bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium
Authors: Miller S I , Pulkkinen W S , Selsted M E , Mekalanos J J
Published: January 2020

The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages.
Use of transposon Tn916 to inactivate and isolate a mutacin-associated gene from Streptococcus mutans
Authors: Caufield P W , Shah G R , Hollingshead S K
Published: January 2020

Among the attributes thought to contribute to the virulence of Streptococcus mutans is its ability to elaborate bacteriocinlike substances, which may provide a selective force enhancing its colonization potential. One such inhibitory substance, mutacin II, is produced by certain plasmid-containing strains of S. mutans. We introduced insertional mutations into a mutacin II-producing strain of S. mutans (UA96) by transformation with a plasmid carrying Tn916, resulting in transformants bearing single inserts of the transposon at different sites within the chromosome. The insertions identify five different EcoRI fragments required for production of mutacin II (Bac phenotype; bac-1 to bac-5 genotypes). The EcoRI fragments, containing bac-1::Tn916 was ligated into a cosmid vector, pJC74, and transduced into Escherichia coli DH1, where Tn916 is known to be unstable. The loss of Tn916 resulted in a 30-kb plasmid, pPC974, containing approximately 15 kb of S. mutans DNA. A Bac-associated DNA fragment was then subcloned into the streptococcus-E. coli shuttle vector pVA838 and transformed into S. mutants, where it was capable of complementing the bac mutation in the Bac- parent. These findings suggest that we have isolated at least one gene associated with mutacin production.
An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin
Authors: Schiavo G , Papini E , Genna G , Montecucco C
Published: January 2020

Tetanus toxin is composed of a heavy chain (100 kDa) and a light chain (50 kDa) held together by a single interchain disulfide bridge. An additional intrachain disulfide is present in the carboxy-terminal part of the heavy chain. Reduction of the two disulfide bonds in tetanus toxin with both chemical and proteinaceous reducing agents was studied. Dithiothreitol and 2-mercaptoethanol cleaved both the inter- and intrachain disulfide bridges of the toxin, while glutathione and cysteine were ineffective. Specific reduction of the single interchain disulfide link was achieved with the thioredoxin-thioredoxin reductase system, thus indicating that this bond is exposed at the protein surface. Also, dead or permeabilized cells were able to reduce the toxin. Such reduced toxin bound to neuronal membranes as well as the native toxin but was not neurotoxic. These findings open the possibility that reduction by cytoplasmic agents released by dead cells contributes to detoxification of tetanus toxin. Moreover, together with the notion that the light chain is the active form of the toxin in the cytoplasm, these results suggest that the interchain disulfide bond of tetanus toxin plays a role in nerve cell penetration.
Effects of recombinant human interleukin-1 beta on accumulation of inflammatory peritoneal macrophages in mice treated with pertussis toxin
Authors: Torre D , Speranza F , Pugliese A , Tambini R
Published: January 2020

In this study we report that treatment with recombinant human interleukin-1 beta (rIL-1 beta) (10 U per mouse, intraperitoneally) significantly increased the number of inflammatory macrophages in the peritoneal cavity of mice treated with pertussis toxin (PT) (1 micrograms per mouse, intravenously). The administration of rIL-1 beta in a single intraperitoneal dose (10 U per mouse) 1 or 2 days before challenge with PT did not prevent the decrease in the number of inflammatory macrophages in the peritoneal cavity of mice. On the other hand, the simultaneous administration of rIL-1 beta and PT, as well as the administration of rIL-1 beta 24 h after injection of PT, significantly counteracted the inhibitory effect of PT on inflammatory peritoneal macrophages.
Protection of mice against the lethal toxicity of a lipopolysaccharide (LPS) by immunization with anti-idiotype antibody to a monoclonal antibody to lipid A from Eikenella corrodens LPS
Authors: Kato T , Takazoe I , Okuda K
Published: January 2020

We produced anti-idiotype antibodies to antibody to lipid A from Eikenella corrodens. The ALA-1 monoclonal antibody (immunoglobulin M [IgM] isotype), which had already been produced in our laboratory (T. Kato, I. Takazoe, and K. Okuda, Infect. Immun. 57:656-659, 1989), had reacted strongly with lipid A from E. corrodens, Escherichia coli, and Salmonella minnesota. Four anti-idiotype monoclonal antibodies to ALA-1 (Ab1), designated A2LA-1 (IgG1 isotype), A2LA-2 (IgG2a isotype), A2LA-3 (IgG2a isotype), and A2LA-4 (IgG3 isotype), which recognized the idiotype Ab1, were produced. A2LA-1, A2LA-2, and A2LA-3 were capable of over 61% inhibition of ALA-1 reactivity to E. coli J5 lipid A in an enzyme-linked immunosorbent assay system. The sera of mice and rabbits immunized with the anti-idiotype antibodies revealed that the internal image anti-idiotype antibody induced the production of IgG antibodies that cross-reacted with or bound to lipid A. These studies indicate that A2LA-1 and A2LA-2 contained an antigenic epitope that mimicked lipid A. Immunization of mice with A2LA-1 resulted in prevention of lethal toxicity from E. coli J5 lipopolysaccharide.
Restriction fragment length polymorphisms among uropathogenic Escherichia coli isolates: pap-related sequences compared with rrn operons
Authors: Arthur M , Arbeit R D , Kim C , Beltran P , Crowe H , Steinbach S , Campanelli C , Wilson R A , Selander R K , Goldstein R
Published: January 2020

