bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative toxicity and virulence of Escherichia coli clones expressing variant and chimeric Shiga-like toxin type II operons
Authors: Paton A W , Bourne A J , Manning P A , Paton J C
Published: January 2020

Shiga-like toxin (SLT)-producing strains of Escherichia coli are known to cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. The SLTs, particularly those related to type II (SLT-II), are a diverse family of toxins which may have differing in vitro or in vivo properties. To examine the impact of naturally occurring SLT-II sequence variation on the capacity of a given E. coli strain to cause disease, operons encoding four different SLT-II-related toxins, designated SLT-II/O111, SLT-II/OX3a, SLT-II/OX3b, and SLT-II/O48, were cloned in the same orientation in pBluescript. French pressure cell lysates of E. coli DH5 alpha derivatives carrying these plasmids differed markedly in cytotoxicity for Vero cells, with 50% cytotoxic doses ranging from 20 to 328,000/ml. The strains also differed in oral virulence for streptomycin-treated mice, as judged by survival rate and/or median survival time, but virulence did not necessarily correlate with in vitro cytotoxicity. The SLT-II type associated with the lowest oral virulence was SLT-II/O111. Both the overall survival rate and the median survival time of mice challenged with clones producing this toxin were significantly greater than that for mice challenged with a clone producing the closely related SLT-II/OX3a. Experiments with clones carrying chimeric O111/OX3a SLT-II operons indicated that the reduced virulence was associated with an Arg-176-->Gly substitution in the mature A subunit. Clones producing SLT-II/O48 and SLT-II/OX3b had similarly high cytotoxicities for Vero cells, but the latter was more virulent when fed to streptomycin-treated mice, as judged by median survival time. Experiments with clones carrying chimeric O48/OX3b SLT-II operons indicated that the increased virulence was a function of the A subunit of SLT-II/OX3b, which differs from the A subunit of SLT-II/O48 by only two amino acids (Met-4-->Thr and Gly-102-->Asp, respectively). These findings raise the possibility that naturally occurring SLT-II sequence variations may impact directly on the capacity of a given SLT-producing E. coli strain to cause disease.
Borrelia burgdorferi binds plasminogen, resulting in enhanced penetration of endothelial monolayers
Authors: Coleman J L , Sellati T J , Testa J E , Kew R R , Furie M B , Benach J L
Published: January 2020

Several strains of Borrelia burgdorferi and Borrelia hermsii can bind human Lys-plasminogen specifically. Affinity blots using 125I-labeled plasminogen showed that numerous polypeptides of all the strains and species tested could bind via lysine residues to the plasminogen molecule since binding could be completely inhibited by the lysine analog epsilon-aminocaproic acid. Binding analysis using 125I-labeled plasminogen on live intact organisms showed that the organisms possess two binding sites for plasminogen: a high-affinity site with a Kd of 24 +/- 12 pM and 106 +/- 14 binding sites per spirochete and a low-affinity site with a Kd of 20 +/- 4 nM and 2,683 +/- 36 binding sites per spirochete. Indirect immunofluorescence and confocal microscopy showed a generalized but punctate pattern of plasminogen binding to the spirochete surface. Exogenously provided urokinase-type plasminogen activator converted B. burgdorferi surface-bound plasminogen to enzymatically active plasmin as demonstrated by the breakdown of the chromogenic plasmin substrate S2251. Plasmin-coated organisms showed an enhanced ability to penetrate endothelial cell monolayers grown on connective tissue substrates compared to untreated controls (P < 0.001). This functional assay demonstrated that enzymatically active plasmin on the surface of spirochetes can lead to greater invasion of tissues.
Cloning and genetic characterization of the flagellum subunit gene (flaA) of Legionella pneumophila serogroup 1
Authors: Heuner K , Bender-Beck L , Brand B C , Lück P C , Mann K H , Marre R , Ott M , Hacker J
Published: January 2020

The gene flaA, encoding the flagellum subunit protein of Legionella pneumophila serogroup 1, has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12 by using an antiflagellin specific polyclonal antiserum. DNA sequence analysis of the flaA gene revealed the presence of a 1,428-bp open reading frame encoding a protein of 475 amino acids with an apparent molecular mass of 48 kDa that is expressed independently of an E. coli vector promoter. Peptide sequencing of the N terminus of the isolated flagellum subunit protein confirmed that this open reading frame encodes the flagellin. By comparing the FlaA amino acid sequence with those of flagellins of various other bacteria, high degrees of homology in the N-terminal and C-terminal amino acids could be observed. The flaA-specific mRNA was determined to be 1.6 kb in size, the expected size of a monocistronic mRNA. Temperature-dependent expression of flagellin was found to be regulated at the transcriptional level. Sequence analysis and primer extension experiments indicated that the transcription of the gene flaA is directed by a sigma 28-like RpoF-FliA factor. By using fliA and fliA+ E. coli K-12 mutants, it was shown that flaA expression in E. coli required the sigma 28 factor. A flaA-specific DNA probe hybridizes with genomic DNA isolated from L. pneumophila and with most of the genomic DNAs from non-L. pneumophila Legionella strains. Two L. pneumophila strains and isolates of Legionella bozemanii and Legionella feeleii (serogroup 1) carry flaA-specific sequences but were not able to produce flagella.
Suppressive effect of interleukin-4 neutralization differs for granulomas around Schistosoma mansoni eggs injected into mice compared with those around eggs laid in infected mice
Authors: Eltoum I A , Wynn T A , Poindexter R W , Finkelman F D , Lewis F A , Sher A , Cheever A W
Published: January 2020

