bluuebunny/crossref_metadata_2025 huggingface dataset:
Priming of human monocytes for enhanced lipopolysaccharide responses: expression of alpha interferon, interferon regulatory factors, and tumor necrosis factor
Authors: Hayes M P , Zoon K C
Published: January 2020

Culture of human monocytes with either granulocyte-macrophage colony-stimulating factor or gamma interferon (IFN-gamma) results in a primed state, during which these cells express heightened responses to bacterial lipopolysaccharide (LPS). The production of IFN-alpha in response to LPS by human monocytes has an absolute requirement for priming. Tumor necrosis factor (TNF) expression is also greatly enhanced in primed monocytes after LPS stimulation, but unlike IFN-alpha, TNF is readily expressed in unprimed monocytes as well. In an effort to determine the molecular events associated with IFN-alpha induction in this system, freshly isolated human monocytes were primed by culture with either IFN-gamma or granulocyte-macrophage colony-stimulating factor and then treated with LPS; expression of IFN-alpha subtype 2 (IFN-alpha 2), IFN regulatory factors (IRFs), and TNF was assessed by Northern (RNA blot) analysis. IRF-1 mRNA is expressed at high levels in monocytes and is regulated by both LPS and priming cytokines, but its expression alone does not correlate with the induction of IFN-alpha 2 expression. IRF-2 mRNA is expressed in a more gradual manner following LPS stimulation, implying a possible feedback mechanism for inhibiting IFN-alpha expression. However, nuclear run-on analysis indicates that IFN-alpha 2 is not transcriptionally modulated in this system, in striking contrast to TNF, which is clearly regulated at the transcriptional level. In addition, IFN-alpha 2 mRNA accumulation is superinduced when primed monocytes are treated with LPS plus cycloheximide, while TNF mRNA is relatively unaffected. The results demonstrate that priming can affect subsequent LPS-induced gene expression at different levels in human monocytes.
Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages
Authors: Han Y , van Rooijen N , Cutler J E
Published: January 2020

We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.
Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e
Authors: Sandlin R C , Apicella M A , Stein D C
Published: January 2020

An isogenic set of gonococcal lipooligosaccharide (LOS) mutants derived from pyocin treatment of Neisseria gonorrhoeae 1291 was used to identify cloned gonococcal DNA fragments. A gene bank from N. gonorrhoeae 1291c chromosomal DNA was made in pLEE10, a shuttle vector that replicates in the gonococcus and Escherichia coli. A plasmid (pSG30) that could transform the LOS mutants 1291d and 1291e to reactivity with monoclonal antibody 3F11 and to production of an LOS component with migration identical to that of the parent, 1291, was identified. pSG30 contains a 9-kb EcoRI fragment. Curing studies indicate that pSG30 encodes gene products that affect LOS biosynthesis in trans. Subcloning identified a 2.6-kb HincII fragment (pSG38) that retained the ability to modify the LOS of 1291d and 1291e. The DNA regions involved in modification of 1291d and 1291e were named lsi-4 and lsi-5, respectively. The region of pSG38 that was involved in LOS modification was further localized by the construction of exonuclease III deletion plasmids. Transformation of these constructs identified a 750-bp fragment that retains the ability to modify 1291e and a 540-bp fragment which retains the ability to modify 1291d.
Isolation and characterization of two Campylobacter glycine-extracted proteins that bind to HeLa cell membranes
Authors: Kervella M , Pagès J M , Pei Z , Grollier G , Blaser M J , Fauchère J L
Published: January 2020

Two immunogenic proteins of 27 (CBF1) and 29 (CBF2) kDa from enteropathogenic Campylobacter species appear to bind to mammalian cells. We purified these two proteins from a pathogenic and adherent Campylobacter jejuni strain to homogeneity by using acid extraction, preparative gel electrophoresis, and electroelution. Polyclonal rabbit antisera to these proteins were prepared. Immunologic studies indicate that CBF1 corresponds to the PEB1 and CBF2 corresponds to the PEB4 described by Pei et al. (Z. Pei, R. T. Ellison, and M. Blaser, J. Biol. Chem. 226:16363-16369, 1991). Immunogold labeling of a C. jejuni adherent strain with anti-CBF1, anti-CBF2, and anti-PEB1 suggested that CBF1 (PEB1) is surface exposed while CBF2 (PEB4) is not. Analysis of whole-cell extracts from 14 strains by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with 7 M urea and immunoblotting with antisera to CBF1 and CBF2 suggests that CBF proteins from adherent and nonadherent strains are different. Use of purified proteins in a microassay of adherence to cellular membranes indicated that CBF1 was much more adherent than CBF2. Adherence of C. jejuni to viable HeLa cells was markedly reduced with the antiserum to CBF1, whereas the CBF2 antiserum was a poor inhibitor. Purified CBF1 competitively inhibited adherence of whole bacteria to HeLa cells, whereas purified CBF2 was no better a competitor than bovine serum albumin. Adherence of CBF2 was markedly reduced in the presence of Tween 20 or SDS, whereas adherence of CBF1 was reduced only by SDS. We conclude that (i) CBF1 (PEB1) is surface exposed and may be the key protein for C. jejuni adhesion and (ii) CBF2 (PEB4) may be complexed with CBF1 and may passively coadhere with CBF1 under certain experimental conditions. Adherent and nonadherent strains contain different isotypes of these two proteins which could be useful markers of C. jejuni adhesion.
Experimental model of congenital syphilis
Authors: Kajdacsy-Balla A , Howeedy A , Bagasra O
Published: January 2020

