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Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an ‘inactive’ low-affinity state (LAS) to an ‘active’ high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH’s ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function., Author summary To bind their ligands, allosteric proteins shift between ‘inactive’ and ‘active’ states, but molecular details of the conformational changes during the transition are often unclear. We describe a monoclonal antibody against the mannose-specific bacterial adhesin, FimH, that blocks the conformational transition in both directions. The antibody-trapped LAS and HAS conformations of FimH are unable to mediate bacterial adhesion under dynamic shear conditions. We propose that the conformational trapping involves a steric block of the core-to-surface switching of certain residues which is critical for the allosteric transitions. Furthermore, we demonstrate that the allosteric switches are structurally and functionally conserved across a broad spectrum of bacterial fimbrial adhesins.

This paper describes challenges and lessons learned throughout the concept, design, assembling, integrating, and testing for hardware and software of Virginia Tech (VT) ThickSat, a testbed for lightweight deployable space structures designed by engineering students at Virginia Tech. The project started in 2017 as part of the senior design undergraduate team at VT in collaboration with the Virginia Space and Near Space Launch Systems. The project's mission is to prove passive deployment of a spring table boom in low earth orbit, obtain deployment confirmation and transmit a picture back to Earth. To develop this project, over 25 different undergraduate and graduate students participated. In this process, they reached many breaking points and tough technical decisions. Throughout its development, the mission faced significant design reviews. It a maximum allowed 100mA power draw from the bus and a top 150 kiloByte packet size transmission for its entire 28-hour mission. The resulting design can be replicated and easily scalable for much more significant roles under the same requirements. This paper builds the challenges and lessons learned from the redesign, assembling, integrating, and testing of hardware and software. Furthermore, it describes the restrictive design characteristics of the ThinSat program in detail, which led the students to come with smart solutions for its many different design interactions. The ThickSat solution includes a custom low power PCB for an STM32 microcontroller, a servo actuated release mechanism, and a versatile chassis for the ThinSat Program. This study comprises an analytical point of view from the senior monitoring group and other engineers from the Center for Space Science and Engineering Research, known as (Space@VT), summarizing the experience from a student-led ThinSat project. The outcome of this paper is to share an experience that leads to bolster future SmallSat missions at Virginia Tech and other institutions.

This paper describes challenges and lessons learned throughout the assembling, integrating, and testing for hardware and software of VCC Ceres, the first Virginia Tech CubeSat built and designed by undergraduates. The project started in 2016 as part of the Virginia CubeSat Constellation (VCC), Virginia Tech (VT), Old Dominion University (ODU), University of Virginia (UVA), and Hampton University in collaboration with the Virginia Space Grant Consortium (VSGC). In July of 2019, the three CubeSats were successfully launched from the International Space Station (ISS). The project's mission is to obtain measurements of properties of the Earth's atmosphere in low earth orbit as well as to collect orbital data throughout their lifespan to develop a drag profile for CubeSats launched from the ISS. To develop the Virginia Tech's spacecraft, VCC Ceres, over 50 different undergraduate students participated. In this process, they reached many breaking points and tough decisions. This paper builds the challenges and lessons learned from assembling, integrating, and testing hardware and software. Furthermore, it describes the initial period of the operations phase, right after deployment, where the students had the opportunity to attempt contact with their satellite. This study comprises of an analytical point of view from the senior monitoring group and other engineers that work at the Center for Space Science and Engineering Research, known as (Space@VT), summarizing the experience from an undergraduate CubeSat project. The outcome of this paper is to share an experience that leads to bolster future CubeSat missions at Virginia Tech and other institutions.

