bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective vaccination with promastigote surface antigen 2 from Leishmania major is mediated by a TH1 type of immune response
Authors: Handman E , Symons F M , Baldwin T M , Curtis J M , Scheerlinck J P
Published: January 2020

Leishmania major promastigote surface antigen-2 complex (PSA-2) comprises a family of three similar but distinct polypeptides. The three PSA-2 polypeptides were purified from cultured promastigotes by a combination of detergent phase separation and monoclonal antibody affinity chromatography. Intraperitoneal vaccination of C3H/He mice with PSA-2 with Corynebacterium parvum as an adjuvant resulted in complete protection from lesion development after challenge infection with virulent L. major. Significant protection was also obtained in the genetically susceptible BALB/cH-2k and BALB/c mice. One of the PSA-2 genes was cloned and expressed in both Escherichia coli and Leishmania mexicana promastigotes. Vaccination with the recombinant PSA-2 purified from E. coli did not confer protection, in contrast to the L. mexicana-derived recombinant PSA-2, which provided excellent protection. CD4+ T cells isolated from the spleens of vaccinated mice produced large amounts of gamma interferon but no detectable interleukin 4 upon stimulation with PSA-2 in vitro. Limiting dilution analysis showed a marked increase in the precursor frequency of PSA-2-specific gamma interferon-secreting CD4+ T cells. No substantial change in precursor frequency was observed for interleukin 4-secreting T cells in the vaccinated mice. A CD4+ PSA-2 specific T-cell line generated from splenocytes of a vaccinated mouse produces a cytokine pattern consistent with a TH1 phenotype. Intravenous injection of this line into naive mice reduced significantly the parasite burden upon challenge infection. Taken together, the data suggest that vaccination with PSA-2 induces a TH1 type of immune response which protects mice from L. major infection. Moreover, a single recombinant PSA-2 polypeptide derived from a genomic clone can also vaccinate, provided that the structural form of the antigen is near native.
A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli
Authors: Leão S C , Rocha C L , Murillo L A , Parra C A , Patarroyo M E
Published: January 2020

Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans.
Distinct antigenic and genetic properties of the immunoglobulin A1 protease produced by Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever in Brazil
Authors: Lomholt H , Kilian M
Published: January 2020

All examined Haemophilus influenzae biogroup aegyptius isolates of the clone associated with Brazilian purpuric fever (the BPF clone) produced type 2 immunoglobulin A1 (IgA1) proteases encoded by identical iga genes that were distinct from the iga genes of other Brazilian H. influenzae biogroup aegyptius isolates. A partial nucleotide sequence analysis revealed close similarities to the iga genes of H. influenzae serotype c and one noncapsular H. influenzae biotype III strain isolated from a case of conjunctivitis in Tunisia, suggesting an evolutionary relationship. Epitopes recognized by neutralizing antibodies differed for the IgA1 proteases of the BPF clone and of other H. influenzae strains, including Brazilian H. influenzae biogroup aegyptius isolates from patients with noninvasive conjunctivitis. The low probability of developing cross-reacting neutralizing antibodies to the IgA1 protease of the BPF clone may contribute to the pathogenic potential of this virulent phenotype in Brazil.
Overexpression of the T-cell receptor V beta 3 in transgenic mice increases mortality during infection by enterotoxin A-producing Staphylococcus aureus
Authors: Zhao Y X , Abdelnour A , Kalland T , Tarkowski A
Published: January 2020

We have previously demonstrated that staphylococcal enterotoxins contribute to arthritis and mortality during staphylococcal infection. To further explore the mechanism by which bacterial superantigens contribute to the pathogenesis of Staphylococcus aureus septicemia, T-cell receptor V beta 3 transgenic (TGV beta 3) mice and nontransgenic (non-TG) littermates were inoculated intravenously with S. aureus AB-1, which produces large amounts of staphylococcal enterotoxin A, which specifically reacts with T-cell receptor V beta 3. Within 9 days after inoculation, 85% of the TGV beta mice died, compared with 31% of their non-TG littermates (P < 0.01). The high mortality of TGV beta 3 mice was accompanied by elevated bacterial burdens in the blood, spleen, and kidneys. The in vivo kinetics of cytokine mRNA expression was studied by an in situ hybridization technique. Staphylococcal infection gave rise to increased expression of interleukin 1 beta (IL-1 beta) mRNA and sparsely expressed tumor necrosis factor alpha (TNF-alpha), IL-4, and IL-10 mRNAs in both groups. Gamma interferon mRNA expression increased on day 3 and was maintained at a detectable level in the late phase of infection in TGV beta 3 mice, in contrast to non-TG mice. Impressively, significantly higher expression of TNF-beta mRNA in TGV beta 3 mice was noted throughout the course of infection than in non-TG littermates. These findings suggest that overproduction of TNF-beta and gamma interferon, the Th1 cytokines, may play a crucial role in the pathogenesis of septicemia caused by enterotoxin-secreting staphylococci.
Human and rat macrophages mediate fungistatic activity against Rhizopus species differently: in vitro and ex vivo studies
Authors: Jorens P G , Boelaert J R , Halloy V , Zamora R , Schneider Y J , Herman A G
Published: January 2020