Female LSH hamsters infected with Treponema pallidum subsp, endemicum before pregnancy or during early pregnancy transmit a form of syphilis to the fetus that is similar to human congenital syphilis. The offspring develops rhinitis, skin rash, failure to thrive, and hepatosplenomegaly. T. pallidum is detectable in their livers, spleens, and nasal secretions. Immunoglobulin M antibodies are detected in the serum.
Unexpectedly high levels of some presumably protective secretory immunoglobulin A antibodies to dental plaque bacteria in salivas of both caries-resistant and caries-susceptible subjects
Authors: Hocini H , Iscaki S , Bouvet J P , Pillot J
Published: January 2020

The role of salivary antibodies in protection against cariogenic bacteria is actually a matter of debate. Correlation between caries experience and naturally occurring antibodies was extensively investigated. Comparison of salivary antibodies from 21 caries-resistant and 22 caries-susceptible subjects was carried out by using a new quantitative method. Secretory immunoglobulin A (S-IgA) antibodies to Streptococcus sobrinus and Streptococcus sanguis cells were detected in all salivas and at similar levels in both groups. When assayed with two major antigens from S. sobrinus, i.e., protein antigen I/II and cell wall carbohydrates, only specific activities of antibodies to the protein component were increased (P < 0.01), but this occurred unexpectedly in the caries-susceptible group. Western blot (immunoblot) analysis with the culture supernatant and cell wall proteins from S. sobrinus showed the same antibody specificity in both groups. No selective increase of the protease-resistant S-IgA2 subclass was found, and avidities of antibodies to both antigen I/II and cell wall carbohydrates were similar. Our results demonstrate that naturally induced S-IgA antibodies against S. sanguis, S. sobrinus, and the major antigens of the latter are not sufficient to inhibit caries development.
The K88 fimbrial adhesin of enterotoxigenic Escherichia coli binds to beta 1-linked galactosyl residues in glycosphingolipids
Authors: Payne D , O'Reilly M , Williamson D
Published: January 2020

The K88 fimbrial adhesin enables certain strains of enterotoxigenic Escherichia coli to adhere to the porcine small intestine. In this study, the ability of the K88 adhesin to bind to glycosphingolipids was monitored by modified enzyme-linked immunosorbent assay and thin-layer chromatography overlay binding analysis. The binding of the K88 adhesin to glycosphingolipid-coated microtiter plates was saturable, with 50% maximal binding occurring with gangliotriaosylceramide, gangliotetraosylceramide, and lactosylceramide at 67 +/- 21, 117 +/- 21, and 73 +/- 22 pM, respectively. Thin-layer chromatography overlay binding analysis demonstrated that serotype O8:K87:K88ab:H19 E. coli bound to hydroxylated galactosylceramide, gangliotriaocylceramide, gangliotetraosylceramide, and lactosylceramide but not to globotriaosylceramide, nonhydroxylated galactosylceramide, glucosides, glucosylceramide, or a mixture of ceramides. The K88 adhesin did not bind by either assay to globoside, the Forssman glycolipid, GM1, GM2, GM3, GD1a, GD2, GD3, GQ1b, or GT1b. The binding pattern observed with the K88 adhesin suggests that beta 1-linked galactosyl residues are the minimum determinant required for binding, provided they are presented correctly. It is suggested that beta 1-linked galactose residues may form the molecular basis of both glycoprotein and glycolipid receptors for the K88 fimbrial adhesin in the porcine small intestine.
Purification and characterization of polyphosphate kinase from Neisseria meningitidis
Authors: Tinsley C R , Manjula B N , Gotschlich E C
Published: January 2020

The important human pathogens Neisseria meningitidis and Neisseria gonorrhoeae accumulate phosphate in the form of polyphosphate (A. Noegel and E. C. Gotschlich, J. Exp. Med. 157:2049-2060, 1983), and the localization of more than half of this long-chain polymer on the exterior of the cells suggests a function as a protective, capsule-like coating. To enable further genetic investigation of the role of polyphosphate in Neisseria spp., the enzyme polyphosphate kinase (PPK), which catalyzes the synthesis of polyphosphate from ATP, was purified from N. meningitidis BNCV. The activity is dependent on Mg2+ and phosphate or polyphosphate and is inhibited by ADP. The Km for ATP is 1.5 mM, and the turnover number is 47 phosphate residues per polypeptide per s. Analysis of PPK labelled with [gamma-32P]ATP indicates that the enzyme is phosphorylated during the reaction, probably at an arginine residue. N-terminal and two internal amino acid sequences were derived from the purified protein and will allow the design of synthetic oligonucleotides for cloning and genetic manipulation of the ppk gene.
Inactivation of the streptococcal erythrogenic toxin B gene (speB) in Streptococcus pyogenes
Authors: Chaussee M S , Gerlach D , Yu C E , Ferretti J J
Published: January 2020

