Plasmodium sporozoites require the protein B9 to invade hepatocytes

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite in the liver. This leads to the release of thousands of merozoites into the blood circulation and initiation of the pathogenic blood stages of malaria. Merozoite invasion of erythrocytes has been well characterized at the molecular and structural levels. In sharp contrast, the molecular mechanisms of sporozoite invasion of hepatocytes are poorly characterized. Here we report a new role during sporozoite entry for the B9 protein, a member of the 6-cysteine domain protein family. Using genetic tagging and gene deletion approaches in rodent malaria parasites, we show that B9 is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. Structural modelling indicates that the N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an invasion complex with Rh5 and RIPR in P. falciparum merozoites. We provide evidence that the beta-propeller domain of B9 is essential for protein function during sporozoite entry and interacts with P36 and P52, both also essential for productive invasion of hepatocytes. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

Download Full-text

Anaplasma phagocytophilum Outer Membrane Protein A Interacts with Sialylated Glycoproteins To Promote Infection of Mammalian Host Cells

Infection and Immunity ◽

10.1128/iai.00654-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3748-3760 ◽

Cited By ~ 36

Author(s):

Nore Ojogun ◽

Amandeep Kahlon ◽

Stephanie A. Ragland ◽

Matthew J. Troese ◽

Juliana E. Mastronunzio ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Anaplasma Phagocytophilum ◽

Protein A ◽

Host Cells ◽

Mammalian Host ◽

Content Type ◽

Outer Membrane Protein A ◽

Sialylated Glycoproteins

ABSTRACTAnaplasma phagocytophilumis the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA).A. phagocytophilumbinding to sialyl Lewis x (sLex) and other sialylated glycans that decorate P selectin glycoprotein 1 (PSGL-1) and other glycoproteins is critical for infection of mammalian host cells. Here, we demonstrate the importance ofA. phagocytophilumouter membrane protein A (OmpA) APH_0338 in infection of mammalian host cells. OmpA is transcriptionally induced during transmission feeding ofA. phagocytophilum-infected ticks on mice and is upregulated during invasion of HL-60 cells. OmpA is presented on the pathogen's surface. Sera from HGA patients and experimentally infected mice recognize recombinant OmpA. Pretreatment ofA. phagocytophilumorganisms with OmpA antiserum reduces their abilities to infect HL-60 cells. The OmpA N-terminal region is predicted to contain the protein's extracellular domain. GlutathioneS-transferase (GST)-tagged versions of OmpA and OmpA amino acids 19 to 74 (OmpA19-74) but not OmpA75-205bind to, and competitively inhibitA. phagocytophiluminfection of, host cells. Pretreatment of host cells with sialidase or trypsin reduces or nearly eliminates, respectively, GST-OmpA adhesion. Therefore, OmpA interacts with sialylated glycoproteins. This study identifies the firstA. phagocytophilumadhesin-receptor pair and delineates the region of OmpA that is critical for infection.

Download Full-text

Characterization of Sec-Translocon-Dependent Extracytoplasmic Proteins of Rickettsia typhi

Journal of Bacteriology ◽

10.1128/jb.00794-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6234-6242 ◽

Cited By ~ 19

Author(s):

Nicole C. Ammerman ◽

M. Sayeedur Rahman ◽

Abdu F. Azad

Keyword(s):

