bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutropenia Restores Virulence to an Attenuated Cu,Zn Superoxide Dismutase-Deficient
Haemophilus ducreyi
Strain in the Swine Model of Chancroid
Authors: San Mateo Lani R. , Toffer Kristen L. , Orndorff Paul E. , Kawula Thomas H.
Published: December 2019

Haemophilus ducreyi
causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus.
H. ducreyi
expresses a periplasmic copper-zinc superoxide dismutase (Cu,Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect
H. ducreyi
from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in
H. ducreyi
pathogenesis, we compared a Cu,Zn SOD-deficient
H. ducreyi
strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (

sodC
+
sodC

) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient
H. ducreyi
. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in
H. ducreyi
, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.
Development of Antibodies against Chondroitin Sulfate A-AdherentPlasmodium falciparumin Pregnant Women
Authors: Maubert Bertrand , Fievet Nadine , Tami Germaine , Cot Michel , Boudin Christian , Deloron Philippe
Published: December 2019

In areas wherePlasmodium falciparumis endemic, pregnant women are at increased risk for malaria, and this risk is greatest during the first pregnancy. The placenta sequesters parasites that are able to cytoadhere to chondroitin sulfate A (CSA), a molecule expressed by the placental syncytiotrophoblast, while parasites from a nonpregnant host do not bind to CSA. Cytoadherence is mediated by the expression of variants of theP. falciparum-erythrocyte membrane protein 1 family. Each member of this molecule family induces antibodies that specifically agglutinate infected erythrocytes and inhibit their cytoadherence ability. We investigated whether the higher susceptibility of primigravidae was related to the lack of immune response towards CSA-binding parasites. In a cross-sectional study, primigravidae delivering with a noninfected placenta were less likely to have antibodies agglutinating CSA-binding parasites than multigravidae (P< 0.01). In contrast, parasites from nonpregnant hosts were as likely to be recognized by the sera from women of various parities. In a longitudinal study, at 6 months of pregnancy, antibodies against CSA-binding parasites were present in 31.8% of primigravidae and in 76.9% of secundigravidae (P= 0.02). The antibodies against CSA-binding parasites inhibited the cytoadherence of a CSA-adherent parasite strain to the human placental trophoblast. Our data support the idea that the higher susceptibility of primiparae is related to a lack of a specific immune response to placental parasites.
Functional Characterization of Type IV Pili Expressed on Diarrhea-Associated Isolates of
Aeromonas
species
Authors: Kirov Sylvia M. , O’Donovan Lisa A. , Sanderson Kevin
Published: December 2019

Our past work has shown that long, flexible type IV pili (single or in bundles) are the predominant pili expressed on fecal isolates of diarrhea-associated species of
Aeromonas
(
Aeromonas veronii
biovar sobria and
A. caviae
). They represent a family of type IV pili which we have designated Bfp (for bundle-forming pili). Reports from Japan suggest that Bfp are intestinal colonization factors. This study presents compelling evidence to support this conclusion.
Aeromonas
bacteria and/or Bfp purified from a strain of
A. veronii
biovar sobria were shown to adhere to epithelial and intestinal cell lines, freshly isolated human enterocytes, and fresh and fixed human and rabbit intestinal tissues, as determined by light and electron microscopy and immunohistochemical detection. Removal of Bfp by mechanical means decreased adhesion to cell lines by up to 80%. Purified Bfp blocked adhesion of the test strain to intestinal cells in a dose-dependent manner. Adhesion was also blocked by the Fab fraction of anti-Bfp immunoglobulin G. Moreover, ultrastructural studies (ruthenium red staining and transmission and scanning electron microscopy) demonstrated for the first time that
Aeromonas
adhesion to human enterocytes is pilus mediated and suggested that Bfp may also promote colonization by forming bacterium-to-bacterium linkages. Bfp-positive isolates examined for type IV pilus-mediated twitching motility in agar and slide culture assays developed for
Pseudomonas aeruginosa
did not, however, exhibit this function.
The Endogenous Balance of Soluble Tumor Necrosis Factor Receptors and Tumor Necrosis Factor Modulates Cachexia and Mortality in Mice Acutely Infected withTrypanosoma cruzi
Authors: Truyens Carine , Torrico Faustino , Lucas Rudolf , De Baetselier Patrick , Buurman Wim A. , Carlier Yves
Published: December 2019

To better understand the role of tumor necrosis factor (TNF) duringTrypanosoma cruziinfection in BALB/c mice, we have investigated the kinetics of circulating tumor necrosis factor (TNF), soluble TNF receptor 1 (sTNR1), and sTNFR2 levels, as well as the interactions between such factors, in relation to parasitemia, cachexia, and mortality of acutely infected animals. Our data show that the parasitemic phase ofT. cruziinfection in mice is associated with high levels of circulating TNF and sTNFR2, resulting in the formation of cytokine-receptor complexes and some degree of neutralization of TNF bioactivity. Although sTNR2 levels always exceeded TNF levels, low sTNFR/TNF circulating ratios were associated with cachexia in all infected mice, whereas the lowest ratios were observed in dying animals harboring the highest parasitemia. We also studied the modulation of sTNFR/TNF ratios induced by anti-TNF antibodies administered to infected animals and their consequences on the outcome of the infection. The injection of anti-TNF monoclonal antibody (MAb) TN3 into infected mice resulted in a paradoxical overproduction of TNF (associated with a higher parasitemia), lowered the sTNFR/TNF circulating ratios, and considerably worsened cachexia and mortality of animals. Another anti-TNF MAb (1F3F3) decreased the in vivo availability of TNF as well as parasite levels and reduced cachexia. Altogether, such results highlight that, besides playing a beneficial role early in infection, TNF also triggers harmful effects in the parasitemic phase, which are limited by the in vivo simultaneous endogenous production of soluble receptors.
Temporal Sequence of Pulmonary and Systemic Inflammatory Responses to Graded Polymicrobial Peritonitis in Mice
Authors: Stamme Cordula , Bundschuh Daniela Sophie , Hartung Thomas , Gebert Ulla , Wollin Lutz , Nüsing Rolf , Wendel Albrecht , Uhlig Stefan
Published: December 2019

