bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen
Authors: Yang C , Zhu Y , Magee D M , Cox R A
Published: January 2020

Detection of anti-Coccidioides complement-fixing (CF) antibody is a valuable diagnostic and prognostic aid in coccidioidomycosis. The CF antibody response is directed against a heat-labile antigen that has chitinase activity, hereafter referred to as the CF/chitinase protein. To identify and clone this immunoreactive enzyme, we constructed a Coccidioides immitis cDNA lambda ZAP expression library from spherule RNA and detected fusion peptides expressing CF epitopes by immunoscreening. A cDNA clone consisting of 1,623 bp was identified, sequenced, and found to contain a single open reading frame that encodes a protein of 47 kDa with 427 amino acids. Deduced amino acid sequence analyses showed that the cloned CF/chitinase cDNA contains a 35-amino-acid region, beginning at Ser-18 and ending at and ending at Arg-52 which has 92% homology with the reported N-terminal amino acid sequence of authentic CF/chitinase protein. The first 17 amino acids in the deduced sequence of the cloned cDNA are not present on the mature CF/chitinase protein, suggesting that it may be a signal peptide. Expression of the CF/chitinase cDNA insert by using the pGEX-4T-3 vector yields a fusion peptide that bears CF-specific epitopes and shows chitinase activity. The CF/chitinase clone will enable large-scale production of the recombinant CF antigen for use in immunoassays and facilitate studies on the role of chitinase in the morphogenesis of C. immitis.
Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion
Authors: Rosenthal L A , Sutterwala F S , Kehrli M E , Mosser D M
Published: January 2020

It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to mediate the initial complement-dependent adhesion of metacyclic promastigotes to human monocyte-derived macrophages and that Mac-1 is the predominant complement receptor responsible for the phagocytosis of complement-opsonized metacyclic promastigotes.
Role of manganese superoxide dismutase in a mucoid isolate of Pseudomonas aeruginosa: adaptation to oxidative stress
Authors: Polack B , Dacheux D , Delic-Attree I , Toussaint B , Vignais P M
Published: January 2020

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is a leading cause of morbidity among cystic fibrosis (CF) patients. In the lungs of CF patients, the bacteria are exposed to activated oxygen species produced by the phagocytes of the host or resulting from the metabolism of oxygen. Two isoforms of superoxide dismutase are synthesized by P. aeruginosa; they differ by the metal present at their active site, which is either iron or manganese. To evaluate the role of manganese-containing superoxide dismutase (MnSOD), encoded by sodA, we have isolated a sodA mutant of the mucoid P. aeruginosa strain CHA isolated from the bronchopulmonary tract of a CF patient. The sodA mutant exhibited an increased sensitivity to oxidative stress generated by paraquat and was less resistant to oxidative stress in the stationary phase of growth compared with its parental strain. It was observed that MnSOD was expressed in the parental strain solely during the stationary phase of growth and that cells of the sodA mutant taken at the stationary phase resumed growth with a longer delay than the sodA+ cells when reinoculated in a new medium, especially in the presence of paraquat. These results suggest that MnSOD may participate in the adaptation of mucoid strains of P. aeruginosa to the stationary phase of growth in the lungs of CF patients.
Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae
Authors: Baumgart K W , McKenzie K R , Radford A J , Ramshaw I , Britton W J
Published: January 2020

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.
Haemophilus ducreyi hemolysin acts as a contact cytotoxin and damages human foreskin fibroblasts in cell culture
Authors: Alfa M J , DeGagne P , Totten P A
Published: January 2020

Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, produces several factors that damage human cells. We used isogenic mutants of H. ducreyi 35000 to demonstrate that the hemolytic activity and the cytotoxic effect of H. ducreyi on human foreskin fibroblasts are due to the same toxin.
Interleukin-12-mediated resistance to Trypanosoma cruzi is dependent on tumor necrosis factor alpha and gamma interferon
Authors: Hunter C A , Slifer T , Araujo F
Published: January 2020

