Structural remodeling of bacteriophage T4 and host membranes during infection initiation

The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinnyE. coliminicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection.

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ssRNA phage penetration triggers detachment of the F-pilus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2011901117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25751-25758

Author(s):

Laith Harb ◽

Karthik Chamakura ◽

Pratick Khara ◽

Peter J. Christie ◽

Ry Young ◽

...

Keyword(s):

Cell Surface ◽

Selective Advantage ◽

Type Iv Secretion ◽

Host Cells ◽

Early Step ◽

Host Cytoplasm ◽

Type Iv ◽

Maturation Protein ◽

Coupling Protein ◽

Multistep Model

Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

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Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1425008112 ◽

2015 ◽

Vol 112 (8) ◽

pp. 2449-2454 ◽

Cited By ~ 31

Author(s):

Chuan Hong ◽

Maija K. Pietilä ◽

Caroline J. Fu ◽

Michael F. Schmid ◽

Dennis H. Bamford ◽

...

Keyword(s):

Structural Changes ◽

Electron Tomography ◽

Host Cells ◽

Biochemical Treatment ◽

Archaeal Virus ◽

Virus Life Cycle ◽

Cryo Electron Tomography ◽

Empty Tube ◽

Subtomogram Averaging ◽

Unusual Shape

Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.

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Nanometer-resolution in situ structure of the SARS-CoV-2 postfusion spike protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2112703118 ◽

2021 ◽

Vol 118 (48) ◽

pp. e2112703118

Author(s):

Linhua Tai ◽

Guoliang Zhu ◽

Minnan Yang ◽

Lei Cao ◽

Xiaorui Xing ◽

...

Keyword(s):

Transmembrane Domain ◽

Structural Information ◽

Electron Tomography ◽

Host Cells ◽

Spike Protein ◽

Fusion Pore ◽

Nanometer Resolution ◽

Glycosylation Sites ◽

Entry Mechanism

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates membrane fusion to allow entry of the viral genome into host cells. To understand its detailed entry mechanism and develop a specific entry inhibitor, in situ structural information on the SARS-CoV-2 spike protein in different states is urgent. Here, by using cryo-electron tomography, we observed both prefusion and postfusion spikes in β-propiolactone–inactivated SARS-CoV-2 virions and solved the in situ structure of the postfusion spike at nanometer resolution. Compared to previous reports, the six-helix bundle fusion core, the glycosylation sites, and the location of the transmembrane domain were clearly resolved. We observed oligomerization patterns of the spikes on the viral membrane, likely suggesting a mechanism of fusion pore formation.

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Automated electron tomography: from data collection to image processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100136507 ◽

1995 ◽

Vol 53 ◽

pp. 26-27

Author(s):

Weiping Liu ◽

Jennifer Fung ◽

W.J. de Ruijter ◽

Hans Chen ◽

John W. Sedat ◽

...

Keyword(s):

Image Processing ◽

Data Collection ◽

Structural Information ◽

Imaging Technique ◽

Electron Tomography ◽

Ccd Camera ◽

Automated Data Collection ◽

Full Control ◽

Transmission Electron ◽

Amorphous Structures

Electron tomography is a technique where many projections of an object are collected from the transmission electron microscope (TEM), and are then used to reconstruct the object in its entirety, allowing internal structure to be viewed. As vital as is the 3-D structural information and with no other 3-D imaging technique to compete in its resolution range, electron tomography of amorphous structures has been exercised only sporadically over the last ten years. Its general lack of popularity can be attributed to the tediousness of the entire process starting from the data collection, image processing for reconstruction, and extending to the 3-D image analysis. We have been investing effort to automate all aspects of electron tomography. Our systems of data collection and tomographic image processing will be briefly described.To date, we have developed a second generation automated data collection system based on an SGI workstation (Fig. 1) (The previous version used a micro VAX). The computer takes full control of the microscope operations with its graphical menu driven environment. This is made possible by the direct digital recording of images using the CCD camera.

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Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

Infection and Immunity ◽

10.1128/iai.72.10.5983-5992.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5983-5992 ◽

Cited By ~ 56

Author(s):

Jessica A. Sexton ◽

Jennifer L. Miller ◽

Aki Yoneda ◽

Thomas E. Kehl-Fie ◽

Joseph P. Vogel

Keyword(s):

Cell Surface ◽

Legionella Pneumophila ◽

Bacterial Species ◽

Wide Distribution ◽

Host Cells ◽

Growth Defect ◽

Intracellular Growth ◽

Homologous Proteins ◽

Type Iv ◽

Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

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A Single-Pass Type I Membrane Protein from the Apicomplexan Parasite Cryptosporidium parvum with Nanomolar Binding Affinity to Host Cell Surface

Microorganisms ◽

10.3390/microorganisms9051015 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1015

Author(s):

Tianyu Zhang ◽

Xin Gao ◽

Dongqiang Wang ◽

Jixue Zhao ◽

Nan Zhang ◽

...

