bluuebunny/crossref_metadata_2025 huggingface dataset:
Gamma Interferon Modulates CD95 (Fas) and CD95 Ligand (Fas-L) Expression and Nitric Oxide-Induced Apoptosis during the Acute Phase ofTrypanosoma cruziInfection: a Possible Role in Immune Response Control
Authors: Martins Gislâine A. , Vieira Leda Q. , Cunha Fernando Q. , Silva João S.
Published: December 2019

We have previously shown that splenocytes from mice acutely infected withTrypanosoma cruziexhibit high levels of nitric oxide (NO)-mediated apoptosis. In the present study, we used the gamma interferon (IFN-γ)-knockout (IFN-γ−/−) mice to investigate the role of IFN-γ in modulating apoptosis induction and host protection duringT. cruziinfection in mice. IFN-γ−/−mice were highly susceptible to infection and exhibited significant reduction of NO production and apoptosis levels in splenocytes but normal lymphoproliferative response compared to the infected wild-type (WT) mice. Furthermore, IFN-γ modulates an enhancement of Fas and Fas-L expression after infection, since the infected IFN-γ−/−mice showed significantly lower levels of Fas and Fas-L expression. The addition of recombinant murine IFN-γ to spleen cells cultures from infected IFN-γ−/−mice increased apoptosis levels, Fas expression, and NO production. In the presence of IFN-γ and absence of NO, although Fas expression was maintained, apoptosis levels were significantly reduced but still higher than those found in splenocytes from uninfected mice, suggesting that Fas–Fas-L interaction could also play a role in apoptosis induction inT. cruzi-infected mice. Moreover, in vivo, the treatment of infected WT mice with the inducible nitric oxide synthase inhibitor aminoguanidine also led to decreased NO and apoptosis levels but not Fas expression, suggesting that IFN-γ modulates apoptosis induction by two independent and distinct mechanisms: induction of NO production and of Fas and Fas-L expression. We suggest that besides being of crucial importance in mediating resistance to experimentalT. cruziinfection, IFN-γ could participate in the immune response control through apoptosis modulation.
Evidence of Thymus-Independent Local and Systemic Antibody Responses toCryptosporidium parvumInfection in Nude Mice
Authors: Adjei Andrew A. , Jones Janet T. , Riggs Michael W. , Enriquez F. Javier
Published: December 2019

Differences in susceptibility to persistent cryptosporidial infection between two strains of adult athymic nude mice prompted us to investigate the immune mechanism(s) that may control resistance to infection in these T-cell-deficient mice. We studied fecal oocyst shedding, serum and fecal parasite-specific antibody responses, and fecal immunoglobulin levels in athymic C57BL/6J nude and athymic BALB/cJ nude mice following oral inoculation withCryptosporidium parvumoocysts at 8 to 9 weeks of age. C57BL/6J nude mice had significantly higher fecal parasite-specific immunoglobulin A (IgA) (days 27, 31, 35, and 42 postinoculation) and IgM (days 10, 17, 24, 28, 31, 38, 42, and 48 postinoculation) levels than BALB/cJ nude mice (P< 0.05) and significantly higher serum parasite-specific IgA levels at 63 days postinoculation (P< 0.03). Moreover, C57BL/6J nude mice shed significantly fewerC. parvumoocysts than BALB/cJ nude mice from days 52 to 63 postinoculation (P< 0.05). In contrast, BALB/cJ nude mice had higher levels of non-parasite-specific IgA (days 38 to 63 postinoculation) and IgM (days 24, 35, 38, and 52 postinoculation) than C57BL/6J nude mice in feces (P< 0.05). These data suggest that parasite-specific fecal antibodies may be associated with resistance toC. parvumin C57BL/6J nude mice.
Molecular and Functional Characteristics of a Protective Human Monoclonal Antibody to Serotype 8Streptococcus pneumoniaeCapsular Polysaccharide
Authors: Zhong Z. , Burns T. , Chang Q. , Carroll M. , Pirofski L.
Published: December 2019