Streptococcal proteinase precursor (SPP) is a zymogen secreted by Streptococcus pyogenes that becomes activated to a cysteine proteinase. SPP has been shown to be immunologically identical to streptococcal erythrogenic toxin B (SPE B), and sequence comparison has shown a high degree of homology between the two proteins. In this study, we have constructed a speB mutant strain of S. pyogenes by insertional inactivation. An internal fragment of the cloned speB gene in plasmid pCR1000 was replaced with an erythromycin resistance determinant, and the recombinant plasmid was introduced into strain NZ131 by electrotransformation. Following the selection of erythromycin-resistant clones, Southern hybridization experiments confirmed the presence of the recombinant plasmid containing the erm gene in the chromosome of the resistant strains. Analysis of extracellular proteins produced by the wild-type and speB mutant strains by Ouchterlony immunodiffusion and isoelectric focusing revealed the presence of SPE B in the wild-type strain but not the speB mutant. Additionally, SPP, which has an isoelectric focusing pattern similar to that of SPE B and reacts with SPE B antiserum, was not detected among the extracellular proteins of the speB mutant strain. Proteinase activity as assayed by two different methods was present in the extracellular proteins produced by the wild-type strain, but the speB mutant strain had no extracellular proteinase activity. The mutant strain had a growth rate similar to that of the wild-type strain and produced normal levels of other extracellular products, suggesting that proteinase was not essential for viability as previously suggested. Our data are consistent with the view that a single gene (speB) produces a single protein that has been identified and/or assayed as either SPE B or SPP.
Biochemical analysis of the membrane and soluble forms of the complement regulatory protein of Trypanosoma cruzi
Authors: Norris K A , Schrimpf J E
Published: January 2020

A developmentally regulated, 160-kDa trypomastigote surface glycoprotein was previously shown to bind the third component of complement and to inhibit activation of the alternative complement pathway, thus providing the parasites a means of avoiding the lytic effects of complement. We now show that this complement regulatory protein (CRP) binds human C4b, a component of the classical pathway C3 convertase, and may therefore also act to restrict classical complement activation. Characterization of the extent of carbohydrate modification of the protein revealed extensive N-linked glycosylation and no apparent O-linked sugars. The CRP purified from parasites treated with an inhibitor of N-linked glycosylation exhibited a decreased binding affinity for C3b compared with that of the fully glycosylated protein. We have previously shown that the protein was anchored to the membrane via a glycosyl phosphatidylinositol linkage and was spontaneously shed from the parasite surface. The spontaneous release of CRP from the parasite surface may augment the protection of the parasites from complement-mediated lysis by the removal of complement-CRP complexes. The majority of the shed CRP had an apparent molecular mass of 160 kDa and lacked the glycolipid anchor, whereas the membrane form was recovered with the glycolipid anchor attached and had an apparent molecular mass of 185 kDa. Both the membrane form (185 kDa) and the soluble form (160 kDa) retained binding affinity for C3b. Evidence is presented to indicate that the conversion of the 185-kDa membrane form to the 160-kDa form is the result of cleavage by an endogenous phospholipase C.
Acid phosphatase purified from Mycoplasma fermentans has protein tyrosine phosphatase-like activity
Authors: Shibata K , Noda M , Sawa Y , Watanabe T
Published: January 2020

Acid phosphatase purified from Mycoplasma fermentans dephosphorylated phosphotyrosine-containing lysozyme and Raytide, a peptide substrate for protein tyrosine phosphatases. The optimum pH for Raytide was about 5.5. Raytide phosphatase activity was inhibited by potassium fluoride, sodium molybdate, and sodium orthovanadate and was found to exist in some mycoplasmas.
Cloning and nucleotide sequence analysis of psaA, the Streptococcus pneumoniae gene encoding a 37-kilodalton protein homologous to previously reported Streptococcus sp. adhesins
Authors: Sampson J S , O'Connor S P , Stinson A R , Tharpe J A , Russell H
Published: January 2020

Gene psaA, which encodes the Streptococcus pneumoniae 37-kDa protein, was cloned in Escherichia coli, and its complete nucleotide sequence was determined. Analysis of the sequence of the 2.4-kb cloned fragment revealed three open reading frames (ORFs). ORF2, which is 933 bp long, was identified as psaA. The two other ORFs identified flank psaA. ORF1, located upstream of psaA, is 836 nucleotides long and encodes a protein with a calculated molecular mass of 29,843 Da. The sequence for ORF3, located downstream of psaA, was only partially determined. Northern (RNA) blot analysis of pneumococcal RNA suggests that psaA is transcribed as part of a polycistronic message. Analysis of the primary structure of the protein encoded by this gene indicated significant similarity to two previously reported streptococcal proteins, SsaB (80% similarity) and FimA (92.3% similarity), from S. sanguis and S. parasanguis, respectively. These two homologous proteins have been shown to be associated with bacterial adhesion, and the possibility of a similar role for PsaA is hypothesized.
C9-mediated killing of bacterial cells by transferred C5b-8 complexes: transferred C5b-9 complexes are nonbactericidal
Authors: Blanchard K P , Dankert J R
Published: January 2020

