Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.Flagellated bacteria have tailored their motility to diverse habitats. For example, the enteric model organisms Salmonella enterica serovar Typhimurium and Escherichia coli colonize animal digestive tracts and can reside outside a host, assembling flagella over their cell body to swim. However, a diverse spectrum of flagellar swimming ability is seen across the bacterial kingdom. Caulobacter crescentus inhabits low-nutrient freshwater environments where it swims using a high-efficiency flagellar motor (1, 2), whereas Vibrio species produce high-speed, sodium-driven polar flagella to capitalize on the high sodium gradient of their marine habitat (3). On the other hand, the ε-proteobacteria and spirochetes, many of which thrive exclusively in association with a host, have evolved characteristically rapid and powerful swimming capabilities that enable them to bore through mucous layers coating epithelial cells or between tissues. Indeed, the ε-proteobacteria Campylobacter jejuni and Helicobacter pylori are capable of continued swimming in high-viscosity media that immobilize E. coli or Vibrio cells (4–6), and similar behavior is observed for spirochetes (7, 8).Despite differences in the organisms’ swimming ability, the flagellar motor is composed of a conserved core of ∼20 structural proteins (9). The mechanism of flagellar motility is conserved (10), with torque generated by rotor and stator components (9). Stator complexes, heterooligomers of four motility A (MotA) and two motility B (MotB) proteins, are thought to form a ring that surrounds the axial driveshaft. Transmembrane helices of MotA and MotB form an ion channel, and MotB features a large periplasmic domain that binds peptidoglycan (11, 12) and the flagellar structural component, the P-ring (13). The stator complex couples ion flux to exertion of force on the cytoplasmic rotor ring (the C-ring), which transmits torque to the axial driveshaft (the rod), universal joint (the hook), and helical propeller (the filament), culminating in propulsion of the bacterium. Biophysical (14) and freeze-fracture (15) studies together with modeling (16) have proposed that a tight ring of ∼11 stator complexes dynamically assembles around the rod above the outer lobe of the C-ring in closely related Salmonella and E. coli motors (which we collectively refer to as the “enteric motor”). However, despite these conclusions, and although the structures observed in subtomogram averages have been proposed to be the stator complexes (17–19), the locations and stoichiometries of the stator complexes remain to be confirmed.How can we explain the wide diversity in flagellar swimming abilities in the context of a conserved core flagellar motor? Biophysical studies suggest that the source of the difference lies, at least in part, in variations in the mechanical output of the motors themselves. Torques of motors from different bacteria have been shown to range over an order of magnitude, and torque correlates with swimming speed and the ability of bacteria to propel themselves through different viscosities, indicating that adaptations are likely to be at the level of the motor itself. [Torque also varies within a single species, up to a maximum value, as a function of the number of stator complexes incorporated into the motor (14)]. For example, C. crescentus motors have been measured to produce torques of 350 pN⋅nm (2). Estimates for the torque of the enteric motor ranges from ∼1,300 to ∼2,000 pN⋅nm (20, 21). The ε-proteobacterium H. pylori has been estimated to swim with torque of 3,600 pN⋅nm (22), and spirochetes are capable of swimming with 4,000 pN⋅nm of torque (21, 23). Sodium-driven motor torques in Vibrio spp. have been measured between ∼2,000 and 4,000 pN⋅nm (24), depending on the magnitude of the sodium gradient. It is noteworthy, however, that an estimated sodium motive force in Vibrio spp. that is lower than the standard E. coli proton motive force nevertheless drives the Vibrio motor with higher torque than the E. coli motor (24, 25), further suggesting that torque differences likely exist at the level of the motor. However, the molecular mechanism by which different motors might produce different torques has not been investigated.The simplest scenario for tuning motor torque would be evolved adaptation of motor architecture. In support of this scenario, we recently showed that many motors have evolved additional structures not found in the well-studied enteric motors (18), and we observed that the C-ring radius varies among species (17, 18). One of the most widespread novel structures is a periplasmic basal disk directly beneath the outer membrane, often co-occurring with varied uncharacterized additional structures, which we collectively term “disk complexes.” Consistently, disk complexes have been seen only in motors that produce torque higher than that in E. coli or Salmonella. For example, the sodium-driven ∼2,000+ pN⋅nm torque motors of Vibrio species assemble a disk complex featuring a basal disk beneath the outer membrane (18) in addition to smaller H- and T-rings composed of FlgOT (flagella O, T) and MotXY (motility X, Y), respectively (26, 27). It has been shown that the T-ring interacts with stator complexes in Vibrio spp. (28), although the exact location and number of stator complexes in Vibrio spp. remains unclear. ε-Proteobacteria such as Helicobacter species, C. jejuni, and Wolinella succinogenes also assemble disk complexes composed of large basal disks beneath the outer membrane together with additional smaller disks (18, 29). Although these and other cases of additional disks have been reported (18, 30), their relation to flagellar function remains enigmatic, and it is unclear if these widespread disk complexes are homologous or analogous.In this study, we hypothesized that bacteria have tuned their swimming abilities by evolving structural adaptations to their flagellar motors that would result in altered torque generation. Using electron cryo-tomography and subtomogram averaging, we found that Vibrio polar γ-proteobacterial and Campylobacter ε-proteobacterial flagellar motors incorporate 13 and 17 stator complexes, respectively, compared with the ∼11 in enteric bacteria. In both cases, these stator complexes are scaffolded into wider stator rings relative to the enteric motor by components of their respective disk complexes. The wider C. jejuni stator ring is further reflected in a considerably wider rotor C-ring. Further analysis of the components of the Vibrio and C. jejuni disk complexes reveals that they share a core protein, FlgP, but each has acquired diverse additional components to form divergent disk-complex architectures. We conclude by showing that our structural data of wider stator rings featuring additional stator complexes can quantitatively account for the differences in torque between different flagellar motors.     

