bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous interleukin-12 is involved in resistance of mice to Mycobacterium avium complex infection
Authors: Saunders B M , Zhan Y , Cheers C
Published: January 2020

Acquired cellular resistance against Mycobacterium avium complex (MAC) infections involves the induction of Th1 type gamma interferon (IFN-gamma)-producing T cells. Interleukin-12 (IL-12) is a cytokine involved in the control of IFN-gamma production by T cells and NK cells. The role of IL-12 in the response to MAC infection was investigated. Depletion of endogenous IL-12 by injection of monoclonal antibody prior to and during intranasal infection with MAC resulted in an 150- to 550-fold increase in bacterial load in lung, spleen, and liver homogenates by 10 weeks postinfection. Depletion of IL-12 abrogated the ability of spleen cell cultures to produce IFN-gamma in response to stimulus with live MAC. IL-12-depleted mice showed a 75% decrease in the number of inflammatory cells entering the lungs following intranasal infection with MAC, with significant reductions in cytotoxic activity and nitric oxide production by lung cells. This work suggests that IL-12 plays a major role in the activation of IFN-gamma-producing cells during MAC infection.
Relationship between maternally derived anti-Plasmodium falciparum antibodies and risk of infection and disease in infants living in an area of Liberia, west Africa, in which malaria is highly endemic
Authors: Høgh B , Marbiah N T , Burghaus P A , Andersen P K
Published: January 2020

In areas where Plasmodium falciparum is endemic, immunoglobulin G is acquired by the fetus in utero, mainly during the third trimester of pregnancy. The potential protective effect of transferred anti-P. falciparum maternal antibodies was examined in a longitudinal study of 100 infants from birth to 1 year of age. The probability of acquiring a P. falciparum infection and developing an episode of clinical malaria was determined in relation to the P. falciparum-specific antibody level of the infant at birth against P. falciparum schizont antigen or recombinant merozoite surface protein MSP1(19) antigen. The risk of acquiring an episode of clinical malaria increased from birth to 6 months of age, after which it decreased. The overall prevalence of P. falciparum parasitemia was highest (48.9%) in the 6-month-old infants. The age-specific hematocrit value showed the lowest mean value (30.2) from 6 to 9 months, and the spleen rate was the highest (69.8%) at the same age. There was a lower risk of developing an episode of clinical malaria during the first year of life in the infants with high levels of anti-MSP1(19) antibodies at birth. The level of maternally derived overall anti-schizont antigen antibodies did not seem to play a role in the relative risk of developing malaria infection or disease during the first year of life, though the level of specific anti-MSP1(19) antibodies may be associated with protection.
Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae
Authors: Connell T D , Metzger D J , Wang M , Jobling M G , Holmes R K
Published: January 2020

The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis.
Chronic respiratory mycoplasmosis in C3H/HeN and C57BL/6N mice: lesion severity and antibody response
Authors: Cartner S C , Simecka J W , Lindsey J R , Cassell G H , Davis J K
Published: January 2020

Mycoplasma pneumoniae is a leading, worldwide cause of death and disability due to pneumonia. Mycoplasma pulmonis infection in mice is an invaluable model for the study of host defenses against respiratory mycoplasmas in vivo. C3H/HeN mice are much more susceptible to acute inflammatory lung disease due to M. pulmonis than C57BL/6N mice, but little is known about the chronic disease in these mouse strains. We infected C3H/HeN and C57BL/6N mice with 10(4) CFU of M. pulmonis UAB CT and evaluated them at weekly intervals by quantitative mycoplasma culture of nasal passages, trachea, and lungs, assessment of lesion severity in nasal passages, trachea, and lungs, and determination of serum immunoglobulin classes and subclasses by enzyme-linked immunosorbent assay. We found that C3H/HeN mice had 2 to 5 logs more organisms in their lungs and far more severe lung disease than C57BL/6N mice through 63 days postinfection. Although both strains of mice developed the same classes of antibody, C3H/HeN mice had much greater anti-M. pulmonis immunoglobulin G (IgG) responses in the IgG1 and IgG2a subclasses than C57BL/6N mice. These results suggest that adaptive immunity does not effect resolution of chronic mycoplasma infection and disease in the lungs.
Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis
Authors: Li E , Yang W G , Zhang T , Stanley S L
Published: January 2020