The aim of this study was to determine if interleukin-12 (IL-12) has a role in the immune response to Trypanosoma cruzi. Infection of BALB/c mice with the virulent Tulahuen strain of T. cruzi is characterized by a high-level parasitemia, pathology in the heart associated with the presence of amastigotes, and death during the acute phase of the disease. Administration of IL-12 to BALB/c mice infected with T. cruzi resulted in a reduced parasitemia and a significant delay in the time to death compared with those for infected controls. This protective effect was correlated with increased levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in serum. To determine if these cytokines were involved in the protective effects of IL-12, we treated infected mice with IL-12 alone or in combination with monoclonal antibodies specific for IFN-gamma or TNF-alpha. These antibodies antagonized the protective effect of exogenous IL-12. Treatment of infected mice with a polygonal antibody specific for IL-12 resulted in a significant increase in parasitemia but did not affect the time to death. These latter studies demonstrate a role for endogenous IL-12 in resistance to T. cruzi. Together, our data identify an IL-12-mediated mechanism of resistance to T. cruzi, which is dependent on IFN-gamma and TNF-alpha.
Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells
Authors: Meier C , Oelschlaeger T A , Merkert H , Korhonen T K , Hacker J
Published: January 2020

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.
Characterization of T cells that confer a high degree of protective immunity against tuberculosis in mice after vaccination with tumor cells expressing mycobacterial hsp65
Authors: Silva C L , Silva M F , Pietro R C , Lowrie D B
Published: January 2020

Mice vaccinated by injection with tumor cells expressing the Mycobacterium leprae gene for hsp65 acquire a remarkably high degree of protection against challenge with Mycobacterium tuberculosis. We used limiting-dilution analysis to assess the frequency of CD4+ CD8- and CD4- CD8+ splenocytes responding to mycobacterial hsp65 in such vaccinated mice. Cells of both phenotypes were present at very high and equal frequencies (approximately 1:100). Vaccination with live Mycobacterium bovis BCG also increased the frequencies of both phenotypes of hsp65-reactive cells equally (to approximately 1:2,500), whereas vaccination procedures that were not protective, with either dead BCG, hsp65 protein in incomplete Freund's adjuvant, or hsp65 mixed with tumor cells, resulted in preferential increase in CD4+ CD8- cells. Twelve CD4+ CD8- and twelve CD4- CD8+ hsp65-responsive T-cell clones were obtained and characterized. All showed conventional antigen recognition via major histocompatibility complex class II and class I pathways but differed in secretion of gamma interferon and interleukin 4 and cytotoxicity. In tests of antimycobacterial activity against M. tuberculosis, both in infected macrophages in vitro and by adoptive transfer of protection with T-cell clones injected into irradiated mice, the most effective clones were the most cytotoxic and secretion of gamma interferon made only a secondary contribution.
Determinants of activation by complement of group II phospholipase A2 acting against Escherichia coli
Authors: Madsen L M , Inada M , Weiss J
Published: January 2020

Prompt killing of many strains of Escherichia coli during phagocytosis in vitro by isolated polymorphonuclear leukocytes (PMN) requires the presence of nonlethal doses of nonimmune serum (B. A. Mannion, J. Weiss, and P. Elsbach, J. Clin. Invest. 86:631-641, 1990). Because this requirement is bypassed in a phospholipase A (PLA)-rich mutant (pldA ) of E. coli, we have examined the effect of serum on bacteria] phospholipid (PL) degradation during phagocytosis of wild-type (pldA+) and PLA-deficient (pldA) E. coli. In parallel with increased killing, nonlethal doses of serum increased the degradation of prelabeled bacterial PL during phagocytosis by two- to fivefold, to nearly the same levels (ca. 50 to 60%) as those produced during phagocytosis of E. coli pldA in the absence of serum. The effects on the E. coli pldA mutant imply that there is a serum-mediated enhancement of granule-associated group II PMN PLA2 activity. At the same doses, serum promoted action against E. coli in the presence of purified rabbit and human group II PLA2 but did not activate bacterial PLA. Related PLA2s that lack specific structural determinants needed for optimal activity against E. coli treated with the bactericidal/permeability-increasing protein (BPI) of PMN are also less active than wild-type group II PLA2 against serum-treated E. coli. Treatment of E. coli with C7- or C9-depleted serum did not enhance bacterial killing or PL degradation during phagocytosis or the action of purified PLA2. In summary, these findings suggest that (i) nonlethal assemblies of the membrane attack complex promote intracellular killing and destruction of E. coli ingested by PMN, in part by promoting the action of granule-associated PLA2 against ingested bacteria, and (ii) structural determinants first implicated in PLA2 action against BPI-treated E. coli are also important in PLA2 action in concert with other host defense systems, such as complement.
Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products
Authors: Curtis M A , Aduse-Opoku J , Slaney J M , Rangarajan M , Booth V , Cridland J , Shepherd P
Published: January 2020