Among the adhesin-encoding virulence operons associated with uropathogenic Escherichia coli, only pap (pyelonephritis-associated pilus)-related gene clusters typically exhibit variation in their structure and chromosomal copy number. To access further such variability, we compared pap restriction fragment length polymorphisms (RFLPs) with those detected among rRNA (rrn) operons, which encode an essential host function unrelated to virulence. To place such findings in a phylogenetic perspective, the E. coli isolates were also characterized by using multilocus enzyme electrophoresis. Variation in the rrn RFLP profiles correlated with evolutionary divergence resolved by multilocus enzyme electrophoresis; isolates with identical rrn profiles represented the same or closely related electrophoretic types. In contrast, such isolates frequently had different pap-related RFLPs, indicating that these genetic variations have developed recently relative to the changes associated with essential rrn operons or metabolic enzymes. Despite such fluctuations, two lines of evidence indicate conditions under which the pap-related RFLPs can be stably maintained. First, for each of 20 patients with urosepsis, both the primary urinary tract isolate and the concurrent blood isolate were identical. Second, although obtained from different patients, some isolates representing the same electrophoretic type also had identical pap-related RFLPs. Thus, the genotypic diversity of this virulence adhesin operon was not generated during the course of acute infection or during laboratory manipulations. Since fecal E. coli isolates frequently carry chromosomally encoded pap-related gene clusters, these findings suggest that the intra- and interchromosomal recombination events generating the polymorphisms associated with the pap-related sequences likely occur among the E. coli of the commensal reservoir.
Cloning and expression of the gene for the cross-reactive alpha antigen of Mycobacterium kansasii
Authors: Matsuo K , Yamaguchi R , Yamazaki A , Tasaka H , Terasaka K , Yamada T
Published: January 2020

The gene for the extracellular alpha antigen of Mycobacterium kansasii was cloned by using the alpha-antigen gene fragments of M. bovis BCG as probes. Gene analysis revealed that this gene encodes 325 amino acid residues, including 40 amino acids for the signal peptide, followed by 285 amino acids for the mature protein. A comparison of the nucleotide sequences of the genes isolated from these two mycobacterial species showed that the levels of DNA and amino acid homology were 84.8 and 89.1%, respectively. The hydropathy profiles were also compared, and two highly changed hydrophilic regions were observed, which might account for the antigenic diversity of this antigen or its acquirement of antigenic specificity.
Stimulation of oxidative burst in human monocytes by lipoteichoic acids
Authors: Levy R , Kotb M , Nagauker O , Majumdar G , Alkan M , Ofek I , Beachey E H
Published: January 2020

Lipoteichoic acid isolated from Streptococcus faecalis or Streptococcus pyogenes caused direct activation of the respiratory burst in human peripheral blood monocytes. This activity appears to be related to the ability of lipoteichoic acid to bind to the monocyte membrane and trigger the polarization of receptors (capping).
Immunocytochemical localization of neuraminidase in Trypanosoma cruzi
Authors: Souto-Padrón T , Harth G , de Souza W
Published: January 2020

A polyclonal antibody obtained against neuraminidase purified from Trypanosoma cruzi was used for the localization of the protein in whole cells by immunofluorescence microscopy and in thin sections of parasites (epimastigote, amastigote, and trypomastigote forms) embedded at a low temperature in Lowicryl K4M resin. The intensity of labeling, as evaluated by the number of gold particles associated with the parasite, varied according to the protozoan developmental stage. In the noninfective epimastigote forms, labeling of the cell surface was very weak. However, an intense labeling of some cytoplasmic vacuoles was observed. Labeling of the surfaces of most of the trypomastigote forms was weak, while gold particles were seen in association with the flagellar pockets of these forms, which suggests that the enzyme is secreted through this region. Intense labeling of the surfaces of many, but not all, transition forms between trypomastigote and amastigote forms was observed. Amastigote forms found in the supernatant of infected cell cultures had their surfaces intensely labeled, while few particles were seen on the surfaces of intracellular amastigotes. The results obtained are discussed in relation to the role played by T. cruzi neuraminidase in the process of parasite-host cell interaction.
Identification of an immunostimulating protein from Mycobacterium leprae
Authors: Mohagheghpour N , Munn M W , Gelber R H , Engleman E G
Published: January 2020

Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.
Effective vaccination of mice against leprosy bacilli with subunits of Mycobacterium leprae
Authors: Gelber R H , Brennan P J , Hunter S W , Munn M W , Monson J M , Murray L P , Siu P , Tsang M , Engleman E G , Mohagheghpour N
Published: January 2020

Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.
Construction and characterization in vivo of Bordetella pertussis aroA mutants
Authors: Roberts M , Maskell D , Novotny P , Dougan G
Published: January 2020

A DNA fragment encoding a kanamycin resistance determinant was used to insertionally inactivate the cloned aroA gene of Bordetella pertussis in Escherichia coli K-12, and a conjugative shuttle vector system based on the suicide vector pRTP1 was used to deliver the mutations from E. coli back into B. pertussis CN2992FS and BP1. The aroA mutation was introduced by allelic exchange into the chromosome of B. pertussis, resulting in otherwise isogenic parental and aroA mutant pairs. The B. pertussis aroA mutants grew well on laboratory medium supplemented with aromatic compounds but failed to grow on unsupplemented medium. The B. pertussis aroA mutants expressed the normal B. pertussis extracellular, virulence-associated proteins; inactivated, whole-cell vaccines prepared from the mutants protected mice as efficiently as vaccines made from the parent strains against intracerebral challenge with the virulent B. pertussis 18323. Live B. pertussis aroA bacteria inefficiently colonized the lungs of NIH/S mice after they were challenged with aerosol, unlike the wild-type B. pertussis organism. Mice exposed to three separate aerosols of live B. pertussis aroA bacteria were protected against lung colonization after being exposed to an aerosol containing the virulent parental B. pertussis strain. High-level antibodies against B. pertussis rapidly appeared in the sera of mice immunized by aerosol with the B. pertussis aroA strains and challenged with the virulent parent.
Tumor necrosis factor enhances neutrophil-mediated killing of Plasmodium falciparum
Authors: Kumaratilake L M , Ferrante A , Rzepczyk C M
Published: January 2020

We developed a radiometric assay by which the antiplasmodial effects of phagocytic cells can be quantitated. This assay was used to examine the effects of recombinant human tumor necrosis factor alpha (TNF-alpha) on the killing of Plasmodium falciparum by human neutrophils. Data presented demonstrated that neutrophils engulf and destroy P. falciparum, but substantial killing of parasites required the presence of either heat-labile or heat-stable opsonins. While recombinant TNF-alpha at concentrations of 5 to 50,000 U/ml showed no direct effects on the parasite, this cytokine augmented the antimalarial activity of neutrophils at doses of 20 to 250 U/10(6) neutrophils. The results suggest that TNF-alpha is an important component of the immune phagocytic effector mechanisms which are involved in destruction of the malarial parasite.
Extramedullar proliferation of eosinophil granulocytes in chronic schistosomiasis mansoni is mediated by a factor secreted by inflammatory macrophages
Authors: el-Cheikh M C , Borojevic R
Published: January 2020

In chronic murine schistosomiasis mansoni, extramedullar myelopoiesis of eosinophils has been described, associated with tissue inflammatory infiltrates and periovular granulomas. It may be experimentally induced by intraperitoneal glass implants in mice with chronic schistosomiasis but not in normal mice or in mice with acute schistosomiasis. In vivo studies showed that this induction depended on macrophages adherent to the implants. In an in vitro test with bone marrow cells, we demonstrated that this eosinophil proliferation was mediated by a factor secreted by the mobilized macrophages. In contrast to murine interleukin-5 (IL-5), which induced both proliferation and differentiation of eosinophils, the studied monokine did not induce their maturation. A potent neutralizing monoclonal antibody for murine IL-5 did not abrogate the stimulatory activity of the monokine, indicating that it is a cytokine distinct from IL-5. These data, together with a quantitation of bone marrow, blood, and peripheral eosinophils, indicated that in chronic schistosomiasis, the systemic medullar supply of eosinophils is supplemented in tissues by their local proliferation, mediated by macrophages mobilized in local granulomatous and diffuse inflammatory reactions.
Thermoregulation of protein synthesis in Borrelia burgdorferi
Authors: Cluss R G , Boothby J T
Published: January 2020