Both rat alveolar macrophages and a human macrophages cell line with characteristics of human tissue (e.g., alveolar) macrophages (THP-1) were found to inhibit the germination of Rhizopus spores. However, the conditions under which fungistatic activity occurs are different for these two cell types. The inhibition of Rhizopus spore germination by rat alveolar macrophages requires the activation of macrophages and the presence of serum and L-arginine. During rat alveolar macrophage-mediated fungistatic activity, L-arginine is oxidized to nitric. Human macrophage-mediated fungistatic activity is similar to that mediated by rat macrophages in terms of the serum requirement, but it does not require L-arginine. Human macrophages did not produce any nitrite detectable by the colorimetric assay. Their ability to inhibit germination was enhanced by the combination of endotoxin and gamma interferon. The inhibition of Rhizopus spore germination by rat alveolar macrophages is thus mediated by the generation of nitric oxide, whereas the mechanism of similar inhibition by human macrophages remains poorly understood. Serum samples from diabetic rats as well as from patients with diabetes or uremia decreased the inhibitory effect of macrophages on spore germination. Dialysis of the serum samples against a buffered salt solution antagonized this phenomenon, indicating that a low-molecular-weight factor in the sera of patients with diabetes or uremia may modulate local antifungal defense mechanisms. The absence of L-arginine-dependent nitrogen oxidation in human macrophages, compared with its presence in rat alveolar macrophages, under conditions during which fungistatic activity occurs suggests that this phenomenon is species specific.
Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells
Authors: Batterman H J , Peek J A , Loutit J S , Falkow S , Tompkins L S
Published: January 2020

Bartonella henselae expresses pili phenotypically similar to type 4 pili. B. henselae pilus expression undergoes phase variation with multiple passages. Low-passage-number, piliated B. henselae adhered to and invaded HEp-2 cells to a greater extent than did multiply passaged B. henselae with reduced pilus expression. Pili may be a pathogenic determinant for Bartonella species.
Influence of site specifically altered Mip proteins on intracellular survival of Legionella pneumophila in eukaryotic cells
Authors: Wintermeyer E , Ludwig B , Steinert M , Schmidt B , Fischer G , Hacker J
Published: January 2020

Legionella pneumophila, the causative agent of Legionnaires' disease, is able to survive intracellularly in eukaryotic cells such as monocytes, macrophages, and protozoan organisms. The Mip (macrophage infectivity potentiator) protein represents a factor of L. pneumophila necessary for optimal intracellular survival. Interestingly, Mip belongs to the substance class of FK 506-binding proteins and exhibits peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506. In order to identify amino acids most likely to be involved in the enzymatic activity of Mip, site-directed mutagenized Mip proteins were constructed and characterized. It was shown that an Asp-142 to Leu-142 mutation and a Tyr-185 to Ala-185 substitution resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% of the activity of the wild-type Mip, respectively). Genes coding for the wild-type and for site-directed-mutagenized Mip proteins were used to complement three different Mip-negative mutants of the L. pneumophila Corby, Philadelphia I, and Wadsworth. While Mip protein expression could be restored in the corresponding complementants, significant Mip-specific PPIase activity could be detected only in Mip mutants complemented with wild-type mip genes. To investigate the influence of the PPIase activity of Mip on intracellular survival of L. pneumophila, invasion assays were performed using the macrophage-like cell line U937, human blood monocytes, and Acanthamoeba castellanii. The Mip-negative mutants were approximately 50- to 100-fold less infective for A. castellanii and for human mononuclear phagocytes in vitro compared with their isogenic Mip-positive parental strains. The wild-type invasion rate could be restored by introducing an intact copy of the mip gene into Mip-negative strains. In addition, no differences in intracellular survival were observed between the wild-type isolates and the Legionella strains exhibiting strongly reduced PPIase activity. These data indicated that the enzymatic activity of Mip does not contribute to intracellular survival of L. pneumophila.
An antigenic peptide inducing cross-reacting antibodies inhibiting the interaction of Streptococcus mutans PAc with human salivary components
Authors: Senpuku H , Miyauchi T , Hanada N , Nisizawa T
Published: January 2020

