Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

Pyocin efficacy in a murine model of Pseudomonas aeruginosa sepsis.

BACKGROUND: Bloodstream infections with antibiotic-resistant Pseudomonas aeruginosa are common and increasingly difficult to treat. Pyocins are naturally occurring protein antibiotics produced by P. aeruginosa that have potential for human use. OBJECTIVES: To determine if pyocin treatment is effective in a murine model of sepsis with P. aeruginosa. METHODS: Recombinant pyocins S5 and AP41 were purified and tested for efficacy in a Galleria mellonella infection model and a murine model of P. aeruginosa sepsis. RESULTS: Both pyocins produced no adverse effects when injected alone into mice and showed good in vitro antipseudomonal activity. In an invertebrate model of sepsis using G. mellonella, both pyocins significantly prolonged survival from 1/10 (10%) survival in controls to 80%-100% survival among groups of 10 pyocin-treated larvae. Following injection into mice, both showed extensive distribution into different organs. When administered 5 h after infection, pyocin S5 significantly increased survival from 33% (2/6) to 83% (5/6) in a murine model of sepsis (difference significant by log-rank test, P < 0.05). CONCLUSIONS: Pyocins S5 and AP41 show in vivo biological activity and can improve survival in two models of P. aeruginosa infection. They hold promise as novel antimicrobial agents for treatment of MDR infections with this microbe.

Targeted Killing of Pseudomonas aeruginosa by Pyocin G Occurs via the Hemin Transporter Hur.

Pseudomonas aeruginosa is a priority pathogen for the development of new antibiotics, particularly because multi-drug-resistant strains of this bacterium cause serious nosocomial infections and are the leading cause of death in cystic fibrosis patients. Pyocins, bacteriocins of P. aeruginosa, are potent and diverse protein antibiotics that are deployed during bacterial competition. Pyocins are produced by more than 90% of P. aeruginosa strains and may have utility as last resort antibiotics against this bacterium. In this study, we explore the antimicrobial activity of a newly discovered pyocin called pyocin G (PyoG). We demonstrate that PyoG has broad killing activity against a collection of clinical P. aeruginosa isolates and is active in a Galleria mellonella infection model. We go on to identify cell envelope proteins that are necessary for the import of PyoG and its killing activity. PyoG recognizes bacterial cells by binding to Hur, an outer-membrane TonB-dependent transporter. Both pyocin and Hur interact with TonB1, which in complex with ExbB-ExbD links the proton motive force generated across the inner membrane with energy-dependent pyocin translocation across the outer membrane. Inner-membrane translocation of PyoG is dependent on the conserved inner-membrane AAA+ ATPase/protease, FtsH. We also report a functional exploration of the PyoG receptor. We demonstrate that Hur can bind to hemin in vitro and that this interaction is blocked by PyoG, confirming the role of Hur in hemin acquisition.

Screening of the Enterocin-Encoding Genes and Their Genetic Determinism in the Bacteriocinogenic Enterococcus faecium GHB21.

Enterococci are well-known for their ability to produce a variety of antimicrobial peptides called enterocins. Most of these enterocins withstand extreme conditions and are very effective against a broad spectrum of undesirable bacteria including some Gram-negative bacteria. The same enterococci strain can produce multiple enterocins simultaneously. The genetic determinants of these bacteriocins can either be located on plasmids or on bacterial chromosome. Digestion of Enterococcus faecium GHB21 plasmids with various restriction endonucleases suggests the presence of two plasmids named pGHB-21.1 and pGHB-21.2 whose respective sizes are ~ 10.0 kb and ~ 3.3 kb. The screening of enterocin-encoding genes among E. faecium GHB21 genome by PCR followed by amplicon sequencing indicated the presence of three different enterocin structural genes similar to entA, entB, and entP genes previously detected in other E. faecium strains. These enterocin genes were, subsequently, localized on the bacterial chromosome based on PCR-targeted screening using total DNA and plasmids of E. faecium GHB21 as separate templates.