The structural characteristics and biological activity of human antibodies that are reactive with the capsular polysaccharides of most serotypes ofStreptococcus pneumoniae, including serotype 8, are unknown. This paper describes the generation, molecular structure, and protective efficacy of a human monoclonal antibody (MAb) reactive with the capsular polysaccharide of serotype 8Streptococcus pneumoniae. We generated the immunoglobulin M(κ) [IgM(κ)] MAb D11 by Epstein-Barr virus transformation of peripheral lymphocytes from a Pneumovax recipient. Nucleic acid sequence analysis revealed that MAb D11 uses V3-15/VH3 and A20/Vκgene segments with evidence of somatic mutation. In vitro studies revealed MAb D11-dependent complement deposition on the capsule of serotype 8 organisms via either the classical or the alternative complement pathway. In vivo, MAb D11 prolonged the survival of both normal and C4-deficient mice with lethal serotype 8S. pneumoniaeinfection. Our findings demonstrate that a serotype-specific human IgM with certain structural and functional characteristics was protective in mice lacking a functional classical complement pathway and show that alternative complement pathway activation is an important determinant of pneumococcal protection.
Infection of Mice Lacking Interleukin-7 (IL-7) Reveals an Unexpected Role for IL-7 in the Development of the ParasiteSchistosoma mansoni
Authors: Wolowczuk Isabelle , Nutten Sophie , Roye Olivier , Delacre Myriam , Capron Monique , Murray Richard M. , Trottein François , Auriault Claude
Published: December 2019

A single intradermal administration of recombinant interleukin-7 (IL-7) has been shown to aggravate the course of murine schistosomiasis, to favor the development of Th2-associated antibodies specific for the parasite, and to alter migration kinetics and/or migratory route of the parasite within its vertebrate host. Here we show that after infection of IL-7-deficient mice withSchistosoma mansoni, the predominant parasite-specific humoral response follows a Th1 pattern, and the development of the parasite is greatly impaired. In IL-7-deficient mice, increased numbers of larvae reach the lungs and fewer larvae reach the liver, compared to control mice. In the absence of IL-7, female worms show an altered fecundity, leading to decreased numbers of eggs trapped in the tissues and to an amelioration of the pathology of the infected host. The most striking observation is the blockade of parasite growth in an IL-7-defective environment, leading to dwarf male and female worms. The results of this study have important implications for the role of IL-7 in the host-parasite relationship and show how parasites can disable or evade the host immune response.
Attenuated Host Resistance againstMycobacterium bovisBCG Infection in Mice Lacking Osteopontin
Authors: Nau Gerard J. , Liaw Lucy , Chupp Geoffrey L. , Berman Jeffrey S. , Hogan Brigid L. M. , Young Richard A.
Published: December 2019

Expression of the cytokine osteopontin (OPN) is elevated in granulomas caused byMycobacterium tuberculosis. We tested the hypothesis that OPN contributes to host protection in a mouse model of mycobacterial infection. When infected withMycobacterium bovisBCG, mice lacking a functional OPN gene had more severe infections characterized by heavier bacterial loads and a delayed clearance of the bacteria. The OPN-null mice had greater granuloma burdens consistent with the elevated bacterial load. The ability of osteopontin to facilitate the clearance of mycobacteria was most pronounced early after infection and appeared to be independent of known mediators of resistance to infection by mycobacteria: antigen-specific T-cell immunity, gamma interferon production, and nitric oxide production. BCG grew more rapidly in macrophages derived from OPN-null mice than in those from wild-type mice, demonstrating that the null phenotype was due to an intrinsic macrophage defect. These results indicate that osteopontin augments the host response against a mycobacterial infection and that it acts independently from other antimycobacterial resistance mechanisms.
Protection againstMycobacterium aviumby DNA Vaccines Expressing Mycobacterial Antigens as Fusion Proteins with Green Fluorescent Protein
Authors: Velaz-Faircloth Maria , Cobb Alison J. , Horstman Amanda L. , Henry Stanley C. , Frothingham Richard
Published: December 2019

Mycobacterium aviumcauses disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed theM. avium65-kDa antigen, theMycobacterium bovisBCG 85A antigen, and theM. avium85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally withM. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleenM. aviumCFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination againstM. avium.
Detection of Als Proteins on the Cell Wall of
Candida albicans
in Murine Tissues
Authors: Hoyer L. L. , Clevenger J. , Hecht J. E. , Ehrhart E. J. , Poulet F. M.
Published: December 2019