The Entamoeba histolytica 27-kDa cysteine proteinases exhibit striking binding specificities for immobilized laminin over other components of the extracellular matrix, such as collagen and fibronectin. Inactivation of the proteinase with the active-site inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane abolishes laminin binding by the enzyme, and conversely, laminin inhibits cleavage of a fluorogenic dipeptide substrate of the amebic cysteine proteinase, suggesting that the substrate binding pocket of the enzyme is involved in the binding of laminin. The addition of laminin but not fibronectin or collagen to E. histolytica trophozoites significantly reduces amebic liver abscess formation in severe combined immunodeficient mice, further supporting the hypothesis that E. histolytica cysteine proteinases play an important role in amebic pathogenesis. The specific interaction of amebic proteinases with laminin may be exploited in designing new inhibitors of these enzymes.
Avirulence of a Pseudomonas aeruginosa algC mutant in a burned-mouse model of infection
Authors: Goldberg J B , Coyne M J , Neely A N , Holder I A
Published: January 2020

The virulence of wild-type Pseudomonas aeruginosa PAO1 and that of a genetically defined algC mutant, PAO1 algC::tet, were compared in a burned-mouse model of infection. Unlike PAO1, PAO1 algC::tet was avirulent, grew less well in the eschar, and did not disseminate to the liver of challenged animals. We have previously shown that the P. aeruginosa algC gene is required for biosynthesis of alginate and lipopolysaccharide (M.J. Coyne, Jr., K.S. Russell, C.L. Coyle, and J.B. Goldberg, J. Bacteriol. 176:3500-3507, 1994). In order to determine whether the alginate or lipopolysaccharide (LPS) defect was responsible for the avirulence of this strain, we constructed a strain with a mutation in an alginate-specific gene, algD. PAO1-algD was virulent in the burned-mouse model, thus implicating the LPS defect in PAO1 algC::tet as the relevant alteration responsible for the avirulence of this strain.
Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes
Authors: Fadul C E , Channon J Y , Kasper L H
Published: January 2020

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.
Development of an improved animal model of shigellosis in the adult rabbit by colonic infection with Shigella flexneri 2a
Authors: Rabbani G H , Albert M J , Rahman H , Islam M , Mahalanabis D , Kabir I , Alam K , Ansaruzzaman M
Published: January 2020

Rabbits are not usually susceptible to intestinal Shigella infection without extensive pretreatment, including starvation and administration of antimicrobial, antimotility, and toxic agents (carbon tetrachloride). Most animals infected this way die rapidly and do not always develop colonic lesions and signs of dysentery. We describe here a successful experimental infection in the adult rabbit which does not require preparatory treatment and which reproduced characteristic features of human shigellosis. Unstarved, untreated adult rabbits were infected by direct inoculation of virulent Shigella flexneri 2a (10 ml of 10(7) bacteria per ml) into the proximal colon after ligation of the distal cecum (cecal bypass). Within 24 h of infection, most inoculated animals consistently developed clinical dysentery, characterized by liquid stool mixed with mucus and blood, leukocytosis, anorexia, and weight loss. Histologically, there were edema, exudation, superficial ulceration, and polymorphonuclear infiltrations in the lamina propria; crypt abscess formation; focal hemorrhages; and the presence of immunohistochemically stained S. flexneri in the colonic mucosa. Successful bacterial colonization was indicated by the isolation of the challenge strain of S. flexneri 2a from the colonic contents. None of the control rabbits challenged with nonvirulent S. flexneri or without cecal bypass developed dysentery or colitis. We conclude that successful Shigella infection can be induced by direct colonic inoculation with virulent S. flexneri 2a in adult rabbits without starvation and pretreatment. The colitis is dependent on the virulence of the bacteria and on the cecal bypass. This model should be useful in studies of the immunopathogenesis of Shigella infection.
The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases
Authors: Chu L , Burgum A , Kolodrubetz D , Holt S C
Published: January 2020