This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface. The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus. Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT. Western blot (immunoblot) analysis of P. gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000. Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P. gingivalis DNA. These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P. gingivalis in periodontal disease.
Influence of adjuvants on protection induced by a recombinant fusion protein against malarial infection
Authors: Daly T M , Long C A
Published: January 2020

Previously, we described a protective immune response induced by the carboxyl-terminal region of the merozoite surface protein-1 (MSP-1) from the rodent malarial parasite Plasmodium yoelii yoelii 17XL, expressed as a fusion protein and designated glutathione S-transferase (GST)-PYC2. We also demonstrated that the humoral response induced by GST-PYC2 was the primary mechanism by which immunized animals controlled their blood-stage infections. We have now examined the influence of several adjuvants on the immune response to the GST-PYC2 fusion protein. While alum, Freund's adjuvant, Ribi adjuvant system, and TiterMax were efficacious in eliciting a protective response with GST-PYC2 in BALB/c mice, saponin failed to induce protection, although significant levels of PYC2-specific antibodies were produced in all immunized animals. This protection depended on the mouse strain since immunization of Swiss Webster mice with GST-PYC2 in alum did not produce levels of PYC2-specific antibodies comparable to those in BALB/c mice nor did it induce any demonstrable level of protection against parasite challenge. Swiss Webster mice were protected, however, when immunized with GST-PYC2 in other adjuvants. Immunization with PYC2, isolated free of GST induced lower levels of antigen-specific antibody; only those animals given PYC2 in Freund's adjuvant demonstrated a significant degree of protection, suggesting the possibility (of additional cellular effector mechanisms. These findings demonstrate that adjuvant, host genotype, and the fine specificity of the response significantly influence the protection induced by the carboxyl terminus of MSP-1 in vivo and illustrate the need to consider these factors in evaluating MSP-1 as a vaccine component.
A Babesia bovis 225-kilodalton spherical-body protein: localization to the cytoplasmic face of infected erythrocytes after merozoite invasion
Authors: Dowling S C , Perryman L E , Jasmer D P
Published: January 2020

A 225-kDa Babesia bovis protein occurs on the cytoplasmic side of infected-erythrocyte membranes. Here it is demonstrated that the 225-kDa protein localizes to spherical-body organelles of merozoites. Organelles consistent in size and shape with spherical bodies were isolated between 1.17 and 1.21 g/cm(3) in a sucrose density gradient. Organelles consistent with rhoptries and micronemes were also present in fractions from 1.17 to 1.19 g/cm(3). Antisera generated by immunizing mice with the fraction (1.20 to 1.21 g/cm(3)) most enriched for spherical bodies reacted predominantly with spherical bodies in B. bovis merozoites. A monoclonal antibody generated from this immunization (70/97.14) recognized an epitope that occurs in the repeat region of the 225-kDa protein (now referred to as SBP2). Monoclonal antibody 70/97.14 bound to merozoite spherical bodies, vesicles in infected-host cytoplasm, and the cytoplasmic face of the infected-erythrocyte membrane. These results indicate that spherical-body proteins become associated with the host membrane via transport through the erythrocyte cytoplasm after intracellular invasion.
Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli
Authors: Valvatne H , Sommerfelt H , Gaastra W , Bhan M K , Grewal H M
Published: January 2020

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.
Molecular basis of size and antigenic variation of a Mycoplasma hominis adhesin encoded by divergent vaa genes
Authors: Zhang Q , Wise K S
Published: January 2020

