bluuebunny/crossref_metadata_2025 huggingface dataset:
Timing of lymphocyte activation in neonates infected with human immunodeficiency virus
Authors: Gallagher K , Gorre M , Harawa N , Dillon M , Wafer D , Stiehm E R , Bryson Y , Song D , Dickover R , Plaeger S
Published: January 2020

Human immunodeficiency virus (HIV) infection in children is associated with qualitative and quantitative changes in the peripheral lymphocyte surface phenotype beyond the normal maturational changes. Neonates, however, have been reported to have a delayed immune response to HIV compared to HIV-infected adults. We prospectively performed immunophenotyping of T lymphocytes by three-color immunofluorescent labeling and laser flow cytometry to determine the timing of phenotypic alterations in 112 neonates born to HIV-infected mothers. Serial testing was performed at birth (cord blood) and at 2, 6, and 12 weeks of age. Data were divided retrospectively for analysis into those for HIV-infected (n = 14) infants and those for exposed, uninfected infants. Our results show that both infected and uninfected infants had a decline in the percentages and numbers of CD4 cells beginning at 2 weeks of age but that the decline was greater in the HIV-infected group. The activation and differentiation of CD8 T cells in HIV+ infants were shown by a significant increase in CD45RA- CD45RO+ CD8+ cells by 6 weeks of age and by increases in CD8+ S6F1+ CD3+ cells and HLA-DR+ CD38+ CD8+ cells by 2 weeks of age. These results indicate that HIV-infected neonates show alterations in T-cell phenotype reflecting those reported for older HIV-infected children. Most importantly, neonatal T cells are able to respond to HIV within the first weeks of life.
Afipia clevelandensis antibodies and cross-reactivity with Brucella spp. and Yersinia enterocolitica O:9
Authors: Drancourt M , Brouqui P , Raoult D
Published: January 2020

Afipia clevelandensis is a recently described gram-negative bacterium whose potential pathogenic role in human disease is under investigation. Only one strain, from the pretibial lesion of a patient hospitalized with necrotizing pancreatitis for 5 months, has been isolated. Using an indirect immunofluorescence assay to detect anti-A. clevelandensis antibodies, we found a seroprevalence of 1.5% among 30,194 sera routinely submitted for laboratory diagnosis of rickettsial diseases. However, among the 52 patients who were clinically evaluable and who exhibited detectable antibodies against A. clevelandensis, 42% were eventually diagnosed as certainly or probably having brucellosis and 15% were eventually diagnosed as certainly or probably having Yersinia enterocolitica O:9 infection, which is the serotype most often encountered in Europe. Western immunoblotting and cross-adsorption tests showed that an 11.5-kDa proteinase K-labile band and a 21-kDa proteinase-stable band, presumably lipopolysaccharide, were responsible for cross-reactivity among A. clevelandensis, Brucella abortus, and Y. enterocolitica O:9. Other diagnoses included nosocomial infections and various community-acquired diseases for which the role of A. clevelandensis remains undefined. Physicians and clinical microbiologists should be aware of this cross-reactivity in future assessments of the role of A. clevelandensis in human pathology.
Salmonella infection increases porcine antibacterial peptide concentrations in serum
Authors: Zhang G , Ross C R , Dritz S S , Nietfeld J C , Blecha F
Published: January 2020

PR-39 is a multifunctional neutrophil peptide involved in host defense and inflammation. To investigate the involvement of PR-39 in a Salmonella choleraesuis infection, a PR-39 enzyme immunoassay was developed. The concentrations of PR-39 in serum were 13.6 +/- 1.9 ng/ml before challenge and increased (P < 0.01) threefold by 10 to 14 days postinfection. Peripheral blood neutrophil counts paralleled the changes in the concentrations of PR-39 in serum, both returned to basal values by 4 weeks postinfection. These findings suggest that the concentrations of serum PR-39 reflect the involvement of this antibacterial peptide in the host's response to an S. choleraesuis infection.
The enterotoxin of Bacteroides fragilis is a metalloprotease
Authors: Moncrief J S , Obiso R , Barroso L A , Kling J J , Wright R L , Van Tassell R L , Lyerly D M , Wilkins T D
Published: January 2020

