bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential induction of tumor necrosis factor alpha in murine and human leukocytes by Mycoplasma arthritidis-derived superantigen
Authors: Rink L , Nicklas W , Alvarez-Ossorio L , Koester M , Kirchner H
Published: January 2020

Mycoplasma arthritidis-derived superantigen (MAS) is exclusively produced by M. arthritidis, which is the only known mycoplasma to produce a superantigen. As a superantigen, MAS shows properties similar to those of the staphylococcal enterotoxins and related substances, such as binding to major histocompatibility complex (MHC) class II and V beta-specific stimulation of T cells. In this series of experiments, we demonstrate some differences between MAS and other superantigens. MAS induced the production of tumor necrosis factor alpha (TNF-alpha) mRNA in human as well as in murine leukocytes. However, only in murine leukocytes was the mRNA adequately translated into the protein. In human peripheral blood mononuclear cells, we found only small amounts of TNF, whereas in murine spleen cells we detected levels more than three times higher. The proliferative response to MAS has been shown to be restricted to I-E alpha in the murine MHC. Furthermore, TNF was induced in I-E alpha+ bone marrow-derived macrophages by MAS. In these cells, MAS rapidly induced very high levels of TNF and the amounts of mRNA detected correlated to the amount of protein produced. In comparison with other superantigens, including the staphylococcal enterotoxins, toxic shock syndrome toxin 1, and exfoliative toxin A, the failure of MAS to induce TNF-alpha in human peripheral blood mononuclear cells is specific for MAS and not common to all superantigens. The direct activation of bone marrow-derived macrophages also seems to be specific for MAS. These data suggest that the induction of TNF-alpha by MAS is dependent on the strength of binding to the MHC class II molecule.
The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality
Authors: Lindgren S W , Samuel J E , Schmitt C K , O'Brien A D
Published: January 2020

Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-like toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytotoxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related toxins have been described. Because EHEC O91:H21 strain B2F1, which produces two SLT-II-related toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD50], < 10 organisms), we asked whether the pathogenicity of strain B2F1 was a consequence of SLT-II-related toxin production. For this purpose, we compared the lethality of orally administered E. coli DH5 alpha (Strr) strains that produced different cytotoxic levels of SLT-II, SLT-IIvha (cloned from B2F1), SLT-IIvhb (also cloned from B2F1), or SLT-IIc (cloned from EHEC O157:H7 strain E32511) on Vero cells. We also calculated the specific activities of purified SLT-IIvhb and SLT-II in intraperitoneally injected mice and on Vero cells. The two purified toxins were equally toxic for mice, but SLT-IIvhb was approximately 100-fold less active than SLT-II on Vero cells and bound to the glycolipid receptor Gb3 with lower affinity than did SLT-II. In addition, characterization of SLT-II-related toxin-binding (B) subunit mutants generated in this study revealed that the reduced in vitro cytotoxic levels of the SLT-II-related toxins were due to Asn-16 in the B subunit. Taken together, these findings do not support the idea that B2F1 is uniquely virulent because of the in vivo toxicity of SLT-II-related toxins but do demonstrate differences in in vitro cytotoxic activity among the SLT-II group produced by human EHEC isolates.
Sequence determination and mutational analysis of the lly locus of Legionella pneumophila
Authors: Wintermeyer E , Flügel M , Ott M , Steinert M , Rdest U , Mann K H , Hacker J
Published: January 2020

The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus.
Identification and characterization of a Candida albicans-binding proteoglycan secreted from rat submandibular salivary glands
Authors: Hoffman M P , Haidaris C G
Published: January 2020

A previously identified Candida albicans-binding glycoprotein secreted from rat submandibular glands (RSMG) has been further purified from an aqueous RSMG extract by ion-exchange chromatography and gel filtration. Biochemical analysis of the glycoprotein revealed high levels of uronic acid and sulfate, suggesting that it was a proteoglycan. Its amino acid and carbohydrate compositions were similar to those observed for other proteoglycans and differed significantly from those of RSMG mucin, the major secretory glycoprotein of RSMG. In addition, the apparent molecular weight of the glycoprotein was reduced following treatment with either chondroitinase ABC or heparitinase, demonstrating the presence of chondroitin sulfate and heparan sulfate. On the basis of its structure and anatomical source, the glycoprotein is referred to as submandibular gland secreted proteoglycan 1 (SGSP1). SGSP1 also binds monoclonal antibody 1F9, which recognizes the human blood group A carbohydrate epitope found on RSMG mucin. Hence, SGSP1 appears to be a hybrid molecule with carbohydrate structures found in both proteoglycans and RSMG mucin. Enzymatic digestion of SGSP1, followed by its interaction with a radiolabelled C. albicans strain in a filter-binding assay, demonstrated that binding to this strain appears to be mediated primarily via the heparan sulfate side chains of SGSP1 and not via the blood group A oligosaccharide.
Cloning of TH0- and TH2-type helper lymphocytes from liver granulomas of Schistosoma mansoni-infected mice
Authors: Zhu Y , Lukacs N W , Boros D L
Published: January 2020

