The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.

Linking topography of its potential surface with the dynamics of folding of a protein model

The “3-color, 46-bead” model of a folding polypeptide is the vehicle for adapting to proteins a mode of analysis used heretofore for atomic clusters, to relate the topography of the potential surface to the dynamics that lead to formation of selected structures. The analysis is based on sequences of stationary points—successive minima, joined by saddles—that rise monotonically in energy from basin bottoms. Like structure-seeking clusters, the potential surface of the model studied here is staircase-like, rather than sawtooth-like, with highly collective motions required for passage from one minimum to the next. The surface has several deep basins whose minima correspond to very similar structures, but which are separated by high energy barriers.

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the β cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Inhibition of gene expression in human cells through small molecule-RNA interactions

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5′ end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from d-and l-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC50 of ≈50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (Eδ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by Eδ and the TCR α enhancer, and that it prevents Eδ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.

Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers

A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated ≈4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.

ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (Jahraus et al., 1998). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (Defacque et al., 2000a), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Sequence-Specific Interaction between the Disintegrin Domain of Mouse ADAM 3 and Murine Eggs: Role of β1 Integrin-associated Proteins CD9, CD81, and CD98

ADAM 3 is a sperm surface glycoprotein that has been implicated in sperm-egg adhesion. Because little is known about the adhesive activity of ADAMs, we investigated the interaction of ADAM 3 disintegrin domains, made in bacteria and in insect cells, with murine eggs. Both recombinant proteins inhibited sperm-egg binding and fusion with potencies similar to that which we recently reported for the ADAM 2 disintegrin domain. Alanine scanning mutagenesis revealed a critical importance for the glutamine at position 7 of the disintegrin loop. Fluorescent beads coated with the ADAM 3 disintegrin domain bound to the egg surface. Bead binding was inhibited by an authentic, but not by a scrambled, peptide analog of the disintegrin loop. Bead binding was also inhibited by the function-blocking anti-α6 monoclonal antibody (mAb) GoH3, but not by a nonfunction blocking anti-α6 mAb, or by mAbs against either the αv or β3 integrin subunits. We also present evidence that in addition to the tetraspanin CD9, two other β1-integrin-associated proteins, the tetraspanin CD81 as well as the single pass transmembrane protein CD98 are expressed on murine eggs. Antibodies to CD9 and CD98 inhibited in vitro fertilization and binding of the ADAM 3 disintegrin domain. Our findings are discussed in terms of the involvement of multiple sperm ADAMs and multiple egg β1 integrin-associated proteins in sperm-egg binding and fusion. We propose that an egg surface “tetraspan web” facilitates fertilization and that it may do so by fostering ADAM–integrin interactions.

Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads.

The folding mechanism of larger model proteins: role of native structure.

The folding mechanism of a 125-bead heteropolymer model for proteins is investigated with Monte Carlo simulations on a cubic lattice. Sequences that do and do not fold in a reasonable time are compared. The overall folding behavior is found to be more complex than that of models for smaller proteins. Folding begins with a rapid collapse followed by a slow search through the semi-compact globule for a sequence-dependent stable core with about 30 out of 176 native contacts which serves as the transition state for folding to a near-native structure. Efficient search for the core is dependent on structural features of the native state. Sequences that fold have large amounts of stable, cooperative structure that is accessible through short-range initiation sites, such as those in anti-parallel sheets connected by turns. Before folding is completed, the system can encounter a second bottleneck, involving the condensation and rearrangement of surface residues. Overly stable local structure of the surface residues slows this stage of the folding process. The relation of the results from the 125-mer model studies to the folding of real proteins is discussed.

Multicomponent diffusion in polymeric liquids.

It is shown how the phase-space kinetic theory of polymeric liquid mixtures leads to a set of extended Maxwell-Stefan equations describing multicomponent diffusion. This expression reduces to standard results for dilute solutions and for undiluted polymers. The polymer molecules are modeled as flexible bead-spring structures. To obtain the Maxwell-Stefan equations, the usual expression for the hydrodynamic drag force on a bead, used in previous kinetic theories, must be replaced by a new expression that accounts explicitly for bead-bead interactions between different molecules.

Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.

Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.

The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.

Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, including exotoxin A, elastase, alkaline protease, alginate, phospholipases, and extracellular rhamnolipids. The previously characterized rhlABR gene cluster encodes a regulatory protein (RhlR) and a rhamnosyltransferase (RhlAB), both of which are required for rhamnolipid synthesis. Another gene, rhII, has now been identified downstream of the rhlABR gene cluster. The putative RhlI protein shares significant sequence similarity with bacterial autoinducer synthetases of the LuxI type. A P. aeruginosa rhlI mutant strain carrying a disrupted rhlI gene was unable to produce rhamnolipids and lacked rhamnosyltransferase activity. Rhamnolipid synthesis was restored by introducing a wild-type rhlI gene into such strains or, alternatively, by adding either the cell-free spent supernatant from a P. aeruginosa wild-type strain or synthetic N-acylhomoserine lactones. Half-maximal induction of rhamnolipid synthesis in the rhlI mutant strain required 0.5 microM N-butyrylhomoserine lactone or 10 microM N-(3-oxohexanoyl)homoserine lactone. The P. aeruginosa rhlA promoter was active in the heterologous host Pseudomonas putida when both the rhlR and rhlI genes were present or when the rhlR gene alone was supplied together with synthetic N-acylhomoserine lactones. The RhlR-RhlI regulatory system was found to be essential for the production of elastase as well, and cross-communication between the RhlR-RhlI rhamnolipid regulatory system and the LasR-LasI elastase regulatory system was demonstrated.