During the past decade, strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in animals and humans. The extracellular enterotoxin has been purified and characterized as a single polypeptide (M(r), approximately 20,000). Single specific primer-PCR was used to clone a portion of the B. fragilis enterotoxin gene. The recombinant protein expressed by the cloned gene fragment reacted with monospecific antibodies to B. fragilis enterotoxin by enzyme-linked immunosorbent assay and immunoblot analysis. The deduced amino acid sequence revealed a signature zinc-binding consensus motif (HEXXHXXGXXH/Met-turn) characteristic of metalloproteases termed metzincins. Sequence comparisons showed close identity to matrix metalloproteases (e.g., human fibroblast collagenase) within the zinc-binding and Met-turn region. Purified enterotoxin contained 1 g-atom of Zn2+ per molecule and hydrolyzed gelatin, azocoll, actin, tropomyosin, and fibrinogen. The enterotoxin also underwent autodigestion. The N-terminal amino acid sequences of two autodigestion products were identical to the deduced amino acid sequence of the recombinant enterotoxin and revealed cleavage at Cys-Leu and Ser-Leu peptide bonds. Gelatinase (type IV collagenase) activity comigrated with the toxin when analyzed by gel fractionation and zymography, indicating that protease activity is due to the enterotoxin and not to a contaminating protease(s). Optimal proteolytic activity occurred at 37 degrees C and pH 6.5. Primary proteolytic cleavage sites in actin were identified, revealing cleavage at Gly-Met and Thr-Leu peptide bonds. Enzymatic activity was inhibited by metal chelators but not by inhibitors of other classes of proteases. Additionally, cytotoxic activity of the enterotoxin on human carcinoma HT-29 cells was inhibited by acetoxymethyl ester EDTA. The metalloprotease activity of the enterotoxin suggests a possible mechanism for enterotoxicity and may have additional implications in the study of disease caused by B. fragilis.
Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis
Authors: Wilson M J , Brandon M R , Walker J
Published: January 2020

A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40).
Biologic activities of antibodies to the neutral-polysaccharide component of the Pseudomonas aeruginosa lipopolysaccharide are blocked by O side chains and mucoid exopolysaccharide (alginate)
Authors: Hatano K , Goldberg J B , Pier G B
Published: January 2020

Virulent strains of Pseudomonas aeruginosa are either of a nonmucoid, lipopolysaccharide (LPS)-smooth or mucoid, LPS-rough phenotype, and immunity to these different variants is efficiently mediated by antibodies specific to O antigens or mucoid exopolysaccharide (also called alginate), respectively. In addition to O side chains and core polysaccharide components, the LPS of P. aeruginosa also contains neutral-polysaccharide components that express antigenic determinants common to many clinical isolates. We evaluated antibodies specific to neutral polysaccharides for the ability to mediate opsonic killing and protective immunity. Antibodies to these antigens mediated opsonic killing of poorly virulent nonmucoid LPS-rough isolates but not of isogenic strains with either a LPS-smooth or a mucoid phenotype. Antibodies to neutral-polysaccharide antigens also failed to protect neutropenic mice from challenge with modest doses of LPS-smooth P. aeruginosa strains (< 10(3) CFU per mouse), whereas O-antigen-specific antibodies were highly protective. Antibodies to neutral polysaccharides deposited significantly (P = 0.002) more C3 onto LPS-rough strains than did antibodies to O side chains, but this situation was reversed when isogenic LPS-smooth strains were tested. Given that protective immunity against P. aeruginosa must be directed against either nonmucoid LPS-smooth strains or mucoid LPS-rough strains, it appears that antibodies specific to neutral-polysaccharide antigens do not protect against P. aeruginosa infection. Lack of protection is likely due to the ability of both O side chains and mucoid exopolysaccharide (alginate) to interfere with the opsonic killing activity of neutral-polysaccharide-specific antibodies.
Persistence of local cytokine production in shigellosis in acute and convalescent stages
Authors: Raqib R , Lindberg A A , Wretlind B , Bardhan P K , Andersson U , Andersson J
Published: January 2020