The formation of the C5b-9 complex on the outer membrane of complement-sensitive cells of Escherichia coli results in inhibition of inner membrane function and the death of the cell. Cells bearing a precursor of the C5b-9 site, the C5b-8 complex, suffer no loss in viability. Antibiotic-sensitive, complement-sensitive donor cells bearing precursor C5b-8 complexes were incubated with equal numbers of antibiotic-resistant, complement-sensitive acceptor cells that had not been exposed to a complement source. This cell mixture was incubated with 5 mM EDTA for 5 min and then with calcium chloride (20 mM) for various times. The excess calcium ion concentration was effectively reduced with additional EDTA, and the cell mixture was washed and resuspended in buffer. The viability of the acceptor cells was assayed by plating on antibiotic-containing media. C9 was added to the mixture, and the mixture was incubated for 10 min at 37 degrees C and then plated as described above. It was found that the acceptor cells were killed by the addition of purified C9 only after incubation with donor cells bearing C5b-8 sites during the transfer procedure. This indicates that precursor C5b-8 sites that support C9-mediated killing could be transferred between cells. No loss in viability was detected for acceptor cells subjected to the procedure described above in the presence of donor cells bearing complete C5b-9 complexes, formed prior to mixing with acceptor cells for the transfer procedure.
Role of trypsin-like cleavage at arginine 192 in the enzymatic and cytotonic activities of Escherichia coli heat-labile enterotoxin
Authors: Grant C C , Messer R J , Cieplak W
Published: January 2020

Previous studies of cholera toxin and Escherichia coli heat-labile enterotoxin have suggested that proteolytic cleavage plays an important role in the expression of ADP-ribosyltransferase activity and toxicity. Specifically, several studies have implicated a trypsin-like cleavage at arginine 192, which lies within an exposed region subtended by a disulfide bond in the intact A subunit, in toxicity. To investigate the role of this modification in the enzymatic and cytotonic properties of heat-labile enterotoxin, the response of purified, recombinant A subunit to tryptic activation and the effect of substituting arginine 192 with glycine on the activities of the holotoxin were examined. The recombinant A subunit of heat-labile enterotoxin exhibited significant levels of ADP-ribosyltransferase activity that were only nominally increased (approximately twofold) by prior limited trypsinolysis. The enzymatic activity also did not appear to be affected by auto-ADP-ribosylation that occurs during the high-level synthesis of the recombinant A subunit in E. coli. A mutant form of the holotoxin containing the arginine 192-to-glycine substitution exhibited levels of cytotonic activity for CHO cells that were similar to that of the untreated, wild-type holotoxin but exhibited a marked delay in the ability to increase intracellular levels of cyclic AMP in Caco-2 cells. The results indicate that trypsin-like cleavage of the A subunit of E. coli heat-labile enterotoxin at arginine 192 is not requisite to the expression of enzymatic activity by the A subunit and further reveal that this modification, although it enhances the biological and enzymatic activities of the toxin, is not absolutely required for the enterotoxin to elicit cytotonic effects.
Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette
Authors: Kolenbrander P E , Andersen R N , Ganeshkumar N
Published: January 2020

Human oral viridans group streptococci that coaggregate with Actinomyces naeslundii PK606 express surface proteins related to ScaA, the coaggregation-mediating adhesin of Streptococcus gordonii PK488 (R. N. Andersen, N. Ganeshkumar, and P. E. Kolenbrander, Infect. Immun. 61:981-987, 1993). The nucleotide sequence of the 6,125-bp EcoRI insert of pRA1, containing scaA, the gene encoding ScaA, was determined. Six open reading frames (ORFs) were identified. The orientation of four ORFs, two upstream (ORF 1 and ORF 2) and one downstream (ORF 4) of scaA (ORF 3), indicated transcription in one direction, whereas ORF 5 and ORF 6 were transcribed divergently. Computer analysis of the deduced amino acid sequences identified a consensus binding site for ATP (GxxGxGKS) in the putative 28,054-Da protein encoded by ORF 1. ORF 2 potentially encoded a hydrophobic protein of 29,705 Da with six potential membrane-spanning regions. ScaA was 310 amino acids, 34,787 Da, and contained the lipoprotein consensus sequence LxxC, also reported for the ScaA-related proteins SsaB, FimA, and PsaA from Streptococcus sanguis 12, Streptococcus parasanguis FW213, and Streptococcus pneumoniae R36A, respectively. ORF 4 potentially encoded a 163-amino-acid protein of 17,912 Da, which was nearly identical to the downstream adjacent gene products of ssaB, fimA, and psaA. No significant homology with other proteins was found with the putative ORF 5 gene product, a 229-amino-acid protein of 25,107 Da. ORF 6 was incomplete and encoded a protein larger than 564 amino acids. This putative protein had a consensus Zn2+ binding motif, HExxH, found among bacterial thermolysins and mammalian neutral endopeptidases and was 40% identical to a homologous 210-amino-acid region of human enkephalinase. The genetic organization of ORFs 1, 2, and 3 was similar to those of the bacterial periplasmic-binding protein-dependent transport systems of gram-negative bacteria and binding-lipoprotein-dependent transport systems of gram-positive bacteria, and these genes appeared to encode ABC (ATP-binding cassette) proteins. This report describes a cell-to-cell adherence function associated with an ATP-binding cassette.
Protective role of sialophorin (CD43)-expressing cells in experimental Staphylococcus aureus infection
Authors: Bremell T , Holmdahl R , Tarkowski A
Published: January 2020