The lungs are the remote organ most commonly affected in human peritonitis. The major goals of this study were to define the dose- and time-dependent relationship between graded septic peritonitis and systemic and pulmonary inflammatory responses in mice. BALB/c mice were treated with intraperitoneal polymicrobial inoculi and sacrificed at 3, 12, and 24 h. The treatment protocol resulted in distinct groups of animals with respect to mortality rate, kinetics, and concentrations of a broad spectrum of pro- and anti-inflammatory endogenous mediators, intrapulmonary bacterial accumulation, and static lung compliance. In sublethally infected mice, pulmonary bacterial proliferation was controlled. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-10, interleukin-6, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor (TNF) in plasma were elevated 3 h after infection exclusively. At 3 h, MCP-1, gamma interferon, and TNF were detected in extracts of pulmonary tissue or in bronchoalveolar lavage (BAL) fluid. Static lung compliance (Cst) was transiently decreased at 12 h. In contrast, in lethally infected mice pulmonary bacterial proliferation was not contained. Concentrations of MCP-1, G-CSF, and TNF in plasma were maximal at 24 h, as were pulmonary MCP-1 levels. Lung myeloperoxidase activity was increased at 3, 12, and 24 h.Cstwas reduced after 3 h and did not reach control values at 24 h. Pulmonary cyclooxygenase-2 mRNA and eicosanoids in BAL fluid and plasma were elevated at 3 and 24 h. This study shows that polymicrobial peritonitis in mice leads to dose-dependent systemic and pulmonary inflammation accompanied by a decrease in lung compliance.
Repeated Administration of Synthetic Oligodeoxynucleotides Expressing CpG Motifs Provides Long-Term Protection against Bacterial Infection
Authors: Klinman Dennis M. , Conover Jackie , Coban Cevahir
Published: December 2019

Synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs stimulate an innate immune response characterized by the production of polyreactive immunoglobulin M antibodies and immunomodulatory cytokines. This immune response has been shown to protect mice from challenge byListeria monocytogenesandFrancisella tularensisfor up to 2 weeks. By repeatedly administering CpG ODN two to four times/month, we found that this protection could be maintained indefinitely. Protection was associated with a significant increase in the number of spleen cells that could be triggered by subsequent pathogen exposure to secrete gamma interferon and interleukin-6 in vivo (P< 0.01). ODN-treated animals remained healthy and developed neither macroscopic nor microscopic evidence of tissue damage or inflammation. Thus, repeated administration of CpG ODN may provide a safe means of conferring long-term protection against infectious pathogens.
Lysogenic Conversion of Environmental
Vibrio mimicus
Strains by CTXΦ
Authors: Faruque Shah M. , Rahman M. Mostafizur , Asadulghani  , Islam K. M. Nasirul , Mekalanos John J.
Published: December 2019

The filamentous bacteriophage CTXΦ, which encodes cholera toxin (CT) in toxigenic
Vibrio cholerae
, is known to propagate by infecting susceptible strains of
V. cholerae
by using the toxin coregulated pilus (TCP) as its receptor and thereby causing the origination of new strains of toxigenic
V. cholerae
from nontoxigenic progenitors. Besides
V. cholerae
,
Vibrio mimicus
strains which are normally TCP negative have also been shown to occasionally produce CT and cause diarrhea in humans. We analyzed nontoxigenic
V. mimicus
strains isolated from surface waters in Bangladesh for susceptibility and lysogenic conversion by CTXΦ and studied the expression of CT in the lysogens by using genetically marked derivatives of the phage. Of 27
V. mimicus
strains analyzed, which were all negative for genes encoding TCP but positive for the regulatory gene
toxR
, 2 strains (7.4%) were infected by CTX-KmΦ, derived from strain SM44(P27459
ctx
::km), and the phage genome integrated into the host chromosome, forming stable lysogens. The lysogens spontaneously produced infectious phage particles in the supernatant fluids of the culture, and high titers of the phage could be achieved when the lysogens were induced with mitomycin C. This is the first demonstration of lysogenic conversion of
V. mimicus
strains by CTXΦ. When a genetically marked derivative of the replicative form of the CTXΦ genome carrying a functional
ctxAB
operon, pMSF9.2, was introduced into nontoxigenic
V. mimicus
strains, the plasmid integrated into the host genome and the strains produced CT both in vitro and inside the intestines of adult rabbits and caused mild-to-severe diarrhea in rabbits. This suggested that in the natural habitat infection of nontoxigenic
V. mimicus
strains by wild-type CTXΦ may lead to the origination of toxigenic
V. mimicus
strains which are capable of producing biologically active CT. The results of this study also supported the existence of a TCP-independent mechanism for infection by CTXΦ and showed that at least one species of
Vibrio
other than
V. cholerae
may contribute to the propagation of the phage.
Interleukin-6 and Interleukin-12 Participate in Induction of a Type 1 Protective T-Cell Response during Vaccination with a Tuberculosis Subunit Vaccine
Authors: Leal Irene S. , Smedegård Birgitte , Andersen Peter , Appelberg Rui
Published: December 2019