Bacterial iron acquisition mediated by outer membrane translocation and cleavage of a host protein.

Iron is an essential micronutrient for most bacteria and is obtained from iron-chelating siderophores or directly from iron-containing host proteins. For Gram-negative bacteria, classical iron transport systems consist of an outer membrane receptor, a periplasmic binding protein, and an inner membrane ABC transporter, which work in concert to deliver iron from the cell surface to the cytoplasm. We recently showed that Pectobacterium spp. are able to acquire iron from ferredoxin, a small and stable 2Fe-2S iron sulfur cluster containing protein and identified the ferredoxin receptor, FusA, a TonB-dependent receptor that binds ferredoxin on the cell surface. The genetic context of fusA suggests an atypical iron acquisition system, lacking a periplasmic binding protein, although the mechanism through which iron is extracted from the captured ferredoxin has remained unknown. Here we show that FusC, an M16 family protease, displays a highly targeted proteolytic activity against plant ferredoxin, and that growth enhancement of Pectobacterium due to iron acquisition from ferredoxin is FusC-dependent. The periplasmic location of FusC indicates a mechanism in which ferredoxin is imported into the periplasm via FusA before cleavage by FusC, as confirmed by the uptake and accumulation of ferredoxin in the periplasm in a strain lacking fusC The existence of homologous uptake systems in a range of pathogenic bacteria suggests that protein uptake for nutrient acquisition may be widespread in bacteria and shows that, similar to their endosymbiotic descendants mitochondria and chloroplasts, bacteria produce dedicated protein import systems.

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic ß Taco Fold.

The translocation and assembly module (TAM) plays a role in the transport and insertion of proteins into the bacterial outer membrane. TamB, a component of this system spans the periplasmic space to engage with its partner protein TamA. Despite efforts to characterize the TAM, the structure and mechanism of action of TamB remained enigmatic. Here we present the crystal structure of TamB amino acids 963-1,138. This region represents half of the conserved DUF490 domain, the defining feature of TamB. TamB963-1138 consists of a concave, taco-shaped ß sheet with a hydrophobic interior. This ß taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic ß strand, potentially allowing TamB to chaperone nascent membrane proteins from the aqueous environment. In addition, sequence analysis suggests that the structure of TamB963-1138 is shared by a large portion of TamB. This architecture could allow TamB to act as a conduit for membrane proteins.

Structure of the bacterial plant-ferredoxin receptor FusA.

Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These receptors acquire iron either in concert with soluble iron-scavenging siderophores or through direct interaction and extraction from host proteins. Characterization of these receptors provides invaluable insight into pathogenesis. However, only a subset of virulence-related TonB-dependent receptors have been currently described. Here we report the discovery of FusA, a new class of TonB-dependent receptor, which is utilized by phytopathogenic Pectobacterium spp. to obtain iron from plant ferredoxin. Through the crystal structure of FusA we show that binding of ferredoxin occurs through specialized extracellular loops that form extensive interactions with ferredoxin. The function of FusA and the presence of homologues in clinically important pathogens suggests that small iron-containing proteins represent an iron source for bacterial pathogens.

The Potassium Binding Protein Kbp Is a Cytoplasmic Potassium Sensor.

Escherichia coli possesses a number of specific K(+) influx and efflux systems that maintain an appropriate intracellular K(+) concentration. Although regulatory mechanisms have been identified for a number of these transport systems, the exact mechanism through which K(+) concentration is sensed in the cell remains unknown. In this work we show that Kbp (K(+) binding protein, formerly YgaU), a soluble 16-kDa cytoplasmic protein from Escherichia coli, is a highly specific K(+) binding protein and is required for normal growth in the presence of high levels of external K(+). Kbp binds a single potassium ion with high specificity over Na(+) and other metal ions found in biological systems, although, in common with K(+) transporters, it also binds Rb(+) and Cs(+). Dissection of the K(+) binding determinants of Kbp suggests a mechanism through which Kbp is able to sense changes in K(+) concentration over the relevant range of intracellular K(+) concentrations.

Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment.

Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

Methods in capillary electrophoresis coupled to mass spectrometry for the identification of clinical proteomic/peptidomic biomarkers in biofluids.

Proteomic biomarkers hold the promise of enabling assessment of patients according to a pathological condition aiming at improvements in diagnosis, prognosis, in general clinical patient management. Capillary electrophoresis coupled to an electrospray ionization time-of-flight mass spectrometer (CE-MS) allows the detection of thousands of small proteins and peptides in various biofluids, in a single, reproducible and time-limited step, enabling the simultaneous comparison of multiple individual proteins and peptides in biomarker discovery, but also in clinical applications. The reliability of the CE-MS platform, together with the use of a validated approach for data processing and mining is, to date, the most advanced technique for biomarker discovery of clinical significance. In this chapter, we report on the materials, methods and protocols used for CE-MS-based clinical proteomics allowing the reproducible profiling of biofluids.

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490(963-1138) domain of TamB from Escherichia coli.

TamB is a recently described inner membrane protein that, together with its partner protein TamA, is required for the efficient secretion of a subset of autotransporter proteins in Gram-negative bacteria. In this study, the C-terminal DUF490963-1138 domain of TamB was overexpressed in Escherichia coli K-12, purified and crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to the primitive trigonal space group P3121, with unit-cell parameters a = b = 57.34, c = 220.74 Å, and diffracted to 2.1 Šresolution. Preliminary secondary-structure and X-ray diffraction analyses are reported. Two molecules are predicted to be present in the asymmetric unit. Experimental phasing using selenomethionine-labelled protein will be undertaken in the future.

Discovery of an archetypal protein transport system in bacterial outer membranes.

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Screening of biomineralization using microfluidics.

Biomineralization is the process where biological systems produce well-defined composite structures such as shell, teeth, and bones. Currently, there is substantial momentum to investigate the processes implicated in biomineralization and to unravel the complex roles of proteins in the control of polymorph switching. An understanding of these processes may have wide-ranging significance in health care applications and in the development of advanced materials. We have demonstrated a microfluidic approach toward these challenges. A reversibly sealed T-junction microfluidic device was fabricated to investigate the influence of extrapallial (EP) fluid proteins in polymorph control of crystal formation in mollusk shells. A range of conditions were investigated on chip, allowing fast screening of various combinations of ion, pH, and protein concentrations. The dynamic formation of crystals was monitored on chip and combined with in situ Raman to reveal the polymorph in real time. To this end, we have demonstrated the unique advantages of this integrated approach in understanding the processes involved in biomineralization and revealing information that is impossible to obtain using traditional methods.

TPR-Mediated self-association of plant SGT1.

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.

The role of electrostatics in colicin nuclease domain translocation into bacterial cells.

The mechanism(s) by which nuclease colicins translocate distinct cytotoxic enzymes (DNases, rRNases, and tRNases) to the cytoplasm of Escherichia coli is unknown. Previous in vitro investigations on isolated colicin nuclease domains have shown that they have a strong propensity to associate with anionic phospholipid vesicles, implying that electrostatic interactions with biological membranes play a role in their import. In the present work we set out to test this hypothesis in vivo. We show that cell killing by the DNase toxin colicin E9 of E. coli HDL11, a strain in which the level of anionic phospholipid and hence inner membrane charge is regulated by isopropyl beta-D-thiogalactopyranoside induction, is critically dependent on the level of inducer, whereas this is not the case for pore-forming colicins that take the same basic route into the periplasm. Moreover, there is a strong correlation between the level and rate of HDL11 cell killing and the net positive charge on a colicin DNase, with similar effects seen for wild type E. coli cells, data that are consistent with a direct, electrostatically mediated interaction between colicin nucleases and the bacterial inner membrane. We next sought to identify how membrane-associated colicin nucleases might be translocated into the cell. We show that neither the Sec or Tat systems are involved in nuclease colicin uptake but that nuclease colicin toxicity is instead dependent on functional FtsH, an inner membrane AAA(+) ATPase and protease that dislocates misfolded membrane proteins to the cytoplasm for destruction.