The pathological manifestations of schistosomiasis mansoni are primarily induced by circumovum hepatic granuloma formation and fibrosis. The growth and modulation of the granulomas are regulated by T lymphocytes and their products. In the present study, we isolated T-lymphocyte clones from lesions at the acute and chronic stages of murine infection. All of the T-cell clones were characterized by immunofluorescence as CD4+CD8- helper cells. Three T-cell clones derived from vigorous granulomas produced interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-gamma) and were therefore classified as TH0-type T lymphocytes. Of the three clones derived from immunomodulated lesions, two produced IL-2 or IL-4 and/or IFN-gamma (TH0 type) and the other one did not produce IFN-gamma but did produce IL-4 and was therefore characterized as a TH2-type helper clone. The clones were further characterized by their responsiveness to soluble egg antigen fractions. The acute infection-derived clones responded to the lower-molecular-mass (60- to 66-kDa and 25- to 30-kDa) fractions, whereas the immunomodulated-granuloma-derived clones responded to the 60- to 66-kDa and higher-molecular-mass (70- to 90-, 93- to 125-, and > 200-kDa) fractions. Upon adoptive transfer into naive mice, both the TH0- and TH2-type clones were capable of inducing granuloma formation of similar magnitude around antigen-coated beads and/or freshly isolated parasite eggs. The present study revealed the presence of TH0-type precursor helper cells within the liver granulomas. The findings underscore the complexity of the granulomatous response at the T-cell level and demonstrate that both TH0- and TH2-type granuloma lymphocytes play a role in parasite egg-induced granuloma formation.
Genetic regulation of fructosyltransferase in Streptococcus mutans
Authors: Kiska D L , Macrina F L
Published: January 2020

Streptococcus mutans possesses several extracellular sucrose-metabolizing enzymes which have been implicated as important virulence factors in dental caries. This study was initiated to investigate the genetic regulation of one of these enzymes, the extracellular fructosyltransferase (Ftf). Fusions were constructed with the region upstream of the S. mutans GS5 Ftf gene (ftf) and a promoterless chloramphenicol acetyltransferase (CAT) gene. The fusions were integrated at a remote site in the chromosome, and transcriptional activity in response to the addition of various carbohydrates to the growth medium was measured. A significant increase in CAT activity was observed when glucose-grown cells were shifted to sucrose-containing medium. Sucrose-induced expression was repressed immediately upon addition of phosphoenolpyruvate phosphotransferase system sugars to the growth media. Deletion analysis of the ftf upstream region revealed that an inverted repeat structure was involved in the control of ftf expression in response to carbohydrate. However, the control of the level of ftf transcription appeared to involve a region distinct from that mediating carbohydrate regulation. CAT gene fusions also were constructed with the ftf upstream region from S. mutans V403, a fructan-hyperproducing strain which synthesizes increased levels of Ftf. Sequence analysis of the upstream ftf region in this strain revealed several nucleotide sequence changes which were associated with high-level ftf expression. Comparison of the GS5 and V403 ftf expression patterns suggested the presence of a trans-acting factor(s) involved in modulation of ftf expression in response to carbohydrate. This factor(s) was either absent or altered in V403, resulting in the inability of this organism to respond to the presence of carbohydrate. The sequences of the ftf regions from three additional fructan-hyperproducing strains were determined and compared with that of V403. Only one strain displayed nucleotide changes similar to those of V403. Two additional strains did not have these changes, suggesting that several mechanisms for up-regulation of ftf expression exist.
In vivo association of Actinobacillus pleuropneumoniae serotype 2 with the respiratory epithelium of pigs
Authors: Dom P , Haesebrouck F , Ducatelle R , Charlier G
Published: January 2020