The 46-kDa hemolysin produced by Treponema denticola may be involved in the etiology of periodontitis. In order to initiate a genetic analysis of the role of this protein in disease, its gene has been cloned. Synthetic oligonucleotides, designed on the basis of the previously reported amino-terminal amino acid sequence of the 45-kDa hemolysin, were used as primers in a PCR to amplify part of the hemolysin (hly) gene. This PCR product was then used to clone the entire hly gene from libraries of T. denticola genomic DNA. Constructs containing the entire cloned region on plasmids in Escherichia coli produced both hemolysis and hemoxidation activities either on sheep blood agar plates or in liquid assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed that the constructs synthesized a protein with molecular size of about 46 kDa which was reactive with anti-T. denticola hemolysin. Nucleotide sequence analysis indicated that the largest open reading frame could encode a protein with a calculated molecular size of 46.2 kDa. The first 31 amino acids encoded by this open reading frame were identical to the experimentally determined amino-terminal sequence of the 45-kDa hemolysin. These results indicate that the entire hly gene has been cloned. The deduced amino acid sequence of the T. denticola hly gene is homologous (23 to 37% identity) to those of proteins that are members of a family of pyridoxal-phosphate-dependent aminotransferases. This suggests that the 46-kDa hemolysin may be related to an aminotransferase and have a novel mechanism of hemolysis. However, the functional aspects of this relationship remain to be investigated.
Functional properties of isotype-switched immunoglobulin M (IgM) and IgG monoclonal antibodies to Pseudomonas aeruginosa lipopolysaccharide
Authors: Pollack M , Koles N L , Preston M J , Brown B J , Pier G B
Published: January 2020

Controversy exists regarding isotype-related differences in the antibacterial and protective properties of lipopolysaccharide (LPS)-specific antibodies of the immunoglobulin M (IgM) class and various IgG subclasses. To clarify this issue, a murine hybridoma secreting IgM monoclonal antibody (MAb) specific for the O polysaccharide of Pseudomonas aeruginosa serogroup O6 LPS was class switched, by sib selection, to produce an IgG3 MAb with identical specificity and variable region heavy and light chain nucleotide sequences. This IgG3-secreting cell line was further switched to the production of O-specific, variable region-identical IgG1, IgG2b, and IgG2a MAbs. Functional comparisons of these LPS-specific IgM and IgG MAb isotypes revealed similar LPS binding, opsonic, and protective activities. Relatively minor isotype-related differences in levels of efficiency of MAb-mediated, complement-dependent opsonophagocytic killing (IgM > IgG2a > IgG3 > IgG2b > > IgG1) were not associated with corresponding differences in in vivo functions. These findings, in conjunction with previously published data, support a cautious approach to generic conclusions regarding the immunotherapeutic superiority of LPS-specific antibodies belonging to either the IgM or IgG class or to a particular IgG subclass.
Structure and regulation of the HSP90 gene from the pathogenic fungus Candida albicans
Authors: Swoboda R K , Bertram G , Budge S , Gooday G W , Gow N A , Brown A J
Published: January 2020

Candida albicans HSP90 sequences were isolated by screening cDNA and genomic libraries with a probe derived from the Saccharomyces cerevisiae homolog, HSP82, which encodes a member of the heat shock protein 90 family of molecular chaperones. Identical sequences were obtained for the 2,197-bp overlap of the cDNA and gene sequences, which were derived from C. albicans 3153A and ATCC 10261, respectively. The C. albicans HSP90 gene contained no introns, and it showed strong homology (61 to 79% identity) to HSP90 sequences from other fungi, vertebrates, and plants. The C-terminal portion of the predicted Hsp90 amino acid sequence was identical to the 47-kDa protein which is thought to be immunoprotective during C. albicans infections (R. C. Matthews, J. Med. Microbiol. 36:367-370, 1992), confirming that this protein represents the C-terminal portion of the 81-kDa Hsp90 protein. Quantitative Northern (RNA) analyses revealed that C. albicans HSP90 mRNA was heat shock inducible and that its levels changed during batch growth, with its maximum levels being reached during the mid-exponential growth phase. HSP90 mRNA levels increased transiently during the yeast-to-hyphal transition but did not correlate directly with germ tube production per se. These data do not exclude a role for Hsp90 in the dimorphic transition. Southern blotting revealed only one HSP90 locus in the diploid C. albicans genome. Repeated attempts to disrupt both alleles and generate a homozygous C. albicans delta hsp90/delta hsp90 null mutant were unsuccessful. These observations suggest the existence of a single HSP90 locus which is essential for viability in C. albicans.
Reduced virulence of Candida albicans mutants affected in multidrug resistance
Authors: Becker J M , Henry L K , Jiang W , Koltin Y
Published: January 2020