Sialophorin (CD43) is a major surface mucin on most hematopoietic cell lineages, including phagocytes. Defects of CD43 expression occur in Wiskott-Aldrich syndrome, a disease characterized by susceptibility to pyogenic infections. In a newly established rat model of septic Staphylococcus aureus arthritis, we have investigated the role of CD43-expressing cells in the progression of the disease. A single injection of a monoclonal antibody specific for CD43 induced a highly erosive course of arthritis and increased mortality in animals exposed to a suboptimal dose of bacteria. Our results demonstrate that sialophorin-expressing cells play a protective role in the early stage of staphylococcal infection.
Mycoplasma arginini TUH-14 membrane lipoproteins induce production of interleukin-1, interleukin-6, and tumor necrosis factor alpha by human monocytes
Authors: Herbelin A , Ruuth E , Delorme D , Michel-Herbelin C , Praz F
Published: January 2020

Mycoplasma arginini TUH-14 partially purified membrane lipoproteins (TUH-14-pp) directly induce secretion of the cytokines involved in the inflammatory response, namely, interleukin 1 (IL-1), tumor necrosis factor alpha, and IL-6, by human monocytes cultured in the absence of serum. The biological activity of each cytokine correlates with its immunoreactivity. Upon stimulation with either TUH-14-pp or lipopolysaccharide, most tumor necrosis factor alpha and IL-6 is secreted in the extracellular compartment, whereas a significant amount of IL-1 remains cell associated. Finally, polymyxin B does not affect secretion of cytokines induced by TUH-14-pp, indicating that mycoplasma lipopolysaccharide does not account for their effects on monocytes. Altogether, our data show that direct interaction of mycoplasma membrane components with human blood monocytes induces secretion of high levels of cytokines known to trigger inflammatory responses. This new concept of membrane-bound active components of mycoplasma may explain its ability to efficiently initiate inflammatory reactions.
Protein D, the glycerophosphodiester phosphodiesterase from Haemophilus influenzae with affinity for human immunoglobulin D, influences virulence in a rat otitis model
Authors: Janson H , Melhus A , Hermansson A , Forsgren A
Published: January 2020

A mutant lacking the ability to express the surface-exposed lipoprotein protein D was constructed by linker insertion and deletion mutagenesis of a cloned DNA insert containing the protein D structural gene from a nontypeable Haemophilus influenzae strain (NTHi). An isogenic NTHi mutant was isolated after transformation of genetically competent bacteria. The transformant was unreactive to a protein D-specific monoclonal antibody in a colony immunoassay. In addition, the mutant lacked the ability to synthesize detectable levels of protein D by protein staining, immunoblot methods, glycerophosphodiester phosphodiesterase activity, and binding studies of radiolabelled immunoglobulin D. The isogenic protein D-deficient mutant was compared with its parental strain for its ability to induce experimental otitis media in rats challenged with bacteria. An approximately 100-times-higher concentration of the mutant compared with that of the wild-type strain was required in order to cause otitis among all rats challenged with that given dose. The protein D mutant exhibited a generation time that was equal to that of the wild-type strain in complex broth medium. No difference in lipopolysaccharide expression was found between the mutant and the parental strain. These results suggest that protein D may influence the pathogenesis of NTHi in the upper respiratory tract.
Cloning, sequencing, and viscometric adhesion analysis of heat-resistant agglutinin 1, an integral membrane hemagglutinin from Escherichia coli O9:H10:K99
Authors: Lutwyche P , Rupps R , Cavanagh J , Warren R A , Brooks D E
Published: January 2020

The gene encoding a mannose-resistant hemagglutinating protein was cloned from Escherichia coli O9:H10:K99. The hemagglutinin is different from two other mannose-resistant hemagglutinins in this strain, K99 and F41. The agglutinin, named heat-resistant agglutinin 1 (HRA1) since heating to 70 degrees C does not destroy its aggregative properties, strongly agglutinates human, pig, and dog erythrocytes, shows little or no affinity towards cow and chicken erythrocytes, but agglutinates human colon adenocarcinoma 201 (COLO 201) cells. The hra1 gene present on the recombinant plasmid pETE1 was localized by subcloning, and its nucleotide sequence was determined. The gene consists of a 792-bp open reading frame coding for a putative protein of 29 kDa with a predicted N-terminal secretory signal sequence. HRA1 shares no significant identity with data base protein sequences. HRA1 is strongly associated with the bacterial membrane, resisting sonication and isolation attempts based upon standard adhesin purification techniques. N-terminal sequencing of a unique 25-kDa band present in polyacrylamide gels of outer membrane preparations of bacteria harboring pETE1 correlated with the predicted N-terminal amino acid sequence of HRA1 after cleavage of the signal peptide. A viscometric agglutination assay sensitive to the strength of bacterial adhesion shows that the agglutination mediated by bacteria expressing HRA1 is weaker than that of bacteria bearing the F41 adhesin, probably because of the high-molecular-weight, multivalent nature of the latter adhesin. Our observations suggest that HRA1 is a monomeric outer membrane agglutinin.
Molecular and functional analysis of the LYS1 gene of Candida albicans
Authors: Garrad R , Schmidt T M , Bhattacharjee J K
Published: January 2020

