Pneumonic Plague Protection Induced by a Monophosphoryl Lipid A Decorated Yersinia Outer-Membrane-Vesicle Vaccine.

A new Yersinia pseudotuberculosis mutant strain, YptbS46, carrying the lpxE insertion and pmrF-J deletion is constructed and shown to exclusively produce monophosphoryl lipid A (MPLA) having adjuvant properties. Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, are designated OMV46-LcrV, which contained MPLA and high amounts of LcrV (Low Calcium response V) and displayed low activation of Toll-like receptor 4 (TLR4). Intramuscular prime-boost immunization with 30 µg of of OMV46-LcrV exhibited substantially reduced reactogenicity than the parent OMV44-LcrV and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV46-LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which are correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV46-LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV46-LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. The studies strongly demonstrate the feasibility of OMV46-LcrV as a new type of plague vaccine candidate.

Three-dimensional architecture of epithelial primary cilia.

We report a complete 3D structural model of typical epithelial primary cilia based on structural maps of full-length primary cilia obtained by serial section electron tomography. Our data demonstrate the architecture of primary cilia differs extensively from the commonly acknowledged 9+0 paradigm. The axoneme structure is relatively stable but gradually evolves from base to tip with a decreasing number of microtubule complexes (MtCs) and a reducing diameter. The axonemal MtCs are cross-linked by previously unrecognized fibrous protein networks. Such an architecture explains why primary cilia can elastically withstand liquid flow for mechanosensing. The nine axonemal MtCs in a cilium are found to differ significantly in length indicating intraflagellar transport processes in primary cilia may be more complicated than that reported for motile cilia. The 3D maps of microtubule doublet-singlet transitions generally display longitudinal gaps at the inner junction between the A- and B-tubules, which indicates the inner junction protein is a major player in doublet-singlet transitions. In addition, vesicles releasing from kidney primary cilia were observed in the structural maps, supporting that ciliary vesicles budding may serve as ectosomes for cell-cell communication.

AbmR (Rv1265) is a novel transcription factor of Mycobacterium tuberculosis that regulates host cell association and expression of the non-coding small RNA Mcr11.

Gene regulatory networks used by Mycobacterium tuberculosis (Mtb) during infection include many genes of unknown function, confounding efforts to determine their roles in Mtb biology. Rv1265 encodes a conserved hypothetical protein that is expressed during infection and in response to elevated levels of cyclic AMP. Here, we report that Rv1265 is a novel auto-inhibitory ATP-binding transcription factor that upregulates expression of the small non-coding RNA Mcr11, and propose that Rv1265 be named ATP-binding mcr11 regulator (AbmR). AbmR directly and specifically bound DNA, as determined by electrophoretic mobility shift assays, and this DNA-binding activity was enhanced by AbmR's interaction with ATP. Genetic knockout of abmR in Mtb increased abmR promoter activity and eliminated growth phase-dependent increases in mcr11 expression during hypoxia. Mutagenesis identified arginine residues in the carboxy terminus that are critical for AbmR's DNA-binding activity and gene regulatory function. Limited similarity to other DNA- or ATP-binding domains suggests that AbmR belongs to a novel class of DNA- and ATP-binding proteins. AbmR was also found to form large organized structures in solution and facilitate the serum-dependent association of Mtb with human lung epithelial cells. These results indicate a potentially complex role for AbmR in Mtb biology.

Cryo-FIB specimen preparation for use in a cartridge-type cryo-TEM.

Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET. Commercial cryosystems are available for cryo-FIB specimen preparation, however re-engineering and additional fixtures are often essential for reliable results with a particular cryo-FIB and cryo-transmission electron microscope (cryo-TEM). Here, we describe our optimized protocol and modified instrumentation for cryo-FIB milling to produce thin lamellae and subsequent damage-free cryotransfer of the lamellae into our cartridge-type cryo-TEM.