Host Cell ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Host Cells ◽

Transcriptional Analysis ◽

Mammalian Host ◽

Signal Peptides ◽

Cell Infection ◽

Rickettsia Typhi ◽

Sec Translocon

ABSTRACT As obligate intracellular, vector-borne bacteria, rickettsiae must adapt to both mammalian and arthropod host cell environments. Deciphering the molecular mechanisms of the interactions between rickettsiae and their host cells has largely been hindered by the genetic intractability of these organisms; however, research in other gram-negative pathogens has demonstrated that many bacterial determinants of attachment, entry, and pathogenesis are extracytoplasmic proteins. The annotations of several rickettsial genomes indicate the presence of homologs of the Sec translocon, the major route for bacterial protein secretion from the cytoplasm. For Rickettsia typhi, the etiologic agent of murine typhus, homologs of the Sec-translocon-associated proteins LepB, SecA, and LspA have been functionally characterized; therefore, the R. typhi Sec apparatus represents a mechanism for the secretion of rickettsial proteins, including virulence factors, into the extracytoplasmic environment. Our objective was to characterize such Sec-dependent R. typhi proteins in the context of a mammalian host cell infection. By using the web-based programs LipoP, SignalP, and Phobius, a total of 191 R. typhi proteins were predicted to contain signal peptides targeting them to the Sec translocon. Of these putative signal peptides, 102 were tested in an Escherichia coli-based alkaline phosphatase (PhoA) gene fusion system. Eighty-four of these candidates exhibited signal peptide activity in E. coli, and transcriptional analysis indicated that at least 54 of the R. typhi extracytoplasmic proteins undergo active gene expression during infections of HeLa cells. This work highlights a number of interesting proteins possibly involved in rickettsial growth and virulence in mammalian cells.

Download Full-text

Actin-Based Motility of Intracellular Microbial Pathogens

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.65.4.595-626.2001 ◽

2001 ◽

Vol 65 (4) ◽

pp. 595-626 ◽

Cited By ~ 152

Author(s):

Marcia B. Goldberg

Keyword(s):

Molecular Mechanisms ◽

Host Cells ◽

Microbial Pathogens ◽

Mammalian Host ◽

Cell Periphery ◽

Actin Assembly ◽

Cell Cytoplasm ◽

Shigella Spp ◽

The Subject ◽

Actin Rearrangement

SUMMARY A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review.

Download Full-text

pOPIN-GG: A resource for modular assembly in protein expression vectors

10.1101/2021.08.10.455798 ◽

2021 ◽

Author(s):

Adam R Bentham ◽

Mark Youles ◽

Melanie N Mendel ◽

Freya A Varden ◽

Juan Carlos De la Concepcion ◽

...

Keyword(s):

Protein Expression ◽

High Throughput ◽

Protein Function ◽

Molecular Mechanisms ◽

Protein Complexes ◽

Restriction Enzymes ◽

Expression Vectors ◽

Vector Systems ◽

Protein Expression And Purification ◽

Modular Cloning

The ability to recombinantly produce target proteins is essential to many biochemical, structural, and biophysical assays that allow for interrogation of molecular mechanisms behind protein function. Purification and solubility tags are routinely used to maximise the yield and ease of protein expression and purification from E. coli. A major hurdle in high-throughput protein expression trials is the cloning required to produce multiple constructs with different solubility tags. Here we report a modification of the well-established pOPIN expression vector suite to be compatible with modular cloning via Type IIS restriction enzymes. This allows users to rapidly generate multiple constructs with any desired tag, introducing modularity in the system and delivering compatibility with other modular cloning vector systems, for example streamlining the process of moving between expression hosts. We demonstrate these constructs maintain the expression capability of the original pOPIN vector suite and can also be used to efficiently express and purify protein complexes, making these vectors an excellent resource for high-throughput protein expression trials.

Download Full-text

Development and validation of fourLeishmaniaspecies constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies

Parasitology ◽

10.1017/s0031182013001777 ◽

2013 ◽

Vol 141 (4) ◽

pp. 501-510 ◽

Cited By ~ 6

Author(s):

ASHA PARBHU PATEL ◽

ANDREW DEACON ◽

GIULIA GETTI

Keyword(s):

Leishmania Major ◽

Fluorescent Protein ◽

Protein A ◽

Host Cells ◽

Mammalian Host ◽

Disease Pathogenesis ◽

Compound Libraries ◽

Green Fluorescent

SUMMARYGreen fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in severalLeishmaniaspecies, mostly forin vitrostudies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropicaandLeishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved inL. aethiopicaandL. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenicLeishmaniaare confirmed as models forin vitroand possiblyin vivoinfections and represent an ideal tool for medium throughput testing of compound libraries.