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans, in particular the A region of this molecule, may be implicated in the induction of dental caries via an interaction with salivary components. For this reason, it was probably used successfully as an antigenic component for experimental vaccination to prevent dental caries in animals. While developing a synthetic peptide vaccine for dental caries, as reported herein, we have identified a unique peptide, TYEAALKQYEADL, as a candidate vaccinal immunogen. The amino acid sequence of this peptide completely corresponds to the sequence of a B-cell epitope in the A region of PAc and additionally contains its own T-cell epitope for B10.D2 mice within the molecule. This peptide strongly induces the production of only cross-reacting antibodies against PAc. In addition, as demonstrated by surface plasmon resonance analysis using the BIAcore system, these cross-reacting antibodies inhibit approximately 50% of the binding of fluid-phase salivary components to immobilized recombinant PAc.
Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells
Authors: van Schilfgaarde M , van Alphen L , Eijk P , Everts V , Dankert J
Published: January 2020

Haemophilus influenzae penetrates the respiratory epithelium during carriage and invasive disease, including respiratory tract infections. We developed an in vitro model system consisting of lung epithelial NCI-H292 cells on permeable supports to study the passage of H. influenzae through lung epithelial cell layers. The NCI-H292 cells formed tight layers with a Ca(2+)-dependent transepithelial resistance of around 40 omega.cm2. H. influenzae passed through the cell layers without affecting the viability of the cells and [3H]inulin penetration. The passage time was independent of the inoculum of H. influenzae in the apical compartment and was not influenced by the presence of capsule or fimbriae on H. influenzae or by the ability of the bacteria to adhere to the epithelial cells. However, highly adherent strains showed greater paracytosis. Different strains passed through the cell layer independently. The passage time was shorter for rapidly growing strains than for slowly growing strains (10 to 18 h and 30 h, respectively). Microscopic examination revealed the presence of clusters of H. influenzae bacteria between the epithelial cells, indicating that bacterial passage was due to paracytosis. After the addition of chloramphenicol, no bacteria were cultured from the basolateral side, and no bacterial clusters between the epithelial cells were seen, suggesting that de novo bacterial protein synthesis was needed for the bacteria to reach the intercellular space. We conclude that H. influenzae passes through viable cell layers of the human lung epithelial cell line NCI-H292 by paracytosis, requiring bacterial protein synthesis.
An immunodominant 90-kilodalton Aspergillus fumigatus antigen is the subunit of a catalase
Authors: López-Medrano R , Ovejero M C , Calera J A , Puente P , Leal F
Published: January 2020

We have identified, purified, and characterized structurally and functionally a 90-kDa immunodominant antigen associated with the water-soluble fraction of Aspergillus fumigatus. This antigen is recognized by 90.3% of serum samples from patients with aspergilloma and should be considered either by itself or better in combination with other purified antigens as a candidate for developing a standardized immunoassay for the detection of aspergilloma. p90 is a glycoprotein containing at least two two N-linked sugar chains of 2 and 5 kDa, respectively, which are not necessary for its reactivity with aspergilloma serum samples. Using specific anti-p90 rabbit serum, we have demonstrated that under native conditions, p90 exists in oligomeric form and has associated catalase activity. This activity is resistant to extreme temperatures (> 60 degrees C), reducing agents (40 mM dithiothreitol), high concentrations of denaturing agents such as 8 M urea and 8% sodium dodecyl sulfate, and treatments with ethanol-chloroform-water (5:3:10 [vol/vol]) mixtures.
Gonococcal infection in a nonhuman host is determined by human complement C1q
Authors: Nowicki S , Martens M G , Nowicki B J
Published: January 2020

Human C1q displayed a dose-dependent protection of gonococcal cells (GC) from the bactericidal effect of newborn rat serum. All rat pups injected with C1q-preincubated GC developed bacteremia, while none of the animals injected with GC only were infected. After clearance of bacteremia at day 6, live GC could still be recovered from tested organs, including the liver. Preincubation of GC with higher concentrations of C1q was associated with increased morbidity. In contrast to human serum as a source of C1q, rat, rabbit, and mouse sera did not increase the in vivo virulence of Neisseria gonorrhoeae. C1q-deficient human serum, heat-inactivated C1q or human serum, type IV collagen, and complement C3 were inefficient in inducing infection. Experimental infection by C1q-preincubated GC was inhibited by anti-C1q antibodies in a dose-dependent fashion, demonstrating a causal effect of C1q function. This report demonstrates the novel finding that human C1q, a component of the human immune system with a general function for elimination of infection, may increase GC virulence and result in the development of disseminated infection in a nonhuman host.
Variation in antigenicity and infectivity of derivatives of Borrelia burgdorferi, strain B31, maintained in the natural, zoonotic cycle compared with maintenance in culture
Authors: Golde W T , Dolan M C
Published: January 2020