We examined the role of cytokines in the development of gamma interferon (IFN-γ)-secreting protective T cells following immunization with a culture filtrate subunit vaccine againstMycobacterium tuberculosiscontaining the adjuvant dimethyldioctadecylammonium bromide (DDA). Depletion of either interleukin-6 (IL-6) or IL-12 with specific neutralizing antibodies during vaccination reduced the priming of T cells for antigen-specific proliferation and IFN-γ secretion. Such reduction was also observed in IL-6 gene-disrupted mice as compared to wild-type animals. IL-6 was found to play a role in the initial differentiation of Th1 cells but not in their expansion. The defect found after IL-6 depletion or in IL-6-knockout mice was compensated by the inclusion of recombinant mouse IL-12 in the vaccine. The induction of protective immunity against an intravenous or an aerosol challenge with live, virulentM. tuberculosiswas markedly reduced by neutralizing either IL-6 or IL-12 during immunization with the vaccine. Likewise, the effects of IL-6 neutralization were partially reversed by including IL-12 in the vaccine. Our data point to an important role of IL-6 and IL-12 in the generation of cell-mediated immunity to tuberculosis.
Lysine Residue 117 of the FasG Adhesin of Enterotoxigenic
Escherichia coli
Is Essential for Binding of 987P Fimbriae to Sulfatide
Authors: Choi Byung-Kwon , Schifferli Dieter M.
Published: December 2019

The FasG subunit of the 987P fimbriae of enterotoxigenic strains of
Escherichia coli
was previously shown to mediate fimbrial binding to a glycoprotein and a sulfatide receptor on intestinal brush borders of piglets. Moreover, the 987P adhesin FasG is required for fimbrial expression, since
fasG
null mutants are nonfimbriated. In this study,
fasG
was modified by site-directed mutagenesis to study its sulfatide binding properties. Twenty single mutants were generated by replacing positively charged lysine (K) or arginine (R) residues with small, nonpolar alanine (A) residues. Reduced levels of binding to sulfatide-containing liposomes correlated with reduced fimbriation and FasG surface display in four
fasG
mutants (R27A, R286A, R226A, and R368). Among the 16 remaining normally fimbriated mutants with wild-type levels of surface-exposed FasG, only one mutant (K117A) did not interact at all with sulfatide-containing liposomes. Four mutants (K117A, R116A, K118A, and R200A) demonstrated reduced binding to such liposomes. Since complete phenotypic dissociation between the structure and specific function of 987P was observed only with mutant K117A, this residue is proposed to play an essential role in the FasG-sulfatide interaction, possibly communicating with the sulfate group of sulfatide by hydrogen bonding and/or salt bridge formation. Residues K17, R116, K118, and R200 may stabilize this interaction.
Protection of Mice against Brucellosis by Vaccination withBrucella melitensisWR201(16MΔpurEK)
Authors: Hoover David L. , Crawford Robert M. , Van De Verg Lillian L. , Izadjoo Mina J. , Bhattacharjee Apurba K. , Paranavitana Chrysanthi M. , Warren Richard L. , Nikolich Mikeljon P. , Hadfield Ted L.
Published: December 2019

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of the conjunctiva or traumatized skin by infected animal products. A vaccine to protect humans from occupational exposure or from zoonotic infection in areas where the disease is endemic would reduce an important cause of morbidity worldwide. Vaccines currently used in animals are unsuitable for human use. We tested a live, attenuated, purine-auxotrophic mutant strain ofBrucella melitensis, WR201, for its ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge withB. melitensis16M. Mice inoculated intraperitoneally with WR201 made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 (IL-2), gamma interferon, and IL-10 when cultured withBrucellaantigens. Immunization led to protection from disseminated infection but had only a slight effect on clearance of the challenge inoculum from the lungs. These studies suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.
Alternative Mechanism of Cholera Toxin Acquisition by
Vibrio cholerae
: Generalized Transduction of CTXΦ by Bacteriophage CP-T1
Authors: Fidelma Boyd E. , Waldor Matthew K.
Published: December 2019

