The lipopolysaccharide transport (Lpt) machinery : A nonconventional transporter for lipopolysaccharide assembly at the outer membrane of Gram-negative bacteria

The outer membrane (OM) of Gram-negative is a unique lipid bilayer containing LPS in its outer leaflet. Because of the presence of amphipathic LPS molecules, theOMbehaves as an effective permeability barrier that makes Gram-negative bacteria inherently resistant to many antibiotics. This review focuses on LPS biogenesis and discusses recent advances that have contributed to our understanding of how this complex molecule is transported across the cellular envelope and is assembled at the OM outer leaflet. Clearly, this knowledge represents an important platform for the development of novel therapeutic options to manage Gram-negative infections

Trapped lipopolysaccharide and LptD intermediates reveal lipopolysaccharide translocation steps across the Escherichia coli outer membrane

Lipopolysaccharide (LPS) is a main component of the outer membrane of Gram-negative bacteria, which is essential for the vitality of most Gram-negative bacteria and plays a critical role for drug resistance. LptD/E complex forms a N-terminal LPS transport slide, a hydrophobic intramembrane hole and the hydrophilic channel of the barrel, for LPS transport, lipid A insertion and core oligosaccharide and O-antigen polysaccharide translocation, respectively. However, there is no direct evidence to confirm that LptD/E transports LPS from the periplasm to the external leaflet of the outer membrane. By replacing LptD residues with an unnatural amino acid p-benzoyl-L-phenyalanine (pBPA) and UV-photo-cross-linking in E.coli, the translocon and LPS intermediates were obtained at the N-terminal domain, the intramembrane hole, the lumenal gate, the lumen of LptD channel, and the extracellular loop 1 and 4, providing the first direct evidence and “snapshots” to reveal LPS translocation steps across the outer membrane

Self-assembled monolayers of copper sulfide nanoparticles on glass as antibacterial coatings

We developed an easy and reproducible synthetic method to graft a monolayer of copper sulfide nanoparticles (CuS NP) on glass and exploited their particular antibacterial features. Samples were fully characterized showing a good stability, a neat photo-thermal effect when irradiated in the Near InfraRed (NIR) region (in the so called \u201cbiological window\u201d), and the ability to release controlled quantities of copper in water. The desired antibacterial activity is thus based on two different mechanisms: (i) slow and sustained copper release from CuS NP-glass samples, (ii) local temperature increase caused by a photo-thermal effect under NIR laser irradiation of CuS NP\u2013glass samples. This behavior allows promising in vivo applications to be foreseen, ensuring a \u201cstatic\u201d antibacterial protection tailored to fight bacterial adhesion in the critical timescale of possible infection and biofilm formation. This can be reinforced, when needed, by a photo-thermal action switchable on demand by an NIR light

Nanocomposite sprayed films with photo-thermal properties for remote bacteria eradication

Currently there is a strong demand for novel protective materials with effcient antibacterial properties. Nanocomposite materials loaded with photo-thermally active nanoparticles can offer promising opportunities due to the local increase of temperature upon near-infrared (NIR) light exposure capable of eradicating bacteria. In this work, we fabricated antibacterial films obtained by spraying on glass slides aqueous solutions of polymers, containing highly photo-thermally active gold nanostars (GNS) or Prussian Blue (PB) nanoparticles. Under NIR light irradiation with low intensities (0.35W/cm2) these films demonstrated a pronounced photo-thermal effect: 06Tmax up to 26.4 ffC for the GNS-containing films and 06Tmax up to 45.8 ffC for the PB-containing films. In the latter case, such a local temperature increase demonstrated a remarkable effect on a Gram-negative strain (P. aeruginosa) killing (84% of dead bacteria), and a promising effect on a Gram-positive strain (S. aureus) eradication (69% of dead bacteria). The fabricated films are promising prototypes for further development of lightweight surfaces with effcient antibacterial action that can be remotely activated on demand

Dissecting Escherichia coli outer membrane biogenesis using differential proteomics

