ALFRED: the ALelle FREquency Database. Update.

Elaboration of ALFRED (http://alfred.med.yale.edu) is being continued in two directions. One of which is developing tools for efficiently annotating the entries and checking the integrity of the data already in the database while the other is to increase the quantity and accessibility of data. Information contained in ALFRED such as, polymorphic sites, number of populations and frequency tables (one sample typed for one site) has significantly increased.

Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins.

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi, to have no actin tails and short (approximately 1- to 3-micrometer) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii, displayed long actin tails (>10 micrometer) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 micrometer long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria. The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.

Serological evidence of human infection with the protozoan Neospora caninum.

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.

Dynamics of actin-based movement by Rickettsia rickettsii in vero cells.

Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria, Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative to Listeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM of Rickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing beta-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 +/- 0.6 micrometer/min (mean +/- standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 +/- 3.1 micrometers/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 +/- 19.2 s versus 33.0 +/- 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria and Rickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.

Developmental biology of Coxiella burnettii.

The obligate intracellular bacterial agent of human Q fever, Coxiella burnetii, has a remarkable ability to persist in the extracellular environment. It replicates only when phagocytosed and delivered to the phagolysosome, where it resists degradation. Different morphological forms of the bacterium have different resistance properties and appear to be stages of a developmental cycle. Despite the lack of genetic systems, the molecular events surrounding C. burnetii development are now being unraveled.

Origins and functions of the chlamydial inclusion.

Chlamydiae dissociate themselves from the endocytic pathway shortly after internalization by actively modifying the vacuole to become fusogenic with sphingomyelin-containing exocytic vesicles. Interaction with this secretory pathway appears to provide a pathogenic mechanism that allows chlamydiae to establish themselves in a site that is not destined to fuse with lysosomes.

Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion.

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C. psittaci-infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.

The Chlamydia trachomatis parasitophorous vacuolar membrane is not passively permeable to low-molecular-weight compounds.

Chlamydia trachomatis is an obligately intracellular bacterial parasite of eucaryotic cells that undergoes a biphasic life cycle within a parasitophorous vacuole (PV) called an inclusion. The parasitophorous vacuolar membrane (PVM) constitutes a barrier between the replicating bacteria and the nutrient-rich environment of the host cytoplasm. To determine whether the chlamydial PVM contains pores that allow passive diffusion of metabolites between the host cytoplasm and the PV, fluorescent tracer molecules were introduced directly into the cytoplasm of infected cells by transfection or microinjection. Fluorescence microscopy and laser scanning confocal microscopy were subsequently employed to determine whether equilibration of the fluorescent tracers between the cytoplasm and the PV occurred. No movement of tracer molecules as small as 520 Da from the cytoplasm to the PV was observed. These data suggest that the chlamydial PV is not passively permeable to small molecules through open channels in the PVM.

Vesicular interactions of the Chlamydia trachomatis inclusion are determined by chlamydial early protein synthesis rather than route of entry.

Chlamydiae replicate intracellularly within a vacuole that has recently been characterized as intersecting an exocytic pathway. One of the initial events during chlamydial infection is the expression of a chlamydial early gene product(s) that effectively isolates the inclusion from the endocytic-lysosomal pathway and makes it fusogenic with sphingomyelin-containing exocytic vesicles. Associated with this change in vesicular interaction is the delivery of the vacuole to the peri-Golgi region of the host cell. Inhibition of chlamydial early transcription or translation causes Chlamydia trachomatis-containing vesicles to remain dispersed throughout the cytoplasm, where they eventually fuse with lysosomes. Chlamydiae that have been internalized by Fc-mediated endocytosis also avoid lysosomal digestion by a mechanism that requires chlamydial protein synthesis. These results suggest that the vesicular interactions of the chlamydial inclusion are defined by parasite-directed modification of the endocytic vesicle rather than by the route of internalization.

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 pl 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 call 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.

A developmental stage-specific histone H1 homolog of Coxiella burnetii.

Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.

Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane.

Chlamydia trachomatis acquires C6-NBD-sphingomyelin endogenously synthesized from C6-NBD-ceramide and transported to the vesicle (inclusion) in which they multiply. Here we explore the mechanisms of this unusual trafficking and further characterize the association of the chlamydial inclusion with the Golgi apparatus. Endocytosed chlamydiae are trafficked to the Golgi region and begin to acquire sphingolipids from the host within a few hours following infection. The transport of NBD-sphingolipid to the inclusion is energy- and temperature-dependent with the characteristics of an active, vesicle-mediated process. Photo-oxidation of C5-DMB-ceramide, in the presence of diaminobenzidine, identified DMB-lipids in vesicles in the process of fusing to the chlamydial inclusion membrane. C6-NBD-sphingomyelin incorporated into the plasma membrane is not trafficked to the inclusion to a significant degree, suggesting the pathway for sphingomyelin trafficking is direct from the Golgi apparatus to the chlamydial inclusion. Lectins and antibody probes for Golgi-specific glycoproteins demonstrate the close association of the chlamydial inclusion with the Golgi apparatus but do not detect these markers in the inclusion membrane. Collectively, the data are consistent with a model in which C.trachomatis inhabits a unique vesicle which interrupts an exocytic pathway to intercept host sphingolipids in transit from the Golgi apparatus to the plasma membrane.

Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis.

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.

Characterization of the succinate dehydrogenase-encoding gene cluster (sdh) from the rickettsia Coxiella burnetii.

We have identified and sequenced four genes that encode the protein subunits comprising the succinate dehydrogenase enzyme complex (Sdh) of the rickettsia Coxiella burnetii. The Sdh-encoding gene cluster (sdhCDAB) begins 3326 bp upstream from the citrate synthase-encoding gene (gltA) start codon and is read with opposite polarity. An open reading frame encoding the N-terminal 280 amino acids (aa) of 2-oxoglutarate dehydrogenase (SucA) begins 24 bp downstream from the stop codon of the gene specifying the iron-sulfur subunit (sdhB) of Sdh. The deduced aa sequence of Sdh subunits and the N-terminal portion of SucA revealed significant aa identity with the Esherichia coli homologues ranging from a low of 36.6% for SdhD to a high of 61.2% for SdhA and SdhB. Primer extension identified transcription start points (tsp) for sdh and sucA. The region upstream from the sdh tsp, but not the sucA tsp, displayed homology to promoter consensus sequences of E. coli. Further evidence that sucA transcription can occur independent of sdh transcription was provided by demonstrating that a TnphoA insertion disrupting sdhB had no effect on the production of SucA by an E. coli cell-extract-directed in vitro transcription/translation system. The plasmid clone pLPM60, which carries the C. burnetii sdhCDAB coding and upstream regulatory regions, rescued an E. coli sdhA mutant (MOB252), indicating functional expression of the rickettsial locus. A cell extract of MOB252 transformed with pLPM60 showed a sixfold greater level of Sdh enzyme activity over the E. coli wild type. A plasmid clone lacking the sdh upstream regulatory region did not complement nor produce sdh mRNA by dot blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells.

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.

Directional actin polymerization associated with spotted fever group Rickettsia infection of Vero cells.

Members of the spotted fever group (SFG) of rickettsiae spread rapidly from cell to cell by an unknown mechanism(s). Staining of Rickettsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated with one pole of intracellular rickettsiae. F-actin tails greater than 70 microns in length were seen extending from rickettsiae. Treatment of infected cells with chloramphenicol eliminated rickettsia-associated F-actin tails, suggesting that de novo protein synthesis of one or more rickettsial proteins is required for tail formation. Rickettsiae were coated with F-actin as early as 15 min postinfection, and tail formation was detected by 30 min. A survey of virulent and avirulent species within the SFG rickettsiae demonstrated that all formed actin tails. Typhus group rickettsiae, which do not spread directly from cell to cell, lacked F-actin tails entirely or exhibited only very short tails. Transmission electron microscopy demonstrated fibrillar material in close association with R. rickettsii but not Rickettsia prowazekii. Biochemical evidence that actin polymerization plays a role in movement was provided by showing that transit of R. rickettsii from infected cells into the cell culture medium was inhibited by treatment of host cells with cytochalasin D. These data suggest that the cell-to-cell transmission of SFG rickettsiae may be aided by induction of actin polymerization in a fashion similar to that described for Shigella flexneri and Listeria monocytogenes.

Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli.

A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.

DNA probes for detecting Coxiella burnetii strains.

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A plasmid-encoded surface protein found in chronic-disease isolates of Coxiella burnetti.

The cbbE' gene codes for the E' protein of Coxiella burnetii and was detected in genomic DNA from all known human isolates of the biotzere strain but not in DNA from the other five strains of C. burnetti. The biotzere strain is strictly associated with chronic disease in humans. Extrinsic iodination of biotzere strain cells radiolabeled a 55-kDa protein which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the E' protein synthesized in vitro from recombinants containing the cbbE' gene. The 125I-labeled 55-kDa protein was immunoprecipitated with polyclonal anti-E' antiserum, confirming its identity as E'. Predicted secondary structure of the E' polypeptide shows six regions of beta-sheet structure and an alpha-helix near the C terminus with adequate lengths to span a membrane. The predicted hydropathy profile of E' is similar to profiles of known outer membrane proteins and corroborates the biochemical data, indicating that the protein is located in the outer membrane of C. burnetii.