Biomedical subjects

T Hackstadt

Publications and source records attributed to T Hackstadt.

67 records · Page 4Linked to original sources

PubMed1986

Antigenic variation in the phase I lipopolysaccharide of Coxiella burnetii isolates.

Coxiella burnetii isolates from a variety of clinical and geographical sources were screened for antigenic variation of lipopolysaccharides (LPSs) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. All isolates from chronic Q fever or other sources possessed a phase I-type LPS. These LPSs appeared to fall into three groups based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile or on reactivity with rabbit anti-C. burnetti antisera. The LPS of one group was identified on isolates from milk, ticks, or primary Q fever. The two remaining groups were found almost exclusively on isolates from human cases of chronic Q fever.

Antigens, Bacterial↗

PubMed1986

Identification and properties of chlamydial polypeptides that bind eucaryotic cell surface components.

An electroblotting technique was used to identify proteins of Chlamydia that bound surface-radioiodinated and Triton X-100-solubilized HeLa cell extracts. Two proteins, with apparent molecular masses of 18 and 32 kilodaltons (kDa), that bound HeLa cell surface components were identified on Chlamydia trachomatis L2 elementary bodies (EBs). Radioiodinated heparin, which disrupts chlamydial association with cultured cells, was also bound by these proteins. These two proteins were found on EBs but were absent or were present in reduced amounts on the noninfectious reticulate bodies. All C. trachomatis strains tested displayed two such proteins, although the apparent molecular weight of the larger protein varied with serotype in correlation with biotype and the disease that it caused. Two Chlamydia psittaci strains examined displayed only a single binding protein in the range of 17 to 19 kDa. All of the binding proteins stained intensely and distinctively on silver-stained sodium dodecyl sulfate-polyacrylamide gels and displayed an unusual sensitivity to reducing agents. The 32-kDa protein was not seen and did not bind 125I-labeled HeLa cell components if the EBs were solubilized in the presence of 2-mercaptoethanol. The 32-kDa protein was not affected by dithiothreitol, however. Similar to the effect of 2-mercaptoethanol, the 32-kDa protein was not visualized after treatment of EBs with the protease inhibitors tosyl-phenylalanine chloromethyl ketone (TPCK) or tosyl-lysine chloromethyl ketone (TLCK). TPCK and TLCK also abolished infectivity as did the alkylating agents N-ethylmaleimide and iodoacetamide, yet the latter two agents did not affect the appearance of the 32-kDa protein. These proteins were not detected in immunoblots with either rabbit antisera to C. trachomatis L2 EBs or by serum from a patient with lymphogranuloma venereum. The role of these proteins in the interaction of chlamydiae with host cells is not clear, but the binding of eucaryotic cell surface components and heparin, presence only during the infectious stage of the life cycle, variation between serotypes in correlation with disease, and sensitivity to reducing agents or protease inhibitors, collectively, suggest a role for these proteins in parasite-host interactions.

Bacterial Proteins↗

PubMed1985

Lipopolysaccharide variation in Coxiella burnetti: intrastrain heterogeneity in structure and antigenicity.

We isolated lipopolysaccharides (LPSs) from phase variants of Coxiella burnetii Nine Mile and compared the isolated LPS and C. burnetii cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The LPSs were found to be the predominant component which varied structurally and antigenically between virulent phase I and avirulent phase II. A comparison of techniques historically used to extract the phase I antigenic component revealed that the aqueous phase of phenol-water, trichloroacetic acid, and dimethyl sulfoxide extractions of phase I C. burnettii cells all contained phase I LPS, although the efficiency and specificity of extraction varied. Our studies provide additional evidence that phase variation in C. burnetii is analogous to the smooth-to-rough LPS variation of gram-negative enteric bacteria, with phase I LPS being equivalent to smooth LPS and phase II being equivalent to rough LPS. In addition, we identified a variant with a third LPS chemotype with appears to have a structural complexity intermediate to phase I and II LPSs. All three C. burnetii LPS contain a 2-keto-3-deoxyoctulosonic acid-like substance, heptose, and gel Limulus amoebocyte lysates in subnanogram amounts. The C. burnetii LPSs were nontoxic to chicken embryos at doses of over 80 micrograms per embryo, in contrast to Salmonella typhimurium smooth- and rough-type LPSs, which were toxic in nanogram amounts.