Keyword(s):

Membrane Protein ◽

Cell Surface ◽

Host Cell ◽

Binding Affinity ◽

Cryptosporidium Parvum ◽

Host Cells ◽

Watery Diarrhea ◽

Type I ◽

Single Pass ◽

Host Cell Surface

Cryptosporidium parvum is a globally recognized zoonotic parasite of medical and veterinary importance. This parasite mainly infects intestinal epithelial cells and causes mild to severe watery diarrhea that could be deadly in patients with weakened or defect immunity. However, its molecular interactions with hosts and pathogenesis, an important part in adaptation of parasitic lifestyle, remain poorly understood. Here we report the identification and characterization of a C. parvum T-cell immunomodulatory protein homolog (CpTIPH). CpTIPH is a 901-aa single-pass type I membrane protein encoded by cgd5_830 gene that also contains a short Vibrio, Colwellia, Bradyrhizobium and Shewanella (VCBS) repeat and relatively long integrin alpha (ITGA) N-terminus domain. Immunofluorescence assay confirmed the location of CpTIPH on the cell surface of C. parvum sporozoites. In congruence with the presence of VCBS repeat and ITGA domain, CpTIPH displayed high, nanomolar binding affinity to host cell surface (i.e., Kd(App) at 16.2 to 44.7 nM on fixed HCT-8 and CHO-K1 cells, respectively). The involvement of CpTIPH in the parasite invasion is partly supported by experiments showing that an anti-CpTIPH antibody could partially block the invasion of C. parvum sporozoites into host cells. These observations provide a strong basis for further investigation of the roles of CpTIPH in parasite-host cell interactions.

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A graph-based algorithm for detecting rigid domains in protein structures

BMC Bioinformatics ◽

10.1186/s12859-021-03966-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Truong Khanh Linh Dang ◽

Thach Nguyen ◽

Michael Habeck ◽

Mehmet Gültas ◽

Stephan Waack

Keyword(s):

Structural Changes ◽

Viterbi Algorithm ◽

Structural Information ◽

Line Graph ◽

Clustering Algorithms ◽

Protein Structures ◽

Alternative Methods ◽

Structural Transitions ◽

Tuning Parameters ◽

Conformational Ensembles

Abstract Background Conformational transitions are implicated in the biological function of many proteins. Structural changes in proteins can be described approximately as the relative movement of rigid domains against each other. Despite previous efforts, there is a need to develop new domain segmentation algorithms that are capable of analysing the entire structure database efficiently and do not require the choice of protein-dependent tuning parameters such as the number of rigid domains. Results We develop a graph-based method for detecting rigid domains in proteins. Structural information from multiple conformational states is represented by a graph whose nodes correspond to amino acids. Graph clustering algorithms allow us to reduce the graph and run the Viterbi algorithm on the associated line graph to obtain a segmentation of the input structures into rigid domains. In contrast to many alternative methods, our approach does not require knowledge about the number of rigid domains. Moreover, we identified default values for the algorithmic parameters that are suitable for a large number of conformational ensembles. We test our algorithm on examples from the DynDom database and illustrate our method on various challenging systems whose structural transitions have been studied extensively. Conclusions The results strongly suggest that our graph-based algorithm forms a novel framework to characterize structural transitions in proteins via detecting their rigid domains. The web server is available at http://azifi.tz.agrar.uni-goettingen.de/webservice/.

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Effectors of biotrophic fungal plant pathogens

Functional Plant Biology ◽

10.1071/fp10072 ◽

2010 ◽

Vol 37 (10) ◽

pp. 913 ◽

Cited By ~ 12

Author(s):

Pamela H. P. Gan ◽

Maryam Rafiqi ◽

Adrienne R. Hardham ◽

Peter N. Dodds

Keyword(s):

Plant Tissue ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Plant Cells ◽

Host Cells ◽

Plant Proteins ◽

Living Plant ◽

Host Cytoplasm ◽

Fungal Plant Pathogens ◽

Biotrophic Fungi

Plant pathogenic biotrophic fungi are able to grow within living plant tissue due to the action of secreted pathogen proteins known as effectors that alter the response of plant cells to pathogens. The discovery and identification of these proteins has greatly expanded with the sequencing and annotation of fungal pathogen genomes. Studies to characterise effector function have revealed that a subset of these secreted pathogen proteins interact with plant proteins within the host cytoplasm. This review focuses on the effectors of intracellular biotrophic and hemibiotrophic fungal plant pathogens and summarises advances in understanding the roles of these proteins in disease and in elucidating the mechanism of fungal effector uptake into host cells.

