bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTRecently, 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

ABSTRACTSix 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

ABSTRACT

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

ABSTRACTNovel 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

ABSTRACTIn 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

ABSTRACT

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

ABSTRACTPlasmodium 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.