Bacterial Proteins↗

PubMed1985

Effect of proteolytic cleavage of surface-exposed proteins on infectivity of Chlamydia trachomatis.

The proteolytic cleavage of Chlamydia trachomatis LGV-434 surface proteins and resultant effects on infectivity and association with cultured human epithelial (HeLa) cells have been examined. Of several proteases examined, trypsin, chymotrypsin, and thermolysin extensively cleaved the chlamydial major outer membrane protein (MOMP). Two proteases, trypsin and thermolysin, cleaved the MOMP to the extent that monomeric MOMP was not detectable by immunoblotting with monospecific polyclonal antibodies. In the case of thermolysin, not even antigenic fragments were detected. Surprisingly, infectivity toward HeLa cells was not diminished. In addition, the association of intrinsically 14C-radiolabeled elementary bodies (EBs) with HeLa cells or their dissociation by proteinase K was not measurably affected by prior trypsinization of the EBs. Trypsinization of lactoperoxidase surface-iodinated elementary bodies demonstrated that most of the 125I-labeled surface proteins were cleaved. In all cases, however, a number of proteolytic cleavage fragments remained associated with the EB surface after surface proteolysis. When trypsinized EBs were electrophoresed under nonreducing conditions and immunoblotted with either polyclonal or type-specific monoclonal MOMP antibodies, MOMP was found in a large oligomeric form that failed to enter the polyacrylamide stacking gel. Additionally, trypsinized viable EBs bound radioiodinated type-specific MOMP monoclonal antibody as efficiently as did the control nontrypsinized organisms. Taken together, the findings indicate that although the MOMP is highly susceptible to surface proteolysis, the supramolecular structure of the protein on the EB surface is apparently maintained by disulfide interactions. Thus, if surface-exposed chlamydial proteins are involved in the initial interaction of chlamydiae with eucaryotic cells, the functional domains of these proteins which mediate this interaction must be resistant to proteolysis and remain associated with the EB surface.

Antigens, Bacterial↗

PubMed1985

Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?

The effects of exogenous reducing agents on a number of biological properties of purified Chlamydia trachomatis LGV-434 and Chlamydia psittaci meningopneumonitis elementary bodies (EBs) have been examined in an attempt to identify in vitro correlates of early events in the differentiation of the infectious EB to the replicative cell type, the reticulate body (RB). Treatment of EBs with dithiothreitol elicited a number of changes normally associated with differentiation to the RB. EBs in the presence of 10 mM dithiothreitol displayed enhanced rates of [14C]glutamate oxidation, reduced infectivity, and decreased osmotic stability, and their Machiavello staining properties changed to those characteristic of the RB. A true differentiation of EB to RB did not take place under these conditions, since EBs treated in this manner and examined by transmission electron microscopy did not demonstrate increased size or decreased electron density as do isolated RBs. Additional studies were initiated to identify the macromolecules involved in this process. With polyacrylamide gel electrophoresis and immunoblotting procedures with monoclonal and polyclonal monospecific antibodies, the chlamydial major outer membrane protein was found to be the predominant component that varied under reducing versus nonreducing conditions. Furthermore, the extent of disulfide-mediated cross-linking of the major outer membrane protein varied between the infective and replicative forms of the C. trachomatis LGV-434 life cycle. Implications of disulfide interactions in the life cycle of chlamydiae are discussed.

Bacterial Outer Membrane Proteins↗

PubMed1983

Estimation of the cytoplasmic pH of Coxiella burnetii and effect of substrate oxidation on proton motive force.