Horizontal transfer of genes encoding virulence factors has played a central role in the evolution of many pathogenic bacteria. The unexpected discovery that the genes encoding cholera toxin (
ctxAB
), the main cause of the profuse secretory diarrhea characteristic of cholera, are encoded on a novel filamentous phage named CTXΦ, has resulted in a renewed interest in the potential mechanisms of transfer of virulence genes among
Vibrio cholerae
. We describe here an alternative mechanism of cholera toxin gene transfer into nontoxigenic
V. cholerae
isolates, including strains that lack both the CTXΦ receptor, the toxin coregulated pilus (TCP), and
attRS
, the chromosomal attachment site for CTXΦ integration. A temperature-sensitive mutant of the
V. cholerae
generalized transducing bacteriophage CP-T1 (CP-T1ts) was used to transfer a genetically marked derivative of the CTX prophage into four nontoxigenic
V. cholerae
strains, including two
V. cholerae
vaccine strains. We demonstrate that CTXΦ transduced by CP-T1ts can replicate and integrate into these nontoxigenic
V. cholerae
strains with high efficiency. In fact, CP-T1ts transduces the CTX prophage preferentially when compared with other chromosomal markers. These results reveal a potential mechanism by which CTXΦ
+
V. cholerae
strains that lack the TCP receptor may have arisen. Finally, these findings indicate an additional pathway for reversion of live-attenuated
V. cholerae
vaccine strains.
Allelic Diversity and Antibody Recognition ofPlasmodium falciparumMerozoite Surface Protein 1 during Hypoendemic Malaria Transmission in the Brazilian Amazon Region
Authors: Da Silveira Lucimeire A. , Dorta Míriam L. , Kimura Emília A. S. , Katzin Alejandro M. , Kawamoto Fumihiko , Tanabe Kazuyuki , Ferreira Marcelo U.
Published: December 2019

The polymorphic merozoite surface protein (MSP-1) ofPlasmodium falciparumis a major asexual blood-stage malaria vaccine candidate. The impact of allelic diversity on recognition of MSP-1 during the immune response remains to be investigated in areas of hypoendemicity such as the Brazilian Amazon region. In this study, PCR was used to type variable regions, blocks 2, 4, and 10, of themsp-1gene and to characterize major gene types (unique combinations of allelic types in variable blocks) inP. falciparumisolates collected across the Amazon basin over a period of 12 years. Twelve of the 24 possible gene types were found among 181 isolates, and 68 (38%) of them had more than one gene type. Temporal, but not spatial, variation was found in the distribution of MSP-1 gene types in the Amazon. Interestingly, some gene types occurred more frequently than expected from random assortment of allelic types in different blocks, as previously found in other areas of endemicity. We also compared the antibody recognition of polymorphic (block 2), dimorphic (block 6), and conserved (block 3) regions of MSP-1 in Amazonian malaria patients and clinically immune Africans, using a panel of recombinant peptides. Results were summarized as follows. (i) All blocks were targeted by naturally acquired cytophilic antibodies of the subclasses IgG1 and IgG3, but the balance between IgG1 and IgG3 depended on the subjects' cumulative exposure to malaria. (ii) The balance between IgG1 and IgG3 subclasses and the duration of antibody responses differed in relation to distinct MSP-1 peptides. (iii) Antibody responses to variable blocks 2 and 6 were predominantly type specific, but variant-specific antibodies that target isolate-specific repetitive motifs within block 2 were more frequent in Amazonian patients than in previously studied African populations.
Citrobacter rodentium espB
Is Necessary for Signal Transduction and for Infection of Laboratory Mice
Authors: Newman Joseph V. , Zabel Brian A. , Jha Sharda S. , Schauer David B.
Published: December 2019

Citrobacter rodentium
is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic
Escherichia coli
(EPEC). EPEC
espB
is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers. Mice challenged with wild-type
C. rodentium
develop a mucosal immunoglobulin A response to EspB. In this study,
C. rodentium espB
has been cloned and its nucleotide sequence has been determined.
C. rodentium espB
was found to have 90% identity to EPEC
espB
. A nonpolar insertion mutation in
C. rodentium espB
was constructed and used to replace the chromosomal wild-type allele. The
C. rodentium espB
mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers. The
C. rodentium espB
mutant also failed to colonize laboratory mice following experimental inoculation. The
espB
mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice. These studies indicate that
espB
is necessary for signal transduction and for colonization of laboratory mice by
C. rodentium
.
Identification and Molecular Analysis of the Gene Encoding
Rickettsia typhi
Hemolysin
Authors: Radulovic Suzana , Troyer Jill M. , Beier Magda S. , Lau Audrey O. T. , Azad Abdu F.
Published: December 2019

Rickettsia typhi
, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (
R. typhi
and
R. prowazekii
) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by
R. typhi
has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (
tlyC
) encoding a hemolysin from
R. typhi
. DNA sequence analysis of
R. typhi tlyC
revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the
R. typhi tlyC
gene product with hemolytic activity, we performed complementation studies with hemolysin-negative
Proteus mirabilis
WPM111 (a HpmA
−
mutant of BA6163) transformed with
R. typhi tlyC
or
R. typhi GFPuv-tlyC
constructs. We demonstrated that the cloned
tlyC
gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of
P. mirabilis
. The availability of the cloned
R. typhi tlyC
will permit further characterization and definition of its role in rickettsial virulence.
Isolation, Characterization, cDNA Cloning, and Antimicrobial Properties of Two Distinct Subfamilies of α-Defensins from Rhesus Macaque Leukocytes
Authors: Tang Yi-Quan , Yuan Jun , Miller Christopher J. , Selsted Michael E.
Published: December 2019