The ability of an Actinobacillus pleuropneumoniae serotype 2 strain to associate in vivo with the epithelium of the porcine respiratory tract was investigated in a sequential study after intranasal inoculation of hysterectomy-derived and colostrum-deprived pigs. At 30 min postinoculation more than 95% of the bacteria present in the lungs were intimately associated with the epithelium of the alveoli or the cilia of the terminal bronchioli, as observed by light and electron microscopy. At 90 and 180 min postinoculation multiple focal early inflammatory lesions in which histologically different, more or less concentric zones could be distinguished were observed. In the center of these pneumonic areas bacteria were associated with infiltrated cells and exudate. In the zone surrounding the center, approximately 95% of the bacteria were lying with their longest side in close apposition to the epithelial cells of alveoli and the cilia of the terminal bronchioli. Bacteria were only sporadically associated with the cilia or the epithelium of the bronchi and trachea. Bacteria were not observed in tonsils or conchae. In view of the findings presented here, we propose the hypothesis that adherence of the A. pleuropneumoniae serotype 2 strain to epithelial cells of the lower respiratory tract constitutes an important initial step in pathogenesis.
Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium
Authors: van Vliet A H , Jongejan F , van Kleef M , van der Zeijst B A
Published: January 2020

Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAP1; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated map1, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium.
Anti-lipoteichoic acid antibodies enhance release of cytokines by monocytes sensitized with lipoteichoic acid
Authors: Mancuso G , Tomasello F , Ofek I , Teti G
Published: January 2020

Lipoteichoic acid (LTA) from gram-positive bacteria can stimulate monocytes to produce cytokines. To ascertain whether aggregation of LTA receptors can contribute to this effect, human monocytes were sensitized with LTA from Streptococcus pyogenes, washed, and treated with anti-LTA antibodies. The addition of anti-LTA antibodies or F(ab')2 fragments markedly enhanced the aggregation of LTA receptors, as evidenced by indirect immunofluorescence and the release of tumor necrosis factor alpha and interleukin-1 beta. These findings suggest that aggregation of LTA receptors of monocytes is required for triggering marked cytokine responses.
Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning
Authors: Lopes M F , dos Reis G A
Published: January 2020

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.
Protein phosphorylation in murine peritoneal macrophages induced by infection with Salmonella species
Authors: Saito S , Shinomiya H , Nakano M
Published: January 2020

Infection of peritoneal macrophages from C3H/HeN and C3H/HeJ mice with Salmonella typhimurium or S. enteritidis induced extensive phosphorylation in a set of proteins with molecular masses of 85, 72, 35, 30, and 23 kDa, which were different from those induced by bacterial lipopolysaccharide. The phosphorylated proteins of 35, 30, and 23 kDa (pp35, pp30, and pp23, respectively) originated from the infecting bacteria, because living bacteria could induce these phosphorylated proteins themselves, and no induction of the proteins occurred in macrophages after phagocytosis of heat-killed or UV-irradiated organisms. When the infected macrophages were disrupted and separated into bacterial and macrophage debris fractions, pp85 and pp72 remained in the macrophage debris fraction, with none in the bacterial fraction. Induction of pp85 and pp72 in infected macrophages was inhibited in the presence of chloramphenicol but not cytochalasin D, suggesting that bacterial growth in the macrophages is necessary for induction of both proteins. Neither of these proteins could be detected in macrophages infected with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Listeria monocytogenes. These results support the view that phosphorylation of the 85- and 72-kDa proteins occurs in the macrophages during the early phases of the interaction between Salmonella organisms and macrophages. The functions of specific proteins remain to be clarified.
Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination
Authors: Schödel F , Kelly S M , Peterson D L , Milich D R , Curtiss R
Published: January 2020

