Structural Basis of Lipopolysaccharide O-Antigen Chain Length Modality

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

The Gram-negative cell envelope is composed of an inner and outer membrane, each with different lipid compositions. The inner membrane is predominantly made up of phosphatidylglycerol, phosphatidylethanolamine, and cardiolipin, while the outer membrane contains the same lipids but has a predominantly lipopolysaccharide composition in the outer leaflet. The outer membrane forms a permeability barrier that blocks the entry of toxins and antibiotics; however, it is decorated with outer membrane proteins that allow nutrient uptake and the efflux of intracellular toxins. In Escherichia coli, the σE stress response is essential for viability but is upregulated when disruptions to outer membrane protein biogenesis occurs. Here, we used a high-throughput genetic screen to identify genetic interactions with the genes responsible for cardiolipin biosynthesis. We demonstrate that the σE stress response is not essential in a mutant lacking cardiolipin and the major outer membrane protein OmpC. This mutant also became more resistant to batimastat, a selective inhibitor of RseP that causes a lethal decrease in σE activity in E. coli. Finally, we reveal that the toxicity associated with decreased σE activity can be relieved by treatment with an inhibitor of the Sec translocon, which translocates outer membrane proteins across the inner membrane. We conclude that the loss of cardiolipin suppresses Sec activity, reducing the level of misfolded outer membrane proteins in the periplasm, thereby relieving the essential nature of the σE stress response.IMPORTANCEThe process of building the Gram-negative bacterial cell envelope is complex and requires careful coordination of many different pathways. Because of its essential role in maintaining cell viability, the cell envelope is an important target for new antibiotic treatments. The membranes that make up the cell envelope contain three major lipids, but the precise role of these lipids and how they influence the coordination of the different cell envelope pathways is not well understood. Our data indicate that when the synthesis of one of these lipids is abolished, coordination of cell envelope biosynthesis is dysregulated. Importantly, an essential regulatory mechanism for controlling the response to disruption of the cell envelope becomes non-essential. These findings provide new insight into cell envelope biogenesis that could be harnessed for developing antimicrobial strategies.

The bacterial cell envelope provides a protective barrier that is challenging for small molecules and biomolecules to cross. Given the anionic nature of both Gram-positive and Gram-negative bacterial cell envelopes, negatively charged molecules are particularly difficult to deliver into these organisms. Many strategies have been employed to penetrate bacteria, ranging from reagents such as cell-penetrating peptides, enzymes, and metal-chelating compounds to physical perturbations. While cationic polymers are known antimicrobial agents, polymers that promote the permeabilization of bacterial cells without causing high levels of toxicity and cell lysis have not yet been described. Here, we investigate four polymers that display a cationic poly(2-(dimethylamino)ethyl methacrylate (D) block for the internalization of an anionic adenosine triphosphate (ATP)-based chemical probe into Escherichia coli and Bacillus subtilis. We evaluated two polymer architectures, linear and micellar, to determine how shape and hydrophobicity affect internalization efficiency. We found that, in addition to these reagents successfully promoting probe internalization, the probe-labeled cells were able to continue to grow and divide. The micellar structures in particular were highly effective for the delivery of the negatively charged chemical probe. Finally, we demonstrated that these cationic polymers could act as general permeabilization reagents, promoting the entry of other molecules, such as antibiotics.