Disruption of a multidrug resistance gene (CaMDR1) in Candida albicans resulted in mutant strains that colonized mouse kidneys to very high levels but were markedly reduced in their virulence. No obvious differences in several properties related to colonization and dissemination were noted among MDR+ or mdr- strains. These results suggest that specific fungal efflux pumps play a role in fungal pathogenicity.
Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50
Authors: Aduse-Opoku J , Muir J , Slaney J M , Rangarajan M , Curtis M A
Published: January 2020

Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein. Immunochemical analysis of P. gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface. Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P. gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P. gingivalis chromosome. These data indicate that the ArgI protease of P. gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties.
Fusobacterium nucleatum inhibits human T-cell activation by arresting cells in the mid-G1 phase of the cell cycle
Authors: Shenker B J , Datar S
Published: January 2020

Fusobacterium nucleatum has been implicated in the pathogenesis of several diseases, including urinary tract infections, bacteremia, pericarditis, otitis media, and disorders of the oral cavity such as pulpal infections, alveolar bone abscesses, and periodontal disease. We have previously demonstrated that sonic extracts of F. nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens. In this study, we have further characterized this immunosuppressive protein (FIP) and initiated experiments to determine its mode of action. The purified FIP has an apparent molecular mass of 90 to 100 kDa; sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the FIP is actually composed of two subunits with molecular masses of 48 and 44 kDa. Purified FIP retained its biological activity and was capable of inhibiting mitogen-induced proliferation of human T cells. Inhibition was dose dependent, and the FIP exhibited a specific activity approximately 250-fold greater than that of the crude extract. Cell cycle analysis indicates that FIP-treated cells were prevented from exiting the G0/G1 phase of the cell cycle. However, FIP did not alter the expression of activation markers (CD69, CD25, and CD71) or interleukin-2 secretion. The latter observations suggest that the T cells did indeed become activated and had entered the G1 phase of the cell cycle. Analysis of the expression of cyclins indicates that the phase of the cell cycle that is FIP sensitive resides somewhere beyond the restriction point of cyclin D2 (early to mid-G1) but prior to that of cyclins D3 and E (mid- to late G1). Finally, analysis of the expression of the proliferating cell nuclear antigen indicates that this is the earliest detectable defect in T cells exposed to FIP. We propose that if a block in the G1 phase of the cell cycle occurs in vivo in lymphocytes, it may result in a state of local and/or systemic immunosuppression. These suppressive effects could alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of F. nucleatum itself or that of some other opportunistic organisms.
Identification and functional characterization of thioredoxin of Mycobacterium tuberculosis
Authors: Wieles B , Nagai S , Wiker H G , Harboe M , Ottenhoff T H
Published: January 2020

We have previously described a Mycobacterium tuberculosis protein designated MPT46 that was present in culture filtrates. Here we report that the MPT46 protein is thioredoxin of M. tuberculosis. MPT46 is recognized by antibodies to thioredoxin (Trx) of Escherichia coli, and antibodies of MPT46 recognize Mycobacterium leprae Trx. Moreover, MPT46 was shown to have enzymatic activity identical to that of Trx of other species, such as its ability to reduce insulin. These findings identify MPT46 as a functionally active Trx.
Involvement of bactericidal factors from thrombin-stimulated platelets in clearance of adherent viridans streptococci in experimental infective endocarditis
Authors: Dankert J , van der Werff J , Zaat S A , Joldersma W , Klein D , Hess J
Published: January 2020