Borrelia burgdorferi, the etiological agent of Lyme disease, infects humans via the bite of a tick. The microbe survives in at least two vastly different environments: an arthropod vector and a warm-blooded host. We examined protein synthesis in B. burgdorferi B31 in response to sudden heat stress, which is similar to that which occurs during the transmission from vector to host. Proteins synthesized after shifts from 28 degrees C to higher temperatures and in pulse-chase experiments were labeled with 3H-labeled amino acids for 4 h and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The synthesis of four proteins we designated as heat stress proteins (HSPs) was increased by shifts to higher temperatures (HSP-1, 75 kilodaltons [kDa]; HSP-2, 42 kDa; HSP-3, 39 kDa; and HSP-4, 27 kDa); and the amount of one protein we designated as heat-labile protein 1 (29.5 kDa) was decreased at higher temperatures. At 37 to 40 degrees C, the major heat stress protein, HSP-1, represented 14 to 18% of the total cell protein compared with 1 to 2% of the total cell protein at 28 degrees C. HSP-1 was stable during a 4-h chase at either 40 or 28 degrees C. Demonstration of similar HSPs in low-passage, pathogenic strains of B. burgdorferi suggests that the heat stress response may be common among B. burgdorferi strains and may play a role in Lyme disease.
Intracellular and cell-to-cell spread of Listeria monocytogenes involves interaction with F-actin in the enterocytelike cell line Caco-2
Authors: Mounier J , Ryter A , Coquis-Rondon M , Sansonetti P J
Published: January 2020

Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.
Augmentation of GG2EE macrophage cell line-mediated anti-Candida activity by gamma interferon, tumor necrosis factor, and interleukin-1
Authors: Blasi E , Farinelli S , Varesio L , Bistoni F
Published: January 2020

The expression of anti-Candida activity in the GG2EE macrophage cell line, generated by immortalization of fresh bone marrow with v-raf and v-myc oncogenes, was studied. GG2EE cells spontaneously inhibited the growth of an agerminative mutant of Candida albicans in vitro. The anti-Candida activity was maximal after 8 h of coculture and was proportional to the effector-to-target ratio. Gamma interferon (IFN-gamma), interleukin-1 (IL-1), and tumor necrosis factor (TNF) all significantly enhanced the anti-Candida activity of GG2EE cells. In contrast, IL-3, IL-4, and colony-stimulating factor 1 were ineffective. The augmentation of anti-Candida activity was not always concomitant with enhancement of phagocytosis, since IFN-gamma and colony-stimulating factor 1, but not IL-1 or TNF, augmented the phagocytic ability of GG2EE cells. Furthermore, the augmentation of anti-Candida activity in GG2EE cells did not correlate with the acquisition of antitumor activity. In fact, none of the cytokines alone were able to induce antitumor activity in GG2EE cells, which, however, could be activated to a tumoricidal stage by IFN-gamma plus heat-killed Listeria monocytogenes. These findings demonstrate that GG2EE cells exhibit spontaneous anti-Candida activity and that such activity is enhanced by TNF, IL-1, and IFN-gamma.
Phagocyte function in juvenile periodontitis
Authors: Repo H , Saxén L , Jäättelä M , Ristola M , Leirisalo-Repo M
Published: January 2020

We studied the chemotaxis of peripheral blood polymorphonuclear leukocytes (PMNs) and monocytes and the production of tumor necrosis factor alpha by monocytes of patients with juvenile periodontitis (JP). As a group, the patients' PMNs showed significantly increased chemotaxis determined by counting the number of migrating cells within a 3-microns-pore-size filter. Determined as distance of migration within the filter, as chemotactic increment based on checkerboard analysis, as leukotactic index calculated on the basis of distance of migration and cell count at different depths within a 3-microns-pore-size filter, as distance of migration under agarose, and as the number of PMNs migrating across a 5-microns-pore-size filter, the chemotactic migration rates of PMNs of patients were similar to those of controls. Evaluation of the data on an individual basis suggested that in terms of PMN chemotaxis some patients were hyperresponsive and some were hyporesponsive. Chemotaxis, spontaneous migration, and the rates of lipopolysaccharide-induced tumor necrosis factor alpha production by JP monocytes were similar to those of control cells. Our results give credence to the view that there are minor aberrations in the functions of JP phagocytes, but the extent to which these aberrations are relevant to accumulation of PMNs at sites of infection and inflammation in vivo and possibly contribute to the pathogenesis of JP remains unclear.
Cloning of the genes for AF/R1 pili from rabbit enteroadherent Escherichia coli RDEC-1 and DNA sequence of the major structural subunit
Authors: Wolf M K , Boedeker E C
Published: January 2020

AF/R1 pili on the surface of Escherichia coli RDEC-1 promote attachment of the bacteria to rabbit intestinal brush borders. In order to characterize AF/R1 pili and manipulate their expression, we cloned the genes necessary for AF/R1 expression; determined the size of proteins produced in minicells; located the gene encoding the major structural subunit, named AfrA; and determined the DNA sequence of afrA as well as the sequence of 700 additional nucleotides upstream of afrA. Two contiguous EcoRI fragments spanning 7.9 kilobases were cloned from the 86-megadalton plasmid of RDEC-1 into vector pUC19 to make plasmid pW1. Bacteria carrying pW1 produced AF/R1 pili that were recognized by AF/R1-specific antiserum and promoted adherence of bacteria to brush borders prepared from rabbit intestine. Proteins with a molecular weight of 17,000 (17K proteins), which was the size of AfrA, as well as 15K, 15.5K, 26K, 28K, and 80K proteins were detected in minicells carrying pW1. The gene afrA was located by using an oligonucleotide probe, and its DNA sequence was determined. The DNA sequence of 700 additional nucleotides upstream was determined because this sequence may be important in the regulation of AF/R1 expression.
Immunogenicity and antigenicity in rabbits of a repeated sequence of Plasmodium falciparum antigen Pf155/RESA fused to two immunoglobulin G-binding domains of staphylococcal protein A
Authors: Sjölander A , Ståhl S , Nygren P A , Aslund L , Ahlborg N , Wåhlin B , Scherf A , Berzins K , Uhlén M , Perlmann P
Published: January 2020