Shigella infection is accompanied by an intestinal activation of epithelial cells, T cells, and macrophages within the inflamed colonic mucosa. A prospective study was carried out to elucidate the cytokine pattern in Shigella infection linked to development of immunity and eradication of bacteria from the local site and also to correlate the cytokine profile with histological severity. An indirect immunohistochemical technique was used to determine the production and localization of various cytokines at the single-cell level in cryopreserved rectal biopsies from 24 patients with either Shigella dysenteriae type 1 (n = 18) or Shigella flexneri (n = 6) infection. The histopathological profile included presence of chronic inflammatory cells with or without neutrophils and microulcers in the lamina propria, crypt distortion, branching, and less frequently crypt abscesses. Patients had significantly higher (P < 0.005) numbers of cytokine producing cells for all of the cytokines studied, interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-1ra, tumor necrosis factor alpha (TNF-alpha), IL-6, IL-8, IL-4, IL-10, gamma interferon, TNF-beta, and transforming growth factor beta 1-3, in the biopsies than the healthy controls (n = 13). The cytokine production profile during the study period was dominated by IL-1 beta, transforming growth factor beta 1-3, IL-4, and IL-10. Significantly increased frequencies of cytokine-producing cells (P < 0.05) were observed for IL-1, IL-6, gamma interferon, and TNF-alpha in biopsies with severe inflammation in comparison with those with mild inflammation. During the acute stage of the disease, 20 of 24 patients exhibited acute inflammation in the rectal biopsies and the cellular infiltration was still extensive 30 days after the onset of diarrhea, although the disease was clinically resolved. In accordance with the histological findings, cytokine production was also upregulated during the convalescent phase; there was no significant difference (P > 0.05) in the incidence of cytokine-producing cells between acute (2 to 8 days after the onset of diarrhea) and convalescent (30 days after onset) stages.
Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF
Authors: Rawling E G , Martin N L , Hancock R E
Published: January 2020

The Pseudomonas aeruginosa major outer membrane protein OprF has been proposed for use as a vaccine and as a target for immunotherapeutic and diagnostic monoclonal antibodies. The well-conserved epitopes for 10 surface-reactive, OprF-specific monoclonal antibodies were localized by both overlapping peptide analysis and immunodetection of OprF peptides generated by cyanogen bromide and the protease papain. Three of the monoclonal antibodies bound to specific overlapping octapeptides, which had been synthesized on 160 pins to cover the entire 326 amino acids of OprF. The highest reactivities were as follows: MA7-1 to the pin with attached peptide GTYETGNK (amino acids 55 to 62), MA7-2 to NLADFMKQ (amino acids 237 to 244), and MA5-8 to TAEGRAIN (amino acids 307 to 314). The other monoclonal antibodies showed no reactivity, indicating that they do not recognize linear epitopes. Two polyclonal sera were also tested and demonstrated weak reactivity with discrete regions of OprF, suggesting that the majority of antibodies produced might recognize conformational epitopes. Utilizing defined peptides generated with cyanogen bromide and papain, the conformational epitopes recognized by the seven monoclonal antibodies were localized to regions that were 42 to 90 amino acids long. These regions were located on two adjacent loops in the middle of an amended structural model of OprF.
CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro
Authors: Yang Z , Carter C D , Miller M S , Bochsler P N
Published: January 2020

The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).
Analysis of immunization route-related variation in the immune response to heat-killed Salmonella typhimurium in mice
Authors: Thatte J , Rath S , Bal V
Published: January 2020