A murine model of disseminated candidiasis was utilized to determine whether
Candida albicans
Als proteins are produced in vivo. The kidneys, spleen, heart, liver, and lungs were collected from mice inoculated with one of three
C. albicans
strains (SC5314, B311, or WO-1). Immunohistochemical analysis of murine tissues by using a rabbit polyclonal anti-Als serum indicated that Als proteins were produced by each
C. albicans
cell in the tissues examined. Patterns of staining with the anti-Als serum were similar among the
C. albicans
strains tested. These data indicated that Als protein production was widespread in disseminated candidiasis and that, despite strain differences in
ALS
gene expression previously noted in vitro, Als protein production in vivo was similar among
C. albicans
strains. The extensive production of Als proteins in vivo and their presence on the
C. albicans
cell wall position these proteins well for a role in host-pathogen interaction.
The Cloned Locus of Enterocyte Effacement from Enterohemorrhagic
Escherichia coli
O157:H7 Is Unable To Confer the Attaching and Effacing Phenotype upon
E. coli
K-12
Authors: Elliott Simon J. , Yu Jie , Kaper James B.
Published: December 2019

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 possesses the same genes in identical order and orientation as the LEE of enteropathogenic
E. coli
(EPEC) O127:H6 but is unable to form attaching and effacing (A/E) lesions or to secrete Esp proteins when it is cloned in an
E. coli
K-12 background. The A/E phenotype could not be restored by
trans
complementation with a variety of cloned EPEC LEE fragments, suggesting functional and/or regulatory differences between the LEE pathogenicity islands of EPEC O127:H6 and EHEC O157:H7.
Infectivity ofPlasmodium bergheiSporozoites Delivered by Intravenous Inoculation versus Mosquito Bite: Implications for Sporozoite Vaccine Trials
Authors: Vaughan Jefferson A. , Scheller Libia F. , Wirtz Robert A. , Azad Abdu F.
Published: December 2019

Plasmodium bergheisporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.
CpG Motifs inPorphyromonas gingivalisDNA Stimulate Interleukin-6 Expression in Human Gingival Fibroblasts
Authors: Takeshita Akira , Imai Kenichi , Hanazawa Shigemasa
Published: December 2019

We suggest here thatPorphyromonas gingivalisDNA may function as a virulence factor in periodontal disease through expression of inflammatory cytokine. The bacterial DNA markedly stimulated in a dose-dependent manner interleukin-6 (IL-6) production by human gingival fibroblasts. The stimulatory action was eliminated by treatment with DNase but not RNase. The stimulatory effect was not observed in the fibroblasts treated with eucaryotic DNAs. The bacterial DNA also stimulated in dose- and treatment time-dependent manners the expression of the IL-6 gene in the cells. In addition, the stimulatory effect was eliminated when the DNA was methylated with CpG motif methylase. Interestingly, a 30-base synthetic oligonucleotide containing the palindromic motif GACGTC could stimulate expression of the IL-6 gene and production of its protein in the cells. Furthermore, the synthetic oligonucleotide-induced expression of this cytokine gene was blocked by pyrrolidine dithiocarbamate andN-acetyl-l-cystine, potent inhibitors of transcriptional factor NF-κB. Gel mobility shift assay showed increased binding of NF-κB to its consensus sequence in the synthetic oligonucleotide-treated cells. Also, using specific antibody against p50 and p65, which compose NF-κB, we showed the consensus sequence-binding proteins to be NF-κB. These results are the first to demonstrate that the internal CpG motifs inP. gingivalisDNA stimulate IL-6 expression in human gingival fibroblasts via stimulation of NF-κB.
Genetically Detoxified Mutants of Heat-Labile Toxin from
Escherichia coli
Are Able To Act as Oral Adjuvants
Authors: Douce Gill , Giannelli Valentina , Pizza Mariagrazia , Lewis David , Everest Paul , Rappuoli Rino , Dougan Gordon
Published: December 2019

Detoxified mutants of the
Escherichia coli
heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i.n. immunizations were consistently higher than the equivalent p.o.-delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response.
Role ofMycoplasma penetransEndonuclease P40 as a Potential Pathogenic Determinant
Authors: Bendjennat Mourad , Blanchard Alain , Loutfi Mohammed , Montagnier Luc , Bahraoui Elmostafa
Published: December 2019