The LYS1 gene of Candida albicans has been localized to a 1.8-kb DNA fragment present on the plasmid YpBRG2. YpBRG2 has been shown to complement the saccharopine dehydrogenase mutant Stx4-4A of Saccharomyces cerevisiae. Transformants of S. cerevisiae Stx4-4A exhibited significant saccharopine dehydrogenase activity, and cells that had lost YpBRG2 after nonselective growth had no enzyme activity. The DNA sequence of the LYS1 gene has been determined. The LYS1 DNA contains typical yeast upstream regulatory sequences, including the GCN4 motif and candidate sequences responsible for transcription termination within the 3' noncoding region. The fragment contained an open reading frame of 1,146 nucleotides coding for a putative protein of 382 amino acids. The open reading frame has 60% identity at the nucleotide level and 71% similarity at the amino acid level to the LYS5 gene of Yarrowia lipolytica, which is believed to code for saccharopine dehydrogenase. A peptide of 11 amino acids has been found, which is present in S. cerevisiae, Y. lipolytica, and C. albicans. This peptide can be expanded to 16 amino acids when the sequences from Y. lipolytica and C. albicans are compared. A motif responsible for the binding of the adenosine residue of NADH has been described previously and is very similar to this peptide, which may be the site of NADH binding in the saccharopine dehydrogenase of C. albicans.
Superantigenic properties of the group A streptococcal exotoxin SpeF (MF)
Authors: Norrby-Teglund A , Newton D , Kotb M , Holm S E , Norgren M
Published: January 2020

Streptococcal pyrogenic exotoxin F (SpeF), previously referred to as mitogenic factor, is a newly described potent mitogen produced by group A streptococci. To investigate whether this protein belongs to the family of microbial superantigens, we analyzed the cellular and molecular requirements for its presentation to T cells and compared it with the known streptococcal superantigen pyrogenic exotoxin A (SpeA) and the nonspecific polyclonal T-cell mitogen phytohemagglutinin (PHA). SpeF and SpeA were efficiently presented by autologous antigen-presenting cells (APCs) and an allogeneic B lymphoma cell line, Raji. In contrast, the monocytic cell line U937, which does not express major histocompatibility complex (MHC) class II molecules, failed to present SpeF as well as SpeA but supported the response to PHA. Thus, the presentation of SpeF by APCs was class II dependent but not MHC restricted. The requirement for HLA class II was further supported by the ability of anti-HLA-DQ monoclonal antibody to block the SpeF-induced proliferative response by 75 to 100%. Paraformaldehyde (PFA) fixation of autologous APCs resulted in an impaired ability of SpeF and SpeA to induce optimal T-cell proliferation. In contrast, fixation of Raji cells did not affect the induced proliferation. The stimulatory effect of PHA remained unaffected by both the use of PFA-fixed APCs and the addition of the HLA class II-specific monoclonal antibodies. The addition of a supernatant enriched in interleukin 1 and interleukin 6 to fixed autologous APCs resulted in an increased SpeF-induced response; thus, the impairment was not due to a requirement for processing, but, rather, costimulatory factors produced by metabolically active APCs were needed. SpeF was found to preferentially activate T cells bearing V beta 2, 4, 8, 15, and 19, as determined by quantitative PCR. The data presented clearly show that SpeF is a superantigen. We also studied the prevalence of the speF gene in clinical isolates by Southern blot analyses, and the gene could be detected in 42 group A streptococcal strains, which represented 14 serotypes.
A monoclonal antibody that recognizes phosphatidylinositol inhibits induction of tumor necrosis factor alpha by different strains of Plasmodium falciparum
Authors: Bate C A , Kwiatkowski D
Published: January 2020

