Sca1, a previously undescribed paralog from autotransporter protein-encoding genes in Rickettsia species

Background  

Among the 17 genes encoding autotransporter proteins of the "surface cell antigen" (sca) family in the currently sequenced Rickettsia genomes, ompA, sca5 (ompB) and sca4 (gene D), have been extensively used for identification and phylogenetic purposes for Rickettsia species. However, none of these genes is present in all 20 currently validated Rickettsia species. Of the remaining 14 sca genes, sca1 is the only gene to be present in all nine sequenced Rickettsia genomes. To estimate whether the sca1 gene is present in all Rickettsia species and its usefulness as an identification and phylogenetic tool, we searched for sca1genes in the four published Rickettsia genomes and amplified and sequenced this gene in the remaining 16 validated Rickettsia species.     

Ku70, a component of DNA-dependent protein kinase, is a mammalian receptor for Rickettsia conorii

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.     

Identification and characterization of App: an immunogenic autotransporter protein of Neisseria meningitidis

In a search for immunogenic virulence factors in Neisseria meningitidis, we have identified a gene encoding a predicted 160 kDa protein with homology to the autotransporter family of proteins. Members of this family are secreted or surface exposed and are often associated with virulence in Gram-negative bacterial pathogens. We named the gene adhesion and penetration protein (app), because of its extensive homology to the hap gene of Haemophilus influenzae. We reconstructed the gene with reference to genomic sequence data and cloned and expressed the protein in Escherichia coli. Rabbit antiserum raised against recombinant App reacted with proteins in all meningococcal isolates examined, which represented clonal groups responsible for the majority of meningococcal invasive disease. Antibodies to the protein were detected in the sera of patients convalescing from meningococcal infection. Purified App had strong stimulating activity for T cells isolated from a number of healthy donors and from one convalescent patient. We confirmed that App is surface localized, cleaved and secreted by N. meningitidis. Importantly, the rabbit anti-App serum killed the organism in the presence of complement. Thus, App is conserved among meningococci, immunogenic in humans and potentially involved in virulence. It therefore merits further investigation as a component of a future multivalent vaccine.     

Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

Identification and characterization of autotransporter proteins of Yersinia pestis KIM

Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.     

Identification and characterization of a novel mammalian intermediate filament-associated protein

A novel monoclonal antibody, designated M1.4, recognizes the high molecular weight microtubule-associated protein MAP1A (ca. Mr 380 kD) in both bovine and rat brain. In HeLa cells, however, M1.4 binds to a 240 kD polypeptide on immunoblots and co-localizes with both vimentin and cytokeratin filaments using double-label immunofluorescence microscopy. Immunoelectron microscopy indicates that the 240 kD polypeptide localizes along bundled intermediate filaments in a periodic manner. Two-dimensional electrophoretic analysis indicates that the 240 kD polypeptide has a basic pI of 7.7. When HeLa cell intermediate filaments are isolated using standard non-ionic detergent/high-salt conditions the 240 kD polypeptide does not sediment with the intermediate filaments, unlike the established intermediate filament-associated protein plectin. Immunoblot analysis with M1.4 shows the 240 kD polypeptide is expressed in a number of mammalian cell lines. Additionally, double-label immunofluorescence shows the 240 kD polypeptide to associate with vimentin filaments in African Green Monkey kidney (CV-1) and JC neuroblastoma cells. Due to its unique biochemical and biological characteristics, the 240 kD polypeptide is clearly a novel intermediate filament-associated protein for which we have proposed the designation gyronemin (Gr. gyros: around; nemin: filament).     

Identification and characterization of structural domains of human ERp57: association with calreticulin requires several domains

The amino acid sequence of ERp57, which functions in the endoplasmic reticulum together with the lectins calreticulin and calnexin to achieve folding of newly synthesized glycoproteins, is highly similar to that of protein disulfide isomerase (PDI), but they have their own distinct roles in protein folding. We have characterized the domain structure of ERp57 by limited proteolysis and N-terminal sequencing and have found it to be similar but not identical to that of PDI. ERp57 had three major protease-sensitive regions, the first of which was located between residues 120 and 150, the second between 201 and 215, and the third between 313 and 341, the data thus being consistent with a four-domain structure abb'a'. Recombinant expression in Escherichia coli was used to verify the domain boundaries. Each single domain and a b'a' double domain could be produced in the form of soluble, folded polypeptides, as verified by circular dichroism spectra and urea gradient gel electrophoresis. When the ability of ERp57 and its a and a' domains to fold denatured RNase A was studied by electrospray mass analyses, ERp57 markedly enhanced the folding rate at early time points, although less effectively than PDI, but was an ineffective catalyst of the overall process. The a and a' domains produced only minor, if any, increases in the folding rate at the early stages and no increase at the late stages. Interaction of the soluble ERp57 domains with the P domain of calreticulin was studied by chemical cross-linking in vitro. None of the single ERp57 domains nor the b'a' double domain could be cross-linked to the P domain, whereas cross-linking was obtained with a hybrid ERpabb'PDIa'c polypeptide but not with ERpabPDIb'a'c, indicating that multiple domains are involved in this protein-protein interaction and that the b' domain of ERp57 cannot be replaced by that of PDI.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.     

