The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Uropathogenic Escherichia coli P and Type 1 Fimbriae Act in Synergy in a Living Host to Facilitate Renal Colonization Leading to Nephron Obstruction

The progression of a natural bacterial infection is a dynamic process influenced by the physiological characteristics of the target organ. Recent developments in live animal imaging allow for the study of the dynamic microbe-host interplay in real-time as the infection progresses within an organ of a live host. Here we used multiphoton microscopy-based live animal imaging, combined with advanced surgical procedures, to investigate the role of uropathogenic Escherichia coli (UPEC) attachment organelles P and Type 1 fimbriae in renal bacterial infection. A GFP+ expressing variant of UPEC strain CFT073 and genetically well-defined isogenic mutants were microinfused into rat glomerulus or proximal tubules. Within 2 h bacteria colonized along the flat squamous epithelium of the Bowman's capsule despite being exposed to the primary filtrate. When facing the challenge of the filtrate flow in the proximal tubule, the P and Type 1 fimbriae appeared to act in synergy to promote colonization. P fimbriae enhanced early colonization of the tubular epithelium, while Type 1 fimbriae mediated colonization of the center of the tubule via a mechanism believed to involve inter-bacterial binding and biofilm formation. The heterogeneous bacterial community within the tubule subsequently affected renal filtration leading to total obstruction of the nephron within 8 h. Our results reveal the importance of physiological factors such as filtration in determining bacterial colonization patterns, and demonstrate that the spatial resolution of an infectious niche can be as small as the center, or periphery, of a tubule lumen. Furthermore, our data show how secondary physiological injuries such as obstruction contribute to the full pathophysiology of pyelonephritis

Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization

Identification of Ligand Binding Sites of Proteins Using the Gaussian Network Model

The nonlocal nature of the protein-ligand binding problem is investigated via the Gaussian Network Model with which the residues lying along interaction pathways in a protein and the residues at the binding site are predicted. The predictions of the binding site residues are verified by using several benchmark systems where the topology of the unbound protein and the bound protein-ligand complex are known. Predictions are made on the unbound protein. Agreement of results with the bound complexes indicates that the information for binding resides in the unbound protein. Cliques that consist of three or more residues that are far apart along the primary structure but are in contact in the folded structure are shown to be important determinants of the binding problem. Comparison with known structures shows that the predictive capability of the method is significant

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems

Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible

Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella

Consistent patterns of common species across tropical tree communities

Trees structure the Earth’s most biodiverse ecosystem, tropical forests. The vast number of tree species presents a formidable challenge to understanding these forests, including their response to environmental change, as very little is known about most tropical tree species. A focus on the common species may circumvent this challenge. Here we investigate abundance patterns of common tree species using inventory data on 1,003,805 trees with trunk diameters of at least 10 cm across 1,568 locations1,2,3,4,5,6 in closed-canopy, structurally intact old-growth tropical forests in Africa, Amazonia and Southeast Asia. We estimate that 2.2%, 2.2% and 2.3% of species comprise 50% of the tropical trees in these regions, respectively. Extrapolating across all closed-canopy tropical forests, we estimate that just 1,053 species comprise half of Earth’s 800 billion tropical trees with trunk diameters of at least 10 cm. Despite differing biogeographic, climatic and anthropogenic histories7, we find notably consistent patterns of common species and species abundance distributions across the continents. This suggests that fundamental mechanisms of tree community assembly may apply to all tropical forests. Resampling analyses show that the most common species are likely to belong to a manageable list of known species, enabling targeted efforts to understand their ecology. Although they do not detract from the importance of rare species, our results open new opportunities to understand the world’s most diverse forests, including modelling their response to environmental change, by focusing on the common species that constitute the majority of their trees.Publisher PDFPeer reviewe