The clinical symptoms of human malaria are mediated, at least in part, by the release of tumor necrosis factor alpha (TNF) by monocytes and macrophages. We have found that lysates of Plasmodium falciparum-infected erythrocytes stimulate the secretion of TNF from human mononuclear cells, and we have generated several immunoglobulin M monoclonal antibodies (MAbs) that inhibit the induction of TNF by such lysates. Here we describe the properties of MAb 5AB3-11, which causes dose-dependent inhibition of the TNF-inducing factors derived from P. falciparum-infected erythrocytes, with a 50% reduction in TNF secretion at nanomolar concentrations (1 to 2 micrograms/ml). The inhibitory effect appears to be malaria specific in that the induction of TNF by either lipopolysaccharide or lipoteichoic acid is not affected. MAb 5AB3-11 binds to liposomes containing phosphatidylinositol but not to other phospholipid liposomes, showing that it recognizes a phosphatidylinositol-like epitope. MAb 5AB3-11 inhibits the induction of TNF by whole lysates from several strains of P. falciparum which originated from different parts of the tropics, indicating that all of the major TNF-inducing factors derived from Plasmodium-infected erythrocytes contain a common epitope. A phosphatidylinositol-like epitope expressed by Plasmodium-infected erythrocytes may be a suitable immunological target for the prevention or treatment of severe malaria.
Mapping of multiple HLA class II-restricted T-cell epitopes of the mycobacterial 70-kilodalton heat shock protein
Authors: Oftung F , Geluk A , Lundin K E , Meloen R H , Thole J E , Mustafa A S , Ottenhoff T H
Published: January 2020

By combining a DNA subclone and synthetic-peptide approach, we mapped epitopes of the immunogenic mycobacterial 70-kDa heat shock protein (HSP70) recognized by human CD4+ T-cell clones and lines. In addition, we identified the respective HLA-DR molecules used in antigen presentation. The donor groups used were healthy persons immunized with killed Mycobacterium leprae and tuberculoid leprosy patients. The results show that the N-terminal part of the HSP70 molecule contains three different T-cell epitopes, of which two were presented by DR7 (amino acids [aa] 66 to 82 and 210 to 226) and one was presented by DR3 (aa 262 to 274). The C-terminal part contains one epitope (aa 413 to 424) presented by HLA-DR2. The C-terminal epitope shows extensive homology to the corresponding region of the human HSP70 sequence. All of the T-cell epitopes identified were presented by only one particular HLA-DR molecule. We also found that HLA-DR5 and DRw53 can present HSP70 to T cells, demonstrating the presence of additional epitopes not yet defined at the peptide level. On the basis of the donors used in this study, recognition of HSP70 at the epitope level seems to be ruled by the restriction elements expressed by the donor rather than by any difference in reactivity between healthy individuals and patients. In conclusion, mycobacterial HSP70 is relevant to subunit vaccine design since it contains a variety of T-cell epitopes presented in the context of multiple HLA-DR molecules.
Development and evaluation of an experimental vaccination program using a live avirulent Salmonella typhimurium strain to protect immunized chickens against challenge with homologous and heterologous Salmonella serotypes
Authors: Hassan J O , Curtiss R
Published: January 2020

A stable live avirulent, genetically modified delta cya delta crp Salmonella typhimurium vaccine strain, chi 3985, was used in several vaccination strategies to evaluate its use in the control of Salmonella infection in chickens. Oral vaccination of chickens at 1 and at 14 days of age with 10(8) CFU of chi 3985 protected against invasion of spleen, ovary, and bursa of Fabricius and colonization of the ileum and cecum in chickens challenged with 10(6) CFU of virulent homologous Salmonella strains from group B. Chickens challenged with heterologous Salmonella strains from groups C, D, and E were protected against visceral invasion of spleen and ovary, while invasion of the bursa of Fabricius and colonization of ileum and cecum was reduced in vaccinated chickens. Oral vaccination at 2 and at 4 weeks of age induced an excellent protection against challenge with virulent group B Salmonella serotypes and very good protection against challenge with group D or E Salmonella serotypes, while protection against challenge with group C Salmonella serotypes was marginal but significant. Vaccination at 2 and at 4 weeks of age also protected vaccinated chickens against challenge with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains. The protection of chickens vaccinated with chi 3985 against challenge with homologous and heterologous Salmonella serotypes is outstanding, and the complete protection against ovarian invasion in chickens challenged with 10(8) CFU of highly invasive S. typhimurium or S. enteritidis strains suggests that vaccination of chickens with chi 3985 can complement the present hygiene- and sanitation-based Salmonella control measures. This paper reports a breakthrough in the use of live avirulent vaccine to control Salmonella carriers in chickens.
Invasion and intracellular survival of Bordetella bronchiseptica in mouse dendritic cells
Authors: Guzman C A , Rohde M , Bock M , Timmis K N
Published: January 2020

We have studied the interaction between the respiratory pathogen Bordetella bronchiseptica and murine spleen dendritic cells, important antigen-presenting cells that are found in the airway epithelium. Wild-type B. bronchiseptica 5376 attached very efficiently to dendritic cells, whereas the bvg mutant ATCC 10580, wild-type strain BB7865, and its spontaneous delta bvgS mutant BB7866 bound less efficiently. However, all tested B. bronchiseptica strains were able to invade dendritic cells and survive intracellularly for at least 72 h. These results suggest that bvg-independent or bvg-downregulated products are involved in the uptake and intracellular survival. Transmission electron microscopic analysis revealed that bacteria grew and replicated intracellularly and were present in typical phagosomes, which fused with lysosomes during the initial infection period. However, in later infection stages some bacteria seemed to escape into an unfused endocytic compartment, where individual bacteria were tightly surrounded by a membrane. The in vitro interaction of B. bronchiseptica with dendritic cells reported here may be relevant to natural infections caused by this organism that lead to chronicity or an altered immune response.
Synthesis and some immunologic properties of an O-acetyl pectin [poly(1-->4)-alpha-D-GalpA]-protein conjugate as a vaccine for typhoid fever
Authors: Szu S C , Bystricky S , Hinojosa-Ahumada M , Egan W , Robbins J B
Published: January 2020

