Bacterial Cell Envelopes

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life. In plants, bacteria and fungi, ammonium represents a vital source of nitrogen, which is scavenged from the external environment. In contrast, in animal cells ammonium is a cytotoxic metabolic waste product and must be excreted to prevent cell death. Transport of ammonium is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. In addition to their function as transporters, Amt/Mep/Rh proteins play roles in a diverse array of biological processes and human physiopathology. Despite this clear physiological importance and medical relevance, the molecular mechanism of Amt/Mep/Rh proteins has remained elusive. Crystal structures of bacterial Amt/Rh proteins suggest electroneutral transport, whilst functional evidence supports an electrogenic mechanism. Here, focusing on bacterial members of the family, we summarize the structure of Amt/Rh proteins and what three decades of research tells us concerning the general mechanisms of ammonium translocation, in particular the possibility that the transport mechanism might differ in various members of the Amt/Mep/Rh superfamily.

Adhesive interactions between   Staphylococcus aureus   and the host rely on cell-wall-anchored proteins such as fibronectin-binding protein B (FnBPB). Recently we showed that the FnBPB protein expressed by clonal complex (CC) 1 isolates of   S. aureus   mediates bacterial adhesion to corneodesmosin. The proposed ligand-binding region of CC1-type FnBPB shares just 60 % amino acid identity with the archetypal FnBPB protein from CC8. Here we investigated ligand binding and biofilm formation by CC1-type FnBPB. We found that the A domain of FnBPB binds to fibrinogen and corneodesmosin and identified residues within the hydrophobic ligand trench in the A domain that are essential for the binding of CC1-type FnBPB to ligands and during biofilm formation. We further investigated the interplay between different ligands and the influence of ligand binding on biofilm formation. Overall, our study provides new insights into the requirements for CC1-type FnBPB-mediated adhesion to host proteins and FnBPB-mediated biofilm formation in   S. aureus  .

The type IX secretion system (T9SS) is a multiprotein machine distributed in   Bacteroidota   and responsible for the secretion of various proteins across the outer membrane. Secreted effectors can be either delivered into the medium or anchored to the cell surface. The T9SS is composed of a transenvelope complex consisting of proton-motive force-dependent motors connected to a membrane-associated ring and outer membrane translocons, and a cell-surface anchoring complex that processes effectors once translocated. The T9SS is involved in pathogenesis, metal acquisition, carbohydrate degradation, S-layer biogenesis and gliding motility. The broad spectrum of functions is linked to a highly versatile repertoire of effectors including metallophores, enzymes, toxins and adhesins, that all possess specific signatures to be recruited and transported by the apparatus. This review summarizes the current knowledge on T9SS substrate secretion signals, transport, processing and activities.

Bacteria produce an array of diverse, dynamic and often complex lipid structures, some of which function beyond their typical role in membrane structure. The model organism,   E. coli  , has three major membrane lipids, which are glycerophosphoglycerol (phosphatidylglycerol), glycerophosphoethanolamine (phosphatidylethanolamine) and cardiolipin. However, it is now appreciated that some bacteria have the capacity to synthesize a range of lipids, including glycerophosphocholines, glycerophosphoinositols, ‘phosphorous-free’ N-acyl amines, sphingolipids and plasmalogens. In recent years, some of these bacterial lipids have emerged as influential contributors to the microbe–host molecular dialogue. This review outlines our current knowledge of bacterial lipid diversity, with a focus on the membrane lipids of microbiome-associated bacteria that have documented roles as signalling molecules.

To prevail in the interaction with eukaryotic hosts, many bacterial pathogens use protein secretion systems to release virulence factors at the host–pathogen interface and/or deliver them directly into host cells. An outstanding example of the complexity and sophistication of secretion systems and the diversity of their protein substrates, effectors, is the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) Type IVB secretion system (T4BSS) of   Legionella pneumophila   and related species.   Legionella   species are facultative intracellular pathogens of environmental protozoa and opportunistic human respiratory pathogens. The Dot/Icm T4BSS translocates an exceptionally large number of effectors, more than 300 per   L. pneumophila   strain, and is essential for evasion of phagolysosomal degradation and exploitation of protozoa and human macrophages as replicative niches. Recent technological advancements in the imaging of large protein complexes have provided new insight into the architecture of the T4BSS and allowed us to propose models for the transport mechanism. At the same time, significant progress has been made in assigning functions to about a third of   L. pneumophila   effectors, discovering unprecedented new enzymatic activities and concepts of host subversion. In this review, we describe the current knowledge of the workings of the Dot/Icm T4BSS machinery and provide an overview of the activities and functions of the to-date characterized effectors in the interaction of   L. pneumophila   with host cells.

The outer membrane (OM) is a formidable permeability barrier that protects Gram-negative bacteria from detergents and antibiotics. It possesses exquisite lipid asymmetry, requiring the placement and retention of lipopolysaccharides (LPS) in the outer leaflet, and phospholipids (PLs) in the inner leaflet. To establish OM lipid asymmetry, LPS are transported from the inner membrane (IM) directly to the outer leaflet of the OM. In contrast, mechanisms for PL trafficking across the cell envelope are much less understood. In this review, we summarize and discuss recent advances in our understanding of PL transport, making parallel comparisons to well-established pathways for OM lipoprotein (Lol) and LPS (Lpt). Insights into putative PL transport systems highlight possible connections back to the ‘Bayer bridges’, adhesion zones between the IM and the OM that had been observed more than 50 years ago, and proposed as passages for export of OM components, including LPS and PLs.

The lipopolysaccharide (LPS) is a characteristic molecule of the outer leaflet of the Gram-negative bacterial outer membrane, which consists of lipid A, core oligosaccharide, and O antigen. The lipid A is embedded in outer membrane and provides an efficient permeability barrier, which is particularly important to reduce the permeability of antibiotics, toxic cationic metals, and antimicrobial peptides. LPS, an important modulator of innate immune responses ranging from localized inflammation to disseminated sepsis, displays a high level of structural and functional heterogeneity, which arise due to regulated differences in the acylation of the lipid A and the incorporation of non-stoichiometric modifications in lipid A and the core oligosaccharide. This review focuses on the current mechanistic understanding of the synthesis and assembly of the lipid A molecule and its most salient non-stoichiometric modifications.

The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins – acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O-acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.

The polymyxin and lipopeptide classes of antibiotics are membrane-targeting drugs of last resort used to treat infections caused by multi-drug-resistant pathogens. Despite similar structures, these two antibiotic classes have distinct modes of action and clinical uses. The polymyxins target lipopolysaccharide in the membranes of most Gram-negative species and are often used to treat infections caused by carbapenem-resistant species such as   Escherichia coli  ,   Acinetobacter baumannii   and   Pseudomonas aeruginosa  . By contrast, the lipopeptide daptomycin requires membrane phosphatidylglycerol for activity and is only used to treat infections caused by drug-resistant Gram-positive bacteria such as methicillin-resistant   Staphylococcus aureus   and vancomycin-resistant enterococci. However, despite having distinct targets, both antibiotic classes cause membrane disruption, are potently bactericidal in vitro and share similarities in resistance mechanisms. Furthermore, there are concerns about the efficacy of these antibiotics, and there is increasing interest in using both polymyxins and daptomycin in combination therapies to improve patient outcomes. In this review article, we will explore what is known about these distinct but structurally similar classes of antibiotics, discuss recent advances in the field and highlight remaining gaps in our knowledge.