The molecular basis for the size and antigenic diversity of the variable adherence-associated (Vaa) antigen, a major surface protein and a putative adhesin (of Mycoplasma hominis, is described. Size-variant alleles of the single-copy vaa gene encode abundant surface lipoproteins containing one to four nearly identical, tandem repetitive units of 121 amino acids in the central region of the mature Vaa product. Gain or loss of central repeats in vaa genes gives rise to distinct size-variant Vaa antigens in clonal populations of this organism. The N-terminal and repeat regions of Vaa contain highly conserved sequences, while the C-terminal region, implicated as the adherence-mediating module, is highly variable and divergent among different strains of this pathogen. Sequence variation in this region may underlie the strain-dependent binding of some monoclonal antibodies to Vaa products. The Vaa antigen is expressed in vivo during chronic, active arthritis associated with M. hominis infection and is highly immunogenic in the human host. Size variation and C-terminal antigenic divergence of Vaa could affect the adherence of M. hominis and evasion of antibody-mediated immunity, thereby contributing to the organism's adaptive capability in the human host. Variation in vaa genes reveals a distinct pattern of mutations generating mycoplasma surface variation.
Characterization of the acid resistance phenotype and rpoS alleles of shiga-like toxin-producing Escherichia coli
Authors: Waterman S R , Small P L
Published: January 2020

Shiga-like toxin-producing Escherichia coli (SLTEC) strains are an important group of enteric pathogens. In this study we have examined the abilities of 58 SLTEC isolates to survive at pH 2.5 and found 13 of these isolates to be defective in acid resistance. Introduction of rpoS on a plasmid conferred acid resistance to the majority of the acid-sensitive isolates. The rpoS genes from two of these isolates were sequenced; both isolates contained lesions in the rpoS gene resulting in a nonfunctional RpoS. These results show that mutant rpoS alleles exist in natural populations of E. coli. Such mutations may play an important role in determining the infective dose of SLTEC and suggest that isolates may vary in infectivity.
T- and B-lymphocyte-independent formation of alveolar macrophage-derived multinucleated giant cells in murine Pneumocystis carinii pneumonia
Authors: Hanano R , Reifenberg K , Kaufmann S H
Published: January 2020

Multinucleated giant cells developed in Pneumocystis carinii-diseased gene disruption mutant mice deficient in major histocompatibility complex class II molecules, T-cell receptor alpha beta cells, or all mature T and B lymphocytes. These findings demonstrate lymphocyte-independent fusion of alveolar macrophages under morbid conditions. Pulmonary parasite burden seems to be a decisive factor in multinucleation.
Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharide-flagellin protein conjugates
Authors: Brett P J , Woods D E
Published: January 2020

The O-polysaccharide moiety of Burkholderia pseudomallei 319a lipopolysaccharide was covalently linked to flagellin protein isolated from the same strain. A glycoconjugate incorporating adipic acid dihydrazide as a spacer molecule elicited high-titer immunoglobulin G responses to both the protein and carbohydrate components of the construct. This immunoglobulin G was capable of protecting diabetic rats from challenge with a heterologous B. pseudomallei strain.
Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA
Authors: Ricci V , Ciacci C , Zarrilli R , Sommi P , Tummuru M K , Del Vecchio Blanco C , Bruni C B , Cover T L , Blaser M J , Romano M
Published: January 2020

Helicobacter pylori infection is associated with inflammation of the gastric mucosa and with gastric mucosal damage. In this study, we sought to test the hypothesis that two H. pylori virulence factors (VacA and CagA) impair gastric epithelial cell migration and proliferation, the main processes involved in gastric mucosal healing in vivo. Human gastric epithelial cells (MKN 28) were incubated with undialyzed or dialyzed broth culture filtrates from wild-type H. pylori strains or isogenic mutants defective in production of VacA, CagA, or both products. We found that (i) VacA specifically inhibited cell proliferation without affecting cell migration, (ii) CagA exerted no effect on either cell migration or proliferation, and (iii) undialyzed H. pylori broth culture filtrates inhibited both cell migration and proliferation through a VacA- and CagA-independent mechanism. These findings demonstrate that, in addition to damaging the gastric mucosa, H. pylori products may also impair physiological processes required for mucosal repair.
Entry of Borrelia burgdorferi into macrophages is end-on and leads to degradation in lysosomes
Authors: Montgomery R R , Malawista S E
Published: January 2020