Download Full-text

Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

Scientific Reports ◽

10.1038/s41598-020-75216-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Fernando Hinostroza ◽

Alan Neely ◽

Ingrid Araya-Duran ◽

Vanessa Marabolí ◽

Jonathan Canan ◽

...

Keyword(s):

Conformational Changes ◽

Protein Function ◽

Molecular Mechanisms ◽

Protein Complexes ◽

Coarse Grained ◽

Cell Physiology ◽

Missense Mutations ◽

Small Change ◽

Dynamics Simulations ◽

Dynamin 2

Abstract High order oligomers are crucial for normal cell physiology, and protein function perturbed by missense mutations underlies several autosomal dominant diseases. Dynamin-2 is one of such protein forming helical oligomers that catalyze membrane fission. Mutations in this protein, where R465W is the most frequent, cause dominant centronuclear myopathy, but the molecular mechanisms underpinning the functional modifications remain to be investigated. To unveil the structural impact of this mutation in dynamin-2, we used full-atom molecular dynamics simulations and coarse-grained models and built dimers and helices of wild-type (WT) monomers, mutant monomers, or both WT and mutant monomers combined. Our results show that the mutation R465W causes changes in the interactions with neighbor amino acids that propagate through the oligomer. These new interactions perturb the contact between monomers and favor an extended conformation of the bundle signaling element (BSE), a dynamin region that transmits the conformational changes from the GTPase domain to the rest of the protein. This extended configuration of the BSE that is only relevant in the helices illustrates how a small change in the microenvironment surrounding a single residue can propagate through the oligomer structures of dynamin explaining how dominance emerges in large protein complexes.

Download Full-text

Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens

Journal of Bacteriology ◽

10.1128/jb.186.12.3903-3910.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3903-3910 ◽

Cited By ~ 36

Author(s):

Stephen R. Shouldice ◽

Robert J. Skene ◽

Douglas R. Dougan ◽

Gyorgy Snell ◽

Duncan E. McRee ◽

...

Keyword(s):

Molecular Mechanisms ◽

Binding Protein ◽

Iron Acquisition ◽

Protein A ◽

Structural Basis ◽

Mammalian Host ◽

Iron Binding ◽

Gram Negative ◽

Shipping Fever ◽

Iron Binding Proteins

ABSTRACT We have determined the 1.35- and 1.45-Å structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe3+) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

Download Full-text

The Periplasmic HrpB1 Protein from Xanthomonas spp. Binds to Peptidoglycan and to Components of the Type III Secretion System

Applied and Environmental Microbiology ◽

10.1128/aem.01226-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6312-6324 ◽

Cited By ~ 11

Author(s):

Jens Hausner ◽

Nadine Hartmann ◽

Christian Lorenz ◽

Daniela Büttner

Keyword(s):

Type Iii Secretion ◽

Protein Function ◽

Xanthomonas Campestris ◽

Protein Complexes ◽

Structural Similarity ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Type Iii ◽

Inner Membrane Protein

ABSTRACTThe plant-pathogenic bacteriumXanthomonas campestrispv. vesicatoria employs a type III secretion (T3S) system to translocate bacterial effector proteins into eukaryotic host cells. The membrane-spanning secretion apparatus consists of 11 core components and several associated proteins with yet unknown functions. In this study, we analyzed the role of HrpB1, which was previously shown to be essential for T3S and the formation of the extracellular T3S pilus. We provide experimental evidence that HrpB1 localizes to the bacterial periplasm and binds to peptidoglycan, which is in agreement with its predicted structural similarity to the putative peptidoglycan-binding domain of the lytic transglycosylase Slt70 fromEscherichia coli. Interaction studies revealed that HrpB1 forms protein complexes and binds to T3S system components, including the inner membrane protein HrcD, the secretin HrcC, the pilus protein HrpE, and the putative inner rod protein HrpB2. The analysis of deletion and point mutant derivatives of HrpB1 led to the identification of amino acid residues that contribute to the interaction of HrpB1 with itself and HrcD and/or to protein function. The finding that HrpB1 and HrpB2 colocalize to the periplasm and both interact with HrcD suggests that they are part of a periplasmic substructure of the T3S system.