Avirulent salmonellae expressing foreign genes are attractive for use as oral vaccine carriers. To facilitate the stable expression of heterologous genes without conferring antibiotic resistance, a deletion of the asdA1 gene was introduced into Salmonella typhimurium and S. typhi delta cya delta crp mutant vaccine strains. An asd-complementing plasmid expressing hybrid hepatitis B virus nucleocapsid-pre-S (HBcAg-pre-S) particles was constructed. These hybrid HBcAg-pre-S particle genes were stably expressed in S. typhimurium and S. typhi delta cya delta crp mutant vaccine strains in this balanced, lethal host-vector combination. A single oral immunization of BALB/c mice with a recombinant S. typhimurium delta cya delta crp mutant synthesizing hybrid HBcAg-pre-S elicited potentially virus-neutralizing anti-pre-S serum immunoglobulin G antibodies. In addition, serum immunoglobulin G recognizing S. typhimurium lipopolysaccharide was induced. Distribution in tissue after oral immunization was analyzed in one plasmid-strain combination. The recombinant S. typhimurium colonized the gut-associated lymphoid tissue and the spleen and persisted for over 4 weeks, retaining the HBcAg-pre-S expression plasmid. An isogenic virulence plasmid-cured S. typhimurium delta cya delta crp strain expressing the same HBcAg-pre-S gene had reduced immunogenicity for the carried antigen after oral immunization.
Construction and characterization of a fimA mutant of Porphyromonas gingivalis
Authors: Hamada N , Watanabe K , Sasakawa C , Yoshikawa M , Yoshimura F , Umemoto T
Published: January 2020

Although fimbriae of Porphyromonas gingivalis have been implicated as playing a major role in adherence to gingival tissue surfaces, no conclusive genetic evidence has yet been obtained. The fimA gene, the determinant for the major fimbrial subunit protein, was cloned and sequenced (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988). We undertook to inactivate the fimA gene by a homologous recombination technique and examined the fimA mutant for changes in surface properties, including production of fimbriae, adherence to human gingival fibroblasts and epithelial cells, hemagglutinating activity, and surface hydrophobicity. To inactivate the fimA gene, we disrupted a fimA clone by insertion of a DNA segment containing an erythromycin resistance (Emr) gene. This was then delivered into P. gingivalis ATCC 33277 from an Escherichia coli K-12 strain, SM10 lambda pir, by using a mobilizable suicide vector, pGP704; recombination at the fimA locus led to the isolation of a fimA mutant. Disruption of the fimA locus and disappearance of FimA production were confirmed by Southern hybridization with a fimA-specific DNA probe and Western immunoblotting with a monoclonal antibody against the FimA protein, respectively. The fimA mutant constructed failed to express long (0.5- to 1.0-micron) fimbriae from the bacterial surface and had a diminished adhesive capacity to tissue-cultured human gingival fibroblasts and epithelial cells. Observation of the bacteria adhering to human gingival fibroblasts by scanning electron microscopy revealed that the wild-type strain had dramatic local changes in the appearance of the microvilli at the point of contact with large bacterial clumps, whereas the fimA mutant did not. In contrast, neither the hemagglutinating activity nor the surface hydrophobicity was changed in the fimA mutant. These data thus constitute the first direct genetic evidence demonstrating that the FimA protein of P. gingivalis is essential for the interaction of the organism with human gingival tissue cells through a function(s) encoded by the fimA gene.
Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae
Authors: Jarvis G A
Published: January 2020

The deposition and degradation of human complement component C3 on the cell surfaces of Neisseria meningitidis and Neisseria gonorrhoeae were studied. Bacteria were incubated in human serum, and ester-linked C3 fragments were analyzed by hydroxylamine release and immunoblot detection. Similar patterns of C3 degradation were found for both serum-resistant and serum-sensitive meningococcal strains of serogroups A, B, C, Y, and W135, as well as for serum-sensitive gonococcal strains and their sialylated serum-resistant variants. The predominant fragments in all cases were the 40-kDa alpha' 2 chain of iC3b and the 75-kDa beta chain common to both C3b and iC3b. The 67-kDa alpha' 1 chain of iC3b was also detected. The 105-kDa alpha' chain of intact C3b represented a minor proportion of deposited C3. Capsule-specific immunoglobulin G or immunoglobulin A1 did not alter the observed degradation patterns, nor did incubation of meningococci in properdin-deficient serum. The degradation of C3 in C5-, C6-, or C8-deficient serum was the same as that in normal serum, although the deposition of C3 was severely limited, based as indicated by the intensity of the fragments. With the use of an enzyme-linked immunosorbent assay that measured total iC3b and C3, I found that both iC3b deposition and C3 deposition varied among meningococcal and gonococcal strains and that the amounts of iC3b and C3 were independent of the relative quantities of cell surface sialic acid and of serum sensitivity for meningococci but not for gonococci. I conclude that complement activation on neisserial cell surface results in the formation of an identical repertoire of predominantly iC3b fragments of ester-linked C3b molecules regardless of the presence of sialic acid in either the capsule or the lipooligosaccharide or of the sensitivity of the organism to complement-mediated lysis but that the quantities of both ester- and amide-linked iC3b molecules deposited exhibit strain variability.
Identification of two functional forms of immunoglobulin G3-binding protein expressed by group A streptococci
Authors: Pack T D , Otten R A , Raeder R H , Boyle M D
Published: January 2020