The original isolate of Borrelia burgdorferi, strain B31, can be maintained in vitro indefinitely. A number of studies have demonstrated that there are recognizable changes in the genetic composition of the spirochete after more than 60 passages. We have maintained B31 in the natural zoonotic cycle of transmission of infection between laboratory mice and laboratory-reared Ixodes ticks. To determine whether similar changes occur in the natural transmission cycle, we reisolated strain B31 from mouse skin at the fifth zoonotic cycle. This reisolated derivative had the same infectivity as the parent B31 strain, had lost the 8-kb supercoiled plasmid present in B31, and induced a gross serum antibody response indistinguishable from the B31 immune response. Analysis of antigen expression with monoclonal antibodies generated against B31, however, showed differential expression of a subset of antigens between B31 and the isolated derivative.
Antibody-independent binding of complement component C1q by Legionella pneumophila
Authors: Mintz C S , Arnold P I , Johnson W , Schultz D R
Published: January 2020

Incubation of Legionella pneumophila Philadelphia 1 in normal human serum depleted of either classical-pathway component C1q or alternative-pathway component factor B resulted in activation of the complement system. Experiments focused on the role of the classical pathway in complement activation revealed that legionellae bound C1q independently of antibody. Purified preparations of L. pneumophila major outer membrane protein but not serogroup 1 lipopolysaccharide bound C1q independently of antibody. This suggests that antibody-independent binding of C1q by L. pneumophila can result in activation of the classical pathway in normal human serum and that major outer membrane protein may be a C1q acceptor on the L. pneumophila cell surface.
Sodium deoxycholate facilitates systemic absorption of verotoxin 2e from pig intestine
Authors: Waddell T E , Gyles C L
Published: January 2020

Injection of verotoxin 2e together with sodium deoxycholate, which increases intestinal permeability to macromolecules, into the intestine of pigs resulted in fluid accumulation, intestinal damage, and signs and lesions of edema disease. Intragastric administration of verotoxin 2e to newborn piglets, who normally absorb protein nonspecifically, resulted in systemic verotoxemia. These results suggest that development of natural edema disease requires a state of increased intestinal permeability.
Adherence to and invasion of tissue culture cells by Vibrio hollisae
Authors: Miliotis M D , Tall B D , Gray R T
Published: January 2020

The adherence to and invasion of cultured epithelial cells by Vibrio hollisae were examined by quantitative studies and by light, fluorescent, and electron microscopy. Condensed actin was observed around clustered adherent and intracellular bacteria. Bacteria multiplied intracellularly. Inhibitor studies indicated that internalization occurred by an integrated pleiotropic process involving eukaryotic and prokaryotic protein syntheses, microfilaments, microtubules, and receptor-mediated endocytosis.
Binding of type 1-piliated Escherichia coli to vaginal mucus
Authors: Venegas M F , Navas E L , Gaffney R A , Duncan J L , Anderson B E , Schaeffer A J
Published: January 2020

To better understand the interactions involved in bacterial adherence and the role of mucus in the pathogenesis of urinary tract infections, we developed a system to study the binding of a recombinant Escherichia coli strain, HB101/pWRS1-17, expressing type 1 pili, to vaginal mucus collected from 28 women. Bacteria bound to differing extents to all specimens examined, and preincubation of bacteria with mannose inhibited binding by 50 to 89%. Additionally, all mucus samples showed reactivity with anti-mannose antibody, and the levels of reactivity correlated with the levels of bacterial binding, suggesting that the mannose-terminal saccharides present on these glycoproteins are the receptors for the binding of type 1-piliated bacteria. Mucus specimens collected over periods of 5 days and 12 weeks exhibited significant variation in bacterial binding, indicating temporal differences in the ability of vaginal mucus to act as a receptor for type 1-piliated E. coli. The results show that vaginal mucus can bind bacteria and may thus influence the initial attachment and subsequent colonization of the vaginal and urinary tract epithelium by E. coli.
Acetylation (O-factor 5) affects the structural and immunological properties of Salmonella typhimurium lipopolysaccharide O antigen
Authors: Slauch J M , Mahan M J , Michetti P , Neutra M R , Mekalanos J J
Published: January 2020

The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment. The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism. Antibodies directed against O antigen are vital to the immune response to infection. In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium. Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S. typhimurium infection, both in vivo and in vitro. Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope. Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes. This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants. We have shown that a change in the acetylation state of LPS has no effect on virulence. However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.
Induction of opsonizing antibodies after injection of recombinant Plasmodium falciparum vaccine candidate antigens in preimmune Saimiri sciureus monkeys
Authors: Perraut R , Mercereau-Puijalon O , Mattei D , Bourreau E , Garraud O , Bonnemains B , Pereia de Silva L , Michel J C
Published: January 2020