The principal pathological manifestation of murine Schistosoma mansoni infection is the egg-induced granuloma. Synchronous pulmonary granulomas forming around intravenously injected schistosome eggs are widely used to study the immunopathology of schistosomiasis. A number of anticytokine antibody treatments have a remarkable effect in modulating granulomas in this model but little effect on the size of hepatic granulomas around laid eggs during experimental infection. To examine this discrepancy, we examined the effects of anticytokine antibodies on liver and lung granulomas around injected eggs and around eggs laid during infection in both locations. Anti-interleukin-4 (IL-4) treatment greatly reduced the volume of granulomas around eggs injected into the liver via the portal vein and around eggs injected into the lung via the tail vein. On the contrary, granulomas around eggs laid by worms in either the liver or the lung during the course of infection were not significantly decreased in size by anti-IL-4 treatment. Thus, site is not important for the disparate effects of anti-IL-4 in granuloma formation around injected versus laid eggs. This effect is seen in naive and sensitized animals and is most probably due to differences in the quality of injected eggs versus those laid in situ by the worms.
Role of Yops in inhibition of phagocytosis and killing of opsonized Yersinia enterocolitica by human granulocytes
Authors: Visser L G , Annema A , van Furth R
Published: January 2020

The virulence plasmid of Yersinia enterocolitica codes for the production of the outer membrane protein YadA and the secretion of several proteins, called Yops, which may play a role in the interaction between granulocytes and this bacterium. We investigated whether the expression of YadA or the secretion of Yops affected the phagocytosis and killing of opsonized Y. enterocolitica by human granulocytes. The rates of phagocytosis and killing of Y. enterocolitica by granulocytes in suspension in the presence of rabbit Yersinia antibodies and complement were determined by microbiological assays. In addition, noningested cell-adherent bacteria were differentiated from ingested yersiniae by immunofluorescence microscopy. Plasmid-bearing opsonized Y. enterocolitica was able to inhibit phagocytosis and killing by human granulocytes. The inhibition of phagocytosis was specific for the plasmid-bearing strain of Y. enterocolitica, since granulocytes were still able to phagocytose and kill Staphylococcus aureus in the presence of Y. enterocolitica. Plasmid-cured Y. enterocolitica was readily phagocytosed and killed by these cells. To investigate the role of YadA or Yops in the inhibition of phagocytosis by granulocytes, the phagocytosis of mutant strains unable to express YadA or to secrete Yops was studied. A Y. enterocolitica mutant unable to secrete Yops lost its ability to inhibit phagocytosis; a mutant expressing only YadA was readily ingested by granulocytes. These results indicate that after attachment of opsonized Y. enterocolitica to granulocytes, Yops play an important role in inhibiting the ingestion of Y. enterocolitica by human granulocytes.
Heterologous antigen expression in Vibrio cholerae vector strains
Authors: Butterton J R , Beattie D T , Gardel C L , Carroll P A , Hyman T , Killeen K P , Mekalanos J J , Calderwood S B
Published: January 2020

Live attenuated vector strains of Vibrio cholerae were derived from Peru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin genetic element and attRS1 sequences, which was developed as a live, oral vaccine strain. A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V. cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter. slt-IB was also placed under transcriptional control of the V. cholerae heat shock promoter, htpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respectively. A new technique was used to perform allelic exchange with lacZ. This method uses plasmid p6891MCS, a pBR327 derivative containing cloned V. cholerae lacZ, to insert markers of interest into the V. cholerae chromosome. Recombinants can be detected by simple color screening and antibiotic selection. In vitro measurements of Slt-IB produced by the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene for Slt-IB increased expression over a single copy. The V. cholerae vectors colonized the gastrointestinal mucosa of rabbits after oral immunization, as demonstrated by very high serum antibody responses to V. cholerae antigens. Comparison of the serologic responses to the B subunit of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulated by htpGp, suggested that both the cholera toxin and heat shock promoters were active in vivo, provoking comparable immunologic responses. Orogastric inoculation of rabbits with vector strains evoked serum immunoglobulin G (IgG) responses to Slt-IB in two of the four strains tested; all four strains produced biliary IgA responses. No correlation was observed between the type of promoter expressing slt-IB and the level of serum IgG or biliary IgA response, but the vector strain containing two copies of the gene for slt-IB evoked greater serum IgG responses than strains containing a single copy, consistent with the increased expression of Slt-IB from this strain observed in vitro. A comparison of the serum and biliary antibody responses to Slt-IB expressed from htpGp versus CtxB expressed from the same promoter suggested that CtxB is a more effective orally delivered immunogen.
Active immunization with recombinant V antigen from Yersinia pestis protects mice against plague
Authors: Leary S E , Williamson E D , Griffin K F , Russell P , Eley S M , Titball R W
Published: January 2020