Analysis of group A streptococcal immunoglobulin G (IgG)-binding protein reactivity with different human IgG3-myeloma proteins provided evidence for at least two functional forms of these molecules. Representative IgG3-binding molecules were isolated, biotinylated, and used as tracers in competitive binding assays. Cross-inhibition studies demonstrated the existence of two distinct patterns of IgG3-binding activity. Proteins of one form could be inhibited from binding to an IgG3-myeloma protein by streptococcal protein G while binding of the second form was not inhibited. These studies further underscore the extent of heterogeneity among immunoglobulin-binding proteins expressed by group A streptococci.
M12 protein from Streptococcus pyogenes is a receptor for immunoglobulin G3 and human albumin
Authors: Retnoningrum D S , Cleary P P
Published: January 2020

We previously showed that M12 protein from opacity factor-negative Streptococcus pyogenes (group A streptococci) CS24 is responsible for immunoglobulin G3 (IgG3) binding activity. Here, we report that this M protein binds human serum albumin (HSA). Deletion analysis showed that the C repeats are sufficient for binding HSA, although upstream regions may be required for optimal binding. Like protein G, IgG3 and HSA bind to independent domains in the M protein. Experiments showed that bound IgG3 did not inhibit HSA binding to the M protein. The interaction between M12 protein and HSA is specific. M12 protein does not bind chicken egg and bovine serum albumins. Alignments of C1 and C2 repeats of M12 protein to sequences at the carboxy termini of other M proteins and Ig receptors revealed highly homologous sequences in the FcRV, M5, M6, ML2.1, and M57 proteins, suggesting that all could bind HSA. As predicted from the alignment, M5 protein and M6+ streptococci bound HSA, whereas an isogenic M6- mutant did not bind HSA. Furthermore, M2 protein from an opacity factor-positive strain also bound HSA.
Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers
Authors: Qi H L , Tai J Y , Blake M S
Published: January 2020

Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the inability to easily manipulate the cloned porin genes by modern molecular techniques and then obtain enough of the expressed modified porin protein to purify and use in these biophysical functional assays. In this report, we describe a method by which the genes encoding three different porin proteins, lacking their neisserial promoter and signal sequences, were cloned into an expression plasmid and transformed into Escherichia coli. Upon induction, large amounts of the porin proteins were produced. The expressed porin proteins were then manipulated to regenerate their native trimer structure and purified by standard protein chemistry. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. This sets the stage for the biophysical characterization of these neisserial porin proteins in more detail.
A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b
Authors: Jarosik G P , Sanders J D , Cope L D , Muller-Eberhard U , Hansen E J
Published: January 2020

Haemophilus influenzae is nearly unique among facultatively anaerobic bacteria in its absolute requirement for exogenously supplied heme for aerobic growth. In this study, a mutant analysis strategy was used to facilitate identification of H. influenzae cell envelope components involved in the uptake of heme. Chemical mutagenesis was employed to produce a mutant of a nontypeable H. influenzae strain unable to utilize either protein-bound forms of heme or low levels of free heme. This mutant was transformed with a plasmid shuttle vector-based genomic library constructed from the same wild-type nontypeable H. influenzae strain, and a growth selection technique was used to obtain a recombinant clone that could utilize heme. Analysis of the DNA insert in the recombinant plasmid revealed the presence of several open reading frames, one of which encoded a 28-kDa protein with significant similarity to the TonB protein of Escherichia coli. This H. influenzae gene product was able to complement a tonB mutation in E. coli, allowing the E. coli tonB mutant to form single colonies on minimal medium containing vitamin B12. When this H. influenzae gene was inactivated by insertional mutagenesis techniques and introduced into the chromosome of wild-type strains of H. influenzae type b, the resultant transformants lost their abilities to utilize heme and produce invasive disease in an animal model. Genetic restoration of the ability to express this TonB homolog resulted in the simultaneous acquisition of both heme utilization ability and virulence. These results indicate that the H. influenzae TonB protein is required not only for heme utilization by this pathogen in vitro, but also for virulence of H. influenzae type b in an animal model.
Protection of gerbils from amebic liver abscess by immunization with a recombinant protein derived from the 170-kilodalton surface adhesin of Entamoeba histolytica
Authors: Zhang T , Stanley S L
Published: January 2020