The cell envelope of Gram-negative bacteria is a complex multi-layered structure comprising an inner cytoplasmic membrane and an additional asymmetric lipid bilayer, the outer membrane, which functions as a selective permeability barrier and is essential for viability. Lipopolysaccharide, an essential glycolipid located in the outer leaflet of the outer membrane, greatly contributes to the peculiar properties exhibited by the outer membrane. This complex molecule is transported to the cell surface by a molecular machine composed of seven essential proteins LptABCDEFG that form a transenvelope complex and function as a single device. While advances in understanding the mechanisms that govern the biogenesis of the cell envelope have been recently made, only few studies are available on how bacterial cells respond to severe envelope biogenesis defects on a global scale. Here we report the use of differential proteomics based on Multidimensional Protein Identification Technology (MudPIT) to investigate how Escherichia coli cells respond to a block of lipopolysaccharide transport to the outer membrane. We analysed the envelope proteome of a lptC conditional mutant grown under permissive and non permissive conditions and identified 123 proteins whose level is modulated upon LptC depletion. Most such proteins belong to pathways implicated in cell envelope biogenesis, peptidoglycan remodelling, cell division and protein folding. Overall these data contribute to our understanding on how E. coli cells respond to LPS transport defects to restore outer membrane functionality. \ua9 2014 Martorana et al

Antimicrobial peptides : insights into membrane permeabilization, lipopolysaccharide fragmentation and application in plant disease control

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N-and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections

The Escherichia coli Lpt transenvelope protein complex for lipopolysaccharide export is assembled via conserved structurally homologous domains

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components

The Escherichia coli Lpt Transenvelope Protein Complex for Lipopolysaccharide Export Is Assembled via Conserved Structurally Homologous Domains

Lipopolysaccharide is a major glycolipid component in the outer leaflet of the outer membrane (OM), a peculiar permeability barrier of Gram-negative bacteria that prevents many toxic compounds from entering the cell. Lipopolysaccharide transport (Lpt) across the periplasmic space and its assembly at the Escherichia coli cell surface are carried out by a transenvelope complex of seven essential Lpt proteins spanning the inner membrane (LptBCFG), the periplasm (LptA), and the OM (LptDE), which appears to operate as a unique machinery. LptC is an essential inner membrane-anchored protein with a large periplasm-protruding domain. LptC binds the inner membrane LptBFG ABC transporter and interacts with the periplasmic protein LptA. However, its role in lipopolysaccharide transport is unclear. Here we show that LptC lacking the transmembrane region is viable and can bind the LptBFG inner membrane complex; thus, the essential LptC functions are located in the periplasmic domain. In addition, we characterize two previously described inactive single mutations at two conserved glycines (G56V and G153R, respectively) of the LptC periplasmic domain, showing that neither mutant is able to assemble the transenvelope machinery. However, while LptCG56V failed to copurify any Lpt component, LptCG153R was able to interact with the inner membrane protein complex LptBFG. Overall, our data further support the model whereby the bridge connecting the inner and outer membranes would be based on the conserved structurally homologous jellyroll domain shared by five out of the seven Lpt components.Chemistry and Chemical Biolog

Structural insights into cardiolipin transfer from the Inner membrane to the outer membrane by PbgA in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is a unique asymmetric lipid bilayer in which the outer leaflet is composed of lipopolysaccharide (LPS) and the inner leaflet is formed by glycerophospholipid (GPL). The OM plays a fundamental role in protecting Gram-negative bacteria from harsh environments and toxic compounds. The transport and assembly pathways for phospholipids of bacterial OM are unknown. Cardiolipin (CL) plays an important role in OM biogenesis and pathogenesis, and the inner membrane (IM) protein PbgA, containing five transmembrane domains and a globular domain in periplasm has been recently identified as a CL transporter from the IM to the OM with an unknown mechanism. Here we present the first two crystal structures of soluble periplasmic globular domain of PbgA from S. typhimurium and E. coli, which revealed that the globular domains of PbgA resemble the structures of the arylsulfatase protein family and contains a novel core hydrophobic pocket that may be responsible for binding and transporting CLs. Our structural and functional studies shed an important light on the mechanism of CL transport in Gram-negative bacteria from the IM to the OM, which offers great potential for the development of novel antibiotics against multi-drug resistant bacterial infections