Recently, we reported the purification to homogeneity and characterization of Ca2+- and Mg2+-dependent endonuclease P40 produced byMycoplasma penetrans(M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210–2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10−7to 10−9M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10−7M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10−9M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that125I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of125I-P40-CEM complexes was about 3 × 10−9M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca2+dependent. Our results suggest that the cytotoxicity ofM. penetransobserved in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.
Attenuation and Immunogenicity ofΔcya ΔcrpDerivatives ofSalmonella choleraesuisin Pigs
Authors: Kennedy Michael J. , Yancey Robert J. , Sanchez Margaret S. , Rzepkowski Robert A. , Kelly Sandra M. , Curtiss Roy
Published: December 2019

Six different isogenicΔcya Δcrpderivatives of a strain ofSalmonella choleraesuisvar. kunzendorf-χ3246 virulent for pigs were constructed by transposon-mediated deletion mutagenesis. These strains were evaluated for virulence and ability to elicit a protective immune response in young weaned pigs after oral administration and were compared to a commercially available vaccine which lacks the 50-kb virulence plasmid (vpl−). These derivatives wereΔcya Δcrpvpl+,Δcya Δcrpvpl−,Δcya Δ(crp-cdt) vpl+,Δcya Δ(crp-cdt) vpl−,Δcya Δcrp pmi-3834vpl+, andΔcya Δ(crp-cdt)pmi-3834. In experiments to evaluate safety, no significant adverse effects of any of the vaccine constructs were observed, except that two of the strains which carried the virulence plasmid (vpl+) caused a small, short-term elevation in maximum temperature compared to pretreatment temperature values. Orally immunized animals, except for those vaccinated with theΔcya Δcrp pmi-3834vpl+strain or SC-54, developed significant serum antibody responses 21 days postvaccination as measured by enzyme-linked immunosorbent assay. No cell-mediated immune responses to heat-killedS. choleraesuiswere noted at the same time point as measured with heat-killed bacteria as antigen in a lymphocyte proliferation assay. In an oral challenge exposure model with a highly virulent heterologous strain ofS. choleraesuis, theΔcya Δcrpstrains with deletions inpmiwere not protective. As measured by morbidity scores, the responses to challenge of the pigs vaccinated with the other fourΔcya Δcrpderivatives were significantly better than those of the nonvaccinated, challenged group. With the exception of temperature elevation and slight differences in diarrhea scores postchallenge, none of these strains differed significantly from each other in the other clinical parameters analyzed. While the commercial vaccine was protective by most of the parameters measured, it was not fully protective against challenge with virulentS. choleraesuisas judged by diarrhea scores and temperature elevation. Collectively, these data demonstrate thatΔcya Δcrpderivatives, with or without the virulence plasmid but not with deletions in thepmigene, are candidates for vaccines for protection against salmonellosis in pigs.
Regulation of Protein Phosphorylation and Pathogen Phagocytosis by Surfactant Protein A
Authors: Schagat Trista L. , Tino Michael James , Wright Jo Rae
Published: December 2019

Surfactant protein A (SP-A), a pulmonary member of the collectin family of proteins, facilitates the rapid clearance of pathogens by upregulating immune cell functions in the lungs. SP-A binds to bacteria and targets them for rapid phagocytosis by alveolar macrophages, but the mechanism by which this stimulation occurs is not clear. To characterize the intracellular events that may be involved, we examined the roles of protein phosphorylation and cytoskeletal polymerization in SP-A-stimulated phagocytosis. In rat alveolar macrophages, SP-A stimulated rapid tyrosine phosphorylation of specific proteins in a dose- and time-dependent manner. The pattern of proteins that were phosphorylated in response to SP-A, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar to that observed for immunoglobulin G (IgG)-stimulated macrophages. Both SP-A and IgG stimulated increases in phagocytosis of
Streptococcus pneumoniae
above levels in the absence of added protein by 394% ± 81% and 200% ± 25%, respectively. Phagocytosis in both cases was dependent on tyrosine kinases, protein kinase C, and actin polymerization but not on microtubule activity. These studies show that SP-A utilizes pathways similar to those used by IgG to increase macrophage phagocytosis of bacteria.
Immunogenicity of DNA Vaccines Expressing Tuberculosis Proteins Fused to Tissue Plasminogen Activator Signal Sequences
Authors: Li Zhongming , Howard Angela , Kelley Cynthia , Delogu Giovanni , Collins Frank , Morris Sheldon
Published: December 2019

Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses ofMycobacterium tuberculosisErdman 17 to 21 days after the final immunization. However, the protective response induced by liveMycobacterium bovisBCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates.
β-Chemokines Enhance Parasite Uptake and Promote Nitric Oxide-Dependent Microbiostatic Activity in Murine Inflammatory Macrophages Infected withTrypanosoma cruzi
Authors: Aliberti Júlio C. S. , Machado Fabiana S. , Souto Janeusa T. , Campanelli Ana P. , Teixeira Mauro M. , Gazzinelli Ricardo T. , Silva João S.
Published: December 2019

In the present study, we describe the ability ofTrypanosoma cruzitrypomastigotes to stimulate the synthesis of β-chemokines by macrophages. In vivo infection withT. cruziled to MIP-1α, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection withT. cruziresulted in expression of β-chemokine MIP-1α, MIP-1β, RANTES, and JE mRNA by macrophages. The expression of the β-chemokine MIP-1α, MIP-1β, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms ofT. cruziwas confirmed by immunocytochemistry. Interestingly, macrophage infection withT. cruzialso resulted in NO production, which we found to be mediated mainly by β-chemokines. Hence, treatment with anti-β-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated withT. cruziparasites. Further, the addition of the exogenous β-chemokines MIP-1α, MIP-1β, RANTES, and JE/MCP-1 induced an increasedT. cruziuptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner.l-NMMA, a specific inhibitor of thel-arginine–NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with β-chemokines. Among the β-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-γ). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ. Together, these results suggest that in addition to their chemotactic activity, murine β-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance toT. cruziinfection.
Adhesion of
Escherichia coli
to HeLa Cells Mediated by
Trypanosoma cruzi
Surface Glycoprotein-Derived Peptides Inserted in the Outer Membrane Protein LamB
Authors: Pereira Cátia M. , Favoreto Sílvio , Franco da Silveira José , Yoshida Nobuko , Castilho Beatriz A.
Published: December 2019

Peptides derived from the surface glycoprotein gp82 of
Trypanosoma cruzi
, previously implicated in the parasite’s invasion of host cells, were expressed as fusions to the protein LamB of
Escherichia coli
in a region known to be exposed on the cell surface. Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells. Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by
T. cruzi
, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.
Blood Mononuclear Cell Nitric Oxide Production and Plasma Cytokine Levels in Healthy Gabonese Children with Prior Mild or Severe Malaria
Authors: Perkins Douglas J. , Kremsner Peter G. , Schmid Daniela , Misukonis Mary A. , Kelly Meghan A. , Weinberg J. Brice
Published: December 2019

Plasmodium falciparummalaria is an important cause of morbidity and mortality in children. Factors that determine the development of mild versus severe malaria are not fully understood. Since host-derived nitric oxide (NO) has antiplasmodial properties, we measured NO production and NO synthase (NOS) activity in peripheral blood mononuclear cells (PBMC) from healthy Gabonese children with a history of prior mild malaria (PMM) or prior severe malaria (PSM) caused byP. falciparum. The PMM group had significantly higher levels of NOS activity in freshly isolated PBMC and higher NO production and NOS activity in cultured PBMC. The investigation of NO-modulating cytokines (e.g., interleukin 12, gamma interferon, tumor necrosis factor alpha [TNF-α], and transforming growth factor β1) as an explanation for differing levels of NOS activity revealed that plasma levels of TNF-α were significantly higher in the PSM group. Our results suggest that NOS/ NO and TNF-α are markers for prior disease severity and important determinants of resistance to malaria.
Transcription of the
Rhodobacter sphaeroides cycA
P1 Promoter by Alternate RNA Polymerase Holoenzymes
Authors: MacGregor Barbara J. , Karls Russell K. , Donohue Timothy J.
Published: December 2019