The protozoan parasite Entamoeba histolytica causes extensive morbidity and mortality worldwide through intestinal infection and amebic liver abscess. Here we show that vaccination of gerbils, a standard model for amebic liver abscess, with recombinant proteins derived from the 170-kDa galactose-binding adhesin of E. histolytica and the serine-rich E. histolytica protein or a combination of the two recombinant antigens provides excellent protection against subsequent hepatic challenge with virulent E. histolytica trophozoites.
Inhibition of phospholipase D blocks activation of fibrinogen-adherent neutrophils by tumor necrosis factor
Authors: Frank M O , Sullivan G W , Carper H T , Mandell G L
Published: January 2020

Tumor necrosis factor stimulates fibrinogen-adherent neutrophils to produce a dramatic oxidative burst; the resulting superoxide and other products may contribute to tissue damage in severe infection. Inhibitors of the phospholipase D pathway blocked this activation without affecting cell adherence or nonspecific activation by phorbol myristate acetate. The phospholipase D pathway appears to be involved in the activation of adherent neutrophils by tumor necrosis factor, and this pathway may be a target for modulation of tumor necrosis factor's effects.
Tick transmission of Borrelia burgdorferi to inbred strains of mice induces an antibody response to P39 but not to outer surface protein A
Authors: Golde W T , Kappel K J , Dequesne G , Feron C , Plainchamp D , Capiau C , Lobet Y
Published: January 2020

Natural tick transmission of infection by Borrelia burgdorferi induces a very different serum antibody response than needle inoculation of spirochetes. We present data, obtained by using the mouse model, that show that the OspA response was barely detectable, whereas all animals developed significant anti-P39 titers after exposure to B. burgdorferi-infected ticks.
Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans
Authors: Kiska D L , Macrina F L
Published: January 2020

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.
A family of surface-exposed proteins of 20 kilodaltons in the genus Borrelia
Authors: Carter C J , Bergström S , Norris S J , Barbour A G
Published: January 2020

Relapsing fever and Lyme disease spirochetes of the genus Borrelia display at their surfaces abundant lipoproteins: Vmp proteins in Borrelia hermsii and Osp proteins in Borrelia burgdorferi. Vmp and Osp proteins largely determine serotype specificity, and neutralizing antibodies of infected or immunized animals are directed at them. For the present study, we examined B. hermsii serotype 33, which is unique among strain HS1 serotypes in the low frequency of switches to other serotypes during infections and in vitro cultivation. Failing to clone the complete vmp33 gene, we accomplished its further characterization by (i) determining three partial amino acid sequences, (ii) designing oligonucleotide primers based on these amino acid sequences, (iii) cloning and sequencing the central portion of vmp33, and (iv) using outwardly directed primers and the inverse PCR to clone the 5' and 3' ends of the gene and flanking regions. The transcriptional start site was identified by primer extension analysis. Vmp33 was a polypeptide of 211 amino acids; the three partial amino acid sequences were identified in the open reading frame. Vmp33 was found to be more similar to other 20-kDa Vmp proteins of B. hermsii and to OspC proteins of B. burgdorferi than it was to 35- to 39-kDa Vmp proteins of the same strain. Moreover, OspC proteins were more similar to Vmp33 than they were to OspA, -B, or -D proteins of B. burgdorferi. These sequence similarities were consistent with Western blot (immunoblot) findings of cross-reactions between Vmp33 and OspC with anti-Vmp33 and anti-OspC sera. The promoter for the expressed vmp33 gene was found to be different from the expression site for other active vmp genes characterized to date. These results indicate that Vmp33 and other small Vmp's belong with OspC to a genus-wide family of 20-kDa proteins and that expression of these proteins may be coordinated with expression of other Vmp and Osp proteins in Borrelia spp.
Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan
Authors: Houpt D C , Pfrommer G S , Young B J , Larson T A , Kozel T R
Published: January 2020