The Role of Serotonin (5-HT) in Behavioral Control: Findings from Animal Research and Clinical Implications

The neurotransmitters serotonin and dopamine both have a critical role in the underlying neurobiology of different behaviors. With focus on the interplay between dopamine and serotonin, it has been proposed that dopamine biases behavior towards habitual responding, and with serotonin offsetting this phenomenon and directing the balance toward more flexible, goal-directed responding. The present focus paper stands in close relationship to the publication by Worbe et al. (2015), which deals with the effects of acute tryptophan depletion, a neurodietary physiological method to decrease central nervous serotonin synthesis in humans for a short period of time, on the balance between hypothetical goal-directed and habitual systems. In that research, acute tryptophan depletion challenge administration and a following short-term reduction in central nervous serotonin synthesis were associated with a shift of behavioral performance towards habitual responding, providing further evidence that central nervous serotonin function modulates the balance between goal-directed and stimulus-response habitual systems of behavioral control. In the present focus paper, we discuss the findings by Worbe and colleagues in light of animal experiments as well as clinical implications and discuss potential future avenues for related research.     

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Host-derived amino acids support the proliferation of symbiotic bacteria

Animals are typically colonized by diverse bacterial symbionts, many of which are commensal and, in numerous cases, even essential for their host’s proper development and growth. In exchange, the host must supply a sufficient array and quantity of nutrients to support the proliferation and persistence of its microbial community. In this investigation, we have examined such a nutritional environment by determining the symbiotic competence of auxotrophic mutants of the bioluminescent bacterium Vibrio fischeri, and have demonstrated that the host squid Euprymna scolopes provides at least 9 aa to the growing culture of symbiotic V. fischeri present in its light-emitting organ. We also collected and analyzed the extracellular fluid from this organ, in which the symbionts reside, and confirmed that it contained significant amounts of amino acids. The combined results suggested that host-derived free amino acids, as well as peptides or proteins, are a source of the amino acids that support the growth of the symbionts. This work describes a technique to sample the symbionts and their surrounding environment without contamination by host tissue components and, in combination with molecular genetic studies, allows the characterization of the nutritional conditions that support a cooperative animal–bacterial symbiosis.     