Platelets activated with thrombin release bactericidal factors. We studied the role of the susceptibility of viridans streptococci to these bactericidal factors in the development of infective endocarditis (IE). By using the experimental endocarditis rabbit model, the initial adherence and the development of IE were assessed for 10 viridans streptococcal strains differing in their susceptibilities to releasate (material released) from thrombin-activated platelets. Six strains were susceptible and four strains were resistant to these releasates. The numbers of vegetations (VGs) colonized at 5 min and 48 h after intravenous challenge with 10(4) CFU were determined. At 5 min after challenge, significantly more VGs were colonized with bacteria of the six platelet releasate-susceptible strains than with bacteria of the four releasate-resistant strains (P < 0.005). In the releasate-susceptible group of strains, the number of colonized VGs decreased significantly between 5 min and 48 h after intravenous inoculation (P < 0.001). Such a decrease was not observed with the releasate-resistant strains. As a result, the final developments of IE due to releasate-susceptible and -resistant strains were not significantly different. The releasate-susceptible strain 1 and the releasate-resistant strain 2 were selected for more detailed experiments. Rabbits were killed at 5 and 30 min and 2, 4, and 48 h after inoculation. The number of culture-positive VGs as well as the number of adherent bacteria on the individual VGs were determined. The 90% infective dose for each strain was 10(5) CFU. At low inoculum concentrations (10(3) and 10(4) CFU) a larger proportion of the inoculated bacteria of both strains was found to be adherent on VGs than at higher challenge doses. The number of culture-positive VGs as well as the number of adherent bacteria per VG decreased rapidly in the first 30 min after challenge with strain 1 but not after challenge with strain 2. Additional experiments with the platelet releasate-susceptible strain S224 and the platelet releasate-resistant stain S182 confirmed the data obtained with strains 1 and 2 and indicated that releasate-susceptible strains disappeared from the VGs with time, whereas releasate-susceptible strains persisted. In vitro studies with VGs excised 5 min after challenge with stain 1 or 2 showed that clearance of the releasate-susceptible strain 1 was not caused by complement bactericidal activity or surface phagocytosis by polymorphonuclear cells. Bacterial cells of strain 1 adherent on excised VTGs were rapidly cleared by exposure to fresh clotting blood or to releasates from thrombin-stimulated platelet suspension.(ABSTRACT TRUNCATED AT 400 WORDS)
Functional conservation among members of the Salmonella typhimurium InvA family of proteins
Authors: Ginocchio C C , Galán J E
Published: January 2020

InvA, which is essential for Salmonella spp. to enter cultured epithelial cells, is a member of a family of proteins involved in either flagellar biosynthesis or the secretion of virulence determinants by a number of plant and mammalian pathogens. The predicted overall secondary structures of these proteins show significant similarities and indicate a modular construction with a hydrophobic amino-terminal half, consisting of six to eight potential transmembrane domains, and a hydrophilic carboxy terminus which is predicted to reside in the cytoplasm. These proteins can be aligned over the entire length of their polypeptide sequences, with the highest degree of homology found in the amino terminus and clusters of conserved residues in the carboxy terminus. We examined the functional conservation among members of this protein family by assessing the ability of MxiA of Shigella flexneri and LcrD of Yersinia pseudotuberculosis to restore invasiveness to an invA mutant of Salmonella typhimurium. We found that MxiA was able to complement the entry defect of the invA mutant strain of S. typhimurium. In contrast, LcrD failed to complement the same strain. However, a plasmid carrying a gene encoding a chimeric protein consisting of the amino terminus of LcrD and the carboxy terminus of InvA complemented the defect of the Salmonella invA mutant. These results indicate that the secretory systems in which these proteins participate are functionally similar and that the Salmonella and Shigella systems are very closely related. These data also suggest that determinants of specificity may be located at the carboxy termini of these proteins.
Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants
Authors: Petras S F , Chidambaram M , Illyes E F , Froshauer S , Weinstock G M , Reese C P
Published: January 2020

Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections.
Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933
Authors: Schmidt H , Beutin L , Karch H
Published: January 2020