In examinations of the factors regulating the quality and quantity of the immune response to Salmonella typhimurium, we have shown previously that live and heat-killed preparations of S. typhimurium can induce gamma interferon-dominant and interleukin-4-dominant immune responses, respectively, upon intraperitoneal (i.p.) immunization of BALB/c mice. Using this system to investigate the role of the route of immunization in the immune response, we show in the present study that i.p. immunization with heat-killed S. typhimurium generates a quantitatively better immune response than does intradermal (i.d.) immunization. The quantitative differences observed between the i.p. and i.d. routes are apparent in the amount of S. typhimurium-specific antibodies produced, the extent of responses in T-cell proliferation assays, and the quantities of lymphokines generated. However, the ratios of immunoglobulin (Ig) isotypes [IgG1/IgG2a] are comparable and the relative dominance of interleukin-4 over gamma interferon is seen in both i.p.- and i.d.-immunized mice, suggesting that the predominant T-cell effector pathways triggered are not qualitatively dependent on the route of immunization. An examination of the antigenic profile recognised by the B-cell and T-cell responses in i.p.- versus i.d.-immunized mice shows that while the Western immunoblot patterns recognized by serum antibodies from the two groups of mice were not significantly different, T cells from i.p.-immunized mice recognized a broader spectrum of antigens in an immunoblot assay than did those from i.d.-immunized mice. These data suggest that there may be a significant difference in the antigen-processing ability of peritoneal and dermal antigen-presenting cells for complex antigenic formulations such as bacterial vaccines.
Relationship between virulence of Mycobacterium avium strains and induction of tumor necrosis factor alpha production in infected mice and in in vitro-cultured mouse macrophages
Authors: Sarmento A M , Appelberg R
Published: January 2020

We studied the ability of two Mycobacterium avium strains with different virulences to induce tumor necrosis factor alpha (TNF) synthesis by mouse resident peritoneal macrophages (RPM phi) in vitro in an experiment to look for a possible correlation between virulence and this TNF-inducing capacity. The low-virulence strain, 1983, induced significantly higher production of TNF by RPM phi than did the high-virulence strain, ATCC 25291. TNF neutralization during culture of infected RPM phi resulted in enhancement of growth of strain 1983 and had no effect on growth of strain ATCC 25291; TNF treatment of strain ATCC 25291-infected macrophages had no effect on mycobacterial growth. The extent of M. avium growth and the amount of TNF synthesis were independent of the presence of contaminating T cells or NK cells in the macrophage monolayers. Intraperitoneal administration of anti-TNF monoclonal antibodies to BALB/c mice infected intravenously with M. avium 1983 abrogated the elimination of the bacteria in the liver and caused a slight increase in bacterial growth in the spleen. Neutralization of TNF led to a minor increase in the proliferation of M. avium ATCC 25291 in the liver and spleen of BALB/c mice late in infection. Anti-TNF treatment did not affect the growth of the two M. avium strains in BALB/c.Bcgr (C.D2) mice, suggesting that restriction of M. avium strains to induce TNF production by macrophages may limit their ability to proliferate both in vitro and in vivo.
Mycoplasma hyorhinis molecules that induce tumor necrosis factor alpha secretion by human monocytes
Authors: Kostyal D A , Butler G H , Beezhold D H
Published: January 2020

Mycoplasma hyorhinis has been shown to induce the secretion of tumor necrosis factor alpha (TNF-alpha) from monocytes. To identify the molecules responsible for this activity, we separated sonicated M. hyorhinis lysate material by centrifugation at 100,000 x g into soluble (S) and particulate (P) fractions. The fractions were assayed for TNF-alpha-inducing activity by the L929 bioassay. Both the soluble and particulate fractions were able to induce TNF-alpha in roughly equal amounts. The optimum dose for both fractions was 1 micrograms/ml. Proteinase K treatment of either fraction eliminated the activity, suggesting that a protein component is involved in induction. Phase partitioning into Triton X-114 aqueous (A) and detergent (D) phases showed that the soluble fraction was composed of 80% aqueous-phase proteins, while the particulate fraction was > 75% detergent-phase proteins. All four fractions (SA, SD, PA, and PD) were able to induce TNF-alpha release. Treatment with NaIO4 to remove carbohydrate reduced the inducing activity of the SA phase by 80%, whereas that of the other fractions was unaffected by this treatment. The M(r)S of the inducing activity were determined by the monocyte Western (immunoblot) technique. The SA phase activity was associated with a single periodate-sensitive peak of 69 to 75 kDa. The two detergent phases had similar profiles of inducing activity, containing four peaks of activity. These peaks corresponded to 48 to 52, 43 to 45, 39 to 40, and 31 to 32 kDa. The PA fraction also contained four peaks of activity, 69 to 75, 55 to 57, 48 to 52, and 39 to 40 kDa. Thus, both a protein and glycan moiety from M. hyorhinis are capable of inducing TNF-alpha release from human monocytes.
Porphyromonas gingivalis invasion of gingival epithelial cells
Authors: Lamont R J , Chan A , Belton C M , Izutsu K T , Vasel D , Weinberg A
Published: January 2020

