coli

Cited by 42 publications

References 45 publications

Proteomic analysis of Vibrio cholerae outer membrane vesicles

Proteomic analysis of Vibrio cholerae outer membrane vesicles

Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport

Fat Matters for Bugs: How Lipids and Lipid Modifications Make the Difference in Bacterial Life

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