Experiments to isolate and characterize rhesus macaque myeloid α-defensins (RMADs) were conducted. Seven RMAD peptides were isolated and sequenced, and the cDNAs encoding six of these peptides and one other α-defensin from bone marrow were also characterized. Four of the RMADs were found to be highly similar to human neutrophil α-defensins HNP-1 to HNP-3, while the remaining four peptides were much more similar to human enteric α-defensin HD-5. Two α-defensin pairs differed only by the presence or absence of an additional arginine at the amino termini of their mature peptides, indicative of alternate posttranslational processing. The primary translation products of RMAD-1 to -8 are 94- and 96-amino-acid prepropeptides that are highly similar to those of human α-defensins. Immunolocalization experiments revealed a granular cytoplasmic pattern in the cytoplasms of neutrophils, indistinguishable from the pattern observed after immunostaining of human myeloid α-defensins in polymorphonuclear leukocytes. Each of the purified peptides was tested for its in vitro activities againstStaphylococcus aureus502a,Listeria monocytogenesEGD,Escherichia coliML35, andCryptococcus neoformans271A. Several of the peptides were microbicidal for the gram-positive bacteria andC. neoformansat defensin concentrations in the range of 2 to 5 μM. All of the peptides were bacteriostatic againstE. coli, but none were bactericidal for this organism. This study is the first to characterize the sequences and activities of α-defensins from nonhuman primates, data that should aid in delineating the role of these peptides in rhesus macaque host defense.
Purification and Immunoreactivity of Three Components from the 30/32-Kilodalton Antigen 85 Complex inMycobacterium tuberculosis
Authors: Lim Jae-Hyun , Park Jeong-Kyu , Jo Eun-Kyeong , Song Chang-Hwa , Min Dullei , Song Young-Ja , Kim Hwa-Jung
Published: December 2019

The three proteins of the antigen 85 complex (85A, 85B, and 85C), which are major secretory products ofMycobacterium tuberculosis, were purified to homogeneity in large amounts by a combination of chromatography on hydroxylapatite, DEAE-Sepharose, and DEAE-Sephacel and gel filtration fromM. tuberculosisculture filtrate. Then we examined the immunological reactivity of the three proteins in tuberculosis patients and healthy controls. Antibody responses to the 85B and 85A proteins in patients were significantly greater than responses to the 85C protein. In contrast, all three antigens induced significant lymphoproliferation and gamma interferon production in peripheral blood mononuclear cells from healthy tuberculin reactors.
Anatomic Segmentation of the Intestinal Immune Response in Nonhuman Primates: Differential Distribution of B Cells after Oral and Rectal Immunizations to Sites Defined by Their Source of Vascularization
Authors: Eriksson Kristina , Quiding-Järbrink Marianne , Osek Jacek , Nordström Inger , Hjulström Maria , Holmgren Jan , Czerkinsky Cecil
Published: December 2019

We show that the distribution of specific antibodies and antibody-secreting cells in the intestine after oral and rectal immunizations corresponds to the vascularization and lymph drainage patterns of the gut. Oral immunizations induce antibody responses along the parts of the intestine connected to the superior mesenteric vessels and lymph ducts, whereas rectal immunizations induce antibody responses along the parts of the intestine associated with the inferior mesenteric vessels and ducts.
Essential Functional Role of the Polysaccharide Intercellular Adhesin of
Staphylococcus epidermidis
in Hemagglutination
Authors: Mack Dietrich , Riedewald Joachim , Rohde Holger , Magnus Tim , Feucht Hubert H. , Elsner Holger-A. , Laufs Rainer , Rupp Mark E.
Published: December 2019

Hemagglutination of erythrocytes is a common property of
Staphylococcus epidermidis
strains, which is related to adherence and biofilm formation and may be essential for the pathogenesis of biomaterial-associated infections caused by
S. epidermidis
. In three independent biofilm-producing, hemagglutination-positive
S. epidermidis
isolates, interruption of the
icaADBC
operon essential for polysaccharide intercellular adhesin (PIA) synthesis by Tn
917
insertions led to a hemagglutination-negative phenotype. An immunoglobulin G fraction of antiserum to PIA greatly reduced hemagglutination. Purified PIA led to a 64-fold decrease of hemagglutination titers of these strains; however, it did not mediate hemagglutination by itself. These observations define PIA as the hemagglutinin of
S. epidermidis
or at least as its major functional component.
Enteropathogenic
Escherichia coli
Inhibits Phagocytosis
Authors: Goosney Danika L. , Celli Jean , Kenny Brendan , Finlay B. Brett
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) interacts with intestinal epithelial cells, activating host signaling pathways leading to cytoskeletal rearrangements and ultimately diarrhea. In this study, we demonstrate that EPEC interacts with the macrophage-like cell line J774A.1 to inhibit phagocytosis by these cells. Antiphagocytic activity was also observed in cultured RAW macrophage-like cells upon EPEC infection. The EPEC antiphagocytic phenotype was dependent on the type III secretion pathway of EPEC and its secreted proteins, including EspA, EspB, and EspD. Intimin and Tir mutants displayed intermediate antiphagocytic activity, suggesting that intimate attachment mediated by intimin-Tir binding may also play a role in antiphagocytosis. Tyrosine dephosphorylation of several host proteins was observed following infection with secretion-competent EPEC but not with secretion-deficient mutants. Dephosphorylation was detectable 120 min after infection with EPEC, directly correlating with the onset of the antiphagocytic phenotype. Inhibition of protein tyrosine phosphatases by pervanadate treatment increased the number of intracellular wild-type EPEC organisms to levels seen with secretion-deficient mutants, suggesting that dephosphorylation events are linked to the antiphagocytic phenotype. No tyrosine phosphatase activity was detected with the EPEC-secreted proteins, suggesting that EPEC induces antiphagocytosis via a different mechanism than
Yersinia
species. Taken together, the present findings demonstrate a novel function for EPEC-secreted proteins in triggering macrophage protein tyrosine dephosphorylation and inhibition of phagocytosis.
Therapy with a Combination of Low Doses of Interleukin 12 and Chloroquine Completely Cures Blood-Stage Malaria, Prevents Severe Anemia, and Induces Immunity to Reinfection
Authors: Mohan Karkada , Sam Hakeem , Stevenson Mary M.
Published: December 2019

