Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

DeLucia, Angela M.; Six, David A.; Caughlan, Ruth E.; Gee, Patricia; Hunt, Ian; Lam, Joseph S.; Dean, Charles R.

doi:10.1128/mbio.00142-11

Cited by 56 publications

(64 citation statements)

References 54 publications

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“…Since LptD mediates the distal transport of mature LPS, its absence should cause some accumulation of varied forms of LPS in the membrane(s), since the machinery to synthesize intact LPS is present in lptD::Km r mutant cells. However, electron microscopy did not reveal any gross membrane defects in the lptD::Km r mutant like those described previously for P. aeruginosa upon interference with LPS transport (13,14,48). Since A. baumannii ATCC 19606 tolerates the absence of LptD and can apparently compensate somewhat for the lack of normal LPS transport, this defect itself may not be expected to manifest such pronounced morphological changes.…”

Section: Discussionmentioning

confidence: 77%

“…Interference with LptD-mediated LPS translocation in P. aeruginosa caused aberrant membrane-like material to accumulate within the periplasm (13). Similarly, depletion of WaaP, an essential kinase required for LPS core synthesis, caused the accumulation of truncated LPS in the IM, attributed to an inability to transport this aberrant LPS to the OM (48).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, although we focused on the first six steps of the pathway, we cannot rule out accumulation and/or mislocalization of other pathway intermediates, including more mature forms of LPS. In P. aeruginosa, disruption of LPS transport via depletion or inhibition of LptD or depletion of WaaP, an inner-core kinase required for efficient LPS transport to the OM, led to dramatic changes in membrane morphology and the accumulation of aberrant LPS (13,48). Although A. baumannii ATCC 19606 deleted Table S2 and Fig.…”

Section: Disruption Of Lps Transport Causes Accumulation Of Lipid IV Amentioning

confidence: 99%

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Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

et al. 2016

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Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted In most Gram-negative bacteria, many of the proteins responsible for lipopolysaccharide (LPS) synthesis and transport are essential, making them potential targets for antibacterial drug development. In particular, the nine conserved Lpx enzymes are considered promising for the development of novel antibiotics, since (3-deoxy-D-manno-oct-2-ulosonic acid) 2 -lipid A (Kdo 2 -lipid A) is the minimal LPS structure that supports a functional outer membrane (OM) and cell viability for most Gram-negative bacteria (1, 2). Indeed, the optimization of LpxC inhibitors has been an ongoing effort in antimicrobial research for over 2 decades, which has yielded compounds with impressive antibacterial activity against Gram-negative organisms such as Pseudomonas aeruginosa (1,(3)(4)(5)(6)(7)(8)(9). The identification of RJPXD33, an antimicrobial peptide that inhibits both Escherichia coli LpxA and LpxD, and a recently identified LpxH inhibitor suggests that other LPS biosynthetic steps could also be successfully targeted (10-12). POL7001, a peptidomimetic antibiotic that inhibits LptD, the final essential step of the LPS transport (Lpt) system, has potent and specific antibacterial activity against P. aeruginosa, which, importantly, indicates that the LPS transport and OM assembly machinery may be attractive targets for antibacterial discovery (13)(14)(15).Acinetobacter baumannii is an emerging opportunistic bacterial pathogen of increasing concern due to multidrug resistance (16). A. baumannii is noted for its ability to develop resistance against most conventional antibiotics through mechanisms such as the upregulation of efflux pumps and horizontal transfer of resistance genes (17)(18)(19). Because of this, clinical resistance is becoming a serious issue for this organism (as well as for other Gram-negative pathogens), often necessitating the use of antibiotics of last resort, such as polymyxins (19,20). Understanding resistance to polymyxins, which are cationic and utilize LPS to gain access to cells, has therefore become a focus of renewed in-

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Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

Section: Disruption Of Lps Transport Causes Accumulation Of Lipid IV Amentioning

confidence: 99%

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Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

et al. 2016

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“…The low similarity to pyruvate kinase, a phosphotransferase, suggests that this enzyme is involved in the transfer of phosphates to or from the O-antigen-lipid carrier molecule as part of the ligation process. Alternatively, this enzyme may phosphorylate lipid A-core inside the cell, as occurs in the lipid A of other bacteria, including Pseudomonas aeruginosa, where phosphorylation of lipid A-core is required for LPS transport to the outer membrane (50). The presence of phosphorus has already been demonstrated in the LPS of Anacystis nidulans KM (18), a strain since considered S. elongatus.…”

Section: Discussionmentioning

confidence: 99%

Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

Simkovsky

Effner

Iglesias-Sánchez

et al. 2016

Appl Environ Microbiol

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In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongatus PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678 -16683, 2012, http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide of S. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains. L ipopolysaccharide (LPS) serves as a critical interface between aGram-negative bacterium's internal physiology and its abiotic and biotic environments, protecting the cell from specific antimicrobials, evading the innate immune and complement systems, enabling symbiosis with eukaryotic hosts, acting as a recognition factor for phage infection, and protecting bacteria against digestion by predatory amoebae (1-5). Alterations to any of the three primary components of the LPS structure (the endotoxin lipid A, the core oligosaccharide, or the O-antigen polysaccharide), through changes in sugar content or sequence, acylation, phosphorylation, or other molecular modifications, can alter these interactions, modulate the toxicity of the endotoxin, or prevent cellular behaviors such as motility (6, 7).While much is known about the structure and synthesis of LPS in enteric and pathogenic proteobacteria, little is known about either aspect of LPS in photosynthetic cyanobacteria and how the cyanobacterial LPS affects interactions with the environment (8-11). To date, only three cyanobacterial LPS structures have been reported: the partial structural determination of the filamentous freshwater cyanobacterium Oscillatoria planktothrix LPS (12) and the complete structural determination of the LPSs of the unicellular marine Synechococcus strains WH8102 and CC9311 (9). Beyond these structures, the remaining...

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“…Our results also indicated that the rfaC (waaC) deletion strain 10031 was highly sensitive to polymyxin B (Table 3). This may be in part due to the loss of stabilizing LPS cross-links mediated through the core, leading to a defect in the permeability barrier (62).…”

Section: Discussionmentioning

confidence: 99%

Pathogenicity of Yersinia pestis Synthesis of 1-Dephosphorylated Lipid A

et al. 2013

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Lipopolysaccharide (LPS) Inner-Core Phosphates Are Required for Complete LPS Synthesis and Transport to the Outer Membrane in Pseudomonas aeruginosa PAO1

Cited by 56 publications

References 54 publications

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis

Mutations in Novel Lipopolysaccharide Biogenesis Genes Confer Resistance to Amoebal Grazing in Synechococcus elongatus

Pathogenicity of Yersinia pestis Synthesis of 1-Dephosphorylated Lipid A

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