In this study, we determined the nucleotide sequence of the 5.4-kb SalI restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933. This revealed two open reading frames which shared approximately 60% homology to the hlyC and hlyA genes of the E. coli alpha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary sequence analysis indicated that another open reading frame homolog to the hlyB gene is located close to the 3' end of EHEC-hlyA. The predicted molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 107 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) was not secreted into the culture supernatant by the strain EDL 933. However, hemolytic activity was found in the broth culture supernatant after transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The EHEC hemolysin was precipitated and used as an antigen for immunoblot analysis. This demonstrated that 19 of 20 reconvalescent-phase serum samples from patients with hemolytic uremic syndrome reacted specifically with the antigen; conversely, only 1 of 20 control serum samples demonstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically detect EHEC-hlyA. All Shiga-like toxin-producing O157 strains and 12 of 25 Shiga-like toxin-producing non-O157 strains were PCR positive; strains of other categories of diarrheagenic E. coli were PCR negative. All PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasmid pEO40 carrying most of the EHEC-hlyA gene and a part of the putative EHEC-hlyB gene. In this study, the newly discovered EHEC hemolysin was shown to be responsible for the enterohemolytic phenotype and demonstrated to be related but not identical to alpha-hemolysin. The EHEC hemolysin appears to have clinical importance because it occurs in all O157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.
Nitric oxide production does not directly increase macrophage candidacidal activity
Authors: Vazquez-Torres A , Jones-Carson J , Balish E
Published: January 2020

Some activated murine macrophages produced nitrite but were unable to kill Candida albicans. Furthermore, a nitric oxide (NO) generator inhibited C. albicans growth but was not candidacidal. Our results suggest that NO is candidastatic and that No is not directly involved but is associated with or induces other macrophage candidacidal mechanisms.
Evaluation of transferrin-binding protein 2 within the transferrin-binding protein complex as a potential antigen for future meningococcal vaccines
Authors: Lissolo L , Maitre-Wilmotte G , Dumas P , Mignon M , Danve B , Quentin-Millet M J
Published: January 2020

Because the meningococcal transferrin receptor was shown to elicit bactericidal and protective antibodies in laboratory animals, we undertook a study of the protective role of each of the polypeptides within the Tbp1-Tbp2 complex. We developed a procedure to purify from Neisseria meningitidis B16B6 the two proteins in milligram amounts and raised specific antisera in rabbits and mice. Only antisera specific for Tbp2 displayed bactericidal activity against the parent strain. Mice immunized with purified Tbp2 survived a lethal challenge to a similar degree as animals immunized with the Tbp1-Tbp2 complex, demonstrating that Tbp2 played an important role in the protective activity observed with the complex. Both Tbp1- and Tbp2-specific antisera inhibited transferrin binding to the purified receptor in a solid-phase binding assay, suggesting that the antibodies were able to interact with the Tbp1 molecule only when it was removed from its membrane environment. Finally, Tbp2-specific immunoglobulins were able to lower the growth rate of the meningococci when human transferrin was their sole iron source. Therefore, in all four different systems tested, Tbp2 or antibodies specific for Tbp2 displayed biological characteristics close to those of the Tbp1-Tbp2 complex. This suggests that Tbp2 plays an important role in the protective activity of the complex, eliciting antibodies that are not only bactericidal but also inhibitory for meningococcal growth.
Small repeating units within the Ureaplasma urealyticum MB antigen gene encode serovar specificity and are associated with antigen size variation
Authors: Zheng X , Teng L J , Watson H L , Glass J I , Blanchard A , Cassell G H
Published: January 2020

Ureaplasma urealyticum is a common commensal of the female lower urogenital tract, yet it has been shown to be an important cause of chorioamnion infection, respiratory and central nervous system disease, and death in premature infants. It has been suggested that only certain serovars are capable of producing invasive disease. However, we previously showed that many serotypes are invasive and that perhaps antigen variability and host factors are more important determinants of ureaplasma infections than are different serotypes per se. The molecular characterization in this report describes a mechanism available to ureaplasmas for producing antigen variation. That antigen, designated MB and previously identified on U. urealyticum, contains serovar-specific and cross-reactive epitopes, is produced both in vitro and in vivo, is a predominant antigen recognized during ureaplasma infections of humans, undergoes a high rate of size variation in vitro, and is size variable on invasive ureaplasma isolates. In the present study, we cloned and sequenced the gene of the MB antigen from serovar 3, the serovar most commonly isolated from humans. The 3' two-thirds of the gene was shown to contain identical 18-nucleotide tandem repeats. PCR analysis and direct sequencing of two variants indicated that alterations within this repeat region are responsible for the size variation of the MB antigen. Intact recombinant serovar 3 MB antigen and truncated products, expressed by coupled in vitro transcription and translation of the cloned gene, were immunoprecipitated by both a serovar-specific monoclonal antibody and the serum of a U. urealyticum-infected patient, and these results identified the repeat region of the MB antigen as serovar defining. Resolution of the precise amino acids responsible for specific epitopes and characterization of similar genes in the other serovars should yield reagents useful in elucidating the role of antigen size variants in disease production and the role of specific antibody in protection from ureaplasma disease.
Adhesion of nontypeable Haemophilus influenzae from blood and sputum to human tracheobronchial mucins and lactoferrin
Authors: Kubiet M , Ramphal R
Published: January 2020