Inhibiting antibodies targeting receptor-binding pockets in proteins is a major focus in the development of vaccines and in antibody-based therapeutic strategies. Here, by using a common mannose-specific fimbrial adhesin of Escherichia coli, FimH, we demonstrate that locking the adhesin in a low-binding conformation induces the production of binding pocket-specific, adhesion-inhibiting antibodies. A di-sulfide bridge was introduced into the conformationally dynamic FimH lectin domain, away from the mannose-binding pocket but rendering it defective with regard to mannose binding. Unlike the native, functionally active lectin domain, the functionally defective domain was potent in inducing inhibitory monoclonal antibodies that blocked FimH-mediated bacterial adhesion to epithelial cells and urinary bladder infection in mice. Inhibition of adhesion involved direct competition between the antibodies and mannose for the binding pocket. Binding pocket-specific inhibitory antibodies also were abundant in polyclonal immune serum raised against the functionally defective lectin domain. The monoclonal antibodies elicited against the binding-defective protein bound to the high-affinity conformation of the adhesin more avidly than to the low-affinity form. However, both soluble mannose and blood plasma more strongly inhibited antibody recognition of the high-affinity FimH conformation than the low-affinity form. We propose that in the functionally active conformation the binding-pocket epitopes are shielded from targeted antibody development by ligand masking and that strong immunogenicity of the binding pocket is unblocked when the adhesive domain is in the nonbinding conformation.

This prospective study of cellulitis identified β-hemolytic streptococci as the dominating cause in all investigated subgroups. Group C/G streptococci were more frequently detected than group A streptococci. No single clinical feature substantially increased the probability of confirmed streptococcal etiology., Background. Escherichia coli is a highly clonal pathogen. Extraintestinal isolates belong to a limited number of genetically related groups, which often exhibit characteristic antimicrobial resistance profiles. Methods. We developed a rapid clonotyping method for extraintestinal E coli based on detection of the presence or absence of 7 single nucleotide polymorphisms (SNPs) within 2 genes (fumC and fimH). A reference set of 2559 E coli isolates, primarily of urinary origin, was used to predict the resolving power of the 7-SNP-based typing method, and 582 representative strains from this set were used to evaluate test robustness. Results. Fifty-four unique SNP combinations (“septatypes”) were identified in the reference strains. These septatypes yielded a clonal group resolution power on par with that of traditional multilocus sequence typing. In 72% of isolates, septatype identity predicted sequence type identity with at least 90% (mean, 97%) accuracy. Most septatypes exhibited highly distinctive antimicrobial susceptibility profiles. The 7-SNP-based test could be performed with high specificity and sensitivity using single or multiplex conventional polymerase chain reaction (PCR) and quantitative PCR. In the latter format, E coli presence and septatype identity were determined directly in urine specimens within 45 minutes with bacterial loads as low as 102 colony-forming units/mL and, at clinically significant bacterial loads, with 100% sensitivity and specificity. Conclusions. 7-SNP-based typing of E coli can be used for both epidemiological studies and clinical diagnostics, which could greatly improve the empirical selection of antimicrobial therapy.

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection., Author Summary A common approach in the development of selective inhibitors for ligand-receptor interactions is targeting the receptor binding site with the expectation that inhibitors will sterically interfere with ligand binding and thus block receptor function via a competitive (orthosteric) mechanism. However, using monoclonal antibodies specific for the mannose-binding Escherichia coli adhesin, FimH, we demonstrate that the binding site epitopes allow for non-competitive inhibition that is more effective than orthosteric blocking. FimH, similar to other binding proteins, exhibits conformational flexibility of the ligand-binding pocket shifting between open (inactive) and tight (active) conformations, with relatively low- and high- affinity towards mannose. We show that an antibody that binds just one of the mannose-binding pocket loops prevents the shift from the inactive to the active conformation and hence blocks formation of high-affinity ligand-receptor complexes. This antibody type was more effective in inhibition of bacterial adhesion than anti-FimH antibodies competitively blocking mannose binding, and unlike the latter or a soluble ligand, showed the ability to detach an established bacterial biofilm from a ligand-coated surface. As the newly described antibody can bind the FimH pocket simultaneously with ligand, we refer to it as a parasteric (next-to-ligand) inhibitor that exhibits non-competitive inhibition from within the binding-pocket of the receptor.