The immunoregulatory cytokine interleukin 12 (IL-12) induces host resistance against experimental malaria. In this study, we tested the feasibility of using IL-12 in combination with chloroquine (CQ) to rescue susceptible A/J mice from lethal blood-stagePlasmodium chabaudiAS infection. Combined treatment with low doses of CQ and IL-12 resulted in a >15-fold reduction in the parasite load and 100% survival of A/J mice with established infections. Compared to control mice, which succumbed to severe anemia, CQ-plus-IL-12-treated mice had significantly higher early- and late-stage erythroid-cell progenitors in the bone marrow and spleen, resulting in significantly higher hematocrits, erythrocyte counts, and percentages of reticulocytes. Production of parasite-specific gamma interferon (IFN-γ) by splenocytes from these mice was upregulated >20-fold relative to controls in parallel with enhanced IFN-γ mRNA expression. Further, enhanced responsiveness to IL-12 and increased downstream IFN-γ production in CQ-plus-IL-12-treated mice was evident from increased mRNA expression for the β1 and β2 subunits of IL-12 receptor in the splenocytes. Moreover, this combined therapy induced higher levels of anti-malaria antibodies than did CQ alone as well as sterile immunity against reinfection. Because IL-12 can be used at low doses and is effective even in established infections, it may be feasible to use this immunochemotherapeutic approach in human malaria.
Preliminary Assessment of the Safety and Immunogenicity of a New CTXΦ-Negative, Hemagglutinin/Protease-Defective El Tor Strain as a Cholera Vaccine Candidate
Authors: Benítez Jorge A. , García Luis , Silva Anisia , García Hilda , Fando Rafael , Cedré Barbara , Pérez Antonio , Campos Javier , Rodríguez Boris L. , Pérez JoséL. , Valmaseda Tania , Pérez Oliver , Pérez Alberto , Ramírez Margarita , Ledón Talena , Jidy Manuel Díaz , Lastre Miriam , Bravo Laura , Sierra Gustavo
Published: December 2019

Vibrio cholerae
638 (El Tor, Ogawa), a new CTXΦ-negative hemagglutinin/protease-defective strain that is a cholera vaccine candidate, was examined for safety and immunogenicity in healthy adult volunteers. In a double-blind placebo-controlled study, no significant adverse reactions were observed in volunteers ingesting strain 638. Four volunteers of 42 who ingested strain 638 and 1 of 14 who received placebo experienced loose stools. The strain strongly colonized the human small bowel, as evidenced by its isolation from the stools of 37 of 42 volunteers.
V. cholerae
638, at doses ranging from 4 × 10
7
to 2 × 10
9
vibrios, elicited significant serum vibriocidal antibody and anti-Ogawa immunoglobulin A antibody secreting cell responses.
The Link between Phylogeny and Virulence in
Escherichia coli
Extraintestinal Infection
Authors: Picard Bertrand , Garcia José Sevali , Gouriou Stéphanie , Duriez Patrick , Brahimi Naïma , Bingen Edouard , Elion Jacques , Denamur Erick
Published: December 2019

Previous studies suggesting a link between
Escherichia coli
phylogenetic groups and extraintestinal virulence have been hampered by the difficulty in establishing the intrinsic virulence of a bacterial strain. Indeed, unidentified virulence factors do exist, and the susceptibility of the host to infection is highly variable. To overcome these difficulties, we have developed a mouse model of extraintestinal virulence to test the virulence of the strains under normalized conditions. We then assessed the phylogenetic relationships compared to the
E. coli
reference (ECOR) collection, the presence of several known virulence determinants, and the lethality to mice of 82 human adult
E. coli
strains isolated from normal feces and during the course of extraintestinal infections. Commensal strains belong mainly to phylogenetic groups A and B1, are devoid of virulence determinants, and do not kill the mice. Strains exhibiting the same characteristics as the commensal strains can be isolated under pathogenic conditions, thus indicating the role of host-dependent factors, such as susceptibility linked to underlying disease, in the development of infection. Some strains of phylogenetic groups A, B1, and D are able to kill the mice, their virulence being most often correlated with the presence of virulence determinants. Lastly, strains of the B2 phylogenetic group represent a divergent lineage of highly virulent strains which kill the mice at high frequency and possess the highest level of virulence determinants. The observed link between virulence and phylogeny could correspond to the necessity of virulence determinants in a genetic background that is adequate for the emergence of a virulent clone, an expression of the interdependency of pathogenicity and metabolic activities in pathogenic bacteria.
Vaccination against Anthrax with Attenuated Recombinant Strains of
Bacillus anthracis
That Produce Protective Antigen
Authors: Barnard John P. , Friedlander Arthur M.
Published: December 2019