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.
Expression of the Listeria monocytogenes EGD inlA and inlB genes, whose products mediate bacterial entry into tissue culture cell lines, by PrfA-dependent and -independent mechanisms
Authors: Lingnau A , Domann E , Hudel M , Bock M , Nichterlein T , Wehland J , Chakraborty T
Published: January 2020

Internalization of Listeria monocytogenes into nonphagocytic cell lines in vitro requires the products of the inlAB locus (J.-L. Gaillard, P. Berche, C. Frehel, E. Gouin, and P. Cossart, Cell 65:1127-1141, 1991). By generating isogenic mutants with a chromosomal in-frame deletion in either inlA or inlB, we have identified InlA and InlB as surface-bound proteins of L. monocytogenes with molecular weights of 88,000 and 65,000, respectively. These results were obtained with monoclonal antibodies raised against either protein and corroborated by N-terminal end sequencing of InlA and InlB. By immunoblot analysis, the production of both polypeptides was found to be strongly dependent on growth temperature and, particularly for InlB, on the presence of the PrfA regulator protein. Expression of InlA was not strictly dependent on the presence of the PrfA regulator protein. Transcription analysis of the inlAB locus revealed that the inlA gene was transcribed by several promoters, of which only one is PrfA dependent. This PrfA-dependent inlA promoter, which contains two base substitutions within its putative PrfA DNA-binding palindrome, is responsible for transcription of both inlA and inlB genes. A hitherto unrecognized promoter located 51 bp upstream of the GTG start codon of the inlB gene was also detected. Hence, inlA and inlB are transcribed both individually and in an operon by PrfA-dependent and -independent mechanisms. Tissue culture invasion assays employing various epithelial cell lines demonstrated that both InlA and InlB are required for invasion. In vivo studies using the mouse infection model revealed that both internalin mutants were attenuated for virulence.
Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a
Authors: Turbyfill K R , Joseph S W , Oaks E V
Published: January 2020

The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection.
Production of tumor necrosis factor alpha, interleukin-1 alpha, and interleukin-6 during murine coccidioidomycosis
Authors: Cox R A , Magee D M
Published: January 2020

The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.
Molecular characterization of FrpB, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis
Authors: Pettersson A , Maas A , van Wassenaar D , van der Ley P , Tommassen J
Published: January 2020

The structural gene encoding the 70-kDa outer membrane protein FrpB of Neisseria meningitidis was cloned and sequenced. A mutant lacking FrpB was constructed. No difference in iron utilization between the mutant and the parental strain was observed. A minor effect of the mutation on serum resistance was observed. A topology model for FrpB in the outer membrane is proposed.
Preferential induction of septic arthritis and mortality by superantigen-producing staphylococci
Authors: Bremell T , Tarkowski A
Published: January 2020

Staphylococcal enterotoxins A through D (SEA through SED) and toxic shock syndrome toxin-1 display superantigen properties, i.e., they stimulate a great fraction of T cells expressing certain T-cell receptor V beta sequences. Using a newly established rat model of septic Staphylococcus aureus arthritis, we have recently shown that an S. aureus strain producing SEA showed marked arthritogenic properties. In the present study we decided to employ another five S. aureus strains, each one producing a distinct exotoxin. Almost all rats injected with superantigen-producing strains developed arthritis. In contrast, only 20% of rats injected with an S. aureus strain lacking superantigen production displayed mild and transient arthritis. Mortality was preferentially induced among the rats inoculated with the S. aureus strains producing SEB and SED. This study emphasizes that superantigen production is an important virulence factor in the development of septic S. aureus arthritis. Differences concerning mortality between staphylococcal strains producing different exotoxins may be dependent on the degree of activation of the immune system.
Adenovirus serotype 1 does not act synergistically with Moraxella (Branhamella) catarrhalis to induce otitis media in the chinchilla
Authors: Bakaletz L O , Murwin D M , Billy J M
Published: January 2020