A synthetic gene encoding a tetramer of the repeated subunit EENVEHDA of the Plasmodium falciparum antigen Pf155/RESA was expressed in a dual-expression system. The resulting fusion proteins, designated ZZ-M1 and BB-M1, comprised the EENVEHDA repeats and either two immunoglobulin G-binding domains from staphylococcal protein A or the human serum albumin-binding domains from streptococcal protein G, respectively. The soluble fusion proteins were affinity purified to homogeneity in one-step procedures. ZZ-M1 was used for immunization of rabbits. The rabbit antisera reacted with BB-M1 in an enzyme-linked immunosorbent assay and with Pf155/RESA in immunofluorescence of infected erythrocytes and immunoblotting. Inhibition studies revealed that the antibodies mainly recognized epitopes formed by two or more EENVEHDA subunits and were remarkably specific for Pf155/RESA. Importantly, the antibodies also inhibited P. falciparum merozoite reinvasion in vitro efficiently, indicating that they reacted with biologically important epitopes exposed on the native antigen. Immunization with Freund complete adjuvant resulted in high levels of specific immunoglobulin G antibodies over a 1-year period, whereas the antibody response obtained after immunization without adjuvant was generally weaker, immunoglobulin G and M mediated, and not sustained for longer periods. However, these titers were restored after booster injection. Taken together, the results support the usefulness of recombinant gene constructs of this type as immunogens for malaria vaccines.
Reduced expression of the functionally active complement receptor for iC3b but not for C3d on an avirulent mutant of Candida albicans
Authors: Ollert M W , Wadsworth E , Calderone R A
Published: January 2020

Pseudohyphae of Candida albicans bear surface receptors for iC3b and C3d. In order to determine a possible role for these receptors in the pathogenesis of candidiasis, a spontaneous C. albicans mutant, m-10, which exhibits reduced ability to adhere in vitro to fibrin platelet clots and epithelial cells or to cause endocarditis in a rabbit model, and its parent wild-type (wt) strain were compared for receptor expression in rosetting assays with sheep erythrocytes carrying iC3b (EAC1423bi) or C3d (EAC1423d). An equally high attachment to wt and m-10 was seen with EAC1423d, whereas rosetting with EAC1423bi was reduced by 53% in m-10 compared with wt. In inhibition studies, rosetting of wt with EAC1423bi was markedly inhibited by culture filtrate, hyphal-cell extract, and DEAE-fractionated material prepared from wt (54, 87, and 70% decreases in rosetting, respectively), thus suggesting the presence of the soluble, functionally active iC3b receptor of C. albicans in each of these preparations. Minimal inhibition of iC3b rosetting, however, was seen with the identical materials from m-10 (21, 5, and 12%, respectively). All of the preparations from the two strains were equally effective in their inhibitory activities against rosetting of C3d. A human serum specimen obtained from a patient with chronic mucocutaneous candidiasis blocked iC3b rosetting of the wt strain almost completely. When used in an immunoblot, this serum recognized proteins of 68 to 71, 55, and 50 kilodaltons (kDa) in hyphal-cell extracts of the wt. With the same preparation of the avirulent mutant, only weak reactions with the 68- to 71-kDa and 55-kDa proteins occurred, while the 50-kDa protein was not detectable. Taken together, these results indicate that the expression of the functionally active iC3b receptor on C. albicans may be involved in the virulence of the organism, possibly by mediating adherence to mammalian cells.
Induction of hypersensitivity to endotoxin and tumor necrosis factor by sublethal infection with Salmonella typhimurium
Authors: Matsuura M , Galanos C
Published: January 2020

