Interactions between the Coxiella burnetii parasitophorous vacuole and the endoplasmic reticulum involve the host protein ORP1L

Coxiella burnetii is a gram‐negative intracellular bacterium that forms a large, lysosome‐like parasitophorous vacuole (PV) essential for bacterial replication. Host membrane lipids are critical for the formation and maintenance of this intracellular niche, yet the mechanisms by which Coxiella manipulates host cell lipid metabolism, trafficking and signalling are unknown. Oxysterol‐binding protein‐related protein 1 long (ORP1L) is a mammalian lipid‐binding protein that plays a dual role in cholesterol‐dependent endocytic trafficking as well as interactions between endosomes and the endoplasmic reticulum (ER). We found that ORP1L localized to the Coxiella PV within 12 h of infection through a process requiring the Coxiella Dot/Icm Type 4B secretion system, which secretes effector proteins into the host cell cytoplasm where they manipulate trafficking and signalling pathways. The ORP1L N‐terminal ankyrin repeats were necessary and sufficient for PV localization, indicating that ORP1L binds a PV membrane protein. Strikingly, ORP1L simultaneously co‐localized with the PV and ER, and electron microscopy revealed membrane contact sites between the PV and ER membranes. In ORP1L‐depleted cells, PVs were significantly smaller than PVs from control cells. These data suggest that ORP1L is specifically recruited by the bacteria to the Coxiella PV, where it influences PV membrane dynamics and interactions with the ER.     

Rapid ocular dominance plasticity requires cortical but not geniculate protein synthesis

Synaptic plasticity is a multistep process in which rapid, early phases eventually give way to slower, more enduring stages. Diverse forms of synaptic change share a common requirement for protein synthesis in the late stages of plasticity, which are often associated with structural rearrangements. Ocular dominance plasticity in the primary visual cortex (V1) is a long-lasting form of activity-dependent plasticity comprised of well-defined physiological and anatomical stages. The molecular events underlying these stages remain poorly understood. Using the protein synthesis inhibitor cycloheximide, we investigated a role for protein synthesis in ocular dominance plasticity. Suppression of cortical, but not geniculate, protein synthesis impaired rapid ocular dominance plasticity, while leaving neuronal responsiveness intact. These findings suggest that structural changes underlying ocular dominance plasticity occur rapidly following monocular occlusion, and cortical changes guide subsequent alterations in thalamocortical afferents.     

Microscopy and cytochemistry of the biogenesis of the parasitophorous vacuole

Some parasitic protozoa are able to penetrate into host cells where they multiply. The process of penetration involves steps such as attachment to the host cell surface, internalization of the protozoan through an endocytic process with the formation of a parasitophorous vacuole (PV), and the subsequent interaction of the protozoan with the membrane lining the PV. This review analyzes the biogenesis of the PV from a morphological and cytochemical perspective. Special emphasis is given to (a) the localization of plasma membrane-associated enzymes such as Na⁺-K⁺-ATPase, Ca²⁺-ATPase, 5-nucleotidase, and NAD(P)H-oxidase, (b) glycoconjugates, detected using labeled lectins, (c) anionic sites, detected using cationic particles, and (d) integral membrane proteins, using freeze-fracture replicas, and lipids during the formation of the PV containing Trypanosoma cruzi, Leishmania, Toxoplasma gondii, and Plasmodium.     

The ultrastructure of the parasitophorous vacuole formed by Leishmania major

Protozoan parasites of Leishmania spp. invade macrophages as promastigotes and differentiate into replicative amastigotes within parasitophorous vacuoles. Infection of inbred strains of mice with Leishmania major is a well-studied model of the mammalian immune response to Leishmania species, but the ultrastructure and biochemical properties of the parasitophorous vacuole occupied by this parasite have been best characterized for other species of Leishmania. We examined the parasitophorous vacuole occupied by L. major in lymph nodes of infected mice and in bone marrow-derived macrophages infected in vitro. At all time points after infection, single L. major amastigotes were wrapped tightly by host membrane, suggesting that amastigotes segregate into separate vacuoles during replication. This small, individual vacuole contrasts sharply with the large, communal vacuoles occupied by Leishmania amazonensis. An extensive survey of the literature revealed that the single vacuoles occupied by L. major are characteristic of those formed by Old World species of Leishmania, while New World species of Leishmania form large vacuoles occupied by many amastigotes.     

Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum

Red blood cell (RBC) invasion and parasitophorous vacuole (PV) formation by Plasmodium falciparum are critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to the P. falciparum PVM.     

Genome size of the rickettsia Coxiella burnetii.

The genome size of Coxiella burnetii Nine Mile strain was determined by the method of initial rate of deoxyribonucleic acid renaturation. The mean value obtained was 1.04 X 10(9) daltons.     

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.     

Analysis of the rnc locus of Coxiella burnetii

A 3.2 kb EcoRI genomic DNA fragment of Coxiella burnetii was isolated by virtue of Its ability to suppress mucoidy in Eschertchia coli. Nucleotide sequence analysis revealed the presence of the genes homologous to rnc, era and recO of E. coli. Suppression of capsule synthesis, measured by β-galactosidase expression in Ion cps-lac fusion strains of E. coli, is caused by gene-dosage effects of the plasmid-borne rnc genes of either C. burnetii or E. coli. The rnc gene of C. burnetii complemented rn– E. coli hosts for lambda plaque morphology and stimulation of lambda N gene expression. We also demonstrated heterologous complementation of an E coli strain defective for the expression of Era, an essential protein in E. coli, using the plasmid-borne C. burnetii era. Under the control of the bacteriophage lambda P_L promoter, this 3.2 kb EcoRI DNA fragment directed the synthesis in E. coli of three proteins with approximate molecular masses of 35,27 and 25 kDa. Antibodies against purified E. coli Era protein cross-reacted with the 35 kDa protein of C. burnetii on Western blots.     

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M_r was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (small-cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M_r of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pi of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella cell was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.     

Maturation steps of the Salmonella-containing vacuole.

After uptake, Salmonella resides within a unique organelle, the Salmonella-containing vacuole (SCV) in which it eventually replicates. Here we recapitulate the knowledge about how Salmonella controls SCV maturation and the different steps of its intracellular trafficking.     

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.     

Efficacy of translation in the rickettsia Coxiella burnetii.

The addition of coliphage Q beta ribonucleic acid to a cell-free translation system obtained from Coxiella burnetii cells caused the formation of monosomes and disomes from ribosomal subunits and resulted in synthesis of viral coat protein. Rickettsial ribosomes and associated translation factors functioned together, with fidelity, efficiency, and a specificity similar to those of free-living gram-negative bacteria.     

Enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the N-terminal hydrophobic region but not C-terminal sorting motifs

Severe acute respiratory syndrome coronavirus encodes several accessory proteins of unknown function. We previously showed that one such protein, encoded by ORF6, enhanced the growth of mouse hepatitis virus in tissue culture cells and in mice. Protein 6 consists of an N-terminal hydrophobic peptide and a C-terminal region containing intracellular protein sorting motifs. Herein, we show that mutation of the hydrophobic region but not the sorting motifs affected the ability of protein 6 to enhance virus growth. Collectively, these results support the notion that the 6 protein interacts with membrane-bound viral replication or assembly machinery to directly enhance virus replication and virulence in animals.     

Synthesis of ribonucleotides and their participation in ribonucleic acid synthesis by Coxiella burnetii.

Synthesis of ribonucleic acid (RNA) by the deoxyribonucleic acid-dependent RNA polymerase of Coxiella burnetii required adenosine, uridine, guanosine, and cytidine 5'-triphosphates. Cell-free preparations of this obligate intracellular procaryotic parasite had competence to phosphorylate ribonucleoside mono- and diphosphates in the presence of exogenous adenosine and guanosine 5'-triphosphates to the corresponding di- and triphosphates. C. burnetii contained about 2 nmol of adenosine 5'-triphosphate per mg of protein, which could serve as a approximately P donor for in vivo synthesis of nucleoside triphosphates. The latter were then used as substrates in the synthesis of RNA in a coordinated metabolic system with C. burnetii RNA polymerase. It is suggested that during infection the rickettsiae might obtain the nucleotides necessary for RNA synthesis from the vacuoles in which C. burnetii proliferates.     

Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques.

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.     

Poliovirus replication requires the N‐terminus but not the catalytic Sec7 domain of ArfGEF GBF1

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP‐ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio‐infected cells, p115 is degraded and neither p115 nor Rab1b knock‐down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N‐terminal region of GBF1 lacking the Sec7 domain. Replication of BFA‐resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism.