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T Hackstadt

Publications and source records attributed to T Hackstadt.

At least 55 records · Page 3Linked to original sources
PubMed1993

Diversity in the Chlamydia trachomatis histone homologue Hc2.

Chlamydia trachomatis elementary bodies contain two developmentally expressed histone H1 homologues. An 18-kDa histone homologue, Hc1, is conserved among C. trachomatis serovars and C. psittaci. The other histone homologue, Hc2 (encoded by hctB), varies in size between C. trachomatis serovars but is present in reduced amounts or absent from C. psittaci. The variation in Hc2 size among C. trachomatis serovars was found to be due to internal deletions from a region of the hctB gene encoding lysine- and alanine-rich pentameric repeats.

Amino Acid Sequence↗
PubMed1993

Hc1-mediated effects on DNA structure: a potential regulator of chlamydial development.

Chlamydiae are obligate intracellular bacteria which undergo a unique developmental cycle, alternating between non-replicative elementary bodies (EBs) and replicative reticulate bodies (RBs). The transition from RB to EB is characterized by condensation of the chromosome into a dense nucleoid structure. The chlamydial histone homologue Hc1 is sufficient to induce formation of a similar structure in Escherichia coli. High-level Hc1 expression in E. coli is self-limiting and down-regulates transcription, translation, and replication at concentrations similar to those observed in chlamydial elementary bodies. Expression of Hc1 at sub-structural levels may have specific regulatory functions through its interaction with chromosomal DNA. In E. coli this is reflected in a dramatic shift in the pattern of gene expression. The differential expression of the outer membrane porin proteins OmpC and OmpF and analysis of lacZ fusions with promoter regions sensitive to supercoiling suggests that low-level Hc1 expression results in a net relaxation of chromosomal DNA. Topological analysis of plasmid DNA from both E. coli and Chlamydia trachomatis supports a decrease in superhelicity preceding nucleoid formation. In vitro analysis of purified Hc1-DNA interactions supports preferential binding based upon DNA conformation. These results suggest a dual role in which Hc1-mediated changes in gene expression may precede metabolic inactivity.

Amino Acid Transport Systems↗
PubMed1993

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.

Actin Cytoskeleton↗
PubMed1993

Molecular cloning and expression of hctB encoding a strain-variant chlamydial histone-like protein with DNA-binding activity.

Two DNA-binding proteins with similarity to eukaryotic histone H1 have been described in Chlamydia trachomatis. In addition to the 18-kDa histone H1 homolog Hc1, elementary bodies of C. trachomatis possess an antigenically related histone H1 homolog, which we have termed Hc2, that varies in apparent molecular mass among strains. We report the molecular cloning, expression, and nucleotide sequence of the hctB gene encoding Hc2 and present evidence for in vivo DNA-binding activity of the expressed product. Expression of Hc2 in Escherichia coli induces a compaction of bacterial chromatin that is distinct from that observed upon Hc1 expression. Moreover, isolated nucleoids from Hc2-expressing E. coli exhibit markedly reduced sensitivity to DNase I. These properties of Hc2 are consistent with a postulated role in establishing the nucleoid structure of elementary bodies.

Amino Acid Sequence↗
PubMed1992

Nucleoid condensation in Escherichia coli that express a chlamydial histone homolog.

Chlamydial cell types are adapted for either extracellular survival or intracellular growth. In the transcriptionally inert elementary bodies, the chromosome is densely compacted; in metabolically active reticulate bodies, the chromatin is loosely organized. Condensation of the chlamydial nucleoid occurs concomitant with expression of proteins homologous to eukaryotic histone H1. When the Chlamydia trachomatis 18-kilodalton histone homolog Hc1 is expressed in Escherichia coli, a condensed nucleoid structure similar to that of chlamydiae is observed with both light and electron microscopy. These results support a role for Hc1 in condensation of the chlamydial nucleoid.

Blotting, Western↗
PubMed1992

Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing.

The 120-kDa rickettsial outer membrane protein (rOmpB) is encoded by a gene with the capacity to encode a protein of approximately 168 kDa. The carboxy-terminal end of the molecule is apparently cleaved to yield 120- and 32-kDa products. Both polypeptides are surface exposed and remain associated with the outer membrane of intact rickettsiae. All species of rickettsiae examined display similar cleavage of rOmpB. Comparison of diverse species of rickettsiae demonstrate a conserved N terminus of the 32-kDa fragment, with a predicted procaryotic secretory signal peptide immediately upstream of the proposed cleavage site. Coprecipitation of the 120-kDa rOmpB protein and the 32-kDa peptide by monoclonal antibodies specific for the 120-kDa portion of the molecule suggests that the two fragments remain noncovalently associated on the surface of rickettsiae. Analysis of an avirulent mutant of Rickettsia rickettsii revealed reduced amounts of the 120- and 32-kDa fragments, but with a correspondingly larger rOmpB protein that displayed properties expected of the putative precursor. This avirulent mutant grows intracellularly but fails to cause the lysis of infected cells that is typical of R. rickettsii. DNA sequence analysis of the region of the gene encoding the cleavage site of the avirulent strain revealed no difference from the sequence obtained from virulent R. rickettsii. The 168-kDa putative precursor of the avirulent strain of R. rickettsii was not extracted from the surface by dilute buffers, as is the 120-kDa protein of virulent R. rickettsii or R. prowazekii. These latter results suggest that the 32-kDa C-terminal region of the molecule may serve as a membrane anchor domain.