Clonal dysregulation of the antibody response to tetanus-toxoid after bone marrow transplantation

After bone marrow transplantation (BMT), a prolonged dysregulation of humoral immunity can be observed. In the present study, we investigated whether this is reflected in an abnormal production of specific antibodies (Ab) to the T-cell-dependent recall antigen tetanus-toxoid (TT). The study group consisted of children receiving transplants of an unmodified allogeneic graft and of adults receiving either a T-cell- depleted allogeneic or an unmodified autologous BM graft. Findings were compared with those in healthy controls. In pediatric graft recipients, who were routinely revaccinated early after BMT, the Ab response was quantitatively superior to that in adult graft recipients who did not receive early revaccination. In the majority of graft recipients, the time period after vaccination required to reach the peak level of antibodies was prolonged and the number of responding TT-specific B- cell clones was markedly decreased in comparison with controls. In controls, a low frequency of dominant B-cell clones may produce low quantities of homogeneous Ab components (H-Ab) against a heterogeneous background. However, in BM graft recipients, "overshooting" of Ab production by separate B-cell clones was observed, resulting in the development of H-Ab at a relatively high concentration. These abnormalities were present up to 10 years after BMT, irrespective of either the age of the recipient, the modulation of the graft, or the vaccination schedule used. It is hypothesized that the dysregulated Ab production is the consequence of activation of a restricted number of resting memory B cells, present in germinal centers, repopulating gradually after BMT. Our data show that routine revaccination early after BMT improves the humoral immune response. However, because of a clonally dysregulated Ab production, long-lasting qualitative defects may be present even after normalization of Ab titers.     

Recombinant human interleukin-11 stimulates multilineage hematopoietic recovery in mice after a myelosuppressive regimen of sublethal irradiation and carboplatin

Interleukin-11 (IL-11) is a novel multifunctional hematopoietic cytokine capable of stimulating cells of the myeloid, lymphoid, erythroid, and megakaryocytic lineages in vitro. We have tested the pleiotropic properties of this cytokine on the hematopoietic recovery of mice after a combined regimen of sublethal irradiation and carboplatin administration. This regimen results in severe myelosuppression, characterized by a prolonged period of thrombocytopenia and severe anemia. Administration of recombinant human IL-11 (rhIL-11; 250 micrograms/kg/d) had multilineage effects on bone marrow and spleen hematopoietic activity, increasing the number of megakaryocyte, erythroid, granulocyte, and macrophage progenitors compared with the vehicle-treated controls. This was reflected in the peripheral circulation by a reduction of both the platelet and hematocrit nadirs and a significantly reduced period of thrombocytopenia and anemia in the rhIL-11-treated mice. The results from this study support the broad spectrum of biologic activities that have been attributed to rhIL-11 in vitro and suggest that this cytokine may be an effective agent in the treatment of myelosuppression associated with cancer chemotherapy and bone marrow transplantation.     

Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages

The bone marrow microenvironment supports and regulates the proliferation and differentiation of hematopoietic cells. Dysregulated hematopoiesis in chronic myelogenous leukemia (CML) is caused, at least in part, by abnormalities in CML hematopoietic progenitors leading to altered interactions with the marrow microenvironment. The role of the microenvironment itself in CML has not been well characterized. We examined the capacity of CML stroma to support the growth of long-term culture-initiating cells (LTC-IC) obtained from normal and CML marrow. The growth of normal LTC-IC on CML stroma was significantly reduced compared with normal stroma. This did not appear to be related to abnormal production of soluble factors by CML stroma because normal LTC- IC grew equally well in Transwells above CML stroma as in Transwells above normal stroma. In addition, CML and normal stromal supernatants contained similar quantities of both growth-stimulatory (granulocyte colony-stimulating factor (CSF), interleukin-6, stem cell factor, granulocyte-macrophage CSF, and interleukin-1 beta) and growth- inhibitory cytokines (transforming growth factor-beta, macrophage inflammatory protein-1 alpha, and tumor necrosis factor-alpha). The relative proportion of different cell types in CML and normal stroma was similar. However, polymerase chain reaction and fluorescence in situ hybridization studies showed the presence of bcr-abl-positivo cells in CML stroma, which were CD14+ stromal macrophages. To assess the effect of these malignant macrophages on stromal function, CML and normal stromal cells were separated by fluorescence-activated cell sorting into stromal mesenchymal cell (CD14-) and macrophage (CD14+) populations. CML and normal CD14- cells supported the growth of normal LTC-IC equally well. However, the addition of CML macrophages to normal or CML CD14- mesenchymal cells resulted in impaired progenitor support. This finding indicates that the abnormal function of CML bone marrow stroma is related to the presence of malignant macrophages. In contrast to normal LTC-IC, the growth of CML LTC-IC on allogeneic CML stromal layers was not impaired and was significantly better than that of normal LTC-IC cocultured with the same CML stromal layers. These studies demonstrate that, in addition to abnormalities in CML progenitors themselves, abnormalities in the CML marrow microenvironment related to the presence of malignant stromal macrophages may contribute to the selective expansion of leukemic progenitors and suppression of normal hematopoiesis in CML.     