We have previously shown that Plasmodium falciparum recombinant antigens PfEB200, R23, and Pfi72 inhibit opsonization of infected erythrocytes by hyperimmune Saimiri sera, indicating that they contain target epitopes involved in the phagocytosis of infected erythrocytes. We have investigated in this study the immune response of Saimiri monkeys with previous experience of malaria infections (preimmune monkeys) after injection of these recombinant antigens, administered alone or simultaneously. The humoral response to the recombinant antigens was monitored by radioimmunoassay, and the response to P. falciparum blood stages was assayed by immunofluorescence. The relative proportion of protective versus nonprotective immunoglobulin subtypes was investigated by using 3A2/G6 and 3E4/H8 monoclonal antibodies, and the capacity of the antisera to promote in vitro phagocytosis of infected erythrocytes was evaluated. The antigens evoked in most cases a secondary-type antibody response, resulting in important increases in antigen-specific antibody titers and concomitantly in anti-P. falciparum titers. The ratio of 3A2/G6 to 3E4/H8 immunoglobulin subtypes varied with the immunogen used. Opsonizing antibodies were boosted in several animals, the most promising combination being the mixture of PfEB200 and R23 that induced long-lasting production in five of five animals. The detectable opsonizing activity appearing after immunization of the animals was antigen specific, as it was lost after adsorption of the recombinant antigens. The challenge of the animals with blood stage parasites confirmed previous findings showing a correlation between the presence of detectable opsonizing antibodies in serum and protection.
Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague
Authors: Oyston P C , Williamson E D , Leary S E , Eley S M , Griffin K F , Titball R W
Published: January 2020

An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S. typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence. The recombinant was used to vaccinate mice intragastrically and intravenously. The immunity induced was able to protect mice against challenge with a virulent strain of plague. Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.
High tumor necrosis factor alpha (TNF-alpha) production in Trypanosoma cruzi-infected pregnant mice and increased TNF-alpha gene transcription in their offspring
Authors: Rivera M T , Marques de Araujo S , Lucas R , Deman J , Truyens C , Defresne M P , de Baetselier P , Carlier Y
Published: January 2020

Since tumor necrosis factor alpha (TNF-alpha) is known to be involved in the feto-maternal relationship, this cytokine was studied in Trypanosoma cruzi-infected pregnant BALB/c mice and their fetuses and offspring. Pregnant chronically infected mice displayed significantly higher levels of circulating TNF-alpha than animals either only infected or only pregnant. TNF-alpha was undetectable in sera of uninfected and nonpregnant mice as well as in breast milk obtained from infected and uninfected animals. Fetuses from infected mice exhibited significantly more cells containing TNF-alpha mRNA in their thymus than fetuses from uninfected mothers. When infected 2 months after birth, offspring born to infected and uninfected mothers displayed similar amounts of circulating TNF-alpha during chronic infection, whereas this cytokine was only weakly detectable during the acute phase of the disease. An intravenous injection of lipopolysaccharide during acute infection strongly increased the production of TNF-alpha in offspring born to infected mothers to levels higher than those in progeny from uninfected mice. These results suggest that TNF-alpha is an important cytokine in the feto-maternal relationship during T. cruzi infection and that fetuses and offspring of infected mothers are primed to produce elevated levels of TNF-alpha.
Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci
Authors: Lowe A M , Lambert P A , Smith A W
Published: January 2020

Serum from a patient with Enterococcus faecalis endocarditis was used to identify the gene efaA cloned in Lambda ZapII in Escherichia coli. Nucleotide sequence analysis revealed a 924-bp open reading frame encoding a protein with a predicted molecular weight of 34,768. The amino acid sequence of EfaA shows 55 to 60% homology to a group of streptococcal proteins, FimA from Streptococcus parasanguis, SsaB from Streptococcus sanguis, ScaA from Streptococcus gordonii, and PsaA from Streptococcus pneumoniae. Members of this group have been shown to be adhesins, and we hypothesize that EfaA may function as an adhesin in endocarditis.
Role of a carboxy-terminal site of toxic shock syndrome toxin 1 in eliciting immune responses of human peripheral blood mononuclear cells
Authors: Drynda A , König B , Bonventre P F , König W
Published: January 2020

Staphylococcus aureus toxic shock syndrome toxin 1 (TSST-1) is involved in the pathogenesis of toxic shock syndrome and perhaps other staphylococcal diseases. Recently, the C-terminal part of the TSST-1 toxin has been shown to be responsible for mitogenic activity in animal models. We studied the role of the C-terminal structural unit of TSST-1 with regard to proliferation, cytokine release (tumor necrosis factor alpha [TNF-alpha], interleukin-6 [IL-6], and IL-8), mRNA expression for IL-6, IL-8, IL-10, TNF-alpha, and CD40 ligand (CD40L), synthesis of immunoglobulin E (IgE), IgA, IgG, and IgM, CD23 expression, and soluble CD23 (sCD23) release from human peripheral blood mononuclear cells (PBMC). For this purpose, we used the recombinant wild-type TSST-1 (p17) mutant toxin Y115A (tyrosine residue modified to alanine) and toxin H135A (histidine residue modified to alanine). Unmodified toxin p17 and mutant toxin Y115A, at a concentration below 5 ng, to a lesser degree, induced a strong proliferation. Toxin p17 followed by toxin Y115A was the most pronounced inducer for mRNA expression for IL-10 and CD40L and cytokine generation (mRNA and protein) for TNF-alpha, IL-6, and IL-8. Mutant protein H135A failed to activate human PBMC. Both toxins p17 and, to a lesser degree, Y115A significantly suppressed IL-4- and anti-CD40-induced synthesis of all four Igs as well as IL-4-induced CD23 expression and sCD23 release. Mutant toxin H135A failed to do so. Thus, our data show that a region in the C terminus of TSST-1 is responsible not only for mitogenic activity but also for additional immunomodulating biological activities of TSST-1. More specifically, histidine residue H135A of the 194-amino-acid toxin appears to be critical for the expression of biological activities in a human in vitro model.
Helicobacter-associated gastritis in SCID mice
Authors: Blanchard T G , Czinn S J , Nedrud J G , Redline R W
Published: January 2020