Infectious diseases are becoming a major menace to the state of health worldwide, with difficulties in effective treatment especially of nosocomial infections caused by Gram-negative bacteria being increasingly reported. Inadequate permeation of anti-infectives into or across the Gram-negative bacterial cell envelope, due to its intrinsic barrier function as well as barrier enhancement mediated by resistance mechanisms, can be identified as one of the major reasons for insufficient therapeutic effects. Several in vitro, in silico, and in cellulo models are currently employed to increase the knowledge of anti-infective transport processes into or across the bacterial cell envelope; however, all such models exhibit drawbacks or have limitations with respect to the information they are able to provide. Thus, new approaches which allow for more comprehensive characterization of anti-infective permeation processes (and as such, would be usable as screening methods in early drug discovery and development) are desperately needed. Furthermore, delivery methods or technologies capable of enhancing anti-infective permeation into or across the bacterial cell envelope are required. In this respect, particle-based carrier systems have already been shown to provide the opportunity to overcome compound-related difficulties and allow for targeted delivery. In addition, formulations combining efflux pump inhibitors or antimicrobial peptides with anti-infectives show promise in the restoration of antibiotic activity in resistant bacterial strains. Despite considerable progress in this field however, the design of carriers to specifically enhance transport across the bacterial envelope or to target difficult-to-treat (e.g., intracellular) infections remains an urgently needed area of improvement. What follows is a summary and evaluation of the state of the art of both bacterial permeation models and advanced anti-infective formulation strategies, together with an outlook for future directions in these fields.

Lipopolysaccharides such as the enterobacterial common antigen are important components of the enterobacterial cell envelope that act as a protective barrier against the environment and are often polymerized by the inner membrane bound Wzy-dependent pathway. By employing cryo-electron microscopy we show that WzzE, the co-polymerase component of this pathway that is responsible for the length modulation of the enterobacterial common antigen, is octameric with alternating up-down conformations of its L4 loops. The alternating up-down nature of these essential loops, located at the top of the periplasmic bell, are modulated by clashing helical faces between adjacent protomers that flank the L4 loops around the octameric periplasmic bell. This alternating arrangement and a highly negatively charged binding face create a dynamic environment in which the polysaccharide chain is extended, and suggest a ratchet-type mechanism for polysaccharide elongation.

Homeostasis of the Gram-negative cell envelope

Gram-negative bacterial cell envelope consists of a surface-exposed lipid bilayer (outer membrane or OM) that serves as a permeability barrier to maintain the cellular integrity. Beneath the OM is the periplasmic space that harbours peptidoglycan (PG), a highly cross-linked mesh-like glycan polymer closely encasing the inner membrane (IM). During growth of a bacterium balanced synthesis of the envelope components is required to maintain the cellular integrity, of which little is known. In this study, we identify sanA, an ORF of unknown function encoding a predicted IM-anchored protein as a factor contributing to balanced synthesis of PG in E. coli. Absence of SanA increased the rate of nascent PG strand incorporation, and restored growth and viability to several mutants defective in either cell division or cell elongation. Detailed mutant analysis of sanA showed that it is defective in the envelope barrier properties. Interestingly, overexpression of the periplasmic endopeptidases that cleave the cross-links of the PG mesh was able to alleviate the phenotypes of sanA mutant implying the envelope defects are due to alterations in the PG sacculus. Additionally, a SanA variant (SSDsbA-SanA) targeted to the periplasm, complemented the SanA− phenotypes suggesting it functions in the periplasmic phase of the PG synthesis. Further, we find that SanA functions independently of its paralog, ElyC, known to regulate the synthesis of enterobacterial common antigen (ECA), a surface polysaccharide found in the cell envelopes of most enteric bacteria. Overall, our results suggest a role for SanA in the maintenance of optimal PG synthesis, providing evidence for the existence of an additional layer of regulation in Gram-negative cell envelope biogenesis.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.

Chapter 1 - Overview of the glycosylated components of the bacterial cell envelope