Genome Sequence of Rickettsia conorii subsp. caspia, the Agent of Astrakhan Fever

Rickettsia conorii subsp. caspia is the agent of Astrakhan fever, a spotted fever group rickettsiosis endemic to Astrakhan, Russia. The present study reports the draft genome of Rickettsia conorii subsp. caspia strain A-167.     

Genome Sequence of Rickettsia conorii subsp. israelensis, the Agent of Israeli Spotted Fever

Rickettsia conorii subsp. israelensis is the agent of Israeli spotted fever. The present study reports the draft genome of Rickettsia conorii subsp. israelensis strain ISTT CDC1, isolated from a Rhipicephalus sanguineus tick collected in Israel.     

Generation and characterization of Sca2 (ataxin-2) knockout mice

Ataxin-2, the gene product of the Spinocerebellar Ataxia Type 2 (SCA2) gene, is a protein of unknown function with abundant expression in embryonic and adult tissues. Its interaction with A2BP1/Fox-1, a protein with an RNA recognition motif, suggests involvement of ataxin-2 in mRNA translation or transport. To study the effects of in vivo ataxin-2 function, we generated an ataxin-2 deficient mouse strain. Ataxin-2 deficient mice were viable. Genotypic analysis of litters from mating of heterozygous mice showed segregation distortion with a significant reduction in the birth of Sca-/- females. Detailed macroscopic and microscopic analysis of surviving nullizygous Sca2 knockout mice showed no major histological abnormalities. On a fat-enriched diet, ataxin-2 deficient animals had increased weight gain. Our results demonstrate that ataxin-2, although widely expressed, is not essential in development or during adult survival in the mouse, but leads to adult-onset obesity.     

Genome sequence of Rickettsia conorii subsp. indica, the agent of Indian tick typhus

Rickettsia conorii subsp. indica is the agent of Indian tick typhus. The present study reports the draft genome of Rickettsia conorii subsp. indica strain ITTR (ATCC VR-597).     

Identification of the junction between the glutamine amidotransferase and carbamyl phosphate synthetase domains of the mammalian CAD protein

The hamster CAD gene encodes a protein that catalyzes the first three steps of pyrimidine biosynthesis. We have sequenced a portion of a CAD cDNA and determined the location of the carbamyl phosphate synthetase II coding region. Subdomains coding for the glutamine hydrolyzing and carbamyl phosphate synthesizing functions have been identified through their high degree of similarity to carbamyl phosphate synthetase genes from a variety of organisms. The proline-rich junction between the glutaminase and synthetase domains, however, does not appear to be conserved among carbamyl phosphate synthetases.     

Identification and characterization of a protein containing formin homology (FH1/FH2) domains.

A novel member of the Formin/Diaphanous family of proteins was cloned and characterized. A 4kB mRNA is ubiquitously expressed but is found in abundance in the spleen. FHOS (Formin Homologue Overexpressed in Spleen) contains a 3414bp open reading frame and encodes for an approximately 128kDa protein. FHOS has sequence homology to Diaphanous and Formin proteins within the Formin Homology (FH)1 and FH2 domains. FHOS also contains a coiled-coil, a collagen-like domain, two nuclear localization signals, and several potential PKC and PKA phosphorylation sites. FHOS-specific antiserum was generated and used to determine that FHOS is a predominantly cytoplasmic protein and is expressed in a variety of human cell lines. FHOS was mapped to chromosome 16q22 between framework markers WI-5594 and WI-9392.     

A vector, pSHT, for the expression and secretion of protein domains in mammalian cells.

A phagemid (pSHT) containing the pUC and M13 ori sequences was constructed to facilitate the expression of partial cDNAs or of sequences encoding mammalian membrane- and secretory-protein domains. It provides a start codon and signal sequence flanked upstream by the simian virus 40 and bacteriophage T7 promoters and downstream by cloning sites, stop codons in all three frames, splicing and polyadenylation signals.     

Aminoacylase I from porcine kidney: Identification and characterization of two major protein domains

The domain structure of hog-kidney aminoacylase I was studied by limited proteolytic digestion with trypsin and characterization of the resulting fragments. In the native enzyme, the sequences from residue 6 to 196 and 307 to 406 are resistant to trypsin and remain tightly bound in nondenaturing solvents, while the intervening sequence (197–306) is efficiently degraded by trypsin. We conclude that the N-terminal half of the molecule and its C-terminal fourth form two independently folded domains. Both contain a peculiar PWW(A,L) sequence motif preceded by several strongly polar residues. We propose that these sequences form surface loops that mediate the membrane association of aminoacy clase I. We further show that the three free cysteine residues and the essential Zn²⁺ ion reside in the trypsin-resistant domains, while the intervening sequence contains the only disulfide H bond of the protein.     

Identification, characterization and cloning of myr 1, a mammalian myosin-I

We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.     

CapA, an autotransporter protein of Campylobacter jejuni, mediates association with human epithelial cells and colonization of the chicken gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.