The effect of sublethal infection with Salmonella typhimurium on the sensitivity of mice to the lethal activity of lipopolysaccharide (LPS) was studied in C3H/TifF mice. These mice are more resistant to S. typhimurium infection and survive inocula that are lethal for most other strains of mice. Infection of C3H/TifF mice with 2 x 10(4) CFU of S. typhimurium was without lethal effect. However, administration of LPS at different times after infection revealed that the sensitivity of the animals to the lethal activity of LPS increased exponentially, reaching a maximum by 6 to 8 days after infection. Thereafter, it decreased, reaching preinfection values about 4 weeks after inoculation. At the height of sensitivity, the animals were susceptible to less than 1 microgram of LPS compared with 100 or 200 micrograms in noninfected mice. The sensitization to LPS by infection was paralleled by a sensitization to tumor necrosis factor. The time course of development of sensitization to tumor necrosis factor, as well as the time course of its decrease and disappearance, was almost identical to that of LPS.
Attaching effacement of the rabbit enterocyte brush border is encoded on a single 96.5-kilobase-pair plasmid in an enteropathogenic Escherichia coli O111 strain
Authors: Fletcher J N , Saunders J R , Batt R M , Embaye H , Getty B , Hart C A
Published: January 2020

An enteropathogenic Escherichia coli (EPE) O111 serotype a,b,H- strain carried the following four plasmids: pLV501 (96.5 kilobase pairs [kbp]) specifying resistance to chloramphenicol, tetracycline, and kanamycin; pLV502 (8 kbp) specifying ampicillin resistance; pLV503 (1.9 kbp) specifying streptomycin resistance; and pLV504 (80 kbp) with no resistance markers. This EPEC attached to HEp-2 cells to produce localized clumps of bacteria (localized adhesion) and attached intimately to the enterocyte surface, leading to loss of the brush border (attaching effacement). Plasmid pLV501 was also found to specify the ability to produce localized adhesion on HEp-2 cells and attaching effacement in a rabbit ileal explant model system. Restriction maps showed considerable dissimilarities between pLV501 and pMAR-2, an EPEC plasmid carrying the EPEC adherence factor (EAF) genes. Furthermore, pLV501 did not hybridize with the EAF probe, whereas pLV504 did. There was sequence homology between pLV501 and large plasmids in all seven other well-characterized EPEC, only five of which hybridized with the EAF probe. These findings indicate that pLV501 carries at least one of the genes responsible for production of the brush border damage characteristic of EPEC.
Roles of the disulfide bond and the carboxy-terminal region of the S1 subunit in the assembly and biosynthesis of pertussis toxin
Authors: Antoine R , Locht C
Published: January 2020

A Bordetella pertussis expression system was developed to analyze the structure-function relationship, in vivo assembly, and biosynthesis of pertussis toxin. The toxin structural gene was first deleted from the B. pertussis chromosome; into the resulting B. pertussis strain the toxin gene was introduced on a low-copy-number, broad-host-range plasmid. The amount of pertussis toxin produced and secreted with this expression system was in the same order of magnitude as that produced by B. pertussis Tohama I, indicating that although the plasmid may be present in more than one copy per cell, overproduction of the toxin was not achieved in B. pertussis. Expression of mutant pertussis toxin genes in which the codon for Cys-41 was deleted or altered or in which the carboxy-terminal region was deleted showed that both the single intrachain disulfide bond and the carboxy-terminal region of S1 are essential for the stable expression, assembly, and secretion of S1. On the other hand, the B oligomer was efficiently secreted in the culture medium in the absence of the S1 subunit. The secreted B oligomer contained S2, S3, and S4 subunits as evidenced by enzyme-linked immunosorbent assay and was fully functional with respect to haptoglobin binding. Furthermore, the deletion of the hydrophobic carboxy-terminal region has a drastic effect on S1 subunit solubility; however, inclusion of the hydrophobic region was not sufficient for assembly and secretion, indicating that other interactions involving amino acids beyond residue 207 of the S1 subunit are also required.
A role in vivo for tumor necrosis factor alpha in host defense against Chlamydia trachomatis
Authors: Williams D M , Magee D M , Bonewald L F , Smith J G , Bleicker C A , Byrne G I , Schachter J
Published: January 2020

In a mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]), tumor necrosis factor alpha (TNF-alpha) antigen and bioactivity were demonstrated in vivo in the lung during MoPn infection in both athymic (nude) and heterozygous (nu/+) mice. Antibody to TNF-alpha that was exogenously given neutralized the TNF-alpha in the lung, significantly accelerated mortality, and caused a borderline increase in MoPn counts in the lung by culture in nu/+ mice. Lipopolysaccharide-induced TNF-alpha activity or injections of recombinant murine TNF-alpha significantly but modestly protected nu/+ mice against MoPn-induced mortality. TNF-alpha is produced in vivo during C. trachomatis infection and plays a role in host defense.
Identification of a bacteriophage containing a silent staphylococcal variant enterotoxin gene (sezA+)
Authors: Soltis M T , Mekalanos J J , Betley M J
Published: January 2020