Amino Acid Sequence↗
PubMed1991

DNA polymorphism in the conserved 190 kDa antigen gene repeat region among spotted fever group Rickettsiae.

The 190 kDa protein gene of R. rickettsii (R strain) contains a large region of tandemly arranged repeats. Homologous sequences were discovered within the genomes of all but one species of spotted fever group Rickettsiae. Southern blots showed restriction fragment length polymorphisms between species of Rickettsiae indicating structural differences among repeat regions. Further analysis using polymerase chain reaction techniques indicated the repeat regions vary in size among Rickettsiae. In addition, strains of R. rickettsii that vary in virulence were found to contain polymorphisms in this region.

Antigens, Bacterial↗
PubMed1991

Chlamydia trachomatis developmentally regulated protein is homologous to eukaryotic histone H1.

Chlamydiae are prokaryotic obligate intracellular parasites that undergo a biphasic life cycle involving an infectious, extracellular form known as elementary bodies and an intracellular, replicating form termed reticulate bodies. We have purified from Chlamydia trachomatis a very basic elementary body-specific protein with an apparent molecular mass of 18 kDa, determined its N-terminal amino acid sequence, and cloned the encoding gene. Sequence analysis of the cloned gene revealed some remarkable properties for its expressed product, including a high lysine content (29%), a correspondingly high pI, and significant homology to the H1 class of eukaryotic histones. Furthermore, a monoclonal antibody to this chlamydial histone analog, termed Hc1, displayed immunoblot and antinuclear specificity suggestive of cross-reactivity to H1 histones. The gene was expressed only during the late stages of the chlamydial life cycle concomitant with the reorganization of chlamydial reticulate bodies into elementary bodies, suggesting that the Hc1 protein plays a role in the condensation of chlamydial chromatin during intracellular differentiation.

Amino Acid Sequence↗
PubMed1991

Analysis of the cells involved in the lymphoproliferative response to Coxiella burnetii antigens.

Vaccination with an inactivated, whole cell, Q fever vaccine (Q-vax) induces lasting antibody conversion and a positive delayed-type hypersensitivity (DTH) skin reaction in about 60% of recipients but a long-lasting positive lymphoproliferative or mitogenic response to C. burnetii antigens with peripheral blood mononuclear cells (PBMC) in 85-95% of subjects. Analysis of the lymphoproliferative response to C. burnetii antigens has now been made by fractionation-reconstitution experiments with PBMC from vaccines, from past infections, and from healthy controls. The major contributor to the response in immune subjects proved to be the T lymphocyte. T cells were stimulated by both the phase I and phase II antigens of two prototype strains of C. burnetii and responses were greatly amplified by addition of IL-2. Similar T lymphocyte stimulation profiles were obtained with the 'Priscilla' strain of C. burnetii which represents a different biotype of Coxiella isolated from Q fever endocarditis; Q-vax is therefore likely to protect against endocarditis strains. Fractionation-reconstitution experiments with T and B cells from vaccines and subjects infected in the past, using various antigenic or haptenic fractions from C. burnetii indicate that protein, non-lipopolysaccharide components of the organism are responsible for the mitogenic response of immune T cells. However, the role of the lipopolysaccharide in the protective immunogen has still to be defined.

Animals↗
PubMed1991

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor.

A Rickettsia rickettsii outer surface membrane protein (rOmp B), of an apparent molecular mass of 120 kilodaltons, is a major surface antigen of the Rickettsiae that displays genus, species, and sub-species specific antigenic determinants. The 5' portion of this gene was found to be unstable in plasmids, but was stably cloned in a lambda vector. The nucleotide sequence of the 5' terminus has been determined, thus completing the DNA sequence of the entire gene. Genetic analysis revealed an unusually large open reading frame with the capacity to encode a product much larger than the mature protein. A 32 kilodalton peptide from purified rickettsiae was isolated and the amino terminus was sequenced, which revealed that the peptide is encoded by the 3' portion of this large open reading frame. This suggests a role for post-translational processing of rOmp B from a large precursor molecule.

Amino Acid Sequence↗
PubMed1991

Purification and N-terminal amino acid sequences of Chlamydia trachomatis histone analogs.

DNA-binding proteins specific to Chlamydia trachomatis elementary bodies have been described and recently characterized as procaryotic histone analogs. I have developed an affinity purification procedure for the 18-kDa histone analog, Hc1, based on its affinity for polyanions. The availability of highly purified Hc1 has allowed for determination of its N-terminal amino acid sequence and should prove useful in studies of its biological function. The variable C. trachomatis histone analog not obtained by this procedure was electrophoresed onto Immobilon paper for sequencing. The N terminus of the variable histone was conserved among C. trachomatis serotypes L2, D, and B and was distinct from that of Hc1.

