Khedidja Mosbahi

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 β-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.

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.

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.

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.

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

The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition.

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.

The Structure of a Conserved Domain of TamB Reveals a Hydrophobic β Taco Fold

Summary 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 β sheet with a hydrophobic interior. This β taco structure is of dimensions capable of accommodating and shielding the hydrophobic side of an amphipathic β 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.

Recombinant expression, purification, crystallization and preliminary X-ray diffraction analysis of the C-terminal DUF490963–1138 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 Å resolution. 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.

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

Significance The outer membrane of Gram-negative bacteria is a highly impermeable barrier to a range of toxic chemicals and is responsible for the resistance of these bacteria to important classes of antibiotics. In this work, we show that plant pathogenic Pectobacterium spp. acquire iron from the small, stable, and abundant iron-containing plant protein ferredoxin by transporting ferredoxin across the outer membrane for intracellular processing by a highly specific protease, which induces iron release. The presence of homologous uptake and processing proteins in a range of important animal and plant pathogens suggests an exploitable route through which large molecules can penetrate the outer membrane of Gram-negative bacteria. 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.