bluuebunny/crossref_metadata_2025 huggingface dataset:
Complex Serology and Immune Response of Mice to Variant High-Molecular-Weight O Polysaccharides Isolated from
Pseudomonas aeruginosa
Serogroup O2 Strains
Authors: Hatano Kazue , Pier Gerald B.
Published: December 2019

The O antigen of the
Pseudomonas aeruginosa
lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of
P. aeruginosa
can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on
P. aeruginosa
cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of
P. aeruginosa
serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 μg) than at a high dose (50 μg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to
P. aeruginosa
O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most
P. aeruginosa
strains within a given O-antigen serogroup.
A Live Recombinant Avirulent Oral
Salmonella
Vaccine Expressing Pneumococcal Surface Protein A Induces Protective Responses against
Streptococcus pneumoniae
Authors: Nayak Amiya R. , Tinge Steven A. , Tart Rebecca C. , McDaniel Larry S. , Briles David E. , Curtiss Roy
Published: December 2019

A live oral recombinant
Salmonella
vaccine strain expressing pneumococcal surface protein A (PspA) was developed. The strain was attenuated with Δ
cya Δcrp
mutations. Stable expression of PspA was achieved by the use of the balanced-lethal vector-host system, which employs an
asd
deletion in the host chromosome to impose an obligate requirement for diaminopimelic acid. The chromosomal Δ
asd
mutation was complemented by a plasmid vector possessing the
asd
+
gene. A portion of the
pspA
gene from
Streptococcus pneumoniae
Rx1 was cloned onto a multicopy Asd
+
vector. After oral immunization, the recombinant
Salmonella
-PspA vaccine strain colonized the Peyer’s patches, spleens, and livers of BALB/cByJ and CBA/N mice and stimulated humoral and mucosal antibody responses. Oral immunization of outbred New Zealand White rabbits with the recombinant
Salmonella
strain induced significant anti-PspA immunoglobulin G titers in serum and vaginal secretions. Polyclonal sera from orally immunized mice detected PspA on the
S. pneumoniae
cell surface as revealed by immunofluorescence. Oral immunization of BALB/cJ mice with the PspA-producing
Salmonella
strain elicited antibody to PspA and resistance to challenge by the mouse-virulent human clinical isolate
S. pneumoniae
WU2. Immune sera from orally immunized mice conferred passive protection against otherwise lethal intraperitoneal or intravascular challenge with strain WU2.
Molecular Cloning of a 32-Kilodalton Lipoprotein Component of a Novel Iron-Regulated
Staphylococcus epidermidis
ABC Transporter
Authors: Cockayne Alan , Hill Philip J. , Powell Nick B. L. , Bishop Keith , Sims Cate , Williams Paul
Published: December 2019

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both
Staphylococcus epidermidis
and
Staphylococcus aureus
. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa
S. epidermidis
lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of
S. aureus
was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in
S. epidermidis
and
S. aureus
and of 40 kDa in
Staphylococcus hominis
. No reactivity was detected with
Staphylococcus lugdunensis
,
Staphylococcus cohni
, or
Staphylococcus haemolyticus
. The gene encoding the 32-kDa lipoprotein from
S. epidermidis
has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated
sitA
, -
B
, and -
C
, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of
Streptococcus parasanguis
and ScaA of
Streptococcus gordonii
, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in
S. epidermidis
.
Gelsolin, a Protein That Caps the Barbed Ends and Severs Actin Filaments, Enhances the Actin-Based Motility of
Listeria monocytogenes
in Host Cells
Authors: Laine Roney O. , Phaneuf Katherine L. , Cunningham Casey C. , Kwiatkowski David , Azuma Toshi , Southwick Frederick S.
Published: December 2019

The actin-based motility of
Listeria monocytogenes
requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular
Listeria
. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with
Listeria
. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean ± standard error of the mean, 0.09 ± 0.003 μm/s [
n
= 176] versus 0.05 ± 0.003 μm/s [
n
= 65]). The rate of disassembly of the
Listeria
-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in
Listeria
-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (
r
= 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular
Listeria
motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of
Listeria
contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of
Listeria
to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of
Listeria
because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.
Passive Immunization with Antibodies against Three Distinct Epitopes onPlasmodium yoeliiMerozoite Surface Protein 1 Suppresses Parasitemia
Authors: Spencer Valero Lilian M. , Ogun Solabomi A. , Fleck Suzanne L. , Ling Irene T. , Scott-Finnigan Terry J. , Blackman Michael J. , Holder Anthony A.
Published: December 2019