Nontypeable Haemophilus influenzae strains are the most common pathogens encountered in patients with chronic bronchitis. These organisms chronically colonize the airways of patients and occasionally cause bacteremia. Nontypeable H. influenzae strains have been demonstrated microscopically to bind to mucus, but quantitative studies of adhesion have not been published to date. We have therefore developed a reproducible microtiter plate assay to study mucin binding and have examined the adhesion of sputum and blood strains of nontypeable H. influenzae. The assay is similar to that described for Pseudomonas aeruginosa (S. Vishwanath and R. Ramphal, Infect. Immun. 45:197-202, 1984), but notably 2% Tween 20 is used to desorb bacteria from the wells to quantitate bacterial binding. Using a standard strain, we have established that 1 h of incubation is optimum with an inoculum of < or = 5 x 10(8) CFU/ml. The standard strain binds to bronchitic and cystic fibrosis mucins equally well but binds less to bronchiectasis mucins. It does not bind to bovine serum albumin or fetuin. We have also examined the levels of adhesion of freshly isolated sputum and bacteremia strains and find very significant differences in adhesion. Blood strains bound six to seven times less than sputum strains ([13.8 +/- 7] x 10(2) per well versus [102 +/- 43] x 10(2); P < 0.001). Studies with adhesion to lactoferrin, another glycosylated protein, revealed variable binding of respiratory strains but marked binding of blood strains compared with mucin. An isogenic pair of respiratory and blood isolates was examined by electron microscopy but did not show surface differences. We speculate that bacteremic strains studied may have masked, lost, or downregulated adhesin production to allow them to escape from mucins or upregulated adhesins for lactoferrin to invade the bloodstream.
Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines
Authors: de Velasco E A , Merkus D , Anderton S , Verheul A F , Lizzio E F , Van der Zee R , Van Eden W , Hoffman T , Verhoef J , Snippe H
Published: January 2020

Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.
Loss of the O4 antigen moiety from the lipopolysaccharide of an extraintestinal isolate of Escherichia coli has only minor effects on serum sensitivity and virulence in vivo
Authors: Russo T A , Sharma G , Brown C R , Campagnari A A
Published: January 2020

The O-specific antigen in extraintestinal isolates of Escherichia coli is believed to be an important virulence factor. To assess its role in the pathogenic process, proven isogenic derivatives with either a complete (CP921) or nearly complete (CP920) deficiency of the O4 antigen were obtained by TnphoA'1-mediated transposon mutagenesis of an O4/K54/H5 blood isolate (CP9). By utilizing a previously reported isogenic K54 capsule-deficient derivative (CP9.137), additional isogenic derivatives deficient in both the K54 capsular antigen and either all (CP923) or nearly all (CP922) of the O4 antigen were also constructed. These strains and their wild-type parent were evaluated in vitro for serum sensitivity and in vivo by intraperitoneal challenge of outbred mice. The complete or nearly complete loss of the O4 antigen (CP920 and CP921) resulted in only a minor increase in serum sensitivity. In contrast, CP9.137 had a significant increase in serum sensitivity, and CP922 and CP923 were extremely serum sensitive. When tested in vivo, the complete or nearly complete loss of the O4 antigen resulted in a small but significant increase (P < or = 0.05), not the expected decrease, in virulence compared with its wild-type parent. In contrast, CP9.137 and CP922 were significantly less virulent (P < or = 0.05). These studies do not exclude a role for the O4 antigen moiety of lipopolysaccharide in the pathogenesis of extraintestinal E. coli infection; however, they demonstrate that the O4 antigen plays only a minor role in serum resistance in vitro and that its loss does not diminish and perhaps enhances the virulence of CP9 in vivo after intraperitoneal challenge.
Oral immunization with the dodecapeptide repeat of the serine-rich Entamoeba histolytica protein (SREHP) fused to the cholera toxin B subunit induces a mucosal and systemic anti-SREHP antibody response
Authors: Zhang T , Li E , Stanley S L
Published: January 2020