The Lyme disease spirochete, Borrelia burgdorferi, is ingested rapidly by mouse macrophages in vitro. Spirochetes attach by their ends and become progressively coiled as they move deeper into cells. From the earliest measurements, spirochetes colocalize with a marker of endosomes and lysosomes, and degradation of spirochetes occurs within lysosomes.
The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes
Authors: Mathers D A , Leung W K , Fenno J C , Hong Y , McBride B C
Published: January 2020

The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.
Th1-like antifilarial immune responses predominate in antigen-negative persons
Authors: Dimock K A , Eberhard M L , Lammie P J
Published: January 2020

To characterize immune responses associated with the putatively immune state in bancroftian filariasis (that is, both microfilaria and antigen free), humoral and cellular responses were compared among antigen- and microfilaria-negative, antigen-positive and microfilaria-negative, and microfilaria-positive individuals. Antifilarial isotype levels were measured by enzyme-linked immunosorbent assay. Peripheral blood mononuclear cell responses were measured by proliferation, by bioassay for interleukin-2 (IL-2) and IL-10, and by reverse transcription-PCR for IL-4, IL-5, and gamma interferon. The absence of circulating filarial antigen was associated with Th1-like responses, including significantly higher proliferative (P < 0.001) and IL-2 (P = 0.008) responses and a higher prevalence of gamma interferon (0.02 < P < 0.1) responses. Significantly elevated antifilarial immunoglobulin G4 (IgG4) levels (P = 0.0035) were associated with antigenemia, whereas microfilaremia was associated with significantly decreased antifilarial IgG2 levels (P = 0.0014). IL-4 mRNA levels were not significantly different among the three groups; however, there was a subpopulation of microfilaremic individuals who did not make detectable levels of IL-4 mRNA and who produced low antifilarial IgG4 levels compared with those of individuals who had detectable levels of IL-4 mRNA. IL-5 mRNA levels also were not significantly different among groups; however, more microfilaremic individuals produced IL-5 mRNA in response to adult filarial antigens, and total parasite-specific IL-4 and IL-5 mRNA levels were significantly correlated (P = 0.05). Although longitudinal data are not currently available, the elevated Th1-like responses in antigen- and microfilaria-negative individuals are consistent with the hypothesis that these responses contribute to protection in putatively immune individuals.
Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1
Authors: Ikigai H , Akatsuka A , Tsujiyama H , Nakae T , Shimamura T
Published: January 2020

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.
Identification and characterization of the Yersinia enterocolitica gsrA gene, which protectively responds to intracellular stress induced by macrophage phagocytosis and to extracellular environmental stress
Authors: Yamamoto T , Hanawa T , Ogata S , Kamiya S
Published: January 2020

Yersinia enterocolitica is able to resist the microbicidal mechanisms of macrophages and to grow within phagocytic cells. Some bacteria including Y. enterocolitica have been shown to respond to the hostile environment in macrophages by producing a set of stress proteins which are also induced by environmental stresses. To understand the role of stress proteins in intracellular survival of bacteria, we identified and cloned a Y. enterocolitica gene, called gsrA (global stress requirement). The gsrA gene was identified because its insertional inactivation by a transposon resulted in the inability of the organism to grow at an elevated temperature and to survive within macrophages after phagocytosis. The gsrA gene was sequenced and shown to encode a basic, 49,500-Da protein. The GsrA protein shows significant amino acid sequence homology to the HtrA stress protein which was originally identified in Escherichia coli. Furthermore, the genetically defined Y. enterocolitica gsrA mutant was constructed and characterized. The insertional mutation of gsrA resulted in inhibition of growth at temperatures above 39 degrees C and greatly increased susceptibility to oxidative and osmotic stresses. The mutant additionally lost the ability to survive and replicate within macrophages. These results, taken together, indicate that the gsrA gene is an essential component of the protection mechanism employed by Y. enterocolitica, allowing it to respond to the intracellular stress in macrophages as well as extracellular environmental stress.
Polysaccharide side chains are not required for attaching and effacing adhesion of Escherichia coli O157:H7
Authors: Cockerill F , Beebakhee G , Soni R , Sherman P
Published: January 2020