We have produced monoclonal antibodies againstPlasmodium yoeliimerozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an ∼42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142is further cleaved to an N-terminal ∼33-kDa polypeptide (MSP-133) and a C-terminal ∼19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142but not with either of the constituents MSP-133and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes onP. yoeliiMSP-1 are recognized by antibodies that suppress parasitemia in vivo.
Phospholipase A of
Yersinia enterocolitica
Contributes to Pathogenesis in a Mouse Model
Authors: Schmiel D. H. , Wagar E. , Karamanou L. , Weeks D. , Miller V. L.
Published: December 2019

Some isolates of
Yersinia enterocolitica
exhibit phospholipase activity, which has been linked to lecithin-dependent hemolysis (M. Tsubokura, K. Otsoki, I. Shimohira, and H. Yamamoto, Infect. Immun. 25:939–942, 1979). A gene encoding
Y. enterocolitica
phospholipase was identified, and analysis of the nucleotide sequence revealed two tandemly transcribed open reading frames. The first,
yplA
, has 74% identity and 85% similarity to the phospholipase A found in
Serratia liquefaciens
. Though the other,
yplB
, was less similar to the downstream accessory protein found in
S. liquefaciens
, the organization in both species is similar. Subsequently, a
yplA
-null
Y. enterocolitica
strain, YEDS10, was constructed and demonstrated to be phospholipase negative by plate and spectrophotometric assays. To ascertain whether the phospholipase has a role in pathogenesis, YEDS10 was tested in the mouse model. In experiments with perorally infected BALB/c mice, fewer YEDS10 organisms were recovered from the mesenteric lymph nodes and Peyer’s patches (PP) than the parental strain at 3 or 5 days postinfection. Furthermore, bowel tissue and PP infected with YEDS10 appeared to be less inflamed than those infected with the parental strain. When extremely high doses of both the parental and YEDS10 strains were given, similar numbers of viable bacteria were recovered from the PP and mesenteric lymph nodes on day 3. However, the numbers of foci and the extent of inflammation and necrosis within them were noticeably less for YEDS10 compared to the parental strain. Together these findings suggest that
Y. enterocolitica
produces a phospholipase A which has a role in pathogenesis.
Use of Monoclonal Antibodies To Identify Phospholipase C as the Enterotoxic Factor of the Bifunctional Hemolysin-Phospholipase C Molecule of
Vibrio cholerae
O139
Authors: Pal Sangita , Datta Archana , Nair G. Balakrish , Guhathakurta Bhakti
Published: December 2019

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a
Vibrio cholerae
O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly
−
spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC
−
mutant was not diarrheagenic.
Immune Responses in Ileostomy Fluid and Serum after Oral Cholera Vaccination of Patients Colectomized because of Ulcerative Colitis
Authors: Kilhamn Jan , Brevinge Hans , Svennerholm Ann-Mari , Jertborn Marianne
Published: December 2019

The capacity of an oral inactivated B-subunit–whole-cell cholera vaccine to induce immune responses in patients colectomized due to ulcerative colitis was studied. Two doses of vaccine induced significant mucosal immunoglobulin A (IgA) antibody responses in ileostomy fluid against cholera toxin in 14 of 15 (93%) patients and against whole vibrios in 9 of 15 (60%) cases. The serological responses were lower (but not significantly) than those observed in healthy Swedish volunteers. Increased IgA antitoxin levels were found in ileostomy fluid as late as 2 years after vaccination.
Symbiosis of
Aeromonas veronii
Biovar sobria and
Hirudo medicinalis
, the Medicinal Leech: a Novel Model for Digestive Tract Associations
Authors: Graf Joerg
Published: December 2019

Hirudo medicinalis
, the medicinal leech, is applied postoperatively in modern medicine. Infections by
Aeromonas
occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of
H. medicinalis
. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as
Aeromonas veronii
biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to
H. medicinalis
. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23°C. After 12 h, at a density of 5 × 10
7
CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as
Aeromonas hydrophila
and
A. veronii
biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of
H. medicinalis
is
A. veronii
biovar sobria and that the medicinal leech can be used as a model for digestive tract association of
Aeromonas
species.
Induction of Pro- and Anti-Inflammatory Cytokines byBorrelia burgdorferiLipoproteins in Monocytes Is Mediated by CD14
Authors: Giambartolomei Guillermo H. , Dennis Vida A. , Lasater Barbara L. , Philipp Mario T.
Published: December 2019