A chinchilla model of otitis media in which adenovirus compromise of the tubotympanum facilitates the subsequent induction of middle ear disease was used to investigate Moraxella (Branhamella) catarrhalis pathogenesis. Intranasally inoculated M. catarrhalis did readily colonize the nasopharynx of this host; however, despite evidence of viral infection and tubotympanal compromise, M. catarrhalis did not induce culture-positive otitis media in this model.
Salmonella strain secreting active listeriolysin changes its intracellular localization
Authors: Gentschev I , Sokolovic Z , Mollenkopf H J , Hess J , Kaufmann S H , Kuhn M , Krohne G F , Goebel W
Published: January 2020

We describe the construction of an attenuated Salmonella dublin aroA strain which secretes via the Escherichia coli hemolysin secretion machinery an active hybrid cytolysin consisting of listeriolysin from Listeria monocytogenes and the C-terminal secretion signal of E. coli hemolysin. This hemolytic S. dublin strain is partially released into the cytoplasm of the host cell following uptake by J774 macrophage cells, whereas the nonhemolytic control S. dublin aroA strain remains in the phagosome.
Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila
Authors: James B W , Mauchline W S , Fitzgeorge R B , Dennis P J , Keevil C W
Published: January 2020

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila.
Host restriction phenotypes of Salmonella typhi and Salmonella gallinarum
Authors: Pascopella L , Raupach B , Ghori N , Monack D , Falkow S , Small P L
Published: January 2020

Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems. S. typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse. But, unlike S. typhimurium, S. typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry. S. gallinarum appears to be incapable of entering the murine Peyer's patch epithelium. Our in vitro evidence suggests that S. gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S. typhimurium. S. typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S. gallinarum and S. typhi.
Myosin II is involved in capping and uroid formation in the human pathogen Entamoeba histolytica
Authors: Arhets P , Gounon P , Sansonetti P , Guillén N
Published: January 2020

The redistribution and capping of surface receptors on the human pathogen Entamoeba histolytica was observed in the presence of concanavalin A (ConA). Capping was correlated with plasma membrane folding towards the rear of the amoeba and with uroid formation. The uroid is thought to play a role in the escape of amoebae from the host immune response. To localize myosin II during capping, amoebae were incubated in the presence of ConA and then analyzed by microscopy. Myosin II was three times more concentrated within the uroid compared with the rest of the cell, suggesting that the release of caps may depend upon mechanical contraction driven by myosin II activity. The use of drugs that disrupt cytoskeletal structure or that inhibit myosin heavy chain phosphorylation demonstrated that inhibition of capping prevents uroid formation. Biochemical analysis allowed the identification of two ConA receptors which have been previously described as major pathogenic antigens of this parasite: the 96-kDa antigen, which carries alcohol dehydrogenase 2 activity and binds extracellular matrix proteins, and the Gal-GalNAc-inhibitable surface lectin, which is involved in amoeba-cell interactions and in the degradation of complement particles attached to the parasite.
Characterization of the hemolytic activity of Haemophilus ducreyi
Authors: Totten P A , Norn D V , Stamm W E
Published: January 2020

H. ducreyi is the causative agent of chancroid, a genital ulcer disease most prevalent in developing countries. Chancroid enhances the heterosexual transmission of human immunodeficiency virus and is identified in focal outbreaks in the United States, but little is known about its pathogenesis. We studied the hemolysin produced by H. ducreyi because this molecule might be an important virulence factor in the pathogenesis of chancroid. Ten strains of H. ducreyi were tested on newly devised blood agar plates and were found to have hemolytic activity. We examined the hemolytic activity of H. ducreyi 35000 further and found that it was heat labile, cell associated, greatest at pH 7.0, and produced in logarithmic- but not stationary-phase cultures. Using transposons Tn916 and Tn1545-delta 3, we have isolated three classes of transposon mutants of strain 35000: those with no detectable hemolytic activity, those with reduced hemolytic activity, and those with enhanced hemolytic activity. Transposon insertions in the nonhemolytic mutants were located in a DNA sequence which hybridized to the Proteus mirabilis hemolysin gene. Analysis of clones containing overlapping sections of this region served to further localize the H. ducreyi hemolysin gene and allow its expression in Escherichia coli and complementation of the nonhemolytic defect in an H. ducreyi mutant. These experiments indicate that H. ducreyi 35000 produces a hemolysin that is related to the calcium-independent hemolysin produced by P. mirabilis. Further experiments are needed to define the similarity of the H. ducreyi hemolysin to other calcium-independent hemolysins and to determine its role in the pathogenesis of chancroid.
Borrelia burgdorferi upregulates expression of adhesion molecules on endothelial cells and promotes transendothelial migration of neutrophils in vitro
Authors: Sellati T J , Burns M J , Ficazzola M A , Furie M B
Published: January 2020