The intestinal protozoan parasite Entamoeba histolytica causes amebic dysentery, a major cause of morbidity worldwide. The induction of a mucosal antibody response capable of blocking amebic adhesion to intestinal cells could represent an approach to preventing E. histolytica infection and disease. Here we describe the expression of a chimeric protein containing an immunogenic dodecapeptide derived from the serine-rich E. histolytica protein (SREHP), fused to the cholera toxin B subunit (CtxB). The CtxB-SREHP-12 chimeric protein was purified from Escherichia coli lysates and retained the critical GM1 ganglioside-binding activity of the CtxB moiety. Mice fed the CtxB-SREHP-12 fusion protein along with a subclinical dose of cholera toxin developed mucosal immunoglobulin A and immunoglobulin G and systemic antibody responses that recognized recombinant and native SREHP. Our study confirms the feasibility of inducing mucosal immune responses to immunogenic peptides by their genetic fusion to the CtxB subunit and identifies the CtxB-SREHP-12 chimeric protein as a candidate oral vaccine to prevent E. histolytica infection.
Identification of an immunologically important hypervariable domain of major outer surface protein A of Borrelia burgdorferi
Authors: McGrath B C , Dunn J J , Gorgone G , Guttman D , Dykhuizen D , Luft B J
Published: January 2020

The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent. The hypervariability of these solvent-exposed amino acid residues may contribute to the antigenic variation in OspA. To test this, we have performed site-directed mutagenesis to replace some of the potentially exposed amino acid side chains in the B31 protein with the analogous residues of a Borrelia garinii strain, K48. The altered proteins were then analyzed by Western blot (immunoblot) with monoclonal antibodies which bind OspA on the surface of the intact B31 spirochete. Our results indicate that specific amino acid changes near the tryptophan can abolish the reactivity of OspA to these monoclonal antibodies, which is an important consideration in the design of vaccines based on recombinant OspA.
Prolonged expression of lipopolysaccharide (LPS)-induced inflammatory genes in whole blood requires continual exposure to LPS
Authors: Dedrick R L , Conlon P J
Published: January 2020

Blood-borne lipopolysaccharide (LPS) is thought to be a major inducer of sepsis; however, it remains controversial whether an ongoing exposure to LPS is required to maintain the underlying systemic inflammatory response. To address this question, we have studied the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL-1 beta), and the procoagulant protein tissue factor induced by LPS ex vivo in whole human blood. The addition of a 1-ng/ml bolus of LPS to blood rapidly induced mRNA expression of all three genes. The mRNA levels peaked after 1 to 2 h, depending on the gene, and then declined to baseline after approximately 5 h. The decline in mRNA expression was not caused by a loss of responsiveness of the blood cells to LPS but rather correlated with the neutralization of LPS inflammatory activity by plasma components. Furthermore, administering a 1-ng/ml dose of LPS in six hourly aliquots of 167 pg/ml greatly prolonged the expression of mRNAs and induced a much greater release of TNF-alpha and IL-1 beta protein than did a single bolus. Dosing by repeated additions was more effective than a single bolus in inducing secretion of TNF-alpha and IL-1 beta at LPS levels of < or = 10 ng/ml, which corresponded to the LPS neutralization capacity of plasma. Finally, both mRNA expression and protein secretion induced by repeated administration of LPS were rapidly reversed by the addition of the LPS-neutralizing protein, bactericidal/permeability-increasing protein, even after several hours of stimulation. These results indicate that continuous or repeated exposure to LPS is required to maintain the expression of inflammatory genes and that the activated state is rapidly reversed with LPS neutralization.