Accurate analysis of fusion expression of Pichia pastoris glycosylphosphatidylinositol-modified cell wall proteins.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have diverse intrinsic functions in yeasts, and they also have different uses in vitro. The GPI-modified cell wall proteins GCW21, GCW51, and GCW61 of Pichia pastoris were chosen as anchoring proteins to construct co-expression strains in P. pastoris GS115. The hydrolytic activity and the amount of Candida antarctica lipase B (CALB) displayed on cell surface increased significantly following optimization of the fusion gene dosage and combination of the homogeneous or heterogeneous cell wall proteins. Maximum CALB hydrolytic activity was achieved at 4920 U/g dry cell weight in strain GS115/CALB-GCW (51 + 51 + 61 + 61) after 120 h of methanol induction. Changes in structural morphology and the properties of the cell surfaces caused by co-expression of fusion proteins were observed by transmission electron microscopy (TEM) and on plates containing cell-wall-destabilizing reagent. Our results suggested that both the outer and inner cell layers were significantly altered by overexpression of GPI-modified cell wall proteins. Interestingly, quantitative analysis of the inner layer components showed an increase in ß-1,3-glucan, but no obvious changes in chitin in the strains overexpressing GPI-modified cell wall proteins.

Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B.

To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37 and 109 %, respectively, but decreased by 26 and 43 %, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells.

Effect of fringe-artifact correction on sub-tomogram averaging from Zernike phase-plate cryo-TEM.

Zernike phase-plate (ZPP) imaging greatly increases contrast in cryo-electron microscopy, however fringe artifacts appear in the images. A computational de-fringing method has been proposed, but it has not been widely employed, perhaps because the importance of de-fringing has not been clearly demonstrated. For testing purposes, we employed Zernike phase-plate imaging in a cryo-electron tomographic study of radial-spoke complexes attached to microtubule doublets. We found that the contrast enhancement by ZPP imaging made nonlinear denoising insensitive to the filtering parameters, such that simple low-frequency band-pass filtering made the same improvement in map quality. We employed sub-tomogram averaging, which compensates for the effect of the "missing wedge" and considerably improves map quality. We found that fringes (caused by the abrupt cut-on of the central hole in the phase plate) can lead to incorrect representation of a structure that is well-known from the literature. The expected structure was restored by amplitude scaling, as proposed in the literature. Our results show that de-fringing is an important part of image-processing for cryo-electron tomography of macromolecular complexes with ZPP imaging.

A bacterial vesicle-based pneumococcal vaccine against influenza-mediated secondary Streptococcus pneumoniae pulmonary infection.

Streptococcus pneumoniae (Spn) is a common pathogen causing a secondary bacterial infection following influenza, which leads to severe morbidity and mortality during seasonal and pandemic influenza. Therefore, there is an urgent need to develop bacterial vaccines that prevent severe post-influenza bacterial pneumonia. Here, an improved Yersinia pseudotuberculosis strain (designated as YptbS46) possessing an Asd+ plasmid pSMV92 could synthesize high amounts of the Spn pneumococcal surface protein A (PspA) antigen and monophosphoryl lipid A as an adjuvant. The recombinant strain produced outer membrane vesicles (OMVs) enclosing a high amount of PspA protein (designated as OMV-PspA). A prime-boost intramuscular immunization with OMV-PspA induced both memory adaptive and innate immune responses in vaccinated mice, reduced the viral and bacterial burden, and provided complete protection against influenza-mediated secondary Spn infection. Also, the OMV-PspA immunization afforded significant cross-protection against the secondary Spn A66.1 infection and long-term protection against the secondary Spn D39 challenge. Our study implies that an OMV vaccine delivering Spn antigens can be a new promising pneumococcal vaccine candidate.

Pneumonic plague protection induced by a monophosphoryl lipid A decorated Yersinia outer-membrane-vesicle vaccine.