The accumulation of leukocytic infiltrates in perivascular tissues is a key step in the pathogenesis of Lyme disease, a chronic inflammatory disorder caused by Borrelia burgdorferi. During an inflammatory response, endothelial cell adhesion molecules mediate the attachment of circulating leukocytes to the blood vessel wall and their subsequent extravasation into perivascular tissues. Using cultured human umbilical vein endothelial cells (HUVEC) in a whole-cell enzyme-linked immunosorbent assay, we demonstrated that B. burgdorferi activated endothelium in a dose- and time-dependent fashion as measured by upregulation of the adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). As few as one spirochete per endothelial cell stimulated increased expression of these molecules. Expression of E-selectin peaked after spirochetes and HUVEC were coincubated for 4 h and returned to near-basal levels by 24 h. In contrast, expression of VCAM-1 and ICAM-1 peaked at 12 h and remained elevated at 24 h. HUVEC monolayers cultured on acellular amniotic tissue were used to investigate the consequences of endothelial cell activation by spirochetes. After incubation of HUVEC-amnion cultures with B. burgdorferi, subsequently added neutrophils migrated across the endothelial monolayers. This process was mediated by E-selectin and by CD11/CD18 leukocytic integrins. The extent of migration depended on both the number of spirochetes used to stimulate the HUVEC and the length of the coincubation period. These results raise the possibility that B. burgdorferi induces a host inflammatory response and accompanying perivascular damage through activation of vascular endothelium.
Regulation of virulence by environmental signals in group A streptococci: influence of osmolarity, temperature, gas exchange, and iron limitation on emm transcription
Authors: McIver K S , Heath A S , Scott J R
Published: January 2020

Transcription of the gene encoding the antiphagocytic M protein (emm) of the group A streptococci has been shown to be regulated by CO2 (M. G. Caparon, R. T. Geist, J. Perez-Casal, and J. R. Scott, J. Bacteriol. 174:5693-5701, 1992). We tested the influence of additional environmental growth conditions on emm expression. Increased osmolarity, low temperature, growth with free exchange of gasses, or the restricted availability of iron resulted in decreased transcription from the emm promoter.
Helicobacter pylori expresses a complex surface carbohydrate, Lewis X
Authors: Sherburne R , Taylor D E
Published: January 2020

Monoclonal antibodies (MAbs) specific for Lewis X (Lex) reacted with whole cells of Helicobacter pylori NCTC11637, UA799, UA802, UA825, UA861, UA1182, and UA1206 in immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) experiments. These MAbs have documented specificity to Lex, whereas MAbs for Lea and Leb were negative in both immunoelectron microscopy and ELISA. H. pylori coccoid forms also reacted with the MAbs, whereas the flagellum lacking the sheath showed no reactivity. The Lex structures were associated with membrane fractions in the ELISA experiments, and silver-stained sodium dodecyl sulfate-polyacrylamide gels confirmed the presence of lipopolysaccharides which reacted with the MAbs in immunoblots. Serum from an H. pylori-infected individual contained immunoglobulins which blocked the binding of the Lex MAbs, indicating that part of the host immune response to H. pylori is to the Lex structure. The ability of this gastric pathogen to mimic an oncofetal antigen (self) could explain the down regulation of anti-H. pylori T-cell response seen in H. pylori-infected individuals.
Intergeneric coaggregation of oral Treponema spp. with Fusobacterium spp. and intrageneric coaggregation among Fusobacterium spp
Authors: Kolenbrander P E , Parrish K D , Andersen R N , Greenberg E P
Published: January 2020