Escherichia coli of the serotype O157:H7 is an enterohemorrhagic human pathogen which demonstrates attaching and effacing adhesion to colonocytes in vivo and to epithelial cells grown in tissue culture. Transposon TnphoA mutants of E. coli O157:H7 strain CL-8 were produced. Two of 300 alkaline phosphatase positive mutants, designated JB6 and JB27, did not express O157 side chains as assessed by agglutination with specific polyclonal O157 antiserum, silver staining of lipopolysaccharide extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblots with polyclonal O157-specific antiserum. Both O157-negative mutants and the parent strain demonstrated localized adherence to HEp-2 cells when examined by Giemsa staining and bright-field microscopy. Furthermore, both O157-negative mutants showed enhanced adherence to HEp-2 cells compared with the parent strain when assessed by quantification of adherent bacterial CFUs. The parent strain, CL-8, and both of the mutants produced fluorescent foci when adherent bacteria and HEp-2 cells were stained with fluorescein isothiocyanate-labelled phalloidin. Transmission electron microscopy confirmed attaching and effacing adherence of strain CL-8 and the OO7-negative mutants to HEp-2 cells. These findings indicate that mutants deficient in O157 polysaccharide repeats exhibit adherence to tissue culture cells in vitro and that O157 polysaccharide repeats are not required to produce the attaching and effacing lesion.
Interleukin-6 is required for a protective immune response to systemic Escherichia coli infection
Authors: Dalrymple S A , Slattery R , Aud D M , Krishna M , Lucian L A , Murray R
Published: January 2020

Interleukin-6 (IL-6) is a multipotential cytokine detected in the serum of patients or experimental animals undergoing bacterial sepsis. To date, the role of IL-6 in gram-negative sepsis models has been controversial. We have used IL-6-deficient mice to investigate the role of IL-6 during virulent Escherichia coli infection and in lipopolysaccharide (LPS)-induced mortality. In this report we describe an increased susceptibility of IL-6-deficient mice to E. coli infection in terms of mortality and accumulation of viable bacteria in tissues, indicating a protective role for IL-6 during the immune response against E. coli. In contrast, mortality rates of IL-6-deficient mice and control animals undergoing LPS-induced shock did not differ, indicating that IL-6 was inconsequential for survival in this model. Furthermore, we have shown that neutrophils were crucial for resistance to E. coli in normal mice. IL-6-deficient mice were unable to efficiently induce neutrophilia in the bloodstream immediately following challenge with E. coli, in contrast to a characteristic neutrophilia induced in control animals. Prophylactic treatment of the mutant animals with recombinant IL-6 protein reverted both the deficit of neutrophilia and the accumulation of bacteria in tissues. These data clarify the role of IL-6 as protective in virulent E. coli infection and suggest that the protective effect may be at least partially mediated through neutrophils.
Roles for tumor necrosis factor alpha and nitric oxide in resistance of rat alveolar macrophages to Legionella pneumophila
Authors: Skerrett S J , Martin T R
Published: January 2020