Once-Daily Triple Therapy in Patients with COPD: Patient-Reported Symptoms and Quality of Life

Introduction

Directly recorded patient experience of symptoms and health-related quality of life (HRQoL) can complement lung function and exacerbation rate data in chronic obstructive pulmonary disease (COPD) clinical studies. The FULFIL study recorded daily symptoms and activity limitation together with additional patient-reported outcomes of dyspnea and HRQoL, as part of the prespecified analyses. FULFIL co-primary endpoint data have been previously reported.

Methods

FULFIL was a phase III, 24-week, randomized, double-blind, double-dummy, multicenter study comparing once-daily single inhaler triple therapy [fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI)] 100 µg/62.5 µg/25 µg with twice-daily inhaled corticosteroid/long-acting β₂-agonist therapy [budesonide/formoterol (BUD/FOR)] 400 µg/12 µg in patients with symptomatic COPD at risk of exacerbations. A subset participated for 52 weeks. Patient-reported assessments were: Evaluating Respiratory Symptoms in COPD? (E-RS: COPD), St George’s Respiratory Questionnaire (SGRQ) for COPD, COPD Assessment Test (CAT), baseline and transitional dyspnea indices (TDI) and daily and global anchor questions for activity limitation.

Results

FF/UMEC/VI showed greater reductions from baseline in 4-weekly mean E-RS: COPD total and all subscale scores compared with BUD/FOR; differences were statistically significant (P < 0.05) at each time period. FF/UMEC/VI also demonstrated greater improvements from baseline at weeks 4 and 24 in SGRQ domain scores and TDI focal score compared with BUD/FOR. At weeks 4 and 24, improvements greater than the minimal clinically important difference from baseline were observed in CAT score with FF/UMEC/VI, but not BUD/FOR; differences were statistically significant (P ≤ 0.003).

Conclusion

These findings demonstrate sustained daily symptom and HRQoL benefits of FF/UMEC/VI versus BUD/FOR. The inclusion of the CAT may provide data that are readily generalizable to everyday clinical practice.

Trial registration

ClinicalTrials.gov number: NCT02345161.

Funding

GSK.

     

Theoretical studies of optoelectronic,magnetization and heat transport properties of conductive metal adatoms adsorbed on edge chlorinated nanographenes

The electronic structures, magnetization and quantum transport properties of edge chlorinated nanographenes (Cl NGRs) (C1–C3) functionalized with conductive metal adatoms (Al, Au and Cu) has been investigated by means of density functional theory (DFT) with periodic boundary conditions and plane wave basis functions. The adsorption energy results depict weak chemisorption and strong physisorption for Au adsorption for C1, while C2 and C3 show strong chemisorption towards the studied metals. The role of dispersion forces has also been studied with an empirical classical model. The results show that the metal clusters avoid hollow sites on the Cl NGRs surface and favor atop and bond sites. The net magnetic moment of 0.73 μ_B is observed for the (Cl NGRs–metals) system and is in reasonable agreement with the previous calculations carried out on graphene nanoribbons. The TDDFT calculations predict that the absorption spectra for metal dimer–Cl NGRs lie in the visible region. The predictive electrical conductivity of these systems suggests that the metal adatoms play an important role in the transport properties of devices and can be used for thermoelectric applications.

The electronic structures, magnetization and quantum transport properties of edge chlorinated nanographenes functionalized with conductive metal adatoms have been investigated by means of density functional theory with plane wave basis functions.