Chemical Fixation Creates Nanoscale Clusters on the Cell Surface by Aggregating Membrane Proteins

Chemical fixations have been thought to preserve the structures of the cells or tissues. However, given that the fixatives create crosslinks or aggregate proteins, there is a possibility that these fixatives create nanoscale artefacts by aggregation of membrane proteins which move around freely to some extent on the cell surface. Despite this, little research has been conducted about this problem, probably because there has been no method for observing cell surface structures at the nanoscale. In this study, we have developed a new method to observe cell surfaces stably and with high resolution using atomic force microscopy and a microporous silicon nitride membrane. We demonstrate that the size of the protrusions on the cell surface is increased after treatment with three commonly used fixatives and show that these protrusions were created by the aggregation of membrane proteins by fixatives. These results call attention when observing fixed cell surfaces at the nanoscale.

Glycocalyx Curving the Membrane: Forces Emerging from the Cell Exterior

Morphological transitions are typically attributed to the actions of proteins and lipids. Largely overlooked in membrane shape regulation is the glycocalyx, a pericellular membrane coat that resides on all cells in the human body. Comprised of complex sugar polymers known as glycans as well as glycosylated lipids and proteins, the glycocalyx is ideally positioned to impart forces on the plasma membrane. Large, unstructured polysaccharides and glycoproteins in the glycocalyx can generate crowding pressures strong enough to induce membrane curvature. Stress may also originate from glycan chains that convey curvature preference on asymmetrically distributed lipids, which are exploited by binding factors and infectious agents to induce morphological changes. Through such forces, the glycocalyx can have profound effects on the biogenesis of functional cell surface structures as well as the secretion of extracellular vesicles. In this review, we discuss recent evidence and examples of these mechanisms in normal health and disease.

A repurposed, non-canonical cytochrome c, chaperones calcium binding by PilY1 for type IVa pili formation

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on a non-canonical cytochrome c (TfcP) with an unusual His/Cys heme ligation and calcium. We provide evidence that TfcP is unlikely to participate in electron transport and has been repurposed to promote calcium binding by PilY1.1 at low calcium concentrations, thereby stabilising PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results identify a novel function of cytochromes c and illustrate how incorporating an accessory factor expands the environmental range under which the T4aP system functions.

Key Parameters on the Antibacterial Activity of Silver Camphor Complexes

Nine new complexes with camphor imine or camphor sulfonimine ligands were synthesized and analytically and spectroscopically characterized, aiming to identify the key parameters that drive the antibacterial activity of the complexes with metal cores and imine substituents with distinct electronic and steric characteristics. The antimicrobial activity of all complexes was evaluated by determining their minimum inhibitory concentrations (MIC) against the Gram-negative Escherichia coli ATCC25922, Pseudomonas aeruginosa 477, and Burkholderia contaminans IST408, and the Gram-positive Staphylococcus aureus Newman. Camphor imine complexes based on the hydroxyl silver center ({Ag(OH)}) typically perform better than those based on the nitrate silver center ({Ag(NO3)}), while ligands prone to establish hydrogen bonding facilitate interactions with the bacterial cell surface structures. A different trend is observed for the silver camphor sulfonimine complexes that are almost non-sensitive to the nature of the metal cores {Ag(OH)} or {Ag(NO3)} and display low sensitivity to the Y substituent. The antibacterial activities of the Ag(I) camphor sulfonimine complexes are higher than those of the camphor imine analogues. All the complexes display higher activity towards Gram-negative strains than towards the Gram-positive strain.

Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on β2 Integrins

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

Phage Resistance in Multidrug-Resistant Klebsiella pneumoniae ST258 Evolves via Diverse Mutations That Culminate in Impaired Adsorption

ABSTRACT The evolution of phage resistance poses an inevitable threat to the efficacy of phage therapy. The strategic selection of phage combinations that impose high genetic barriers to resistance and/or high compensatory fitness costs may mitigate this threat. However, for such a strategy to be effective, the evolution of phage resistance must be sufficiently constrained to be consistent. In this study, we isolated lytic phages capable of infecting a modified Klebsiella pneumoniae clinical isolate and characterized a total of 57 phage-resistant mutants that evolved from their prolonged coculture in vitro. Single- and double-phage-resistant mutants were isolated from independently evolved replicate cocultures grown in broth or on plates. Among resistant isolates evolved against the same phage under the same conditions, mutations conferring resistance occurred in different genes, yet in each case, the putative functions of these genes clustered around the synthesis or assembly of specific cell surface structures. All resistant mutants demonstrated impaired phage adsorption, providing a strong indication that these cell surface structures functioned as phage receptors. Combinations of phages targeting different host receptors reduced the incidence of resistance, while, conversely, one three-phage cocktail containing two phages targeting the same receptor increased the incidence of resistance (relative to its two-phage, nonredundant receptor-targeting counterpart). Together, these data suggest that laboratory characterization of phage-resistant mutants is a useful tool to help optimize therapeutic phage selection and cocktail design. IMPORTANCE The therapeutic use of bacteriophage (phage) is garnering renewed interest in the setting of difficult-to-treat infections. Phage resistance is one major limitation of phage therapy; therefore, developing effective strategies to avert or lessen its impact is critical. Characterization of in vitro phage resistance may be an important first step in evaluating the relative likelihood with which phage-resistant populations emerge, the most likely phenotypes of resistant mutants, and the effect of certain phage cocktail combinations in increasing or decreasing the genetic barrier to resistance. If this information confers predictive power in vivo, then routine studies of phage-resistant mutants and their in vitro evolution should be a valuable means for improving the safety and efficacy of phage therapy in humans.