We previously showed that heat-killedBorrelia burgdorferispirochetes and lipidated outer surface protein A (L-OspA) stimulated the in vitro production of interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC) from uninfected humans and rhesus monkeys (G. Giambartolomei et al., Infect. Immun. 66:2691–2697, 1998). Here we demonstrate that uninfected human peripheral blood monocytes, but not B or T cells, are the cells that transcribe the IL-10 cytokine gene in response to heat-killedB. burgdorferi.B. burgdorferisimilarly induced an upregulation of the IL-1β and IL-6 cytokine genes in monocytes and the production of IL-10 and IL-6 in culture supernatants of the human monocytic cell line THP-1. Purified L-OspA (but not unlipidated OspA [U-OspA] or U-OspC) also stimulated the production of both cytokines in THP-1 cells in a dose-dependent fashion, suggesting that acylation of the OspA protein molecule is required for the production of both anti- and pro-inflammatory cytokines in naive monocytes. A lipohexapeptide that contained the tripalmitoyl-modified cysteine motif (Pam3Cys-Hex) ofB. burgdorferilipoproteins but with an arbitrary peptide sequence had the same effect. Monoclonal antibodies (MAbs) MY4 and 60bca, both of which bind to CD14 and are known to block lipopolysaccharide (LPS)-mediated cytokine production, were able to block L-OspA-mediated IL-10 and IL-6 cytokine production. In contrast, MAb 26ic, which also binds to CD14 but does not block LPS function, failed to inhibit L-OspA-mediated cytokine production. These data suggest that activation of monocytes and production of both anti- and pro-inflammatory cytokines induced by lipoproteins proceeds via the CD14 receptor. LPS binding protein was not required for OspA-induced cytokine production. Our results demonstrate that pro- and anti-inflammatory cytokines induced byB. burgdorferilipoproteins in PBMC are produced by monocytes and that lipoprotein and LPS signaling pathways share at least the initial signaling event that involves the CD14 receptor.
Resurgent
Vibrio cholerae
O139: Rearrangement of Cholera Toxin Genetic Elements and Amplification of
rrn
Operon
Authors: Khetawat Gopal , Bhadra Rupak K. , Nandi Suvobroto , Das Jyotirmoy
Published: December 2019

The unprecedented genesis of a novel non-O1
Vibrio cholerae
strain belonging to serogroup O139, which caused an epidemic in late 1992 in the Indian subcontinent, and its subsequent displacement by El Tor O1 vibrios after 18 months initiated a renewed investigation of the aspects of the organism that are related to pathogenesis. The reappearance of
V. cholerae
O139 with altered antibiotic sensitivity compared to O139 Bengal (O139B) in late 1996 has complicated the epidemiological scenario of
V. cholerae
and has necessitated an examination of possible rearrangements in the genome underlying such rapid changes in the phenotypic traits. With a view to investigating whether the phenotypic changes that have occurred are associated with alteration in the genome, the genome of the resurgent
V. cholerae
O139 (O139R) strains were examined. Pulsed-field gel electrophoresis analysis of
Not
I- and
Sfi
I-digested genomic DNA of O139R isolates showed restriction fragment length polymorphism including in the cholera toxin (CTX) genetic element locus and with O139B isolates. Analyses of the organization of the CTX genetic elements in O139R strains showed that in contrast to two copies of the elements connected by two direct-repeat sequences (RS) in most of the genomes of O139B isolates, the genomes of all O139R strains examined, except strain AS192, have three such elements connected by a single RS. While the RS present in the upstream of the CTX genetic elements in the genome of O139R is of O139B origin, the RS connecting the cores of the elements has several new restriction sites and has lost the
Bgl
II site which is supposed to be conserved in all O1 strains and O139B. The endonuclease I-
Ceu
I, which has sites only in the
rrn
operons in the genomes of all organisms examined so far, has 10 sites in the genomes of O139R strains, compared to 9 in the genomes of O139B strains. The recent isolates of
V. cholerae
O139 have thus gained one
rrn
operon. This variation in the number of
rrn
operons within a serogroup has not been reported for any other organism. The results presented in this report suggest that like the pathogenic El Tor O1 strains, the genomes of O139 strains are undergoing rapid alterations.
Bovine CD4+T-Lymphocyte Clones Specific for Rhoptry-Associated Protein 1 ofBabesia bigeminaStimulate Enhanced Immunoglobulin G1 (IgG1) and IgG2 Synthesis
Authors: Brown Wendy C. , McElwain Terry F. , Palmer Guy H. , Chantler Sue Ellen , Estes D. Mark
Published: December 2019