A variant enterotoxin gene, referred to as sezA+, has been identified. Staphylococcus aureus FRI1106, a staphylococcal enterotoxin type D producer (Sed+), contained HindIII fragments of 3.8 and 9.4 kilobase pairs (kbp) that hybridized in Southern blot analysis to a probe containing only staphylococcal enterotoxin type A structural gene sequences. Presumably, probe A-624 hybridized to the 9.4-kbp HindIII fragment because of the sequence homology between sea+ and sed+. This 9.4-kbp HindIII fragment, which was part of a staphylococcal plasmid, was isolated and ligated into an Escherichia coli plasmid vector; Sed+ E. coli recombinant clones were isolated. The 3.8-kbp HindIII fragment was shown to be part of a viable lysogenic bacteriophage, and it contained sezA+. This sezA(+)-containing fragment was cloned into E. coli, and its DNA sequence was determined. Examination of the nucleotide sequence revealed a 771-bp region that contained an open reading frame with 85 and 77% nucleotide and derived amino acid sequence identifies with sea+ and staphylococcal enterotoxin type A, respectively. This open reading frame has 83 to 50% nucleotide sequence identities with the other types of staphylococcal enterotoxin genes. sezA+ was shown to be transcribed into stable mRNA. However, the sezA+ mRNA was not translated into an enterotoxinlike protein because it lacks an appropriate translation initiation codon.
Bovine pulmonary endothelial cell damage mediated by Pasteurella haemolytica pathogenic factors
Authors: Breider M A , Kumar S , Corstvet R E
Published: January 2020

The in vitro effects of Pasteurella haemolytica components on bovine pulmonary endothelial monolayers were investigated to determine the relative role of individual bacterial factors in the pathogenesis of bovine pulmonary pasteurellosis. Bovine pulmonary endothelial monolayers were treated with P. haemolytica bacterial culture supernatant (CS) and P. haemolytica lipopolysaccharide. At 22 h postinoculation, the CS produced severe damage to the endothelial cells, indicated by high 51Cr release, extensive cellular detachment, and morphologic changes characterized by cell contraction, cytoplasmic blebbing, and loss of monolayer confluency. The neutralization of leukotoxin activity of the CS by heat inactivation was ineffective in decreasing the damage to endothelial cells; however, leukotoxin-neutralizing monoclonal antibody slightly diminished the toxic effect. P. haemolytica lipopolysaccharide by itself or as a supplement to CS produced endothelial cell damage similar to that of CS. The preincubation of CS dilutions (10(-1) and 10(-2)) or P. haemolytica lipopolysaccharide with polymyxin B almost completely eliminated cell toxicity. These studies show that P. haemolytica produces a soluble factor that is consistent with bacterial lipopolysaccharide and that is directly toxic to bovine pulmonary endothelial cells in vitro.
Localization and activity of various lysosomal proteases in Leishmania amazonensis-infected macrophages
Authors: Prina E , Antoine J C , Wiederanders B , Kirschke H
Published: January 2020

In mammalian hosts, Leishmania amastigotes are obligatory intracellular parasites of macrophages and multiply within parasitophorous vacuoles of phagolysosomal origin. To understand how they escape the harmful strategies developed by macrophages to kill ingested microorganisms, it is important to obtain information on the functional state of parasitophorous vacuole. For this purpose, we studied the intracellular distribution and activity of host lysosomal proteases in rat bone marrow-derived macrophages infected with Leishmania amazonensis amastigotes. Localization of cathepsins B, H, L, and D was investigated by using specific immunoglobulins. In uninfected macrophages, these enzymes were located in perinuclear granules (most of them were probably secondary lysosomes) which, after infection, disappeared progressively. In infected macrophages, cathepsins were detected mainly in the parasitophorous vacuoles, suggesting that the missing secondary lysosomes had fused with these organelles. Biochemical assays of various proteases (cathepsins B, H, and D and dipeptidyl peptidases I and II) showed that infection was accompanied by a progressive increase of all activities tested, except that of dipeptidyl peptidase II, which remained constant. No more than 1 to 10% of these activities could be attributed to amastigotes. These data indicate that (i) Leishmania infection is followed by an increased synthesis and/or a reduced catabolism of host lysosomal proteases, and (ii) amastigotes grow in a compartment rich in apparently fully active proteases. Unexpectedly, it was found that infected and uninfected macrophages degraded endocytosed proteins similarly. The lack of correlation in infected macrophages between increase of protease activities and catabolism of exogenous proteins could be linked to the huge increase in volume of the lysosomal compartment.
Inoculation of barrier-born pigs with Helicobacter pylori: a useful animal model for gastritis type B
Authors: Engstrand L , Gustavsson S , Jörgensen A , Schwan A , Scheynius A
Published: January 2020