These experiments sought to identify what form of RNA polymerase transcribes the P1 promoter for the
Rhodobacter sphaeroides
cytochrome
c
2
gene (
cycA
). In vitro,
cycA
P1 was recognized by an RNA polymerase holoenzyme fraction that transcribes several well-characterized
Escherichia coli
heat shock (ς
32
) promoters. The in vivo effects of mutations flanking the transcription initiation site (+1) also suggested that
cycA
P1 was recognized by an RNA polymerase similar to
E. coli
Eς
32
. Function of
cycA
P1 was not altered by mutations more than 35 bp upstream of position +1 or by alterations downstream of −7. A point mutation at position −34 that is towards the
E. coli
Eς
32
−35 consensus sequence (G34T) increased
cycA
P1 activity ∼20-fold, while several mutations that reduced or abolished promoter function changed highly conserved bases in presumed −10 or −35 elements. In addition,
cycA
P1 function was retained in mutant promoters with a spacer region as short as 14 nucleotides. When either wild-type or G34T promoters were incubated with reconstituted RNA polymerase holoenzymes,
cycA
P1 transcription was observed only with samples containing either a 37-kDa subunit that is a member of the heat shock sigma factor family (Eς
37
) or a 38-kDa subunit that also allows core RNA polymerase to recognize
E. coli
heat shock promoters (Eς
38
) (R. K. Karls, J. Brooks, P. Rossmeissl, J. Luedke, and T. J. Donohue, J. Bacteriol. 180:10–19, 1998).
Cloning, Sequencing, and Disruption of the
Bacillus subtilis psd
Gene Coding for Phosphatidylserine Decarboxylase
Authors: Matsumoto Kouji , Okada Masahiro , Horikoshi Yuko , Matsuzaki Hiroshi , Kishi Tsutomu , Itaya Mitsuhiro , Shibuya Isao
Published: December 2019

The
psd
gene of
Bacillus subtilis
Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of
pss
, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456–7461, 1994). Introduction of a plasmid containing the
psd
gene into temperature-sensitive
Escherichia coli psd-2
mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the
psd-2
mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of
14
C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in
E. coli psd-2
cells harboring a
Bacillus psd
plasmid.
E. coli
cells harboring the
psd
plasmid, the expression of which was under the control of the T7φ10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The
psd
gene, together with
pss
, was located by Southern hybridization to the 238- to 306-kb
Sfi
I-
Not
I fragment of the chromosome. A
B. subtilis
strain harboring an interrupted
psd
allele,
psd1
::
neo
, was constructed. The null
psd
mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for the
E. coli pssA
null mutant lacking phosphatidylethanolamine. In both the
B. subtilis
null
pss
and
psd
mutants, glucosyldiacylglycerol content increased two- to fourfold. The results suggest that the lack of phosphatidylethanolamine in the
B. subtilis
membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism.
A New Niche for
Vibrio logei
, the Predominant Light Organ Symbiont of Squids in the Genus
Sepiola
Authors: Fidopiastis Pat M. , von Boletzky Sigurd , Ruby Edward G.
Published: December 2019

Two genera of sepiolid squids—
Euprymna
, found primarily in shallow, coastal waters of Hawaii and the Western Pacific, and
Sepiola
, the deeper-, colder-water-dwelling Mediterranean and Atlantic squids—are known to recruit luminous bacteria into light organ symbioses. The light organ symbiont of
Euprymna
spp. is
Vibrio fischeri
, but until now, the light organ symbionts of
Sepiola
spp. have remained inadequately identified. We used a combination of molecular and physiological characteristics to reveal that the light organs of
Sepiola affinis
and
Sepiola robusta
contain a mixed population of
Vibrio logei
and
V. fischeri
, with
V. logei
comprising between 63 and 100% of the bacteria in the light organs that we analyzed.
V. logei
had not previously been known to exist in such symbioses. In addition, this is the first report of two different species of luminous bacteria co-occurring within a single light organ. The luminescence of these symbiotic
V. logei
strains, as well as that of other isolates of
V. logei
tested, is reduced when they are grown at temperatures above 20°C, partly due to a limitation in the synthesis of aliphatic aldehyde, a substrate of the luminescence reaction. In contrast, the luminescence of the
V. fischeri
symbionts is optimal above 24°C and is not enhanced by aldehyde addition. Also,
V. fischeri
strains were markedly more successful than
V. logei
at colonizing the light organs of juvenile
Euprymna scolopes
, especially at 26°C. These findings have important implications for our understanding of the ecological dynamics and evolution of cooperative, and perhaps pathogenic, associations of
Vibrio
spp. with their animal hosts.
Characterization of the
dnaG
Locus in
Mycobacterium smegmatis
Reveals Linkage of DNA Replication and Cell Division
Authors: Klann Amy G. , Belanger Aimee E. , Abanes-De Mello Angelica , Lee Janice Y. , Hatfull Graham F.
Published: December 2019