Download Full-text

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyostelium discoideum

10.1101/2019.12.23.887745 ◽

2019 ◽

Author(s):

Camila Valenzuela ◽

Magdalena Gil ◽

Ítalo M. Urrutia ◽

Andrea Sabag ◽

Jost Enninga ◽

...

Keyword(s):

Dictyostelium Discoideum ◽

Molecular Mechanisms ◽

Intracellular Survival ◽

Effector Proteins ◽

Host Cells ◽

Mammalian Host ◽

Secretion Systems ◽

The Social ◽

Membrane Bound ◽

Type Three Secretion

AbstractThe ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-three secretion systems encoded in SPI-1 and SPI-2 (T3SSSPI-1 and T3SSSPI-2, respectively). Recently, we reported that S. Typhimurium requires T3SSSPI-1 and T3SSSPI-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterized the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.ImportanceThe molecular mechanisms involved in Salmonella survival to predation by phagocytic amoebae, such as D. discoideum, remains poorly understood. Although we established that S. Typhimurium requires two specialized type-three secretion systems to survive in D. discoideum, no effector protein has been implicated in this process so far. Here, we confirmed the presence of a membrane-bound compartment containing S. Typhimurium in D. discoideum, and purified the D. discoideum SCV to characterize the associated proteome. In doing so, we established a key role for effector proteins SopB and SifA in remodeling the protein content of the SCV that ultimately allow the intracellular survival of S. Typhimurium in D. discoideum. We also discuss similarities and differences with the proteomes of the human SCV. These findings contribute to unravel the mechanisms used by Salmonella to survive in the environment exploiting phagocytic amoebae as a reservoir.

Download Full-text

Mechanisms Associated with Trypanosoma cruzi Host Target Cell Adhesion, Recognition and Internalization

Life ◽

10.3390/life11060534 ◽

2021 ◽

Vol 11 (6) ◽

pp. 534

Author(s):

Oscar Hernán Rodríguez-Bejarano ◽

Catalina Avendaño ◽

Manuel Alfonso Patarroyo

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Molecular Mechanisms ◽

Parasitophorous Vacuole ◽

Host Cells ◽

Mammalian Host ◽

Target Cells ◽

Cell Plasma ◽

Insect Bites ◽

Host Target

Chagas disease is caused by the kinetoplastid parasite Trypanosoma cruzi, which is mainly transmitted by hematophagous insect bites. The parasite’s lifecycle has an obligate intracellular phase (amastigotes), while metacyclic and bloodstream-trypomastigotes are its infective forms. Mammalian host cell recognition of the parasite involves the interaction of numerous parasite and host cell plasma membrane molecules and domains (known as lipid rafts), thereby ensuring internalization by activating endocytosis mechanisms triggered by various signaling cascades in both host cells and the parasite. This increases cytoplasmatic Ca2+ and cAMP levels; cytoskeleton remodeling and endosome and lysosome intracellular system association are triggered, leading to parasitophorous vacuole formation. Its membrane becomes modified by containing the parasite’s infectious form within it. Once it has become internalized, the parasite seeks parasitophorous vacuole lysis for continuing its intracellular lifecycle, fragmenting such a vacuole’s membrane. This review covers the cellular and molecular mechanisms involved in T. cruzi adhesion to, recognition of and internalization in host target cells.

Download Full-text