Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyzes their polymerization and mediates pilus translocation across the outer membrane. We report the crystal structure of the full-length FimD usher bound to the FimC:FimH chaperone:adhesin complex and that of the unbound form of the FimD translocation domain. The FimD:FimC:FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC:FimH is held in place through interactions with the two C-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a dramatic conformational change in the β-barrel. The N-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone:subunit complex. The FimD:FimC:FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

P pili are prototypical chaperone-usher pathway-assembled pili used by Gram-negative bacteria to adhere to host tissues. The PapC usher contains five functional domains: a transmembrane β-barrel, a β-sandwich Plug, an N-terminal (periplasmic) domain (NTD), and two C-terminal (periplasmic) domains, CTD1 and CTD2. Here, we delineated usher domain interactions between themselves and with chaperone-subunit complexes and showed that overexpression of individual usher domains inhibits pilus assembly. Prior work revealed that the Plug domain occludes the pore of the transmembrane domain of a solitary usher, but the chaperone-adhesinbound usher has its Plug displaced from the pore, adjacent to the NTD. We demonstrate an interaction between the NTD and Plug domains that suggests a biophysical basis for usher gating. Furthermore, we found that the NTD exhibits high-affinity binding to the chaperone-adhesin (PapDG) complex and low-affinity binding to the major tip subunit PapE (PapDE). We also demonstrate that CTD2 binds with lower affinity to all tested chaperone-subunit complexes except for the chaperone-terminator subunit (PapDH) and has a catalytic role in dissociating the NTD-PapDG complex, suggesting an interplay between recruitment to the NTD and transfer to CTD2 during pilus initiation. The Plug domain and the NTDPlug complex bound all of the chaperone-subunit complexes tested including PapDH, suggesting that the Plug actively recruits chaperone-subunit complexes to the usher and is the sole recruiter of PapDH. Overall, our studies reveal the cooperative, active roles played by periplasmic domains of the usher to initiate, grow, and terminate a prototypical chaperone-usher pathway pilus.biolayer interferometry | macromolecular assembly | urinary tract infections | virulence factor | bacterial pathogenesis

Background Vaccination is an efficient strategy to control the COVID-19 pandemic. In north Cyprus, vaccine distribution started with CoronaVac followed by BNT162b2, and ChAdOx1 vaccines. An option to obtain a third booster dose with BNT162b2 or CoronaVac was later offered to people fully inoculated with CoronaVac. There are few simultaneous and comparative real-world antibody data for these three vaccines as well as boosters after CoronaVac vaccination. Our study was aimed at evaluating antibody responses after these vaccination schemes. Methods We did a prospective, longitudinal population-based study to measure SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG concentrations, assessed by assaying blood samples collected, in participants in north Cyprus who had received the BNT162b2, ChAdOx1, or CoronaVac vaccine at 1 month and 3 months after the second dose. Participants were recruited when they voluntarily came to the laboratory for testing after vaccination, solicited from health-care access points, or from the general population. We also evaluated antibody responses 1 month after a booster dose of BNT162b2 or CoronaVac after primary CoronaVac regimen. Demographics, baseline characteristics, vaccination reactions, and percentage of antibody responders were collected by phone interviews or directly from the laboratory summarised by vaccine and age group. Antibody levels were compared between groups over time by parametric and non-parametric methods. Findings Recruitment, follow-up, and data collection was done between March 1 and Sept 30, 2021. BNT162b2 induced the highest seropositivity and anti-spike RBD IgG antibody titres, followed by ChAdOx1, and then by CoronaVac. In addition, the rate of decline of antibodies was fastest with CoronaVac, followed by ChAdOx1, and then by BNT162b2. For the older age group, the rate of seropositivity at 3 months after the second dose was 100% for BNT162b2, 90% for ChAdOx1, and 60% for CoronaVac. In the multivariate repeated measures model, lower antibody titres were also significantly associated with male sex, older age, and time since vaccination. Boosting a two-dose CoronaVac regimen at 6 months with a single BNT162b2 dose led to significantly increased titres of IgG compared with boosting with CoronaVac; for the 60 years and older age group, the geometric mean fold rise in antibody titre after the booster relative to 1 month post-baseline was 7·9 (95% CI 5·8–10·8) in the BNT162b2 boost group versus 2·8 (1·6–5·0) in the CoronaVac group. Interpretation These longitudinal data can help shape vaccination strategies. Given the low antibody titres and fast decline in the CoronaVac group in individuals 60 years or older, more potent vaccine options could be considered as the primary vaccination or booster dose in these high-risk populations to sustain antibody responses for longer. Funding Crowdfunded in north Cyprus.

Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two Cterminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.outer membrane usher | macromolecular assembly | bacterial pathogenesis

BackgroundNatural products obtained from plants can be potent sources for developing a variety of pharmaceutical products. Allium species have been widely studied for their anti-cancer effects and presented promising results as potential anti-cancer agents. Breast cancer (BCa) is one of the most commonly diagnosed types of cancer in women. In this study, we aimed to investigate the anti-proliferative, cytotoxic and anti-metastatic effects of bulb and stem extracts from Allium autumnale P. H. Davis (Amaryllidaceae), an endemic Allium species to the island of Cyprus, in a comparative approach to weakly metastatic MCF-7 and strongly metastatic MDA-MB-231 breast cancer (BCa) cell lines.MethodsPossible cytotoxic, anti-proliferative and anti-metastatic effects of the Allium extracts on MCF-7 and MDA-MB-231 cells were tested using trypan blue exclusion, MTT and wound heal assays, respectively. Gas Chromatography Mass Spectroscopy (GC-MS) analysis was performed to determine the prominent medically important compounds in Allium autumnale bulb (AAB) and Allium autumnale stem (AAS) extracts. Student unpaired t-test or ANOVA followed by Newman-Keuls post hoc analysis (INSTAT Software) was used where appropriate.ResultsOur results demonstrate that AAB extract (24, 48 and 72 h) exerts significant anti-proliferative effect on both MCF-7 and MDA-MB-231 cells where this effect for AAS extract was observed only at high (5000 and 10,000 μg/mL) concentrations. Cell viability experiments revealed that AAB extract incubations caused more cytotoxicity on both BCa cell lines compared to the AAS. In contrast, there was no effect on lateral motilities of either cell line.ConclusionOverall, our studies demonstrated the anti-cancer activities associated with Allium autumnale, revealing it’s cytotoxic and anti-proliferative potential to be further utilized in in vivo studies.