The gene encoding V antigen from Yersinia pestis was cloned into the plasmid expression vector pGEX-5X-2. When electroporated into Escherichia coli JM109, the recombinant expressed V antigen as a stable fusion protein with glutathione S-transferase. The glutathione S-transferase-V fusion protein was isolated from recombinant E. coli and cleaved with factor Xa to yield purified V antigen as a stable product. Recombinant V antigen was inoculated intraperitoneally into mice and shown to induce a protective immune response against a subcutaneous challenge with 3.74 x 10(6) CFU of virulent Y. pestis. Protection correlated with the induction of a high titer of serum antibodies and a T-cell response specific for recombinant V antigen. These results indicate that V antigen should be a major component of an improved vaccine for plague.
Cryptococcus neoformans melanin and virulence: mechanism of action
Authors: Wang Y , Aisen P , Casadevall A
Published: January 2020

Black melanin-like pigments are produced by several neurotropic fungi, including Cryptococcus neoformans. Pigment production is associated with virulence. In media containing phenolic substrates such as L-dopa, C. neoformans cells become black as a result of pigment accumulation. Pigmented and nonpigmented C. neoformans cells were studied with transmission electron microscopy and electron spin resonance (ESR) spectroscopy. Transmission electron microscopy showed electron-dense cell walls, and ESR spectroscopy revealed a stable free-radical population in pigmented cells. The ESR signals of pigmented cells were increased by light, alkaline pH, and Zn2+ and decreased by acid pH, indicating that the black pigment was a type of melanin. A mutant deficient in melanin synthesis (mel) generated by UV radiation lacked ESR-detectable radicals, was less virulent for mice, was more susceptible to killing by nitrogen- and oxygen-derived radicals, and had 100-foldless phenoloxidase activity than the parent strain. The interaction of melanized C. neoformans, nonmelanized C. neoformans, and the hypomelanotic mel mutant with J774.16 murine macrophage-like cells was studied. Melanized cells were more resistant to antibody-mediated phagocytosis and the antifungal effects of murine macrophages than nonmelanized cells. Small increases in the intensity of the ESR signals of melanized cells in solutions containing chemically generated oxygen- and nitrogen-derived radicals indicated electron transfer to or from melanin. Melanin appears to contribute to virulence by protecting fungal cells against attack by immune effector cells.
Susceptibility of beige mice to Mycobacterium avium: role of neutrophils
Authors: Appelberg R , Castro A G , Gomes S , Pedrosa J , Silva M T
Published: January 2020

The beige mutation in C57BL/6 mice has been shown to increase the susceptibility to infection by Mycobacterium avium. In this study, we confirmed those results and showed that the effect of the beige mutation was most obvious after infection with a strain of lower virulence than with a highly virulent isolate of M. avium. The dissemination of M. avium from the gut was observed with both C57BL/6 and beige mice but was faster in the latter. The expression of gamma interferon (IFN-gamma) and the priming for tumor necrosis factor production during an in vivo infection were similar between beige and immunocompetent C57BL/6 mice. IFN-gamma produced during the infection of beige mice was protective in the spleen, and the administration of recombinant IFN-gamma restored the resistance in the spleen to levels similar to those found in control mice. There were no histological differences between wild-type and beige mice with respect to granuloma formation in the liver. The increased susceptibility of beige mice to M. avium as manifested in the liver was reduced by transfusing neutrophils from wild-type C57BL/6 mice. Likewise, depletion of neutrophils from C57BL/6 mice rendered them as susceptible to M. avium infection of the liver as beige mice. Our results point to the participation of neutrophils in the defect of beige mice in addition to other defects. Furthermore, these results show that neutrophils play a significant role in the defense mechanisms against mycobacterial infections and that beige animals may be a useful model for study of the role of neutrophils in mycobacteriosis.
Intracellular multiplication and toxic destruction of cultured macrophages by Capnocytophaga canimorsus
Authors: Fischer L J , Weyant R S , White E H , Quinn F D
Published: January 2020

Capnocytophaga canimorsus is a gram-negative rod that causes opportunistic infections resulting in bacteremia, septicemia, meningitis, and death in immunocompromised, splenectomized, and alcoholic individuals. Infections caused by a related species, Capnocytophaga cynodegmi, remain localized at the site of the wound where the organism is introduced. Both organisms are part of the normal canine oral flora and are introduced through puncture wounds via dog bites. We found that both C. canimorsus and C. cynodegmi attach, are phagocytized, and multiply intracellularly in J774 mouse macrophage cells. After 48 h of infection by C. canimorsus, large sections of the macrophage cell layer were observed to detach and lyse, while the monolayer infected with C. cynodegmi demonstrated no cytotoxic effects. Tissue culture supernatants from the C. canimorsus-infected J774 cells filtered through a 0.22-micron-pore membrane produced a similar effect on fresh monolayers, while filtrates from C. cynodegmi and uninfected controls produced no effect. No endotoxin release was observed in these supernatants. We conclude that the cytotoxic phenotype of C. canimorsus is the likely result of a toxin produced by this organism.
Entry and survival of Leishmania amazonensis amastigotes within phagolysosome-like vacuoles that shelter Coxiella burnetii in Chinese hamster ovary cells
Authors: Veras P S , Moulia C , Dauguet C , Tunis C T , Thibon M , Rabinovitch M
Published: January 2020