The magnitude of the proton motive force generated during in vitro substrate oxidation by Coxiella burnetii was examined. The intracellular pH of C. burnetii varied from about 5.1 to 6.95 in resting cells over an extracellular pH range of 2 to 7. Similarly, delta psi varied from about 15 mV to -58 mV over approximately the same range of extracellular pH. Both components of the proton motive force increased during substrate oxidation, resulting in an increase in proton motive force from about -92 mV in resting cells to -153 mV in cells metabolizing glutamate at pH 4.2. The respiration-dependent increase in proton motive force was blocked by respiratory inhibitors, but the delta pH was not abolished even by the addition of proton ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone or 2,4-dinitrophenol. Because of this apparently passive component of delta pH maintenance, the largest proton motive force was obtained at an extracellular pH too low to permit respiration. C. burnetii appears, therefore, to behave in many respects like other acidophilic bacteria. Such responses are proposed to contribute to the extreme resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome of eucaryotic cells in which this organism multiplies.

2,4-Dinitrophenol↗

PubMed1983

pH dependence of the Coxiella burnetii glutamate transport system.

The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined. C. burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5. Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete. This transport system is both temperature and energy dependent. Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane. ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport. Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated. Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH). From these results, we propose that L-glutamate transport by C. burnetii is energized via a proton motive force.

Adenosine Triphosphate↗

PubMed1982

Sodium and potassium transport in herpes simplex virus-infected cells.

Sodium (Na+) and potassium (K+) flux in African green monkey kidney cells (Vero) was examined following infection by herpes simplex virus type 1 (HSV-1). A decline in the rate of K+ uptake at 5 h post-infection was shown using 86Rb+ as a K+ tracer. In contrast, host protein synthesis was inhibited by 3 h post-infection. The decrease in rate of K+ transport to levels 70 to 90% of that of mock-infected cells did not, however, reflect an inability of HSV-1-infected cells to maintain normal intracellular concentrations of Na+ and K+. At 7 h post-infection, intracellular Na+ and K+ concentrations were determined to be 26.6 +/- 9.4 mM- and 33.3 +/- 10.3 mM-Na+ and 130.1 +/- 4.7 mM- and 137.1 +/- 3.2 mM-K+ in mock-infected and HSV-1-infected cells respectively. Intracellular Na+ did not increase above control levels over at least a 9 h period following HSV-1 infection. The Michaelis constant (Km) of K+ transport in HSV-1-infected or mock-infected Vero cells at 6 h post-infection was determined to be the same with calculated values of 1.38 +/- 0.51 mM and 1.79 +/- 0.42 mM respectively. A virus-induced alteration of intracellular Na+ and K+ concentrations cannot, therefore, account for the HSV-1 induced inhibition of host protein synthesis at 3 h post-infection as has been suggested in other virus systems.

Animals↗

PubMed1982

Chlamydia trachomatis has penicillin-binding proteins but not detectable muramic acid.

Chlamydia trachomatis LGV-434 was grown in HeLa 229 cells. Benzylpenicillin completely inhibited the formation of infectious elementary bodies (EBs) at a concentration of 19 pmol/ml or higher and produced abnormally large reticulate bodies (RBs) in the inclusions at 30 pmol/ml or higher. The possible targets for penicillin in C. trachomatis were three penicillin-binding proteins (PBPs) which were identified in the Sarkosyl-soluble fractions of both RBs and EBs. The apparent subunit molecular weights were 88,000 (PBP 1), 61,000 (BPB 2), and 36,000 (PBP 3). The 50% binding concentrations of [3H]penicillin for PBPs 1 to 3 in EBs and RBs were between 7 and 70 pmol/ml. Such high susceptibility to penicillin was shown by an organism that did not have detectable muramic acid (less than 0.02% by weight) in preparations of either whole cells or sodium dodecyl sulfate-insoluble residues.

Bacterial Proteins↗

PubMed1981

Biochemical stratagem for obligate parasitism of eukaryotic cells by Coxiella burnetii.

