Opposite evolution of pathogenicity driven by in vivo wzc and wcaJ mutations in ST11-KL64 carbapenem-resistant Klebsiella pneumoniae.

AIMS: To investigate the in vivo evolution of the mucoid-phenotype of ST11-KL64 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from the same patients and gain insights into diverse evolution and biology of these strains. METHODS: Whole genome sequencing and bioinformatic analysis were used to determine the mutation involved in the mucoid phenotype of ST11-KL64 CRKP. Gene knockout, bacterial morphology and capsular polysaccharides (CPS) extraction were used to verify the role of wzc and wcaJ in the mucoid phenotypes. Antimicrobial susceptibility, growth assay, biofilm formation, host cell adhesion and virulence assay were used to investigate the pleiotropic role of CPS changes in ST11-KL64 CRKP strains. RESULTS: Mutation of wzc S682N led to hypermucoid phenotype, which had negative impact on bacterial fitness and resulted in reduced biofilm formation and epithelial cell adhesion; while enhanced resistance to macrophage phagocytosis and virulence. Mutations of wcaJ gene led to non-mucoid phenotype with increased biofilm formation and epithelial cell adhesion, but reduced resistance of macrophage phagocytosis and virulence. Using virulence gene knockout, we demonstrated that CPS, rather than the pLVPK-like virulence plasmid, has a greater effect on mucoid phenotypic changes. CPS could be used as a surrogate marker of virulence in ST11-KL64 CRKP strains. CONCLUSIONS: ST11-KL64 CRKP strains sacrifice certain advantages to develop pathogenicity by changing CPS with two opposite in vivo evolution strategies.

Structural Basis of PER-1-Mediated Cefiderocol Resistance and Synergistic Inhibition of PER-1 by Cefiderocol in Combination with Avibactam or Durlobactam in Acinetobacter baumannii.

Cefiderocol is a novel siderophore cephalosporin that displays activity against Gram-negative bacteria. To establish cefiderocol susceptibility levels of Acinetobacter baumannii strains from China, we performed susceptibility testing and genomic analyses on 131 clinical isolates. Cefiderocol shows high activity against the strains. The production of PER-1 is the key mechanism of cefiderocol resistance. In silico studies predicted that avibactam and durlobactam could inhibit cefiderocol hydrolysis by PER-1, which was confirmed by determining cefiderocol MICs in combination with inhibitors.

Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

A novel NRPS cluster, acquired by horizontal gene transfer from algae, regulates siderophore iron metabolism in Burkholderia seminalis R456.

In an environment with limited iron levels, sufficiently high intracellular iron concentrations are critical for bacterial survival. When iron levels are low, many bacteria including those of the Burkholderia cepacia group secrete chemically diverse siderophores to capture Fe3+. The synthesis of the two main siderophores, ornibactin and pyochelin, is regulated in an iron concentration dependent manner via the regulator protein Fur. In this study, we identified a novel Nonribosomal Peptide Synthetase (NRPS) cluster in strain R456 of Burkholderia seminalis, a member of the B. cepacia group. We show that the NRPS cluster not only allows the production of a so-far undescribed siderophore, but is also required for ornibactin and pyochelin production as it is a crucial component in the signaling pathway targeting the global iron regulating effector Fur which regulates siderophore production. Furthermore, the NRPS cluster is also involved in cell motility and biofilm formation, both of which are directly dependent on iron concentration in various bacteria. Interestingly, our data suggests that this newly discovered NRPS cluster which regulates siderophore iron metabolism in bacteria was obtained by horizontal gene transfer from algae.

High level in vivo mucin-type glycosylation in Escherichia coli.

BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

The Transmembrane Morphogenesis Protein gp1 of Filamentous Phages Contains Walker A and Walker B Motifs Essential for Phage Assembly.

In contrast to lytic phages, filamentous phages are assembled in the inner membrane and secreted across the bacterial envelope without killing the host. For assembly and extrusion of the phage across the host cell wall, filamentous phages code for membrane-embedded morphogenesis proteins. In the outer membrane of Escherichia coli, the protein gp4 forms a pore-like structure, while gp1 and gp11 form a complex in the inner membrane of the host. By comparing sequences with other filamentous phages, we identified putative Walker A and B motifs in gp1 with a conserved lysine in the Walker A motif (K14), and a glutamic and aspartic acid in the Walker B motif (D88, E89). In this work we demonstrate that both, Walker A and Walker B, are essential for phage production. The crucial role of these key residues suggests that gp1 might be a molecular motor driving phage assembly. We further identified essential residues for the function of the assembly complex. Mutations in three out of six cysteine residues abolish phage production. Similarly, two out of six conserved glycine residues are crucial for gp1 function. We hypothesise that the residues represent molecular hinges allowing domain movement for nucleotide binding and phage assembly.