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Identifying and Evaluating Anomalous Structural Change-based Nodes in Generalized Dynamic Social Networks

ACM Transactions on the Web ◽

10.1145/3457906 ◽

2021 ◽

Vol 15 (4) ◽

pp. 1-22

Author(s):

Huan Wang ◽

Chunming Qiao ◽

Xuan Guo ◽

Lei Fang ◽

Ying Sha ◽

...

Keyword(s):

Social Networks ◽

Structural Change ◽

Structural Changes ◽

Structural Information ◽

Similarity Index ◽

Dynamic Social Network ◽

Dynamic Social Networks ◽

Micro Level ◽

Anomaly Analysis ◽

Similarity Method

Recently, dynamic social network research has attracted a great amount of attention, especially in the area of anomaly analysis that analyzes the anomalous change in the evolution of dynamic social networks. However, most of the current research focused on anomaly analysis of the macro representation of dynamic social networks and failed to analyze the nodes that have anomalous structural changes at a micro level. To identify and evaluate anomalous structural change-based nodes in generalized dynamic social networks that only have limited structural information, this research considers undirected and unweighted graphs and develops a multiple-neighbor superposition similarity method ( ), which mainly consists of a multiple-neighbor range algorithm ( ) and a superposition similarity fluctuation algorithm ( ). introduces observation nodes, characterizes the structural similarities of nodes within multiple-neighbor ranges, and proposes a new multiple-neighbor similarity index on the basis of extensional similarity indices. Subsequently, maximally reflects the structural change of each node, using a new superposition similarity fluctuation index from the perspective of diverse multiple-neighbor similarities. As a result, based on and , not only identifies anomalous structural change-based nodes by detecting the anomalous structural changes of nodes but also evaluates their anomalous degrees by quantifying these changes. Results obtained by comparing with state-of-the-art methods via extensive experiments show that can accurately identify anomalous structural change-based nodes and evaluate their anomalous degrees well.

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A light and electron microscope study of the fungal endophytes in the sporophyte and gametophyte of Lycopodium cernuum with observations on the gametophyte–sporophyte junction

Canadian Journal of Botany ◽

10.1139/b92-008 ◽

1992 ◽

Vol 70 (1) ◽

pp. 58-72 ◽

Cited By ~ 40

Author(s):

Jeffrey G. Duckett ◽

Roberto Ligrone

Keyword(s):

Root Nodules ◽

Fungal Endophytes ◽

Fungal Endophyte ◽

Peninsular Malaysia ◽

Epidermal Cells ◽

Host Cells ◽

Wall Material ◽

Intercellular Spaces ◽

Host Cytoplasm ◽

Fungal Associations

The ventral epidermal cells of the photosynthetic, surface-living gametophytes of Lycopodium cernuum, collected from moist shaded banks in Peninsular Malaysia, contain an aseptate fungus. In some cells the hyphae are thick walled and form coils encapsulated by a thin layer of host wall material. In others the fungus is thin walled and shows limited differentiation into larger trunk hyphae and arbuscules. The adjacent host cytoplasm, separated from the fungus by a granular interfacial matrix, contains numerous chloroplasts, mitochondria, and microtubules. The hyphae contact the substratum via the ventral walls of the epidermal cells and the rhizoids are free from infection. In the protocorm and root nodules, aseptate hyphae initially colonize mucilage-filled schizogenous intercellular spaces. Subsequent invasion of the host cells is associated with the development of massive overgrowths of host wall material. The fungal associations in L. cernuum share a mixture of attributes otherwise found in different angiosperm mycorrhizae and in mycotrophic relationships in liverworts. Wall ingrowths are present in both the gametophyte and sporophyte cells in the placenta of L. cernuum. The very limited development of the placenta, compared with L. appressum, certain bryophytes and ferns, the diminutive size, and early senescence of the gametophytes of L. cernuum are all linked to the presence of the protocorm. This massive absorptive organ, homologous to a foot, in terms of its position in sporophyte ontogeny, but external to the parent gametophyte, derives its nutrition partly from photosynthesis and partly from its fungal endophyte. Key words: chloroplasts, Lycopodium, mycorrhiza, pteridophytes, root nodules, symbiosis, transfer cells.

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