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.
Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2
Authors: Khusmith S , Sedegah M , Hoffman S L
Published: January 2020

BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produce antibodies and cytotoxic T lymphocytes against the circumsporozoite protein and against a 140-kDa protein, sporozoite surface protein 2 (PySSP2). Approximately 50% of mice immunized with P815 cells transfected with the gene encoding PySSP2 are protected against malaria, and this protection is reversed by in vivo depletion of CD8+ T cells. To determine if CD8+ T cells against PySSP2 are adequate to protect against malaria in the absence of other malaria-specific immune responses, we produced three CD8+ T-cell clones by stimulating spleen cells from mice immunized with irradiated P. yoelii sporozoites with a mitomycin-treated P815 cell clone transfected with the PySSP2 gene. Adoptive transfer of clone TSLB7 protected 100% of mice against P. yoelii. The second clone protected 58% of mice, and the third clone provided no protection. Clone TSLB7 protected even when administered 3 h after sporozoite inoculation at a time when sporozoites had entered hepatocytes, suggesting that it is recognizing and eliminating infected hepatocytes. These studies demonstrate that cytotoxic T lymphocytes against PySSP2 can protect against P. yoelii sporozoite challenge in the absence of other parasite-specific immune responses.
Immunization with Pseudomonas aeruginosa vaccines and adjuvant can modulate the type of inflammatory response subsequent to infection
Authors: Johansen H K , Espersen F , Cryz S J , Hougen H P , Fomsgaard A , Rygaard J , Høiby N
Published: January 2020

Pseudomonas aeruginosa is the predominant pathogen in patients with cystic fibrosis (CF). To study the possibility of preventing lung inflammation and decreasing the progression of the infection by vaccination, we have developed a rat model of chronic P. aeruginosa lung infection. Rats were immunized with P. aeruginosa whole-cell sonicates, O-polysaccharide toxin A conjugate, an alginate-toxin A conjugate, or native alginate. Control animals received sterile saline or incomplete Freund's adjuvant (IFA). The macroscopic (mean score, 2.4 versus 2.7 to 3.2) (P < 0.05) and microscopic (mean score, 2.0 versus 2.1 to 2.8) pathologic abnormalities were less severe in the control rats injected with sterile saline than in the immunized rats and the IFA group. The more severe lung abnormalities observed in immunized rats could be due to the result of immune complex-mediated lung tissue damage. The histopathologic results in the saline control rats were characterized by acute inflammation dominated by numerous polymorphonuclear leukocytes surrounding the alginate beads (microcolonies), as in CF patients. In contrast, the inflammatory response in the IFA group and in the immunized rats had changed from an acute-type inflammation to a chronic-type inflammation dominated by mononuclear leukocytes and scattered granulomas. Cross-reacting antibodies were induced by the two alginate vaccines, and most immunized animals developed a significant (P < 0.001) antibody titer elevation (in enzyme-linked immunosorbent assay) of the immunoglobulin M (IgM), IgG, and IgA classes against the homologous antigens. The bacterial clearance was significantly (P < 0.05) more efficient in most immunized rats than in the control rats given sterile saline. The present study shows that none of the vaccines could completely prevent chronic lung inflammation 4 weeks after challenge. However, the changed pathologic condition in immunized rats to a chronic-type inflammation might be of great benefit in future management of CF patients since the developing lung tissue damage has been shown to be caused by polymorphonuclear leukocyte-released elastase.
Anticryptococcal resistance in the mouse brain: beneficial effects of local administration of heat-inactivated yeast cells
Authors: Blasi E , Mazzolla R , Barluzzi R , Mosci P , Bistoni F
Published: January 2020

Using a murine model, we have previously shown that brain resistance to local infection with opportunistic fungi is affected by manipulation of the host myelomonocytic compartment. Here, we demonstrate that intracerebral administration of heat-inactivated Cryptococcus neoformans (H-CN) yeast cells results in a consistent enhancement of mouse survival to subsequent local challenge with lethal doses of C. neoformans. The phenomenon, more pronounced upon double H-CN treatment, is associated with (i) massive local inflammatory response, (ii) reduced growth of the fungus within the brain, and (iii) induction of delayed-type hypersensitivity. Moreover, H-CN treatment confers protection against local heterologous challenges. Our data provide initial evidence that intracerebral administration of H-CN results in the establishment of aspecific and specific immune responses; the mechanisms of elicitation and relative contributions to host antimicrobial resistance remains to be elucidated.
Hemagglutinating and chemotactic properties of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis
Authors: Ogawa T , Hamada S
Published: January 2020