Mechanism of the Spontaneous and Directional Membrane Insertion of a 2-Transmembrane Ion Channel.

Protein insertion into membranes is a process occurring in every cell and every cellular compartment. Yet, many thermodynamic aspects of this fundamental biophysical process are not well understood. We investigated physicochemical parameters that influence protein insertion using the model protein KcsA, a 2-transmembrane ion channel. To understand what drives insertion and to identify individual steps of protein integration into a highly apolar environment, we investigated the contribution of electrostatic interactions and lipid composition on protein insertion on a single molecule level. We show that insertion of KcsA is spontaneous and directional as the cytosolic part of the protein does not translocate across the membrane barrier. Surprisingly, not hydrophobic residues but charged amino acids are crucial for the insertion of the unfolded protein into the membrane. Our results demonstrate the importance of electrostatic interactions between membrane and protein during the insertion process of hydrophobic polypeptides into the apolar membrane. On the basis of the observation that negatively charged lipids increase insertion events while high ionic strength in the surrounding aqueous phase decreases insertion events, a two-step mechanism is proposed. Here, an initial electrostatic attraction between membrane and protein represents the first step prior to insertion of hydrophobic residues into the hydrocarbon core of the membrane.

The T7 ejection nanomachine components gp15-gp16 form a spiral ring complex that binds DNA and a lipid membrane.

Bacteriophage T7 initiates infection by ejecting several internal capsid proteins into the host cell; these proteins then assemble into a nanomachine that translocates the viral genome from the phage head into the cytoplasm. The ejected proteins are thought to partially unfold as they pass through the lumen of the portal and the short stubby T7 tail during their entry into the cell. In vivo, the internal proteins gp15 and gp16 assemble into a tubular structure that spans the periplasm and cytoplasmic membrane. We show here that purified gp15 and gp16 can refold from a partially denatured state in vitro, and that gp15 interacts with gp16 to form a spiral ring structure. Purified gp15 binds to DNA, whereas gp16 binds protein-free liposomes; the gp15-gp16 complex binds both DNA and liposomes. Limited proteolysis of the liposome-bound gp16 reveals that its C-terminal region is protected, suggesting a partial membrane insertion of the protein.

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.

BACKGROUND: Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. RESULTS: This work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF. CONCLUSION: In the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

The function of the Na+-driven flagellum of Vibrio cholerae is determined by osmolality and pH.

Vibrio cholerae is motile by its polar flagellum, which is driven by a Na(+)-conducting motor. The stators of the motor, composed of four PomA and two PomB subunits, provide access for Na(+) to the torque-generating unit of the motor. To characterize the Na(+) pathway formed by the PomAB complex, we studied the influence of chloride salts (chaotropic, Na(+), and K(+)) and pH on the motility of V. cholerae. Motility decreased at elevated pH but increased if a chaotropic chloride salt was added, which rules out a direct Na(+) and H(+) competition in the process of binding to the conserved PomB D23 residue. Cells expressing the PomB S26A/T or D42N variants lost motility at low Na(+) concentrations but regained motility in the presence of 170 mM chloride. Both PomA and PomB were modified by N,N'-dicyclohexylcarbodiimide (DCCD), indicating the presence of protonated carboxyl groups in the hydrophobic regions of the two proteins. Na(+) did not protect PomA and PomB from this modification. Our study shows that both osmolality and pH have an influence on the function of the flagellum from V. cholerae. We propose that D23, S26, and D42 of PomB are part of an ion-conducting pathway formed by the PomAB stator complex.

Constructing droplet interface bilayers from the contact of aqueous droplets in oil.