Coxiella burnetii, a rickettsia, and Leishmania amazonensis, a protozoan flagellate, lodge in their host cells within large phagolysosome-like vacuoles. In the present study, C. burnetii-infected Vero or CHO cells were superinfected with L. amazonensis amastigotes to determine if these parasites can home to and survive within heterologous vacuoles. Six hours after superinfection, Leishmania amastigotes were located almost exclusively within large Coxiella-containing vacuoles. Thereafter, the numbers of parasites in the vacuoles increased at the same rate as those in cells infected with L. amazonensis alone. Furthermore, in cultures shifted to 25 degrees C, some of the amastigotes transformed into promastigote-like forms that moved their flagella within the adoptive vacuoles. Thus, L. amazonensis amastigotes not only entered Coxiella vacuoles, most likely by fusion of donor and recipient vacuoles, but temporarily survived, differentiated, and replicated therein. This appears to be the first account of the temporary cohabitation of two living pathogens within the same vacuole in a mammalian cell.
Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes
Authors: Shompole S , Perryman L E , Rurangirwa F R , McElwain T F , Jasmer D P , Musoke A J , Wells C W , McGuire T C
Published: January 2020

To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface-specific immunoprecipitation of [35S]methionine-labeled proteins (200, 28, and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of 200 and 220 kDa were isolated from a MAb 44.18 affinity matrix. Calf serum antibodies to these isolated antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf serum antibodies identified major labeled proteins with sizes of 200 and 72 kDa by surface-specific immunoprecipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationale for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines.
Sulfated polyanions block Chlamydia trachomatis infection of cervix-derived human epithelia
Authors: Zaretzky F R , Pearce-Pratt R , Phillips D M
Published: January 2020

Using a cell line derived from the human cervix and a rapid fluorescence cytotoxicity assay, we have shown that Chlamydia trachomatis infection can be blocked by certain sulfated polysaccharides (carrageenan, pentosan polysulfate, fucoidan, and dextran sulfate) and glycosaminoglycans (heparin, heparan sulfate, and dermatan sulfate) but not by other glycosaminoglycans (chondroitin sulfate A or C, keratan sulfate, and hyaluronic acid). The most negatively charged molecules are the most effective at blocking infection. Results of infection at 4 degrees C suggest that sulfated polyanions act by preventing the adherence of chlamydiae to target cells. These and additional blocking studies with enzymes suggest that a heparan sulfate-like glycosaminoglycan on the surface of elementary bodies is involved in the adherence of chlamydiae to target cells, probably through a nonspecific charge interaction or possibly a heparin-binding protein. We previously observed that the same sulfated polysaccharides inhibit transmission of human immunodeficiency virus in vitro and suggested that these compounds could be used in a vaginal formulation to inhibit infection by human immunodeficiency virus. The results of the present study suggest that the same type of formulation may inhibit sexual transmission of chlamydia.
T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II
Authors: Kelly C G , Todryk S , Kendal H L , Munro G H , Lehner T
Published: January 2020

The T-cell and antibody responses to a cell surface streptococcal antigen (SA I/II) were investigated in naturally sensitized humans. Serum antibody responses were directed predominantly to the N-terminal (residues 39 to 481) and central (residues 816 to 1213) regions of SA I/II which may be involved in bacterial adhesion to salivary receptors. T-cell responses were also directed predominantly towards the central region. The linear peptide relationship of the immunodominant and minor T- and B-cell as well as adhesion epitopes was mapped within residues 816 to 1213. Immunodominant T-cell and B-cell epitopes were identified within residues 803 to 853, which were separated in linear sequence from the adhesion epitopes (residues 1005 to 1044). Adhesion epitopes overlapped with minor B- and T-cell epitopes (residues 1005 to 1054 and 1085 to 1134). An immunodominant promiscuous T-cell epitope (residues 985 to 1004) was adjacent to an adhesion epitope (residues 1005 to 1024). The limited B-cell response to adhesion epitopes is consistent with the success of Streptococcus mutans in colonizing the oral cavity. The strategy of T-cell, adhesion, and B-cell epitope mapping has revealed a general approach for identifying components of subunit vaccines which may focus responses to critical functional determinants. Such epitopes of SA I/II may constitute the components of a subunit vaccine against dental caries.
Factors influencing secondary vibriocidal immune responses: relevance for understanding immunity to cholera
Authors: Losonsky G A , Yunyongying J , Lim V , Reymann M , Lim Y L , Wasserman S S , Levine M M
Published: January 2020