Immunodeficient (SCID) and immunocompetent mice were infected with Helicobacter felis to address the role of autoimmunity in Helicobacter-associated gastritis. The extents of inflammation were equivalent in the two groups. The numbers of H. felis organisms were marginally increased in the SCID mice but did not achieve statistical significance. These results indicate that autoimmunity is not necessary to induce disease and that the presence of an adaptive immune system does not significantly affect H. felis colonization.
Poliovirus hybrids expressing neutralization epitopes from variable domains I and IV of the major outer membrane protein of Chlamydia trachomatis elicit broadly cross-reactive C. trachomatis-neutralizing antibodies
Authors: Murdin A D , Su H , Klein M H , Caldwell H D
Published: January 2020

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes from the variable domains of the major outer membrane protein are candidates for vaccine development. We have constructed hybrid polioviruses expressing sequences from major outer membrane protein variable domains I and IV. Antisera to the hybrids could, in combination, strongly neutralize 8 of the 12 C. trachomatis serovars most commonly associated with oculogenital infections and weakly neutralize the others.
Expression of a nonagglutinating fimbria by Proteus mirabilis
Authors: Tolson D L , Barrigar D L , McLean R J , Altman E
Published: January 2020

We have clarified growth conditions and isolation strategies for the nonagglutinating fimbriae from Proteus mirabilis. Nonagglutinating fimbriae were expressed by all P. mirabilis strains we examined, and the major subunit proteins, which ranged from 23 to 29 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had highly conserved N-terminal sequences.
Roles of nitric oxide in inducible resistance of Escherichia coli to activated murine macrophages
Authors: Nunoshiba T , DeRojas-Walker T , Tannenbaum S R , Demple B
Published: January 2020

Nitric oxide (NO.) is produced as a cytotoxic free radical through enzymatic oxidation of L-arginine in activated macrophages. Pure NO. gas was previously found to induce the Escherichia coli soxRS oxidative stress regulon, which is readily monitored by using a soxS'::lac fusion. The soxRS system includes antioxidant defenses, such as a superoxide dismutase and a DNA repair enzyme for oxidative damage, and protects E. coli from the cytotoxicity of NO.-generating macrophages. Previous experiments involved exposing E. coli to a bolus of NO. rather than the steadily generated gas expected of activated macrophages. We show here detectable induction of soxS transcription by NO. delivered at rates as low as 25 microM/h. Maximal induction was observed at 25 microM NO. per h under anaerobic conditions but at 125 microM/h aerobically. After incubation with murine macrophages, soxS expression was induced in the phagocytosed bacteria up to approximately 30-fold after an 8-h exposure. This in vivo induction was almost completely eliminated by the NO. synthase inhibitor NG-monomethyl-L-arginine. The inhibitor increased the survival of a delta soxRS strain but not that of wild-type E. coli after phagocytosis, which suggests that induction of the soxRS regulon by NO. can counteract most of the cytotoxic effects of NO. production by the macrophages. We show that the soxRS-regulated enzyme glucose-6-phosphate dehydrogenase is an important element of the defense against macrophages.
A 37-kilodalton glycoprotein of Babesia divergens is a major component of a protective fraction containing low-molecular-mass culture-derived exoantigens
Authors: Carcy B , Precigout E , Valentin A , Gorenflot A , Schrevel J
Published: January 2020