Axenic Biofilm Formation and Aggregation bySynechocystissp. Strain PCC 6803 Are Induced by Changes in Nutrient Concentration and Require Cell Surface Structures

ABSTRACTPhototrophic biofilms are key to nutrient cycling in natural environments and bioremediation technologies, but few studies describe biofilm formation by pure (axenic) cultures of a phototrophic microbe. The cyanobacteriumSynechocystissp. strain PCC 6803 (hereSynechocystis) is a model microorganism for the study of oxygenic photosynthesis and biofuel production. We report here that wild-type (WT)Synechocystiscaused extensive biofilm formation in a 2,000-liter outdoor nonaxenic photobioreactor under conditions attributed to nutrient limitation. We developed a biofilm assay and found that axenicSynechocystisforms biofilms of cells and extracellular material but only when cells are induced by an environmental signal, such as a reduction in the concentration of growth medium BG11. Mutants lacking cell surface structures, namely type IV pili and the S-layer, do not form biofilms. To further characterize the molecular mechanisms of cell-cell binding bySynechocystis, we also developed a rapid (8-h) axenic aggregation assay. Mutants lacking type IV pili were unable to aggregate, but mutants lacking a homolog to Wza, a protein required for type 1 exopolysaccharide export inEscherichia coli, had a superbinding phenotype. In WT cultures, 1.2× BG11 medium induced aggregation to the same degree as 0.8× BG11 medium. Overall, our data support that Wza-dependent exopolysaccharide is essential to maintain stable, uniform suspensions of WTSynechocystiscells in unmodified growth medium and that this mechanism is counteracted in a pilus-dependent manner under altered BG11 concentrations.IMPORTANCEMicrobes can exist as suspensions of individual cells in liquids and also commonly form multicellular communities attached to surfaces. Surface-attached communities, called biofilms, can confer antibiotic resistance to pathogenic bacteria during infections and establish food webs for global nutrient cycling in the environment. Phototrophic biofilm formation is one of the earliest phenotypes visible in the fossil record, dating back over 3 billion years. Despite the importance and ubiquity of phototrophic biofilms, most of what we know about the molecular mechanisms, genetic regulation, and environmental signals of biofilm formation comes from studies of heterotrophic bacteria. We aim to help bridge this knowledge gap by developing new assays forSynechocystis, a phototrophic cyanobacterium used to study oxygenic photosynthesis and biofuel production. With the aid of these new assays, we contribute to the development ofSynechocystisas a model organism for the study of axenic phototrophic biofilm formation.

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Bacteria assemble the cell envelope using localized enzymes to account for growth and division of a topologically complicated surface1–3. However, a regulatory pathway has not been identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein coat surrounding the curved 3D surface of a variety of bacteria4,5. By specifically labeling, imaging, and tracking native and purified RsaA, the S-layer protein (SLP) fromC. crescentus, we show that protein self-assembly alone is sufficient to assemble and maintain the S-layerin vivo. By monitoring the location of newly produced S-layer on the surface of living bacteria, we find that S-layer assembly occurs independently of the site of RsaA secretion and that localized production of new cell wall surface area alone is insufficient to explain S-layer assembly patterns. When the cell surface is devoid of a pre-existing S-layer, the location of S-layer assembly depends on the nucleation characteristics of SLP crystals, which grow by capturing RsaA molecules freely diffusing on the outer bacterial surface. Based on these observations, we propose a model of S-layer assembly whereby RsaA monomers are secreted randomly and diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated into growing 2D S-layer crystals. The complicated topology of the cell surface enables formation of defects, gaps, and grain boundaries within the S-layer lattice, thereby guiding the location of S-layer assembly without enzymatic assistance. This unsupervised mechanism poses unique challenges and advantages for designing treatments targeting cell surface structures or utilizing S-layers as self-assembling macromolecular nanomaterials. As an evolutionary driver, 2D protein self-assembly rationalizes the exceptional S-layer subunit sequence and species diversity6.

Multiple phase-variable mechanisms, including capsular polysaccharides, modify bacteriophage susceptibility in Bacteroides thetaiotaomicron

A variety of cell surface structures, including capsular polysaccharides (CPS), dictate interactions between bacteria and their environment including their viruses (bacteriophages). Members of the prominent human gut Bacteroidetes characteristically produce several phase-variable CPS, but their contributions to bacteriophage interactions are unknown. We used engineered strains of the human symbiont Bacteroides thetaiotaomicron, which differ only in the CPS they express, to isolate bacteriophages from two locations in the United States. Testing each of 71 bacteriophages against a panel of strains that express wild-type phase-variable CPS, one of eight different single CPS, or no CPS at all, revealed that each phage infects only a subset of otherwise isogenic strains. Deletion of infection-permissive CPS from B. thetaiotaomicron was sufficient to abolish infection for several individual bacteriophages, while infection of wild-type B. thetaiotaomicron with either of two different bacteriophages rapidly selected for expression of non-permissive CPS. Surprisingly, acapsular B. thetaiotaomicron also escapes complete killing by these bacteriophages, but surviving bacteria exhibit increased expression of 8 distinct phase-variable lipoproteins. When constitutively expressed, one of these lipoproteins promotes resistance to multiple bacteriophages. Finally, both wild-type and acapsular B. thetaiotaomicron were able to separately co-exist with one bacteriophage for over two months in the mouse gut, suggesting that phase-variation promotes resistance but also generates sufficient numbers of susceptible revertants to allow bacteriophage persistence. Our results reveal important roles for Bacteroides CPS and other cell surface structures that allow these bacteria to persist despite bacteriophage predation and hold important implications for using bacteriophages therapeutically to target gut symbionts.