Although serum vibriocidal activity is used extensively as a marker of immunity to O1 Vibrio cholerae, there are limitations in this assay to detect instances of reexposure. We define the conditions operative in producing secondary vibriocidal responses in North American volunteers primed with either wild-type V. cholerae 1, 4, or 6 months later. Secondary serum vibriocidal responses occurred under two distinct secondary challenge conditions. The first occurred when secondary challenge produced a breakthrough in clinical protection. Following secondary exposure, 14 of 22 (64%) and 1 of 29 (3%) subjects with and without vibrio stool excretion, respectively, had secondary responses (P < 0.001); 5 of 6 (83%) and 10 of 45 (22%) subjects with or without diarrhea, respectively, mounted a secondary response (P = 0.006). The second condition occurred in the presence of full clinical protection but was dependent on the time interval between exposure. No subject (0 to 17) vaccinated with CVD 103-HgR and given homologous wild-type challenge within 4 months mounted a secondary vibriocidal response (P = 0.0009). The majority of the serum vibriocidal activity was of the immunoglobulin M (IgM) isotype, seen in 96 and 73% of subjects following primary and secondary exposure, respectively. Vibriocidal activity in the IgG fraction following primary and secondary exposures occurred with < or = 50% of volunteers; lipopolysaccharide (LPS)-specific IgG1 and IgG3 subclass responses supported the vibriocidal isotype data. However, following primary exposure, IgG4 LPS responses predominated, occurring in 81% of responding volunteers. These data suggest that, under certain conditions of secondary exposure to V. cholerae O1 antigens, when there is sufficient active local immunity present, there is a block of vibrio antigen resampling at the M cell level. We discuss the implications of and possible explanations for these findings.
Characterization of an RTX toxin from enterohemorrhagic Escherichia coli O157:H7
Authors: Bauer M E , Welch R A
Published: January 2020

A hemolytic determinant of enterohemorrhagic Escherichia coli O157:H7 is encoded on a 90-kbp plasmid (pO157). This enterohemorrhagic E. coli toxin (Ehx) is a newly described RTX cytotoxin. The prototype RTX toxin is the E. coli hemolysin (Hly) associated with extraintestinal E. coli infections. We expressed Ehx from E. coli K-12 strains harboring either pSK3, a pO157 derivative marked with Tn801 unlinked to Ehx, or a recombinant plasmid containing an 11.9-kbp subclone (pEO40) of pSK3. The Ehx activities and antibody reactivities were compared with those of Hly. Little Ehx was secreted extracellularly from the strain harboring pSK3; however, when the Hly transport genes hlyBD were supplied in trans, both intracellular and extracellular levels of Ehx were enhanced more than 15-fold. The strain harboring pEO40 secreted at least 140-fold more Ehx than did the strain harboring pSK3, and neither intracellular nor extracellular levels were significantly enhanced by the addition of hlyBD in trans. Polyclonal anti-HlyA antiserum and several anti-HlyA monoclonal antibodies, including the monoclonal antibody A10, which is panreactive for nearly all RTX toxins, reacted with EhxA antigen by immunoblot analysis. In hemolysis and 51Cr release assays, Ehx demonstrated similar efficiencies in lysis of BL-3 cells (cells from a bovine lymphoma cell line) and sheep and human erythrocytes. Surprisingly, it demonstrated very little activity against two human lymphoma cell lines. In contrast, Hly lysed all five cell types tested, each to a greater extent than that demonstrated by comparable amounts of Ehx. As with other RTX toxins, Ehx activity was calcium dependent and heat labile.
Human CD4+ and CD8+ T lymphocytes are both cytotoxic to Toxoplasma gondii-infected cells
Authors: Montoya J G , Lowe K E , Clayberger C , Moody D , Do D , Remington J S , Talib S , Subauste C S
Published: January 2020

Studies to determine if Toxoplasma gondii-specific human T cells lyse parasite-infected cells have yielded conflicting results. Furthermore, attempts to obtain human cytotoxic CD8+ T lymphocytes have been difficult because of the lack of a reproducible system for their generation. By using paraformaldehyde-fixed, T. gondii-infected peripheral blood mononuclear cells as antigen-presenting cells, we developed a method whereby T. gondii-specific T-cell lines can be reproducibly generated. Six T. gondii-specific T-cell lines were generated from an individual chronically infected with T. gondii. Cytofluorometric analysis of these lines revealed > 99% CD3+, 85 to 95% CD3+ alpha beta T-cell-receptor-positive (TCR+), 5 to 9% CD3+ gamma delta TCR+, 50 to 70% CD4+, and 20 to 40% CD8+ cells when cells were examined during the first 3 weeks of stimulation and >99% CD3+, >99% CD3+ alpha beta TCR+, < 1% CD3+ gamma delta TCR+, 20 to 40% CD4+, and 60 to 80% CD8+ cells when cells were examined between 5 and 11 weeks. Both CD4+ and CD8+ T cells had remarkable cytotoxic activity against T. gondii-infected target cells (30 to 50% specific Cr release at an effector-to-target ratio of 30:1) but not against uninfected target cells ( < 10% at an effector-to-target ratio of 30:1). Cytotoxic activity by the whole T-cell lines was not T. gondii strain specific. Whole T-cell lines were cytotoxic for target cells infected with the C56 and ME49 strains and the RH strain (which was used to infect peripheral blood mononuclear cells). T. gondii-specific T-cell lines displayed the predominant expression of V beta 7 TCR. The CDR3 regions of the V beta 7 TCRs of these T-cell lines showed a striking degree of sequence identity (oligoclonality). T-cell lines obtained by the method reporter here can be used to characterize functional activity of T-lymphocyte subsets in humans infected with T. gondii.
Protease cleavage of iron-transferrin augments pyocyanin-mediated endothelial cell injury via promotion of hydroxyl radical formation
Authors: Miller R A , Rasmussen G T , Cox C D , Britigan B E
Published: January 2020

Although a number of bacterium- and host-derived factors have been suggested to contribute to the pathogenesis of Pseudomonas aeruginosa-associated tissue injury, the mechanism remains unclear. We have previously shown that protease modification of iron (Fe)-transferrin generates new iron chelates capable of catalyzing hydroxyl radical (.OH) formation from superoxide and hydrogen peroxide. The latter two oxidants are generated during redox cycling of another P. aeruginosa secretory product, pyocyanin. The lung is a major site of P. aeruginosa infection, with damage to local endothelial cells contributing to the pathogenesis of such infections. Endothelial cells are highly susceptible to oxidant-mediated injury. Therefore, we examined whether pseudomonas elastase-cleaved Fe-transferrin and pyocyanin synergistically enhance pulmonary artery endothelial cell injury via .OH formation. By measuring 51Cr release from cultured endothelial cell monolayers, pseudomonas elastase-cleaved Fe-transferrin significantly augmented cell injury resulting from cellular exposure to sublethal concentrations of pyocyanin. This enhancement in injury was not protease specific, as similar results were obtained with pyocyanin in combination with trypsin- or porcine pancreatic elastase-cleaved Fe-transferrin. The association of iron with the transferrin appeared to be necessary in this process. Supporting the involvement of .OH generation via the Haber-Weiss reaction in augmenting cell injury, catalase, dimethyl thiourea, superoxide dismutase, deferoxamine, and dimethyl sulfoxide significantly inhibited cell injury resulting from exposure to pyocyanin and protease-cleaved Fe-transferrin. Furthermore, spin trapping demonstrated the production of .OH in this cellular system. We conclude that .OH formation resulting from the interaction of protease-cleaved Fe-transferrin and endothelial cell redox cycling of pyocyanin may contribute to P. aeruginosa-associated tissue injury via endothelial cell injury.
Responses to vaccination in strains of mice that differ in susceptibility to coccidiosis
Authors: Rose M E , Hesketh P , Wakelin D
Published: January 2020

BALB/c mice are normally resistant to infection with Eimeria vermiformis than C57BL/6 (B6) mice, but these phenotypes were reversed by prior vaccination with crude antigens prepared from developmental stages of the parasite: B6 mice were protected, and BALB/c mice were made more susceptible. Infections with a heterologous species, E. pragensis, were unaffected when this was given either alone or together with E. vermiformis. In both strains of mice, vaccination induced serum antibody responses to E. vermiformis and the levels were boosted by superimposed infection, the highest values being found in BALB/c mice. Cellular responses in the mesenteric lymph nodes (MLN), as indicated by cellularity and proliferation, either unstimulated or restimulated in vitro with E. vermiformis antigen, were decreased in both strains, but markedly more in BALB/c than B6. The capacity of MLN cells to transfer immunity to naive recipients was lowered by vaccination of BALB/c donors but unimpaired in vaccinated B6 mice. Responses to the mitogen, concanavalin A, and to unrelated antigens (human erythrocytes and fowl gamma globulin) were unaffected. Thus, parenteral vaccination, which increased the susceptibility of BALB/c mice to infection with E. vermiformis, had a depressing effect on some specific immune responses in the MLN. It was surprising to find some reduction in the cellular responses of the MLN of B6 mice also, although they were protected by vaccination.
Vibrio cholerae Hcp, a secreted protein coregulated with HlyA
Authors: Williams S G , Varcoe L T , Attridge S R , Manning P A
Published: January 2020

Hcp is a 28-kDa secreted protein of Vibrio cholerae regulated coordinately with the hemolysin, HlyA. Both proteins show a dependence on HlyU for expression, suggesting that Hcp may be secreted by V. cholerae in vivo. We have identified and sequenced two genes for Hcp, designated hcpA and hcpB (hemolysin-coregulated protein). The genes encode identical amino acid sequences. Both express a 28-kDa protein, despite open reading frames with only a 19-kDa capacity, suggesting that the Hcp protein runs aberrantly on polyacrylamide gel electrophoresis. There is no cleavage involved in secretion of Hcp from the cell, suggesting a novel mechanism of secretion. An hcp null mutant was constructed, and this strain displayed no deficiency in virulence or colonization in the infant mouse cholera model. From sequence data and primer extension analysis, we predict that the hcp promoter is the sigma 54 type, with a candidate integration host factor binding site upstream. Although hcp and hlyA are coregulated by HlyU, there are no obvious similarities between their promoters. We predict that an intermediate regulator may be involved in the activation of hcp by HlyU. This raises the possibility that HlyU is part of a regulatory cascade.
Molecular characterization of a common 48-kilodalton outer membrane protein of Actinobacillus pleuropneumoniae
Authors: Cruz W T , Nedialkov Y A , Thacker B J , Mulks M H
Published: January 2020