The supernatants of in vitro cultures of Babesia divergens Rouen 1987 in human erythrocytes, obtained by using a semidefined medium based on human high-density lipoproteins, were fractionated by gel filtration chromatography into four fractions, F1 to F4. The crude supernatant as well as each fraction adjuvanted with Quil-A protected gerbils from mortality due to a homologous infectious challenge. Analysis of the humoral response of the 10 protected gerbils with fraction F4, containing major proteins with molecular masses lower than 50 kDa, showed that a few antigens (from 50 to 17 kDa) could be important candidates for an improved vaccine against B. divergens babesiosis. As an immunodominant response was directed against the 37-kDa antigen (Bd37) in two different B. divergens strains tested, a polyclonal antibody directed against Bd37 was produced in a rabbit. In an immunofluorescence assay, the anti-Bd37 antiserum strongly labelled small internal vesicles of the merozoites and the cell surface was diffusely labelled after fixation, whereas on live merozoites, this labelling was not observed. [3H]glucosamine-radiolabelling experiments demonstrate that Bd37 is a glycoprotein. The Bd37 protein can also be labelled with [14C]palmitate but not with [3H]myristic acid. In Triton X-114 temperature phase partitioning of B. divergens-infected erythrocyte extracts, Bd37 was exclusively found into the detergent phase, indicating that the palmitoylated Bd37 protein was in the membrane fraction. In the in vitro supernatant, the glycoprotein Bd37 was found in a nonpalmitoylated form, indicating excretion and/or release of the glycoprotein from the merozoite.
Lipopolysaccharide regulation of lipoprotein lipase expression in murine macrophages
Authors: Hill M R , Kelly K , Wu X , Wanker F , Bass H , Morgan C , Wang C , Gimble J M
Published: January 2020

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.
Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae
Authors: Hermans P W , Abebe F , Kuteyi V I , Kolk A H , Thole J E , Harboe M
Published: January 2020

Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens. The subsequent use of specific antibodies allowed further determination of antigens by molecular weight. The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis. To characterize Ag84, we screened a lambda gt11 gene library from M. tuberculosis with antibody F126-2 and identified the encoding gene. The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M. tuberculosis gene as a probe. Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system. The derived amino acid sequences of the M. tuberculosis and M. leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens. Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.
Differential susceptibility of yeast and hyphal forms of Candida albicans to proteolytic activity of macrophages
Authors: Blasi E , Pitzurra L , Chimienti A R , Mazzolla R , Puliti M , Barluzzi R , Bistoni F
Published: January 2020

The dimorphic transition of Candida albicans from the yeast (Y-Candida) to the hyphal (H-Candida) form is a complex event whose relevance in fungal pathogenicity is still poorly understood. Using a cloned macrophage (M phi) cell line (ANA-1), we have previously shown that a M phi can discriminate between the two fungal forms, eliciting different secretory responses. In the present study, we investigated the susceptibility of Y-Candida and H-Candida to M phi proteolytic activity. In particular, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) techniques were employed to analyze the patterns of lyticase proteinaceous extracts from cell walls of Y-Candida and H-Candida which had been unexposed or exposed to ANA-1 M phis for 3 h. Silver staining allowed detection of a complex protein pattern in both forms of C. albicans, qualitatively and quantitatively differing from each other, mainly at molecular masses below 106 kDa. Western blot staining with anti-C. albicans mannan antibodies and convalescent-phase sera of mice previously infected systemically or intracerebrally with C. albicans showed that, after contact with M phis, Y-Candida but not H-Candida proteinaceous cell wall components are profoundly modified, with substantial reduction and/or disappearance of many bands. Our experimental approach provides initial insights into the differential susceptibility of Y-Candida and H-Candida to the proteolytic activity of M phis.
Human CAP18: a novel antimicrobial lipopolysaccharide-binding protein
Authors: Larrick J W , Hirata M , Balint R F , Lee J , Zhong J , Wright S C
Published: January 2020

CAP18 (18-kDa cationic antimicrobial protein) is a protein originally identified and purified from rabbit leukocytes on the basis of its capacity to bind and inhibit various activities of lipopolysaccharide (LPS). Here we report the cloning of human CAP18 and characterize the anti-LPS activity of the C-terminal fragment. Oligonucleotide probes designed from the rabbit CAP18 cDNA were used to identify human CAP18 from a bone marrow cDNA library. The cDNA encodes a protein composed of a 30-amino-acid signal peptide, a 103-amino-acid N-terminal domain of unknown function, and a C-terminal domain of 37 amino acids homologous to the LPS-binding antimicrobial domain of rabbit CAP18, designated CAP18(104-140). A human CAP18-specific antiserum was generated by using CAP18 expressed as a fusion protein with the maltose-binding protein. Western blots (immunoblots) with this antiserum showed specific expression of human CAP18 in granulocytes. Synthetic human CAP18(104-140) and a more active truncated fragment, CAP18(104-135), were shown to (i) bind to erythrocytes coated with diverse strains of LPS, (ii) inhibit LPS-induced release of nitric oxide from macrophages, (iii) inhibit LPS-induced generation of tissue factor, and (iv) protect mice from LPS lethality. CAP18(104-140) may have therapeutic utility for conditions associated with elevated concentrations of LPS.
Capsular polysaccharide types 5 and 8 of Staphylococcus aureus bind specifically to human epithelial (KB) cells, endothelial cells, and monocytes and induce release of cytokines
Authors: Soell M , Diab M , Haan-Archipoff G , Beretz A , Herbelin C , Poutrel B , Klein J P
Published: January 2020