We have isolated a UV-induced temperature-sensitive mutant of
Mycobacterium smegmatis
that fails to grow at 42°C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an
M. smegmatis
genomic library and subsequent subcloning reveal that the defect lies within the
M. smegmatis dnaG
gene encoding DNA primase. Sequence analysis of the mutant
dnaG
allele reveals a substitution of proline for alanine at position 496. Thus,
dnaG
is an essential gene in
M. smegmatis
, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the
M. smegmatis dnaG
gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a
dgt
gene encoding dGTPase. The organization of this operon is conserved in
Mycobacterium tuberculosis
and
Mycobacterium leprae
, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.
Mechanistic Properties of the Two-Component Bacteriocin Lactococcin G
Authors: Moll Gert , Hildeng-Hauge Håvard , Nissen-Meyer Jon , Nes Ingolf F. , Konings Wil N. , Driessen Arnold J. M.
Published: December 2019

Lactococcin G is a bacteriocin whose activity depends on the complementary action of two peptides, termed α and β. Biologically active, synthetic lactococcin G was used to study the mode of action on sensitive cells of
Lactococcus lactis
. The α and β peptides can bind independently to the target cell surface, but activity requires the complementary peptide. Once bound to the cell surface, the peptides cannot be displaced to the surfaces of other cells. A complex of α and β peptides forms a transmembrane pore that conducts monovalent cations but not protons. Efflux of potassium ions is observed only above pH 5.0, and the rate of efflux increases steeply with the pH. The consequences of cation fluxes for the viability of the target cells are discussed.
The Periplasmic Cyclodextrin Binding Protein CymE from
Klebsiella oxytoca
and Its Role in Maltodextrin and Cyclodextrin Transport
Authors: Pajatsch Markus , Gerhart Maria , Peist Ralf , Horlacher Reinhold , Boos Winfried , Böck August
Published: December 2019

Klebsiella oxytoca
M5a1 has the capacity to transport and to metabolize α-, β- and γ-cyclodextrins. Cyclodextrin transport is mediated by the products of the
cymE
,
cymF
,
cymG
,
cymD
, and
cymA
genes, which are functionally homologous to the
malE
,
malF
,
malG
,
malK
, and
lamB
gene products of
Escherichia coli
. CymE, which is the periplasmic binding protein, has been overproduced and purified. By substrate-induced fluorescence quenching, the binding of ligands was analyzed. CymE bound α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, with dissociation constants (

K
d

) of 0.02, 0.14 and 0.30 μM, respectively, and linear maltoheptaose, with a

K
d

of 70 μM. In transport experiments, α-cyclodextrin was taken up by the
cym
system of
K. oxytoca
three to five times less efficiently than maltohexaose by the
E. coli
maltose system. Besides α-cyclodextrin, maltohexaose was also taken up by the
K. oxytoca cym
system, but because of the inability of maltodextrins to induce the
cym
system, growth of
E. coli mal
mutants on linear maltodextrin was not observed when the cells harbored only the
cym
uptake system. Strains which gained this capacity by mutation could easily be selected, however.
In Vivo Evidence that
S
-Adenosylmethionine and Fatty Acid Synthesis Intermediates Are the Substrates for the LuxI Family of Autoinducer Synthases
Authors: Val Dale L. , Cronan John E.
Published: December 2019

Many gram-negative bacteria synthesize
N
-acyl homoserine lactone autoinducer molecules as quorum-sensing signals which act as cell density-dependent regulators of gene expression. We have investigated the in vivo source of the acyl chain and homoserine lactone components of the autoinducer synthesized by the LuxI homolog, TraI. In
Escherichia coli
, synthesis of
N
-(3-oxooctanoyl)homoserine lactone by TraI was unaffected in a
fadD
mutant blocked in β-oxidative fatty acid degradation. Also, conditions known to induce the
fad
regulon did not increase autoinducer synthesis. In contrast, cerulenin and diazoborine, specific inhibitors of fatty acid synthesis, both blocked autoinducer synthesis even in a strain dependent on β-oxidative fatty acid degradation for growth. These data provide the first in vivo evidence that the acyl chains in autoinducers synthesized by LuxI-family synthases are derived from acyl-acyl carrier protein substrates rather than acyl coenzyme A substrates. Also, we show that decreased levels of intracellular
S
-adenosylmethionine caused by expression of bacteriophage T3
S
-adenosylmethionine hydrolase result in a marked reduction in autoinducer synthesis, thus providing direct in vivo evidence that the homoserine lactone ring of LuxI-family autoinducers is derived from
S
-adenosylmethionine.
Negative Regulation of IS
2
Transposition by the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex
Authors: Hu Shiau-Ting , Wang Hsuan-Chen , Lei Guang-Sheng , Wang Shao-Hung
Published: December 2019

Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS
2
promoter region. Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS
2
transposition. In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS
2
promoter. A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above. With an IS
2
promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS
2
promoter. IS
2
was found to transpose at a frequency approximately 200-fold higher in an
Escherichia coli
host defective for CRP or adenyl cyclase than in a wild-type host. These results suggest that the cAMP-CRP complex is a negative regulator of IS
2
transposition.
Isolation and Characterization of Three
Streptococcus pneumoniae
Transformation-Specific Loci by Use of a
lacZ
Reporter Insertion Vector
Authors: Pestova Ekaterina V. , Morrison Donald A.
Published: December 2019

Although more than a dozen new proteins are produced when
Streptococcus pneumoniae
cells become competent for genetic transformation, only a few of the corresponding genes have been identified to date. To find genes responsible for the production of competence-specific proteins, a random
lacZ
transcriptional fusion library was constructed in
S. pneumoniae
by using the insertional
lacZ
reporter vector pEVP3. Screening the library for clones with competence-specific β-galactosidase (β-Gal) production yielded three insertion mutants with induced β-Gal levels of about 4, 10, and 40 Miller units. In all three clones, activation of the
lacZ
reporter correlated with competence and depended on competence-stimulating peptide. Chromosomal loci adjacent to the integrated vector were subcloned from the insertion mutants, and their nucleotide sequences were determined. Genes at two of the loci exhibited strong similarity to parts of
Bacillus subtilis com
operons. One locus contained open reading frames (ORFs) homologous to the
comEA
and
comEC
genes in
B. subtilis
but lacked a
comEB
homolog. A second locus contained four ORFs with homology to the
B. subtilis comG
gene ORFs 1 to 4, but
comG
gene ORFs 5 to 7 were replaced in
S. pneumoniae
with an ORF encoding a protein homologous to transport ATP-binding proteins. Genes at all three loci were confirmed to be required for transformation by mutagenesis using pEVP3 for insertion duplications or an
erm
cassette for gene disruptions.
Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of
Agrobacterium tumefaciens
Authors: Lai Erh-Min , Kado Clarence I.
Published: December 2019

Previous studies have implicated the obligatory requirement for the
vir
regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from
Agrobacterium tumefaciens
to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the
virB
operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in
Escherichia coli
. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced
A. tumefaciens
had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production of
vir
gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires other
virB
genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of
A. tumefaciens.
Joint Transcriptional Control ofxpsR, the Unusual Signal Integrator of theRalstonia solanacearumVirulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator
Authors: Huang Jianzhong , Yindeeyoungyeon Wandee , Garg Ram P. , Denny Timothy P. , Schell Mark A.
Published: December 2019

Ralstonia(Pseudomonas)solanacearumis a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of theepsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsRpromoter (PxpsR) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theepspromoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of PxpsRto show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of PxpsR; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of PxpsRrequires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate PxpsR. (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect PxpsRactivation or regulation.
Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily
Authors: Zhang Xiaolan , Zhu Liuqin , Deutscher Murray P.
Published: December 2019

Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of
Escherichia coli
. The purified protein is an α2 dimer of 40 kDa. NH
2
-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as
yjeR
(
o204a
), a previously reported open reading frame located at 94 min on the
E. coli
chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of
yjeR
led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose that
yjeR
be renamed
orn
. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.
Functional Similarity between Archaeal and Bacterial CorA Magnesium Transporters
Authors: Smith Ronald L. , Gottlieb Erik , Kucharski Lisa M. , Maguire Michael E.
Published: December 2019

The constitutively expressed CorA Mg
2+
transporter is the major Mg
2+
influx system of
Salmonella typhimurium
and
Escherichia coli
. Genomic sequence data indicated the presence of a homolog in the archaeal organism
Methanococcus jannaschii
. The putative
M. jannaschii
CorA was expressed in an Mg
2+
-transport-deficient strain of
S. typhimurium
to determine its functional characteristics. The archaeal CorA homolog is a functional Mg
2+
uptake system when expressed in
S. typhimurium
and has properties which are highly similar to those of the normal CorA transporter of
S. typhimurium
despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal. This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.