Previous studies have shown that a vaccine prepared from outer membranes of Actinobacillus pleuropneumoniae serotype 5 can elicit protective immunity in swine against challenge with either serotype 5 or serotype 1. These results suggest the presence of common subcapsular surface antigens, such as outer membrane proteins, that contribute to cross-protective immunity. We have identified a 48-kDa outer membrane protein that is common to all 12 capsular serotypes of A. pleuropneumoniae but is not present in the outer membranes of related species of gram-negative swine pathogens. This protein is immunogenic in swine infected with either A. pleuropneumoniae serotype 5 or 1A, as well as in swine vaccinated with A. pleuropneumoniae serotype 5 outer membranes. This 48-kDa protein is readily detected in outer membranes produced by sucrose density gradient centrifugation, but it is sarcosyl soluble and therefore not found in outer membranes prepared by detergent treatment. The gene encoding the 48-kDa outer membrane protein has been cloned from A. pleuropneumoniae serotype 5 and and has been designated aopA, for Actinobacillus outer membrane protein A. The gene is 1,347 bp in length and encodes a protein, designated AopA, of 449 amino acids with a predicted molecular weight of 48,603. Southern blot analysis under high-stringency conditions showed that strains of all 12 serotypes of A. pleuropneumoniae contain DNA homologous to this gene, as do strains of two closely related species, Actinobacillus suis and Pasteurella multocida. Whether antibodies against the AopA antigen contribute to cross-protective immunity against A. pleuropneumoniae infection remains to be determined.
Fibronectin-binding protein of Streptococcus equi subsp. zooepidemicus
Authors: Lindmark H , Jacobsson K , Frykberg L , Guss B
Published: January 2020

By screening a genomic lambda library of Streptococcus equi subsp. zooepidemicus, we have cloned and sequenced a gene, termed fnz, encoding a fibronectin (Fn)-binding protein called FNZ. On the basis of the deduced amino acid sequence of FNZ, the mature protein has a molecular mass of approximately 61 kDa. Analysis of FNZ reveals a structural organization similar to that of other cell surface proteins from streptococci and staphylococci. The Fn-binding activity is localized to two domains in the C-terminal part of FNZ. One domain is composed of five repeats, which contain a motif similar to what has earlier been found in other Fn-binding proteins in streptococci and staphylococci. The first and second repeats are separated by a short stretch of amino acids, including the motif LAGESGET, which is an important part of the second Fn-binding domain. This motif is also present in an Fn-binding domain (UR) in protein F of Streptococcus pyogenes. A fusion protein covering the Fn-binding domain of FNZ inhibits the binding of the 29-kDa N-terminal fragment of Fn to cells of various streptococcal species as well as to Staphylococcus aureus.
Analysis of proteins encoded by the ptx and ptl genes of Bordetella bronchiseptica and Bordetella parapertussis
Authors: Hausman S Z , Cherry J D , Heininger U , Wirsing von König C H , Burns D L
Published: January 2020

Bordetella pertussis is the only bacteria] species which is known to produce pertussis toxin (PT); however, both Bordetella bronchiseptica and Bordetella parapertussis contain regions homologous to the ptx genes of B. pertussis that encode the toxin subunits. After finding that several children with B. parapertussis infections exhibited modest antibody titers to PT, we examined the ptx genes of both B. parapertussis and B. bronchiseptica to determine whether they would encode stable, functional proteins even though their promoters are thought to be inactive under the conditions that have been examined. We inserted a functional promoter directly upstream of the ptx-ptl region of both species and examined culture supernatants of the resulting strains for PT activity. Biologically active PT was found in the culture supernatants of both engineered species. The toxin encoded by the B. parapertussis ptx genes appeared more labile in culture supernatants than did toxin produced by either B. pertussis or the engineered strain of B. bronchiseptica. This lability might be due to the lack of a full-length S2 subunit. We also investigated the ptl genes of these species, which are necessary for the secretion of this toxin, and found that both B. bronchiseptica and B. parapertussis contain at least certain of these genes, including ptlE and ptlF. Moreover, B. bronchiseptica appeared to contain all essential ptl genes since the introduction of a functional promoter directly upstream of the ptx-ptl region resulted in both production and efficient secretion of toxin. These results indicate that despite a number of amino acid changes in the sequences of the toxins, the toxins encoded by B. bronchiseptica and B. parapertussis are active.
Processing and presentation of an antigen of Mycobacterium avium require access to an acidified compartment with active proteases
Authors: Holsti M A , Allen P M
Published: January 2020

We have generated a murine T-cell hybridoma, 1C9, which recognizes an antigen expressed by a virulent clinical isolate of Mycobacterium avium. Both peritoneal exudate macrophages and bone marrow-derived macrophages infected in vitro with M. avium process and present the antigen to the T-cell hybridoma. Gel filtration chromatography of a sonicate of M. avium followed by T-cell Western blotting (immunoblotting) demonstrated that the antigen recognized by hybridoma 1C9 is approximately 50 kDa. In addition, treatment of macrophages with the lysosomotropic agent chloroquine or with inhibitors of acid proteases inhibits processing and presentation of the antigen. These results indicate that the antigen must encounter an acidic compartment with active proteases for processing and presentation to occur. Our results are discussed in the context of our current understanding of how mycobacterial antigens are processed and presented by infected macrophages to T cells.
Characterizations of neutral lipid fatty acids and cis-9,10-epoxy octadecanoic acid in Pneumocystis carinii carinii
Authors: Kaneshiro E S , Ellis J E , Guo Z , Jayasimhulu K , Maiorano J N , Kallam K A
Published: January 2020

