In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche

Polar delivery of Legionella type IV secretion system substrates is essential for virulence

A recurrent emerging theme is the targeting of proteins to subcellular microdomains within bacterial cells, particularly to the poles. In most cases, it has been assumed that this localization is critical to the protein’s function. Legionella pneumophila uses a type IVB secretion system (T4BSS) to export a large number of protein substrates into the cytoplasm of host cells. Here we show that the Legionella export apparatus is localized to the bacterial poles, as is consistent with many T4SS substrates being retained on the phagosomal membrane adjacent to the poles of the bacterium. More significantly, we were able to demonstrate that polar secretion of substrates is critically required for Legionella’s alteration of the host endocytic pathway, an activity required for this pathogen’s virulence

\u3cem\u3eIn Vivo\u3c/em\u3e Structures of the \u3cem\u3eHelicobacter pylori cag\u3c/em\u3e Type IV Secretion System

The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche

Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons

Polar targeting and assembly of the Legionella Dot/Icm type IV secretion system (T4SS) by T6SS-related components

Legionella pneumophila, the causative agent of Legionnaires′ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells′ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of the Legionella containing vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles of L. pneumophila by themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose that Legionella co-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of the Legionella T4BSS apparatus is mediated by an innovative ″outside-inside″ mechanism

The structural complexity of the Gammaproteobacteria flagellar motor is related to the type of its torque-generating stators

The bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque- generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown. Here we use electron cryo-tomography to determine the in situ macromolecular structures of the flagellar motors of three Gammaproteobacteria species: Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis MR-1, providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the stator system of the motor and its structural complexity. Motors with a single H+-driven stator system have only the core P- and L-rings in their periplasm; those with dual H+-driven stator systems have an extra component elaborating their P-ring; and motors with Na+- (or dual Na+-H+)- driven stator systems have additional rings surrounding both their P- and L-rings. Our results suggest an evolution of structural complexity that may have enabled pathogenic bacteria like L. pneumophila and P. aeruginosa to colonize higher-viscosity environments in animal hosts

Comparative study between the effect of dexmedetomidine and lidocaine infusion on intraoperative analgesic requirement and hemodynamics during craniotomy

Background: Nowadays, anesthesiologists are evaluating several analgesic adjuncts to minimize opioid use during craniotomy. Some studies have evaluated the analgesic-sparing effect of intravenous infusion of dexmedetomidine and lidocaine on intraoperative hemodynamics and post-operative analgesia. There is a paucity of studies focussing on the intraoperative analgesic requirement. Aims and Objectives: The present study compared dexmedetomidine and lidocaine infusion primarily for their effects on intraoperative fentanyl requirements during craniotomy. Materials and Methods: This study was done on 70 patients aged 18–80 years, the American Society of Anesthesiologists physical status I–II, having Glasgow Coma Scale 15, undergoing craniotomies. Patients were randomly allocated to receive either dexmedetomidine (group A, n=35) at a dose of 0.6 mcg/kg bolus over 10 min followed by 0.6 mcg/kg/h infusion or lidocaine (group B, n=35) at a dose of 1.5 mg/kg bolus over 10 min, followed by 1.5 mg/kg/h infusion till the end of skin suture, respectively. Study drugs were started 10 min before the start of surgery. Intraoperative total fentanyl and propofol consumption, intraoperative hemodynamics, recovery from hypnosis, and time to extubation were recorded. Results: The use of dexmedetomidine resulted in considerably less total fentanyl requirement (245.1 vs. 300.7 mcg, P<0.0001) and total propofol requirement (172.7 vs. 236.7 mg, P<0.0001) compared with lidocaine. Comparatively better hemodynamics were observed with the use of dexmedetomidine at all the points of observation. Conclusion: Dexmedetomidine as an analgesic adjunct can be a better alternative to lidocaine in terms of reduced fentanyl consumption, reduced propofol use and favorable hemodynamics, and early recovery from anesthesia

Polar targeting and assembly of the Legionella Dot/Icm type IV secretion system (T4SS) by T6SS-related components

Legionella pneumophila, the causative agent of Legionnaires′ disease, survives and replicates inside amoebae and macrophages by injecting a large number of protein effectors into the host cells′ cytoplasm via the Dot/Icm type IVB secretion system (T4BSS). Previously, we showed that the Dot/Icm T4BSS is localized to both poles of the bacterium and that polar secretion is necessary for the proper targeting of the Legionella containing vacuole (LCV). Here we show that polar targeting of the Dot/Icm core-transmembrane subcomplex (DotC, DotD, DotF, DotG and DotH) is mediated by two Dot/Icm proteins, DotU and IcmF, which are able to localize to the poles of L. pneumophila by themselves. Interestingly, DotU and IcmF are homologs of the T6SS components TssL and TssM, which are part of the T6SS membrane complex (MC). We propose that Legionella co-opted these T6SS components to a novel function that mediates subcellular localization and assembly of this T4SS. Finally, in depth examination of the biogenesis pathway revealed that polar targeting and assembly of the Legionella T4BSS apparatus is mediated by an innovative ″outside-inside″ mechanism