Coxiella burnetti, the etiologic agent of Q fever, is an oligate intracellular parasite of eukaryotes. Unlike the majority of successful bacterial parasites, which escape the bactericidal environment of the phagolysosome by various means, C. burnetii multiplies only in the phagolysosome. In view of the relatively harsh environment inhabited by C. burnetii, we have examined (i) the in vitro metabolism of glucose and glutamate by whole cells of C. burnetii under conditions designed to approximate the pH within the phagolysosome and (ii) the effect of manipulation of the phagolysosomal pH by lysosomotropic amines on the replication of C. burnetii in chicken embryo fibroblasts. The transport, catabolism, and incorporation of both glucose and glutamate were found to be highly stimulated by acidic conditions, whereas at pH 7.0 metabolism of these substrates was minimal. The transport processes were shown to be energy dependent and highly sensitive to inhibition by uncouplers of oxidative phosphorylation. Increasing the phagolysosomal pH of infected chicken embryo fibroblasts by use of the lysosomotropic agents chloroquine, methylamine, or ammonium chloride inhibited the multiplication of C. burnetii, thus demonstrating the in vivo requirement for the acidic conditions of the phagolysosome. This apparent dependence upon phagosome--lysosome fusion to generate pH conditions favorable to C. burnetii replication suggests a unique biochemical mechanism of parasite activation. A pathogenic mechanism based on regulation of microbial metabolism by H+-dependent stimulation of cell function is proposed.

Animals↗

PubMed1981

Stability of the adenosine 5'-triphosphate pool in Coxiella burnetii: influence of pH and substrate.

The ability of Coxiella burnetii to couple oxidation of metabolic substrates to adenosine 5'-triphosphate (ATP) synthesis in axenic reaction buffers was examined. Pyruvate, succinate, and glutamate were catabolized and incorporated at the highest rates of 11 substrates tested. Glutamate oxidation, however, resulted in the greatest stability of the ATP pool and highest intracellular ATP levels over a 48-h period. At pH 4.5, the optimum for metabolism by C. burnetii, glutamate oxidation resulted in maintenance of the ATP pool at a concentration of approximately 0.7 nmol of ATP per mg of dry weight over a 96-h period. In the absence of substrate, ATP declined by 96 h to less than 0.01 nmol/mg of dry weight. When cells were maintained at pH 7.0 in the presence or absence of glutamate, ATP pools were considerably more stable, presumably due to the minimal metabolic activity displayed by C. burnetii at pH 7. The stability of the ATP pool reflected viability as there was greater than an 8-log decrease in viable C. burnetii after incubation for 7 days at pH 4.5 in the absence of glutamate. Viability was retained in the presence of glutamate at pH 4.5 or 7.0 in the absence of any added substrate. The stability of the ATP pool was due to endogenous synthesis of ATP coupled to substrate oxidation as shown by depression of ATP levels in the presence of inhibitors of electron transport or oxidative phosphorylation. In addition, the adenylate energy charge increased from an initial value of 0.57 to 0.73 during glutamate oxidation with a concomitant rise in the total adenylate pool size. C. burnetii therefore appears able to regulate endogenous ATP levels in response to substrate availability and pH, thus effecting a conservation of metabolic energy in neutral or alkaline environments. Such a mechanism has been proposed to play a role in the resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome in which this organism replicates.

Adenosine Monophosphate↗

PubMed1979

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.

Cell Line↗

PubMed1978

Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells.

Noninfected and varicella-zoster virus (VZV)-infected human foreskin fibroblasts were examined for thymidine kinase activity. The specific activity of VZV-infected cell extracts was approximately 7.5-fold greater than that of mock-infected cells and 3-fold greater than that of actively growing cells. The pH optimum of VZV-infected cell thymidine kinase activity was found to be 8.0, whereas thymidine kinase activity in noninfected cells exhibited a sharp pH optimum at 7.4. Electrophoretic analysis of cellular enzymes involved in pyrimidine nucleoside phosphorylation revealed at least three enzymes distinguishable by electrophoretic mobility and substrates used. These enzymes were presumed to be thymidine kinase, deoxycytidine kinase, and uridine kinase. The relative mobilities of these enzymes on 5% polyacrylamide gels were 0.18, 0.91, and 0.54, respectively. In VZV-infected cells, a single band of activity catalyzing the phosphorylation of thymidine, deoxyuridine, deoxycytidine, and cytidine was observed with a relative mobility of 0.48. Cellular pyrimidine-phosphorylating enzymes were not detected in VZV-infected cells. The molecular weight of the VZV-induced enzyme was determined to be 72,000 +/- 7%.

Cell Line↗