The protective efficacy of several live, recombinant anthrax vaccines given in a single-dose regimen was assessed with Hartley guinea pigs. These live vaccines were created by transforming ΔANR and ΔSterne, two nonencapsulated, nontoxinogenic strains of
Bacillus anthracis
, with four different recombinant plasmids that express the anthrax protective antigen (PA) protein to various degrees. This enabled us to assess the effect of the chromosomal background of the strain, as well as the amount of PA produced, on protective efficacy. There were no significant strain-related effects on PA production in vitro, plasmid stability in vivo, survival of the immunizing strain in the host, or protective efficacy of the immunizing infection. The protective efficacy of the live, recombinant anthrax vaccine strains correlated with the anti-PA antibody titers they elicited in vivo and the level of PA they produced in vitro.
A Controlled Clinical Study of the Effect of Nasal Immunization with aStreptococcus mutansAntigen Alone or Incorporated into Liposomes on Induction of Immune Responses
Authors: Childers Noel K. , Tong Giang , Mitchell Stephen , Kirk Katharine , Russell Michael W. , Michalek Suzanne M.
Published: December 2019

Recent attention to mucosal immunization strategies has been focused on the nasal route for vaccine delivery. This study was designed to determine the effectiveness of a liposome-protein vaccine compared to that of a protein-only vaccine in inducing immune responses in humans. Healthy subjects were randomly assigned to two groups and immunized intranasally with a crude antigen preparation rich in glucosyltransferase (C-GTF) fromStreptococcus mutans, alone or in liposomes. Parotid saliva, nasal wash, and serum were collected prior to and at weekly intervals following immunization and were analyzed for anti-C-GTF activity by enzyme-linked immunosorbent assay. The levels of immunoglobulin A (IgA) anti-C-GTF activity in the nasal wash from both groups after immunization increased to a mean peak of fivefold over the baseline level on day 28. Salivary IgA anti-C-GTF responses were induced to a lesser extent. IgG and IgA anti-C-GTF responses in serum were detected on day 14. The IgA responses were predominantly of the IgA1 subclass. These results show that C-GTF vaccines were more effective in inducing a local secretory IgA antibody response than a salivary or serum response when they were given intranasally. The IgA1 anti-C-GTF response in nasal wash samples for liposomal antigen versus antigen only was the only response which was significantly different (P< 0.04). This suggests that the form of the antigen affects the magnitude of the local mucosal response but not that of a disseminated response. These results provide evidence for the effective use of a nasal protein vaccine in humans for the induction of mucosal and systemic responses.
Surface Antigen Exposure by Bismuth Dimercaprol Suppression of
Klebsiella pneumoniae
Capsular Polysaccharide
Authors: Domenico Philip , Tomas J. M. , Merino S. , Rubires X. , Cunha Burke A.
Published: December 2019

The bacterial capsule is an important virulence determinant in animal and plant disease. Bacterial capsule and slime can be inhibited by bismuth compounds, especially when complexed with lipophilic thiol chelators. Bismuth dimercaprol (BisBAL) at 1 ppm of Bi
3+
repressed
Klebsiella pneumoniae
capsule expression in defined medium by nearly 90%, which exposed subsurface structures. The phagocytic index for BisBAL-treated bacteria increased from <10 to 360 bacteria per 100 neutrophils in the presence of complement and anticapsular or anti-O antigen antiserum. BisBAL treatment also enhanced the reactivity of monoclonal antibodies (MAbs) specific for the O1-antigen lipopolysaccharide (LPS) or the LPS core in a dose-dependent manner as indicated by the results of enzyme-linked immunosorbent assays. When anti-O1 MAb was used, the reactivity increased significantly for fully encapsulated O1:K1 or O1:K2 cells but not for O1:K
−
cells. Deposition of C3b also increased significantly for BisBAL-treated O1:K1 or O1:K2 cells but not for O1:K
−
cells. Survival of a serum-sensitive strain was <0.1% when nonimmune human serum absorbed with O1:K1 cells was used and 107% when BisBAL-treated cells were used for absorption. Outer membrane proteins were also more accessible on the surface of
K. pneumoniae
after BisBAL treatment. Thus, at subinhibitory levels, BisBAL inhibited capsule expression, which promoted phagocytosis, enhanced the reactivity of specific antibodies for LPS O antigen, LPS core epitopes, or outer-membrane proteins, and enhanced complement interaction with encapsulated
K. pneumoniae
. By unmasking bacterial surface structures and enhancing the immune system reactivity to bacteria, bismuth thiols may prove useful as adjuncts for vaccination.
Characterization of Candidate Live Oral
Salmonella typhi
Vaccine Strains Harboring Defined Mutations in
aroA
,
aroC
, and
htrA
Authors: Lowe David C. , Savidge Tor C. , Pickard Derek , Eckmann Lars , Kagnoff Martin F. , Dougan Gordon , Chatfield Steven N.
Published: December 2019