A total of 22 strains of Treponema spp. including members of all four named human oral species were tested for coaggregation with 7 strains of oral fusobacteria, 2 strains of nonoral fusobacteria, and 45 strains of other oral bacteria, which included actinobacilli, actinomyces, capnocytophagae, eubacteria, porphyromonads, prevotellae, selenomonads, streptococci, and veillonellae. None of the treponemes coaggregated with any of the latter 45 oral strains or with the two nonoral fusobacteria. All treponemes, eight Treponema denticola strains, eight T. socranskii strains, four oral pectinolytic treponemes, one T. pectinovorum strain, and one T. vincentii strain coaggregated with at least one strain of the fusobacteria tested as partners. The partners consisted of one strain of Fusobacterium periodonticum, five F. nucleatum strains including all four subspecies of F. nucleatum, and a strain of F. simiae obtained from the dental plaque of a monkey. In the more than 100 coaggregations observed, the fusobacterial partner was heat inactivated (85 degrees C for 30 min), while the treponemes were unaffected by the heat treatment. Furthermore, the fusobacteria were usually inactivated by proteinase K treatment, and the treponemes were not affected. Only the T. denticola coaggregations were inhibited by lactose and D-galactosamine. None were inhibited by any of 23 other different sugars or L-arginine. Intragenic coaggregations were seen among the subspecies of F. nucleatum and with F. periodonticum, and none were inhibited by any of the sugars tested or by L-arginine. No intrageneric coaggregations were observed among the treponemes. These data indicate that the human oral treponemes show a specificity for oral fusobacteria as coaggregation partners. Such cell-to cell contact may facilitate efficient metabolic communication and enhance the proliferation of each cell in the progressively more severe stages of periodontal disease.
Escherichia coli-induced activation of neutrophil NADPH-oxidase: lipopolysaccharide and formylated peptides act synergistically to induce release of reactive oxygen metabolites
Authors: Karlsson A , Markfjäll M , Strömberg N , Dahlgren C
Published: January 2020

The prevailing view of neutrophil NADPH-oxidase activation during interaction with bacteria is that the production of toxic oxygen metabolites should be directed into the phagosome containing the engulfed prey. However, in this report we show that a common Escherichia coli strain, HB101, may induce a release of neutrophil oxygen metabolites to the extracellular milieu. This phenomenon is dependent on three factors: (i) the mobilization (upregulation) of neutrophil secretory vesicles prior to interaction with the bacteria, (ii) soluble bacterial factors binding to the formylmethionyl-leucyl-phenylalanine receptor and tentatively identified as formylated peptides, and (iii) a bacterium-associated priming factor identified as lipopolysaccharide.
Contribution of individual disulfide bonds to biological action of Escherichia coli heat-stable enterotoxin B
Authors: Arriaga Y L , Harville B A , Dreyfus L A
Published: January 2020

Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes. The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E2, known mediators of intestinal secretion. In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells. STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds. Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear. Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity. Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity. Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb. Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S. In contrast, the alpha-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb. Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.
Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis
Authors: McDonough K A , Kress Y
Published: January 2020

Dissemination of viable tubercle bacilli from the lung is a critical event in the establishment of Mycobacterium tuberculosis infection. We examined the possibility that M. tuberculosis bacteria could infect and damage lung epithelial cells to determine whether direct penetration of the alveolar epithelium is a plausible route of M. tuberculosis infection. While both virulent H37Rv tubercle bacilli and the attenuated Mycobacterium bovis BCG vaccine strain were able to enter A549 human lung epithelial cells in culture, only the virulent tubercle bacilli were cytotoxic for both polarized and nonpolarized epithelial monolayers and macrophages. In addition, bacterial entry into epithelial cells, but not macrophages, was increased by intracellular passage through macrophages, suggesting enhancement of a bacterially mediated cell entry mechanism in bacteria grown within macrophages. These findings suggest that M. tuberculosis bacteria might have the ability to gain access to the host lymphatics and circulatory system by directly penetrating the alveolar epithelial lining of an infected lung.