In order to examine the possible implication of capsular polysaccharide (CP) types 5 and 8 (CP5 and CP8) from Staphylococcus aureus in the pathological mechanism associated with staphylococcal infections, we tested the immunomodulatory effects of CP5 and CP8 on human epithelial KB cells, endothelial cells, and monocytes. Using biotinylated CP5 and CP8, we provide evidence that both CPs bind to KB cells, endothelial cells, and monocytes in a dose- and calcium-dependent manner through specific interactions. These results were confirmed by competition experiments using soluble cell extracts. Furthermore, we show that CPs bind to identical cell membrane receptors on all three types of human cells and that human normal serum contains a factor(s) which inhibits the binding of both CPs to human KB cells, endothelial cells, and monocytes. The ability of CP5 and CP8 to stimulate the production of cytokines by the human cells was then examined. CP5 and CP8 trigger KB cells to produce interleukin-8 (IL-8); endothelial cells to produce IL-8 and IL-6; and monocytes to produce IL-8, IL-6, IL-1 beta, and tumor necrosis factor alpha. The release of cytokines by all three types of cells is time dependent and dose dependent, and the tumor necrosis factor alpha production by monocytes is not affected by the addition of polymyxin B. We further confirm that human normal serum inhibits the immunomodulatory effects of both polysaccharides on each kind of cell. These results confirm that S. aureus CPs act as bacterial adhesins having immunomodulatory effects for human cells.
Deviation of immune response to Chlamydia psittaci outer membrane protein in lipopolysaccharide-hyporesponsive mice
Authors: Westbay T D , Dascher C C , Hsia R C , Zauderer M , Bavoil P M
Published: January 2020

The outcome of infection is determined by both the quantity and the quality of an induced immune response. In particular, it has been demonstrated for selected pathogens that induction of TH1 or TH2 type helper T-cell subsets determines whether an immune response gives rise to protective immunity or disease-associated immunopathology. The nature of the antigen and the type of antigen-presenting cells recruited in the induction of a response are critical factors that influence the quality of the immune response. Of particular interest in this respect is the immune response to bacterial particles and the impact of cell wall-associated lipopolysaccharide (LPS) on that response. Nonspecific activation of macrophages and B lymphocytes by LPS could skew the phenotype of activated antigen-presenting cells and selectively alter the immunoglobulin isotypes and helper T-cell subsets that are induced following infection. In an initial attempt to detect immune deviation associated with LPS stimulation, we have compared the immunoglobulin isotypes of antibodies specific for the cysteine-rich outer membrane protein Omp2 induced in normal and LPS-hyporesponsive mice following immunization with Chlamydia psittaci strain guinea pig inclusion conjunctivitis whole elementary bodies. We report that there is a dramatic shift of Omp2-specific antibody from predominantly immunoglobulin G2a (IgG2a) isotype in LPS-hyporesponsive mice to high levels of IgG1 isotype in LPS-responder strains. The dependence of the IgG1 isotype shift on the LPS responder status is linked to the structure of the antigen and its natural processing pathway since LPS-hyporesponsive mice are not, in general, deficient in IgG1 antibody production. In particular, the antibody response to purified recombinant Omp2 is predominantly of the IgG1 isotype even in LPS-hyporesponsive mice.
Protein synthesis in Brucella abortus induced during macrophage infection
Authors: Lin J , Ficht T A
Published: January 2020

Brucella abortus is a facultative, intracellular, pathogenic bacterium that replicates within macrophages and resists macrophage microbicidal mechanisms. To study gene expression and to elucidate the defense mechanisms used by B. abortus to resist destruction within macrophages, protein synthesis by B. abortus was examined by pulse-labeling techniques during intracellular growth within J774A.1, a macrophage-like cell line. Prominent changes observed include increased synthesis of Brucella proteins with estimated molecular masses of 62, 28, 24, and 17 kDa. The 62-kDa protein was identified by immunoprecipitation analysis as Hsp62, a GroEL homolog. A protein of 60 kDa was expressed during acid shock and may represent a modified form of Hsp62. The 28- and 17-kDa proteins have not been observed under any in vitro stress condition and presumably represent macrophage-specific induction. The 24-kDa protein comigrates with an unidentified protein induced by acid shock, designated Asp24. Expression of Asp24 is optimal at pH values below 4.0 and within the first 3 h following a shift from pH 7.3 to 3.8. This corresponds directly with a period of optimal bacterial survival at a reduced pH and suggests an active role for this protein in resistance to such environments. The identification of these gene products and the mechanisms controlling their expression is an important step in understanding the resistance of Brucella spp. to intracellular destruction within macrophages.