At the age of 8 weeks, 15 barrier-born pigs, specific pathogen free, were inoculated intragastrically with suspensions of 10(7) to 10(10) CFU of Helicobacter pylori after pretreatment with omeprazole. The pigs were observed for up to 12 weeks, endoscopic biopsy specimens were taken, and serum samples were drawn. H. pylori was identified by routine culturing and by staining with an H. pylori-specific monoclonal antibody on cryostat sections of gastric biopsy specimens. In 11 of 15 inoculated pigs, H. pylori was detected throughout the observation period. In these infected pigs, there was an antibody response to H. pylori, as determined in serum by an enzyme immunoassay. Furthermore, the development of superficial, focal gastritis with infiltrates of mononuclear class II antigen-expressing lymphocytes was observed immunohistologically. H. pylori was never detected and an antibody response to H. pylori was not observed in two control pigs. The development of gastritis and the systemic antibody response to H. pylori support the usefulness of this animal model for studies of H. pylori-related human diseases.
Detection of antigens with affinity for host cell membrane polypeptides in culture supernatants of Trypanosoma cruzi
Authors: Davis C D , Kuhn R E
Published: January 2020

Parasite antigens which bind to host cell molecules of approximately 32 and 34 kilodaltons (kDa) were identified in supernatant fluids obtained from axenic cultures of Trypanosoma cruzi. These parasite components were first detected in culture supernatants obtained after 2 weeks in culture. Immunoblot analysis of culture supernatants exhibiting binding activity revealed the presence of several parasite antigens ranging in molecular mass from approximately 26 to 290 kDa. Gel filtration (Sephacryl S-300) analysis of culture supernatants revealed four major peaks, but only the highest-molecular-mass peak (containing several parasite antigens ranging from 27 to 250 kDa) possessed binding activity for the host cell molecules.
Genetic population structure, clonal phylogeny, and pathogenicity of Salmonella paratyphi B
Authors: Selander R K , Beltran P , Smith N H , Barker R M , Crichton P B , Old D C , Musser J M , Whittam T S
Published: January 2020

Genetic diversity and relationships among 123 strains of Salmonella paratyphi B (serotype 1,4,[5],12:b:[1,2]) were estimated from an assessment of electrophoretically demonstrable allelic variation at 24 chromosomal enzyme gene loci. Fourteen electrophoretic types, marking clones, were distinguished, the phylogeny of the clonal lineages was reconstructed, and biotype and other phenotypic characters were mapped onto this structure. Most d-tartrate-negative strains are members of an abundant, globally distributed clone (Pb 1) that is polymorphic for many biotype characters (including d-tartrate utilization), bacteriophage type, rRNA pattern, and colicin M and phage ES18 sensitivity. This clone is largely responsible for S. paratyphi B enteric fever in humans. In contrast, d-tartrate-positive strains (formerly known as S. java) occurred in all seven of the clonal lineages identified by population genetic analysis, although most d-tartrate-positive isolates belong to only two clones (Pb 3 and Pb 4), which vary in frequency geographically. Monophasic strains represent four closely related clones forming a distinctive phylogenetic lineage. The Kauffmann hypothesis of convergence in serotype among distantly related cell lineages through recombination (via phage transduction or other means) may account for the considerable genotypic diversity among clones of S. paratyphi B. Pb 4, Pb 6, and Pb 7 are more closely allied with clones of S. typhimurium and S. saintpaul than with other clones of S. paratyphi B. Sensitivity or resistance to colicin M and phage ES18 and the electrophoretic pattern of the rRNA, which were incorporated into a recently proposed scheme for the identification of types of S. paratyphi B, individually or in combination fail to mark clones or other meaningful phylogenetic subdivisions.
Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide
Authors: Sanford J E , Lupan D M , Schlageter A M , Kozel T R
Published: January 2020

The in vivo properties of an immunoglobulin isotype-switch family of monoclonal antibodies specific for the polysaccharide capsule of Cryptococcus neoformans were examined in a murine model of cryptococcosis. Subclass-switch variants were isolated by sequential sublining of an immunoglobulin G subclass 1 (IgG1)-secreting cell line. Antibodies of the IgG1, IgG2a, and IgG2b isotypes with identical reactivities with cryptococcal polysaccharide were prepared. The antibodies had the distinct biological properties associated with the heavy chains of each respective isotype. The antibodies were used prophylactically or therapeutically in an attempt to alter the course of cryptococcal infection in mice. Survival of mice and a tissue census of the numbers of viable cryptococci in the lung, spleen, and brain were used as indicators of efficacy. Passive immunization with the IgG2a and IgG2b antibodies effected a reduction in the numbers of cryptococci in lung and spleen. Passive immunization with the IgG1 antibody was markedly less effective. Passive immunization had little or no effect on the numbers of cryptococci in brain tissue, regardless of the immunoglobulin isotype. Despite apparent efficacy with regard to reduction in the numbers of yeast cells in the lung and spleen, the results showed no improvement in survival from murine cryptococcosis. Our results indicate that passive immunization produces a modest effect on the course of murine cryptococcosis in tissues other than brain. However, under the experimental conditions used, such treatment does not have a measurable impact on the ultimate outcome of the infection.