Legionella pneumophila is an intracellular parasite of alveolar macrophages, and recovery from legionellosis is associated with activation of alveolar macrophages to resist intracellular bacterial replication. Gamma interferon (IFN-gamma) is known to activate alveolar macrophages to suppress L. pneumophila, but the role of macrophage-derived cytokines in modulating alveolar macrophage resistance is unknown. To test the hypothesis that macrophage-derived mediators contribute to the resistance of alveolar macrophages to L. pneumophila, we incubated adherent rat alveolar macrophages with Escherichia coli lipopolysaccharide (LPS), recombinant tumor necrosis factor alpha (TNF-alpha), recombinant IFN-gamma, neutralizing anti-TNF-alpha, and/or N(G)-monomethyl-L-arginine (L-NMMA) for 6 h before challenge with L. pneumophila. Monolayers were sonically disrupted and quantitatively cultured on successive days. We also measured bioactive TNF-alpha release by infected macrophages in the presence or absence of IFN-gamma. We found that pretreatment of alveolar macrophages with LPS or, to a lesser degree, TNF-alpha, significantly inhibited intracellular replication of L. pneumophila. Both LPS and TNF-alpha acted synergistically with IFN-gamma at less than the maximally activating concentration to suppress L. pneumophila growth. The independent and coactivating effects of LPS were blocked by anti-TNF-alpha. Killing of L. pneumophila by IFN-gamma at the maximally activating concentration was inhibited by anti-TNF-alpha. The synergistic effects of TNF-alpha. or LPS in combination with IFN-gamma were inhibited by L-NMMA. Infected alveolar macrophages secreted TNF-alpha in proportion to the bacterial inoculum, and secretion of TNF-alpha was potentiated by cocultivation with IFN-gamma. These data indicate that secretion of TNF-alpha is an important autocrine defense mechanism of alveolar macrophages, serving to potentiate the activating effects of IFN-gamma through costimulation of nitric oxide synthesis.
Increased susceptibility to primary infection with Listeria monocytogenes in germfree mice may be due to lack of accumulation of L-selectin+ CD44+ T cells in sites of inflammation
Authors: Inagaki H , Suzuki T , Nomoto K , Yoshikai Y
Published: January 2020

The host defense of germfree (GF) mice against primary infection with Listeria monocytogenes was compared with that of specific-pathogen-free (SPF) mice. In SPF mice, the numbers of bacteria in the peritoneal cavity, liver, and spleen decreased gradually to undetectable levels by day 8 after intraperitoneal infection with a sublethal dose (2 X 10(3) CFU) of L. monocytogenes. On the other hand, the elimination of bacteria in these organs of GF mice was significantly impaired at this stage after inoculation. We have reported previously that T cells coexpressing L-selectin and CD44 play an important role in protection against L. monocytogenes through trafficking to sites of inflammation. Consistent with our previous findings, the number of unique L-selectin+ CD44+ T cells in the peritoneal cavity was remarkably increased on day 8 after infection in SPF mice, whereas such an increase was not evident in GF mice at this stage. Listeria-specific T-cell proliferation was normally detected in the lymph node cells of GF mice inoculated with L. monocytogenes, whereas the T-cell-proliferative response of the peritoneal exudate cells of GF mice was significantly impaired compared with that of SPF mice. These results suggest that the priming of T cells against listerial antigens normally occurs in the peripheral lymphoid organs of GF mice but the trafficking of the activated T cells to the inflamed sites may be severely impaired in GF mice, resulting in increased susceptibility to infection with L. monocytogenes.
Minimal requirements for murine resistance to infection with Francisella tularensis LVS
Authors: Elkins K L , Rhinehart-Jones T R , Culkin S J , Yee D , Winegar R K
Published: January 2020

Intraperitoneal or intravenous infection of mice with Francisella tularensis LVS is lethal, with an intraperitoneal 50% lethal dose (LD50) approaching a single bacterium. Intradermal (i.d.) LVS infection has a much higher LD50, about 10(6) bacteria in BALB/cByJ mice, and survival of i.d. infection leads to solid generation of immunity against lethal challenge. To define the minimal requirements for both initial and long-term survival of i.d. infection, we characterized the nature of i.d. LVS infection in lymphocyte-deficient BALB/cByJ.scid (scid) mice. scid mice infected i.d. with strain LVS survived for about 20 days and then died from overwhelming disseminated infection. However, scid mice treated with monoclonal antibodies to gamma interferon, tumor necrosis factor alpha, or neutrophils-granulocytes all died within 1 week of infection, indicating that these were essential for early control of infection. Studies using GKO (gamma interferon knockout) mice emphasized that gamma interferon is absolutely required for initial survival of i.d. LVS infection. scid mice could be reconstituted for long-term survival of i.d. LVS infection and clearance of bacteria by intravenous transfer of splenic lymphocytes or purified B220-/T+ lymphocytes but not nu/nu lymphocytes. T cells are therefore required for long-term clearance and survival of i.d. LVS infection; efforts to determine whether CD4+ T cells, CD8+ T cells, or both are involved are ongoing.
The Cu,Zn superoxide dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus: purification and biochemical comparison with the Aspergillus fumigatus Cu,Zn superoxide dismutase
Authors: Holdom M D , Hay R J , Hamilton A J
Published: January 2020