The properties of two candidate
Salmonella typhi
-based live oral typhoid vaccine strains, BRD691 (
S. typhi
Ty2 harboring mutations in
aroA
and
aroC
) and BRD1116 (
S. typhi
Ty2 harboring mutations in
aroA
,
aroC
, and
htrA
), were compared in a number of in vitro and in vivo assays. BRD1116 exhibited an increased susceptibility to oxidative stress compared with BRD691, but both strains were equally resistant to heat shock. Both strains showed a similar ability to invade Caco-2 and HT-29 epithelial cells and U937 macrophage-like cells, but BRD1116 was less efficient at surviving in epithelial cells than BRD691. BRD1116 and BRD691 were equally susceptible to intracellular killing within U937 cells. Similar findings were demonstrated in vivo, with BRD1116 being less able to survive and translocate to secondary sites of infection when inoculated into the lumen of human intestinal xenografts in SCID mice. However, translocation of BRD1116 to spleens and livers in SCID mice occurred as efficiently as that of BRD691 when inoculated intraperitonally. The ability of BRD1116 to increase the secretion of interleukin-8 following infection of HT-29 epithelial cells was comparable to that of BRD691. Therefore, loss of the HtrA protease in
S. typhi
does not seem to alter its ability to invade epithelial cells or macrophages or to induce proinflammatory cytokines such as IL-8 but significantly reduces intracellular survival in human intestinal epithelial cells in vitro and in vivo.
YopJ of
Yersinia
spp. Is Sufficient To Cause Downregulation of Multiple Mitogen-Activated Protein Kinases in Eukaryotic Cells
Authors: Palmer Lance E. , Pancetti Alessandra R. , Greenberg Steven , Bliska James B.
Published: December 2019

Pathogenic
Yersinia
spp. utilize a plasmid-encoded type III secretion system to deliver a set of Yop effector proteins into eukaryotic cells. Previous studies have shown that the effector YopJ is required for
Yersinia
to cause downregulation of the mitogen-activated protein (MAP) kinases c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) 1 and 2 in infected macrophages. Here we demonstrate that YopJ is sufficient to cause downregulation of multiple MAP kinases in eukaryotic cells. Cellular fractionation experiments confirmed that YopJ is delivered into the cytoplasmic fraction of macrophages by the type III system. Production of YopJ in COS-1 cells by transfection significantly reduced (5- to 10-fold) activation of JNK, p38, and ERK in response to several different stimuli, including serum and tumor necrosis factor alpha. JNK activation mediated by RacV12, an activated mutant of Rac1, was also blocked by YopJ in COS-1 cells, indicating that YopJ acts downstream of this small GTPase to downregulate MAP kinase signaling. Analysis of transfected COS-1 cells by immunofluorescence microscopy revealed that YopJ is recruited from the cytoplasmic compartment to the cell periphery in response to stimuli (e.g., serum) that induce membrane ruffling. These data indicate that YopJ functions as a “MAP kinase toxin” to selectively block nuclear responses that are triggered by
Yersinia
-host cell interaction.
Antibody Recognition of
Plasmodium falciparum
Erythrocyte Surface Antigens in Kenya: Evidence for Rare and Prevalent Variants
Authors: Bull Peter C. , Lowe Brett S. , Kortok Moses , Marsh Kevin
Published: December 2019

Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) is the name given to a family of parasite proteins that are inserted into the infected erythrocyte surface. Studies using agglutination assays have shown previously that PfEMP1 epitopes are extremely diverse. In a study in Kenya, 21 parasite isolates, including nine from children with severe malaria, were tested for agglutination by 33 pairs of plasma, 21 of which were from the corresponding children. Each plasma pair consisted of a sample taken at the time of disease (acute) and one taken 3 weeks later (convalescent). In agreement with previous studies, infection was generally followed by the induction of antibodies specific to the homologous parasite isolate. In addition however, the results show that (i) some isolates were agglutinated very frequently by heterologous plasma; (ii) unexpectedly, these frequently agglutinated isolates tended to be from individuals with severe malaria; (iii) an inverse relationship existed between the agglutination frequency of each parasite isolate in heterologous plasma and the agglutinating antibody repertoire of the homologous child at the time of disease; and (iv) A 3-month-old child apparently still carrying maternal antibodies was infected by a rarely agglutinated isolate. This child’s plasma agglutinated all isolates at the time of disease, apart from the homologous isolate. These results support the idea that preexisting anti-PfEMP1 antibodies can select the variants that are expressed during a new infection and may suggest the existence of a dominant subset of PfEMP1 variants.
Identification and Characterization of PtlC, an Essential Component of the Pertussis Toxin Secretion System
Authors: Cook David M. , Farizo Karen M. , Burns Drusilla L.
Published: December 2019

PtlC is a member of a set of proteins necessary for the secretion of pertussis toxin (PT) from
Bordetella pertussis
. Using polyclonal antibodies specific for PtlC, we identified PtlC as a protein with an apparent molecular weight of 85,000 that localizes to the membrane fraction of bacterial cell extracts. We found that a mutant strain of
B. pertussis
that contains an in-frame deletion in
ptlC
is unable to secrete PT and that PT secretion is fully restored by expressing
ptlC
in
trans
, indicating that PtlC is essential for transport of PT across the bacterial membrane(s). PT secretion was inhibited in wild-type
B. pertussis
after introduction of a plasmid expressing a mutant
ptlC
altered in the putative nucleotide-binding region, suggesting that this region of PtlC is essential for proper function. Moreover, the observed dominant negative phenotype suggests that PtlC either functions as a multimer or interacts with some other component(s) necessary for secretion of PT.