A newly constructed Yersinia pseudotuberculosis mutant (YptbS46) carrying the lpxE insertion and pmrF-J deletion exclusively synthesized an adjuvant form of lipid A, monophosphoryl lipid A (MPLA). Outer membrane vesicles (OMVs) isolated from YptbS46 harboring an lcrV expression plasmid, pSMV13, were designated OMV 46 -LcrV, which contained MPLA and high amounts of LcrV and displayed low activation of Toll-like receptor 4 (TLR4). Similar to the previous OMV 44 -LcrV, intramuscular prime-boost immunization with 30 µg of OMV 46 -LcrV exhibited substantially reduced reactogenicity and conferred complete protection to mice against a high-dose of respiratory Y. pestis challenge. OMV 46 -LcrV immunization induced robust adaptive responses in both lung mucosal and systemic compartments and orchestrated innate immunity in the lung, which were correlated with rapid bacterial clearance and unremarkable lung damage during Y. pestis challenge. Additionally, OMV 46 -LcrV immunization conferred long-term protection. Moreover, immunization with reduced doses of OMV 46 -LcrV exhibited further lower reactogenicity and still provided great protection against pneumonic plague. Our studies strongly demonstrate the feasibility of OMV 46 -LcrV as a new type of plague vaccine candidate.

Molecular architecture of axonemal microtubule doublets revealed by cryo-electron tomography.

The axoneme, which forms the core of eukaryotic flagella and cilia, is one of the largest macromolecular machines, with a structure that is largely conserved from protists to mammals. Microtubule doublets are structural components of axonemes that contain a number of proteins besides tubulin, and are usually found in arrays of nine doublets arranged around two singlet microtubules. Coordinated sliding of adjacent doublets, which involves a host of other proteins in the axoneme, produces periodic beating movements of the axoneme. We have obtained a three-dimensional density map of intact microtubule doublets using cryo-electron tomography and image averaging. Our map, with a resolution of about 3 nm, provides insights into locations of particular proteins within the doublets and the structural features of the doublets that define their mechanical properties. We identify likely candidates for several of these non-tubulin components of the doublets. This work offers insight on how tubulin protofilaments and accessory proteins attach together to form the doublets and provides a structural basis for understanding doublet function in axonemes.

Sugar, sugar: Glucose activates motion in pancreatic cilia.

Pancreatic primary cilia are active and dynamic, not static antenna-like sensors as previously thought. This movement may be an important mechanism to glucose regulation.

Phenotypic and genetic changes associated with the seesaw effect in MRSA strain N315 in a bioreactor model.

OBJECTIVES: Over the past decade, daptomycin treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections has led to the emergence of daptomycin nonsusceptible (DAP-NS) MRSA strains and a subsequent interest in combinatorial antibiotic therapies. We investigated the phenotypic and genetic changes associated with the seesaw effect, which describes the correlation between daptomycin resistance and increased ß-lactam susceptibility in DAP-NS MRSA and the reverse phenomenon of DAP-NS strains acquiring renewed susceptibility to daptomycin after ß-lactam exposure. METHODS: A continuous bioreactor model was used to study the effects of incremental doses of daptomycin followed by oxacillin on MRSA strain N315. Minimum inhibitory concentrations for daptomycin and oxacillin were determined for the bioreactor-derived samples. Transmission electron microscopy and cytochrome C binding assays were used to measure cell wall thickness and cell membrane charge, respectively, in the bioreactor-derived samples. Whole-genome sequencing was used to identify mutations associated with the seesaw effect. RESULTS: Although daptomycin resistance conferred enhanced susceptibility to oxacillin, oxacillin treatment of DAP-NS strains was accompanied by a lowered minimum inhibitory concentration for daptomycin. Additionally, there was a reduction in relative positive cell surface charge and cell wall thickness. However, the mutations acquired in our DAP-NS populations were not accompanied by additional genomic changes after treatment with oxacillin, implicating alternative mechanisms for the seesaw effect. CONCLUSION: In this study, we successfully produced and characterized the seesaw effect in MRSA strain N315 in a unique bioreactor model.

Structural insights into microtubule doublet interactions in axonemes.

Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of eukaryotic cilia and flagella. Recent structural studies of the axoneme, which forms the core of cilia and flagella, have used cryo-electron tomography to reveal new details of the interactions between some of the multitude of proteins that form the axoneme and regulate its movement. Connections between the several types of dyneins, in particular, suggest ways in which their action might be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding mechanical properties related to the bending of the axoneme, and has also offered insight into the potential role of doublets in the mechanism of dynein activity regulation.

A common partitivirus infection in United States and Czech Republic isolates of bat white-nose syndrome fungal pathogen Pseudogymnoascus destructans.

The psychrophilic (cold-loving) fungus Pseudogymnoascus destructans was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found P. destructans was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family Partitiviridae. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of P. destructans were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus Pseudogymnoascus, as non-pathogenic fungi P. appendiculatus (1 isolate) and P. roseus (6 isolates) tested negative. P. destructans PdPV-1 could be a valuable tool to investigate fungal biogeography and the host-pathogen interactions in bat WNS.

High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy.

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole's distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.

The Developmental Process of the Growing Motile Ciliary Tip Region.

Eukaryotic motile cilia/flagella play vital roles in various physiological processes in mammals and some protists. Defects in cilia formation underlie multiple human disorders, known as ciliopathies. The detailed processes of cilia growth and development are still far from clear despite extensive studies. In this study, we characterized the process of cilium formation (ciliogenesis) by investigating the newly developed motile cilia of deciliated protists using complementary techniques in electron microscopy and image analysis. Our results demonstrated that the distal tip region of motile cilia exhibit progressive morphological changes as cilia develop. This developmental process is time-dependent and continues after growing cilia reach their full lengths. The structural analysis of growing ciliary tips revealed that B-tubules of axonemal microtubule doublets terminate far away from the tip end, which is led by the flagellar tip complex (FTC), demonstrating that the FTC might not directly mediate the fast turnover of intraflagellar transport (IFT).

Unattached kinetochores rather than intrakinetochore tension arrest mitosis in taxol-treated cells.

Kinetochores attach chromosomes to the spindle microtubules and signal the spindle assembly checkpoint to delay mitotic exit until all chromosomes are attached. Light microscopy approaches aimed to indirectly determine distances between various proteins within the kinetochore (termed Delta) suggest that kinetochores become stretched by spindle forces and compact elastically when the force is suppressed. Low Delta is believed to arrest mitotic progression in taxol-treated cells. However, the structural basis of Delta remains unknown. By integrating same-kinetochore light microscopy and electron microscopy, we demonstrate that the value of Delta is affected by the variability in the shape and size of outer kinetochore domains. The outer kinetochore compacts when spindle forces are maximal during metaphase. When the forces are weakened by taxol treatment, the outer kinetochore expands radially and some kinetochores completely lose microtubule attachment, a condition known to arrest mitotic progression. These observations offer an alternative interpretation of intrakinetochore tension and question whether Delta plays a direct role in the control of mitotic progression.

Crystal structure of human calmodulin-like protein: insights into its functional role.

A calmodulin (CaM)-like protein (hCLP) is expressed in human mammary epithelial cells but appears to be limited to certain epithelial cells such as those found in skin, prostate, breast and cervical tissues. A decrease in the expression of this protein is associated with the occurrence of tumors in breast epithelium. The structure of hCLP determined to 1.5 A resolution by X-ray crystallography shows a distinct 30 degrees displacement along the interconnecting central helix, when compared to the highly conserved structure of vertebrate CaM, resulting in a difference in the relative orientation of its two globular domains. Additionally, the electric surface potential landscape at the target protein binding regions on the two globular domains of hCLP is significantly different from those of CaM, indicating that the respective ranges of hCLP and hCaM target proteins do not fully overlap. Observations that hCLP can competitively inhibit CaM activation of target proteins also imply a role for hCLP in which it may also serve as a modulator of CaM activity in the epithelial cells where hCLP is expressed.