The cell surface of the Gram-negative cell envelope contains lipopolysaccharide (LPS) molecules, which form a permeability barrier against hydrophobic antibiotics. The LPS transport (Lpt) machine composed of LptB2FGCADE forms a proteinaceous trans-envelope bridge that allows for the rapid and specific transport of newly synthesized LPS from the inner membrane (IM) to the outer membrane (OM). This transport is powered from the IM by the ATP-binding cassette transporter LptB2FGC. The ATP-driven cycling between closed- and open-dimer states of the ATPase LptB2 is coupled to the extraction of LPS by the transmembrane domains LptFG. However, the mechanism by which LPS moves from a substrate-binding cavity formed by LptFG at the IM to the first component of the periplasmic bridge, the periplasmic β-jellyroll domain of LptF, is poorly understood. To better understand how LptB2FGC functions in Escherichia coli, we searched for suppressors of a defective LptB variant. We found that defects in LptB2 can be suppressed by both structural modifications to the core oligosaccharide of LPS and changes in various regions of LptFG, including a periplasmic loop in LptF that connects the substrate-binding cavity in LptFG to the periplasmic β-jellyroll domain of LptF. These novel suppressors suggest that interactions between the core oligosaccharide of LPS and periplasmic regions in the transporter influence the rate of LPS extraction by LptB2FGC. Together, our genetic data reveal a path for the bi-directional coupling between LptB2 and LptFG that extends from the cytoplasm to the entrance to the periplasmic bridge of the transporter.IMPORTANCEGram-negative bacteria are intrinsically resistant to many antibiotics due to the presence of lipopolysaccharide (LPS) at their cell surface. LPS is transported from its site of synthesis at the inner membrane to the outer membrane by the Lpt machine. Lpt proteins form a transporter that spans the entire envelope and is thought to function similarly to a PEZ candy dispenser. This trans-envelope machine is powered by the cytoplasmic LptB ATPase through a poorly understood mechanism. Using genetic analyses in Escherichia coli, we found that LPS transport involves long-ranging bi-directional coupling across cellular compartments between cytoplasmic LptB and periplasmic regions of the Lpt transporter. This knowledge could be exploited in developing antimicrobials that overcome the permeability barrier imposed by LPS.

The outer membrane (OM) is an essential component of the Gram-negative bacterial cell envelope. Restricted diffusion of integral OM proteins and lipopolysaccharide (LPS) that constitute the outer leaflet of the OM support a model in which the OM is in a semi-crystalline state. The low fluidity of the OM has been suggested to be an important property of this membrane that even contributes to cell rigidity. The LPS characteristics strongly determine the properties of the OM and the LPS layer fluidity has been measured using different techniques that require specific conditions or are technically challenging. Here, we characterize the Escherichia coli LPS fluidity by evaluating the lateral diffusion of the styryl dye FM4-64FX in fluorescence recovery after photobleaching experiments. This technique allowed us to determine the effect of different conditions and genetic backgrounds on the LPS fluidity. Our results show that a fraction of the LPS can slowly diffuse and that the fluidity of the LPS layer adapts by modifying the diffusion of the LPS and the fraction of mobile LPS molecules.

Gram-negative bacteria cause the majority of fatal infections in developed countries, usually in association with septic shock or meningitis. The pathophysiology of gram-negative bacterial infection is thought to be mediated in large part by lipopolysaccharide (LPS), a toxin elaborated by all species of gram-negative bacteria [1]. LPS is a structural component of the gram-negative bacterial cell envelope, and is therefore known as endotoxin. Gram-positive bacteria do not synthesize LPS. The lack of LPS in the cell envelope of grampositive organisms versus its presence in the gram-negative envelope accounts for the difference in staining characteristics of these two major categories of bacteria.

Bacteria are protected by complex molecular architectures known as the cell envelope. The cell envelope is composed of regions with distinct chemical compositions and physical properties, namely, membranes and a cell wall. To develop novel antibiotics to combat pathogenic bacteria, molecular level knowledge of the structure, dynamics, and interplay between the chemical components of the cell envelope that surrounds bacterial cells is imperative. In addition, conserved molecular patterns associated with the bacterial envelope are recognized by receptors as part of the mammalian defensive response to infection, and an improved understanding of bacteria-host interactions would facilitate the search for novel immunotherapeutics. This Perspective introduces an emerging area of computational biology: multiscale molecular dynamics simulations of chemically complex models of bacterial lipids and membranes. We discuss progress to date, and identify areas for future development that will enable the study of aspects of the membrane components that are as yet unexplored by computational methods.

Characterizing the role of phosphatidylglycerol-phosphate phosphatases in Acinetobacter baumannii cell envelope biogenesis and antibiotic resistance

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.