Amino Acid Sequence↗
PubMed1989

Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme.

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.

Animals↗
PubMed1988

Lipopolysaccharide phase variation determines the complement-mediated serum susceptibility of Coxiella burnetii.

Phase variation of Coxiella burnetii is due to variation of the lipopolysaccharide (LPS), a phenomenon analogous to smooth-to-rough LPS variation of gram-negative enteric bacteria. Virulent enterobacteria usually have a smooth LPS and resist serum killing, whereas avirulent rough LPS mutants are sensitive to complement-mediated serum killing. Like gram-negative enterobacteria, smooth LPS phase variants of C. burnetii are virulent, whereas the rough LPS variants are avirulent. We therefore studied the effects of human serum on the LPS variants of the Nine Mile strain of C. burnetii. Analogous to gram-negative enterobacteria, the smooth and intermediate LPS C. burnetii phase variants were resistant to complement-mediated serum killing, whereas the rough LPS variants were killed by serum complement. Although the smooth and intermediate LPS variants were serum resistant, they differed in their interactions with the complement system. The smooth LPS variant activated complement poorly and did not bind C3b; however, the intermediate LPS variant activated complement and bound C3b. The rough LPS variant activated complement via the alternative pathway, whereas the intermediate LPS variant activated the classical pathway. These results provide an explanation for the avirulent nature of the rough LPS variant of C. burnetii and suggest that differences in C. burnetii LPS structure influence the interactions of the LPS phase variants with the complement system.

Animals↗
PubMed1988

Steric hindrance of antibody binding to surface proteins of Coxiella burnetti by phase I lipopolysaccharide.

The exposure of surface protein antigens on virulent phase I Coxiella burnetti was compared with that on avirulent phase II cells. Although anti-phase II antibodies did not bind to the surfaces of native intact phase I cells, they bound to phase I proteins if the proteins were solubilized for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting. In addition, removal of the phase I lipopolysaccharide (LPS) by trichloroacetic acid exposed surface proteins for reactivity with anti-phase II antibodies, as shown by immunofluorescence assays, direct antibody binding, and immunoelectron microscopy using protein A-colloidal gold conjugates. Based on these observations, a simple model of phase variation is proposed to explain the apparently conflicting notions of the identity of the phase II antigen(s). The model suggests that the phase I LPS sterically hinders access of anti-phase II antibodies to a multitude of shared protein antigens, any one of which may confer phase II specificity. Exposure of these shared protein antigens through the appearance of a more truncated LPS (phase II) or extraction of the smooth-type phase I LPS allows antibody accessibility and therefore confers apparent phase II serospecificity.

Antibodies, Bacterial↗
PubMed1987

Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig model.

We compared the relative infectivity and virulence of lipopolysaccharide (LPS) variants of the Nine Mile strain of Coxiella burnetii with those of the Priscilla strain, a representative of endocarditis-type strains. In agreement with results of previous studies, Nine Mile phase I (9mi/I) organisms were highly infectious, eliciting seroconversion and fever with inocula containing as few as four organisms. Viable 9mi/I was recovered from the spleens of infected animals 30 days postinfection. Nine Mile phase II (9mi/II) organisms did not elicit fever or seroconversion except with very large inocula, and viable organisms could not be recovered at 30 days postinfection. The Nine Mile/Crazy variant, bearing the intermediate-type LPS, was also highly infectious, as determined by fever response and seroconversion, although, as with 9mi/II, viable organisms could not be recovered 30 days postinfection. The Priscilla strain in phase I (Pris/I) was as infectious as 9mi/I, as determined by seroconversion and its presence in the spleen 30 days postinfection; but in contrast to 9mi/I, more than 10(5) Pris/I isolates were required to induce fever. The temporal appearances of anti-phase I and II antibodies were similar for the two strains. A variety of serological techniques measuring antibody response against whole-cell and purified LPS antigens in agglutination, immunofluorescence, enzyme-linked immunosorbent, and immunoblot assays did not demonstrate sufficient specificity to distinguish between 9mi/I and Pris/I infections. Results of vaccine cross-challenge experiments showed a significant degree of protection between homologous and heterologous challenge strains. protection between homologous

Animals↗
PubMed1987

Chlamydial hemagglutinin identified as lipopolysaccharide.

Chlamydial lipopolysaccharide (LPS) agglutinated mouse and rabbit erythrocytes but not human, guinea pig, or pronghorn antelope erythrocytes. Hemagglutination was not specific for Chlamydia spp., as rough LPSs from Coxiella burnetii and Escherichia coli also agglutinated erythrocytes from the same animal species. Nonagglutinated and agglutinated erythrocytes bound equivalent amounts of LPS, indicating that hemagglutination was not due to a specific interaction of chlamydial LPS with erythrocytes. Thus, hemagglutination by chlamydial LPS is not mediated by specific receptor-ligand interactions but is a property of the altered surface of the LPS-coated erythrocytes.

Animals↗