Porphyromonas gingivalis 381 fimbriae, their synthetic peptide segments, and lipopolysaccharide (LPS) were examined for hemagglutinating and migration-stimulating activities. P. gingivalis 381 fimbriae clearly caused hemagglutination, and several oligopeptide segments such as FP381(61-80), FP381(171-185), and FP381(302-321), corresponding to the amino acid residue numbers based on the amino acid sequence of fimbrillin proposed by Dickinson et al. (D. P. Dickinson, M. A. Kubiniec, F. Yoshimura, and R. J. Genco, J. Bacteriol. 170:1658-1665, 1988), were also demonstrated to agglutinate erythrocytes although less effectively than the native fimbriae. Furthermore, P. gingivalis 381 LPS but not Escherichia coli O55:B5 LPS definitely exhibited hemagglutination. P. gingivalis fimbriae as well as their synthetic peptides possessing hemagglutinating activity enhanced the chemotaxically induced migration of human peripheral blood monocytes. The results of the analyses using synthetic peptide FP381(61-80), its related compounds, and an analog suggested that the amino acid sequence XLTXXLTXXNXX within fimbrial protein molecules may play an important role structurally in the attachment of the protein to host cells such as erythrocytes and monocytes.
Host specificity of enteropathogenic Escherichia coli from rabbits: lack of correlation between adherence in vitro and pathogenicity for laboratory animals
Authors: Robins-Browne R M , Tokhi A M , Adams L M , Bennett-Wood V
Published: January 2020

The pathogenicity of four attaching and effacing strains of enteropathogenic Escherichia coli originally isolated from diarrheic rabbits was investigated by inoculating them perorally into rabbits, guinea pigs, and mice. The ability of the four strains to adhere to cultured epithelial cells, erythrocytes, and intestinal brush borders from various animal species, including rabbits, guinea pigs, and mice, varied considerably. Only one strain carried AF/R1 fimbriae, which are believed to determine the host specificity of these bacteria. Despite these differences, the pattern of behavior of the four strains in experimentally infected animals was similar. Each strain caused fatal diarrhea in rabbits (although the virulence of individual strains for rabbits differed significantly), and none was virulent for guinea pigs or mice. None of the strains colonized the intestinal tract of guinea pigs, but all were able to cause attaching-effacing lesions in ligated loops of guinea pig small intestine. By contrast, all four strains colonized mice, in particular the distal intestine, but none induced attaching-effacing lesions in mouse intestinal loops. These findings suggest that there may be previously unrecognized host-restricted adhesins in enteropathogenic E. coli and indicate that adherence to erythrocytes or intestinal brush borders in vitro does not necessarily reflect colonizing ability or pathogenicity in vivo.
Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein
Authors: Elsinghorst E A , Weitz J A
Published: January 2020

Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human colon and ileocecum. Two separate loci (tia and tib) that direct noninvasive E. coli HB101 to adhere to and invade intestinal epithelial cells have previously been cosmid cloned from ETEC H10407. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cellular fractions from tib-positive HB101 shows that the tib locus directs the synthesis of a 104-kDa outer membrane protein (the TibA protein). The tib locus was subcloned to a maximum of 6.7 kb and mutagenized with transposon Tn5. Production of TibA was directly correlated with the capacity of the subclones and Tn5 mutants to invade and adhere to epithelial cells, suggesting that TibA was required for these phenotypes. The position and direction of transcription of the tibA gene were identified by complementation and in vivo T7 RNA polymerase-promoter induction experiments. The role of the tib locus in epithelial cell invasion was confirmed by the construction of chromosomal deletion derivatives in H10407. These deletion mutants invaded epithelial cells at about 15% of the parental level and were fully complemented by plasmids bearing the tib locus. The size and function of the TibA protein are similar to those of invasin from Yersinia pseudotuberculosis (103 kDa). However, a tib probe did not hybridize with the gene encoding invasin. Hybridization analyses of genomic DNA from a wide variety of pathogenic and nonpathogenic bacteria, including Salmonella, Shigella, Yersinia, and Escherichia species, indicate that the tib locus is unique to specific ETEC strains.