Pneumocystis carinii causes pneumonitis in immunodeficient individuals and is a prevalent opportunistic infection of patients with AIDS. This pathogen resides extracellularly in the hypophase lining the alveolar epithelium, which is highly enriched in lung surfactant lipids. Procedures yielding highly pure organism preparations that enable reliable biochemical analyses of organisms isolated from the lungs of infected laboratory animals have been developed. The results of the present study revealed that the fatty acid profiles of total lipids, the neutral lipid traction, and individual neutral lipid classes of lungs from normal and immunosuppressed rats as well as P. carinii were grossly similar, although some quantitative differences were detected. One qualitative exception found was the detection in P. carinii of the rare fatty acid cis-9,10-epoxy stearic acid, which was not detected in the lipids of rat lungs. The detection of this fatty acid in P. carinii may also have important taxonomic implications. Unlike phospholipids, many of the fatty acids of nonmembrane neutral lipids may be utilized by P. carinii for other cellular functions, such as stored reserves for energy production and precursors for organism-specific membrane lipids. The present study represents the first report of detailed fatty acid analyses of individual neutral lipid classes of this important opportunistic pathogen.
Expression and immunogenicity of pertussis toxin S1 subunit-tetanus toxin fragment C fusions in Salmonella typhi vaccine strain CVD 908
Authors: Barry E M , Gomez-Duarte O , Chatfield S , Rappuoli R , Pizza M , Losonsky G , Galen J , Levine M M
Published: January 2020

Salmonella typhi vaccine strain CVD 908 can deliver heterologous antigens to the host immune system following mucosal immunization. Stable expression of foreign proteins in Salmonella cells often requires antigen-specific engineering strategies. Fusion of antigens to stabilizing proteins has proven to be a successful strategy for rescuing otherwise unstable proteins. We designed plasmids to allow the fusion of antigens to the amino terminus or carboxyl terminus of fragment C of tetanus toxin, separated by a 4-amino-acid hinge region. Towards the ultimate goal of developing a live oral diphtheria-pertussis-tetanus vaccine, we used these plasmids to stably express the S1 subunit of pertussis toxin in CVD 908. Driven by the anaerobically inducible nirB promoter, the S1 subunit alone was expressed poorly in Salmonella cytoplasm. In contrast, hybrid proteins with S1 fused to either the amino or carboxyl terminus of fragment C were expressed at a high level in CVD 908 and were recognized in Western blot (immunoblot) analysis by monoclonal antibodies directed to S1 and to fragment C. Mice were immunized by the oral or intranasal routes with CVD 908 derivatives harboring these recombinant plasmids. All fusion proteins elicited serum antibody responses to fragment C following intranasal immunization, whereas oral inoculation did not. The configuration of antigens constituting the fusion was critical; S1 fused to the amino terminus of fragment C was less effective than S1 fused to the carboxyl terminus in generating anti-fragment C antibodies. CVD 908 expressing truncated S1 fused to the carboxyl terminus of fragment C elicited neutralizing serum pertussis antitoxin following intranasal immunization of mice.
Absence of tumor necrosis factor alpha, interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor expression but presence of IL-1beta, IL-8, and IL-10 expression in human monocytes exposed to viable or killed Ehrlichia chaffeensis
Authors: Lee E H , Rikihisa Y
Published: January 2020

Ehrlichia chaffeensis is a recently isolated minute gram-negative obligatory intracellular bacterium of monocytes/macrophages and is the etiologic agent of human monocytic ehrlichiosis. It is not known how macrophages respond when they encounter ehrlichiae in terms of cytokine production. In this study, we examined cytokine mRNA expression by incubating E. chaffeensis with THP-1 cells and performing competitive reverse transcription-PCR (RT-PCR). At 2 h postinfection, the levels of interleukin-1beta (IL-1beta), IL-8, and IL-10 mRNAs were significant but lower than those following Escherichia coli lipopolysaccharide (LPS) stimulation. Unlike the situation with E. coli LPS stimulation, however, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha) mRNAs were not induced. Time course and dose-response studies confirmed the absence of IL-6, GM-CSF, and TNF-alpha mRNA induction with E. chaffeensis. Viable E. chaffeensis organisms were not required for IL-1beta IO, IL-8, and IL-10 mRNA induction, since heat-killed E. chaffeensis induced identical time course responses. IL-1beta, IL-8, and IL-10 mRNAs were detected for up to 21, 21, and 24 h postexposure with E. chaffeensis, respectively, which were shut off more rapidly than with LPS stimulation. Although heat treatment of E. chaffeensis had no effect, periodate treatment completely abolished the ability of E. chaffeensis to induce IL-1beta, IL-8, and IL-10 mRNAs. The capture enzyme-linked immunosorbent assay result corresponded with the RT-PCR results, showing that viable and heat-killed E. chaffeensis produced and secreted the same levels of IL-1beta and IL-8. IL-10 production was significantly reduced by heat treatment. Periodate-treated ehrlichiae did not induce production of any of the cytokines tested. Anti-CD14 monoclonal antibody and polymyxin B did not inhibit IL-1beta mRNA expression upon exposure to E. chaffeensis. The absence of TNF-alpha, IL-6, and GM-CSF mRNA induction may delay the development of a protective immune response, thereby allowing E. chaffeensis to set up residence in macrophages.