Global structural rearrangement of the cell penetrating ribonuclease colicin E3 on interaction with phospholipid membranes.

Nuclease type colicins and related bacteriocins possess the unprecedented ability to translocate an enzymatic polypeptide chain across the Gram-negative cell envelope. Here we use the rRNase domain of the cytotoxic ribonuclease colicin E3 to examine the structural changes on its interaction with the membrane. Using phospholipid vesicles as model membranes we show that anionic membranes destabilize the nuclease domain of the rRNase type colicin E3. Intrinsic tryptophan fluorescence and circular dichroism show that vesicles consisting of pure DOPA act as a powerful protein denaturant toward the rRNase domain, although this interaction can be entirely prevented by the addition of salt. Binding of E3 rRNase to DOPA vesicles is an endothermic process (DeltaH=24 kcal mol-1), reflecting unfolding of the protein. Consistent with this, binding of a highly destabilized mutant of the E3 rRNase to DOPA vesicles is exothermic. With mixed vesicles containing anionic and neutral phospholipids at a ratio of 1:3, set to mimic the charge of the Escherichia coli inner membrane, destabilization of E3 rRNase is lessened, although the melting temperature of the protein at pH 7.0 is greatly reduced from 50 degrees C to 30 degrees C. The interaction of E3 rRNase with 1:3 DOPA:DOPC vesicles is also highly dependent on both ionic strength and temperature. We discuss these results in terms of the likely interaction of the E3 rRNase and the related E9 DNase domains with the E. coli inner membrane and their subsequent translocation to the cell cytoplasm.

Destabilization of the colicin E9 Endonuclease domain by interaction with negatively charged phospholipids: implications for colicin translocation into bacteria.

We have shown previously that the 134-residue endonuclease domain of the bacterial cytotoxin colicin E9 (E9 DNase) forms channels in planar lipid bilayers (Mosbahi, K., Lemaître, C., Keeble, A. H., Mobasheri, H., Morel, B., James, R., Moore, G. R., Lea, E. J., and Kleanthous, C. (2002) Nat. Struct. Biol. 9, 476-484). It was proposed that the E9 DNase mediates its own translocation across the cytoplasmic membrane and that the formation of ion channels is essential to this process. Here we describe changes to the structure and stability of the E9 DNase that accompany interaction of the protein with phospholipid vesicles. Formation of the protein-lipid complex at pH 7.5 resulted in a red-shift of the intrinsic protein fluorescence emission maximum (lambda(max)) from 333 to 346 nm. At pH 4.0, where the E9 DNase lacks tertiary structure but retains secondary structure, DOPG induced a blue-shift in lambda(max), from 354 to 342 nm. Changes in lambda(max) were specific for anionic phospholipid vesicles at both pHs, suggesting electrostatics play a role in this association. The effects of phospholipid were negated by Im9 binding, the high affinity, acidic, exosite inhibitor protein, but not by zinc, which binds at the active site. Fluorescence-quenching experiments further demonstrated that similar protein-phospholipid complexes are formed regardless of whether the E9 DNase is initially in its native conformation. Consistent with these observations, chemical and thermal denaturation data as well as proteolytic susceptibility experiments showed that association with negatively charged phospholipids destabilize the E9 DNase. We suggest that formation of a destabilizing protein-lipid complex pre-empts channel formation by the E9 DNase and constitutes the initial step in its translocation across the Escherichia coli inner membrane.

The cytotoxic domain of colicin E9 is a channel-forming endonuclease.

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.