Pectin, a plant polysaccharide, is mostly a linear homopolymer of poly(1-->4)-alpha-D-GalpA with < 5% neutral sugars: its molecular size has a broad distribution around 400 kDa, and the degree of esterification is < 5%. The structure of the capsular polysaccharide of Salmonella typhi (Vi) differs from pectin in that it is N acetylated at C-2 and O acetylated at C-3, and has a molecular size of approximately 2 x 10(3) kDa. There is no serological cross-reaction between pectin and Vi. Pectin, when O acetylated at C-2 and C-3, is antigenically identical to Vi in double immunodiffusion. Unlike Vi, O-acetylated pectin (OAcPec) is not immunogenic in mice, probably because of its comparatively low molecular weight. After storage at 3 to 8 degrees C for 3 months, there was no change in the O-acetyl content or the M(r) of OAcPec. At 60 degrees C, the M(r) of OAcPec declined more rapidly than that of Vi. OAcPec conjugated to tetanus toxoid elicited Vi antibodies in mice, and reinjection elicited a booster response. The levels of Vi antibodies elicited by OAcPec-tetanus toxoid conjugates were lower than those elicited by Vi conjugates, but these differences were not statistically significant. OAcPec has some advantages because it can be measured by standardized colorimetric assays and because it forms more soluble conjugates with proteins than does Vi. One disadvantage is that its glycosidic bond is not as stable as that of Vi. The use of a plant polysaccharide, pectin, as an immunogen for prevention of a systemic infection caused by a capsulated pathogen (S. typhi) provides a novel approach to improve the preparation and immunogenicity of polysaccharide-based vaccines.
Cell-associated collagenolytic activity by group B streptococci
Authors: Jackson R J , Dao M L , Lim D V
Published: January 2020

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.
Inhibition of in vitro erythropoiesis by soluble mediators in Plasmodium chabaudi AS malaria: lack of a major role for interleukin 1, tumor necrosis factor alpha, and gamma interferon
Authors: Yap G S , Stevenson M M
Published: January 2020

By using erythropoietin-dependent proliferation of splenic erythroid cells as an in vitro erythropoiesis model system, we demonstrate that spleen cells from Plasmodium chabaudi AS-infected C57BL/6 mice potently inhibited erythroid cell proliferation. Inhibitory activity was detected in spleen cell conditioned media (SPCM) prepared from infected mice but not from uninfected mice. The inhibitory activity in SPCM was characterized as being heat sensitive, macromolecular, and host derived. The inhibitory activity was not reversed by increasing the erythropoietin concentration and was found to be specific for the late erythroid lineage. Mouse strains, which differ in their resistance to P. chabaudi AS infection, produced and responded to the inhibitory activity to a similar extent. Putative immune mediators, interleukin 1 alpha, interleukin 1 beta, and gamma interferon, were found to be potent inhibitors of erythroid cell proliferation. However, antibody neutralization experiments failed to demonstrate a major role for these cytokines in the inhibitory activity of SPCM. Our results suggest that the elaboration of inhibitor(s) of erythropoiesis in hemopoietic organs of Plasmodium-infected mice may impair erythroid regeneration. The identity of the inhibitory mediator(s) is presently unknown but is distinct from interleukin 1, tumor necrosis factor alpha, and gamma interferon.
Induction of muscle-relaxing factor by staphylococcal alpha-toxin
Authors: Harshman S , Sugg N
Published: January 2020

Brain tissue and serum from mice intracerebrally injected with 1 microgram of staphylococcal alpha-toxin contained elevated amounts of a naturally occurring brain tissue component(s) called muscle-relaxing factor (MRF). MRF induced reversible, generalized, flaccid paralysis of mice after intracerebral but not intraperitoneal or intravenous administration. MRF (i) was soluble in Hanks balanced salt solution and in acidified (pH 2) Hanks balanced salt solution, in which it partitions into ethyl acetate, acetone, and methanol; (ii) was separated from some pigments by thin-layer chromatography on silica gel plates; (iii) did not comigrate with prostaglandin and leukotriene standards during high-pressure liquid chromatography with a mu Bondapak fatty acid column; and (iv) did not contain amino acids, exhibit absorption maxima at a wavelength range of 210 to 600 nm, or fluoresce when exposed to UV light. MRF has been detected in rabbit brain that has been stored frozen at -70 degrees C and has been enhanced in vitro in slices of both mouse and rabbit brain following incubation of the brain slices with staphylococcal alpha-toxin. Studies to identify the chemical nature of MRF and the mechanism by which, in mice, it induces reversible, flaccid paralysis of voluntary muscle are continuing.