We describe a protocol for forming an artificial lipid bilayer by contacting nanoliter aqueous droplets in an oil solution in the presence of phospholipids. A lipid monolayer forms at each oil-water interface, and when two such monolayers touch, a bilayer is created. Droplet interface bilayers (DIBs) are a simple way to generate stable bilayers suitable for single-channel electrophysiology and optical imaging from a wide variety of preparations, ranging from purified proteins to reconstituted eukaryotic cell membrane fragments. Examples include purified proteins from the α-hemolysin pore from Staphylococcus aureus, the anthrax toxin pore and the 1.2-MDa mouse mechanosensitive channel MmPiezo1. Ion channels and ionotropic receptors can also be reconstituted from membrane fragments without further purification. We describe two approaches for forming DIBs. In one approach, a lipid bilayer is created between two aqueous droplets submerged in oil. In the other approach, a membrane is formed between an aqueous droplet and an agarose hydrogel, which allows imaging in addition to electrical recordings. The protocol takes <30 min, including droplet generation, monolayer assembly and bilayer formation. In addition to the main protocol, we also describe the preparation of Ag/AgCl electrodes and sample preparation.

In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers.

The ability to routinely study eukaryotic ion channels in a synthetic lipid environment would have a major impact on our understanding of how different lipids influence ion channel function. Here, we describe a straightforward, detergent-free method for the in vitro reconstitution of eukaryotic ion channels and ionotropic receptors into droplet interface bilayers and measure their electrical activity at both the macroscopic and single-channel level. We explore the general applicability of this method by reconstitution of channels from a wide range of sources including recombinant cell lines and native tissues, as well as preparations that are difficult to study by conventional methods including erythrocytes and mitochondria.

One step at a time: action mechanism of Sushi 1 antimicrobial peptide and derived molecules.

Antimicrobial peptides (AMPs) are a crucial part of the innate immune system of eukaryotes and present a possible alternative to common antibiotics. It is therefore of great importance to understand their modes of action. Using a single-molecule approach in combination with high resolution imaging and bio-functional assays we were able to determine the different steps occurring during the action of the α-helical AMP Sushi 1 during bacterial lysis in spatial and temporal resolution in a biologically relevant context. Furthermore, we comment on the use of Sushi 1 as a template for new peptides to learn more about structure-function relationship of AMPs.

Correlation of charge, hydrophobicity, and structure with antimicrobial activity of S1 and MIRIAM peptides.

Antimicrobial peptides are key elements of the innate immune system. Many of them interact with membranes of bacteria leading to perturbation of the lipid bilayer and eventually to inactivation of the pathogen. The emergence of multidrug-resistant bacteria has necessitated innovations of new and more powerful classes of antimicrobials. Here we present the in-depth study of an antimicrobial peptide, MIRIAM, derived from Sushi1 (S1), a well-characterized peptide from the horseshoe crab. MIRIAM interacts strongly with negatively charged lipids, forming an α-helical structure. MIRIAM was found to neutralize LPS and kill Gram-negative bacteria with high efficiency, while not releasing LPS. The promising therapeutic potential of MIRIAM is shown by hemolytic assays, which demonstrate that eukaryotic membranes are unaffected at bactericidal concentrations. Nanoparticle-conjugated MIRIAM used in single-molecule fluorescence and electron microscopy experiments showed that MIRIAM targets bacterial membranes to kill bacteria similarly to parental S1. Furthermore, fragments derived from MIRIAM and S1 provided insights on their molecular mechanisms of action, in particular, the relationships of functional motifs comprised by charge, hydrophobicity, and structure within each peptide. We conclude that the combination of charge, hydrophobicity, and length of the peptide is important. A close interaction of amino acids in a single molecule in a carefully balanced ensemble of sequence position and secondary structure is crucial.

Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria.

BACKGROUND: Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial. RESULTS: Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level. CONCLUSION: Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process: 1) Binding, mediated mainly by charged residues in the peptide; 2) Peptide association, as peptide concentration increases evidenced by a change in diffusive behavior; 3) Membrane disruption, during which lipopolysaccharide is not released; and 4) Lysis, by leakage of cytosolic content through large membrane defects.

Oncogenic mutations reduce the stability of SRC kinase.

The oncogenic potential of the viral tyrosine kinase v-Src is due to its constitutive activity. Unlike the highly homologous cellular c-Src kinase, a C-terminal deletion of the regulatory tail and numerous point mutations make the viral kinase uncontrollable. To determine the basis of these differences, we analysed the structure and stability of v-Src and c-Src in vitro. We show that the stability of v-Src against unfolding and irreversible aggregation is significantly lower than that of c-Src. Furthermore, in v-Src hydrophobic residues are more exposed already in the native state. In consequence, v-Src was found to be inactive close to physiological temperatures. We thus suggest that the ensemble of mutations that transform c-Src into the oncogenic variant cause a concomitant destabilisation of the kinase.