Cu,Zn superoxide dismutases (SODs) have been purified to homogeneity from Aspergillus flavus and A. niger, which are significant causative agents of aspergillosis, and from A. nidulans and A. terreus, which are much rarer causative agents of disease, using a combination of isoelectric focusing and gel filtration fast protein liquid chromatography. The purified enzymes have been compared with the previously described SOD from the most important pathogen in the genus, A. fumigatus (M. D. Holdom, R. J. Hay, and A. J. Hamilton, Free Radical Res. 22:519-531, 1995). The N-terminal amino acid sequences of the four newly purified enzymes were almost identical and demonstrated homology to known Cu,Zn SODs from a range of organisms including that from the previously described SOD from A. fumigatus. SOD activity was detectable in the culture filtrates of all species, and intracellular Cu,Zn SOD activity as a proportion of total protein was highest in early-log-phase cultures. The specific activities of the purified enzymes were similar, and all four of the newly described enzymes were inhibited by potassium cyanide and diethyldithiocarbamate, known Cu,Zn SOD inhibitors. Sodium azide and o-phenanthroline demonstrated inhibition at concentrations from 5 to 30 mM, and EDTA also exhibited a varying degree of inhibition of SOD activity. However, there were differences in the nonreduced molecular masses, the reduced molecular masses, and the isoelectric points of the four newly described SODs and the A. fumigatus enzyme; these varied from 55 to 123 kDa, 17.5 to 19.5 kDa, and 5.0 to 5.9, respectively. Of particular note was the observation that the A. fumigatus enzyme was thermostable compared with the SODs from the other species; in addition, the A.fiumigatus enzyme retained all of its activity at 37 degrees C relative to 20 degrees C, whereas the SODs of A. nidulans and A. terreus lost significant activity at the higher temperature. Aspergillus Cu,Zn SOD plays a hypothetical role in the avoidance of oxidative killing mechanisms, and our data suggest that the thermotolerant A. fumigatus Cu,Zn SOD would be more effective in such a protective system than, for example, the equivalent enzyme from the more rarely pathogenic A. nidulans.
Stimulation of the ceramide pathway partially mimics lipopolysaccharide-induced responses in murine peritoneal macrophages
Authors: Barber S A , Detore G , McNally R , Vogel S N
Published: January 2020

Recent studies have suggested that lipolysaccharide (LPS) stimulates cells by mimicking the second-messenger function of ceramide, a lipid generated in the cell by the action of sphingomyelinase (SMase). To examine this possibility further, we compared the abilities of LPS, SMase, and/or ceramide analogs to induce cytokine secretion, modulate gene expression, and induce endotoxin tolerance in macrophages. SMase and LPS induced secretion of tumor necrosis factor alpha (TNF-alpha) to comparable degrees; however, unlike LPS, SMase failed to stimulate detectable interferon activity. Cell-permeable analogs of ceramide induced the expression of many LPS-inducible genes; however, the expression of interferon-inducible protein 10 (IP-10) and interferon consensus sequence-binding protein (ICSBP) mRNAs was significantly lower than that induced by LPS. Both SMase-induced TNF-alpha secretion and LPS-induced TNF-alpha secretion were inhibited by pretreatment with a serine/threonine phosphatase inhibitor, calyculin A. Macrophages preexposed in vitro to LPS to induce a well-characterized state of endotoxin tolerance secreted little or no TNF-alpha upon secondary challenge with either LPS or SMase, whereas macrophages preexposed to SMase secreted high levels of TNF-alpha upon secondary stimulation with LPS or SMase. Collectively, these results suggest that ceramide activates a subset of LPS-induced signaling pathways in murine peritoneal exudate macrophages.