Optimal protective immunity against babesial infection is postulated to require both complement-fixing and opsonizing antibodies in addition to gamma interferon (IFN-γ)-mediated macrophage activation. The rhoptry-associated protein 1 (RAP-1) ofBabesia bigeminainduces partial protective immunity and is a candidate vaccine antigen. Previous studies demonstrated that cattle immunized with native protein that were subsequently protected against challenge had a strong IFN-γ and weaker interleukin-4 (IL-4) response in immune lymph node lymphocytes that reflected the cytokine profile of the majority of CD4+T-cell clones obtained from peripheral blood. RAP-1-specific T helper (Th) cell clones that coexpress IFN-γ and IL-4 are typical of numerous parasite-specific clones examined. However, the function of such cells as helper cells to enhance immunoglobulin secretion by bovine B cells has not been reported. In cattle, both immunoglobulin G1 (IgG1) and IgG2 can fix complement, but IgG2 is the superior opsonizing subclass. Therefore, studies were undertaken to ascertain the functional relevance of RAP-1-specific, CD4+Th0 cells as helper cells to enhance IgG1 and/or IgG2 production by autologous B lymphocytes. For comparison, Th0 clones specific for the metazoan parasiteFasciola hepaticathat expressed relatively more IL-4 than theB. bigemina-specific Th cells were similarly assayed.B. bigeminaRAP-1-specific clones could enhance production of both IgG1 and IgG2 by autologous B cells, whereas Th cell clones specific forF. hepaticaenhanced predominantly IgG1 production. The capacity to enhance IgG2 production was associated with production of IFN-γ by Th cells cocultured with B cells, antigen, and IL-2. The in vitro helper T-cell activity of these T-cell clones was representative of the in vivo serologic responses, which were composed of a mixed IgG1-IgG2 response inB. bigeminaRAP-1 immune cattle and a biased IgG1 response inF. hepatica-immune cattle.
Infection-Derived
Enterococcus faecalis
Strains Are Enriched in
esp
, a Gene Encoding a Novel Surface Protein
Authors: Shankar Viswanathan , Baghdayan Arto S. , Huycke Mark M. , Lindahl Gunnar , Gilmore Michael S.
Published: December 2019

We report the identification of a new cell wall-associated protein of
Enterococcus faecalis
. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (
P
< 0.001) enrichment in infection-derived
E. faecalis
isolates. Interestingly, the
esp
gene was not identified in any of 34 clinical
E. faecium
isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various
E. faecalis
isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of ∼202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived
E. faecalis
and group B streptococcal isolates.
Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells
Authors: Kurita-Ochiai Tomoko , Fukushima Kazuo , Ochiai Kuniyasu
Published: December 2019

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3+-T-cell apoptosis followed by similar levels of both CD4+- and CD8+-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3+PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4+and CD8+cells.
Polarized Helper-T-Cell Responses againstLeishmania majorin the Absence of B Cells
Authors: Brown Daniel R. , Reiner Steven L.
Published: December 2019

B-cell-to-T-cell signaling can shape helper T (Th) cell responses. During infection withLeishmania major, Th response is critical in determining the outcome of disease. Resistance depends on the generation of a protective Th1 response, while susceptibility is mediated by the generation of a Th2 response. In this study, we determined whether B cells are required for the development of polarized Th1 and Th2 responses during infection withL. major. Mice lacking B cells due to disruption of the immunoglobulin M locus (μMT) were infected withL. major, and disease progression and Th cell development were assessed. On the genetically resistant C57BL background, both wild-type and μMT mice controlled the infection and mounted a Th1 response. On the genetically susceptible BALB/c background, both wild-type and μMT mice were susceptible to infection and generated Th2 responses. Thus, duringL. majorinfection, neither direct antigen presentation or costimulation by B cells nor antibody-mediated effector functions are essential for the development of polarized Th responses.
Cloning and Characterization of a Novel Membrane-Associated Antigenic Protein ofHelicobacter pylori
Authors: Yoshida Masaru , Wakatsuki Yoshio , Kobayashi Yoshinao , Itoh Toshiyuki , Murakami Kazuhisa , Mizoguchi Akira , Usui Takashi , Chiba Tsutomu , Kita Toru
Published: December 2019

Infection byHelicobacter pylori, a noninvasive bacterium, induces chronic leukocyte infiltration in the stomach by still largely unknown molecular mechanisms. We investigated the possibility that a membrane protein ofH. pyloriinduces an inflammatory reaction in the subepithelial tissue of the stomach. By generating an expression library ofH. pylorichromosomal DNA and screening with rabbit antiserum raised to a membrane fraction ofH. pyloriand sera of infected patients, we cloned a 16.0-kDa protein (HP-MP1) which appeared to attach to the inner membrane of theH. pyloriin a homodimeric form. Anti-HP-MP1 antibodies were detected in the sera of infected patients but not in those of uninfected controls. Coincubation of monocytes with recombinant HP-MP1 led to cell activation and production of interleukin-1α (IL-1α), tumor necrosis factor alpha, IL-8, and macrophage inflammatory protein 1α. The results indicate that HP-MP1 is an antigenic membrane-associated protein ofH. pyloriwhich potentially activates monocytes. This suggests that HP-MP1 may play roles in the pathogenesis of perpetual tissue inflammation associated withH. pyloriinfection.
Mucin-Related Epitopes Distinguish M Cells and Enterocytes in Rabbit Appendix and Peyer’s Patches
Authors: Lelouard Hugues , Reggio Hubert , Mangeat Paul , Neutra Marian , Montcourrier Philippe
Published: December 2019

The biochemical composition of the apical membranes of epithelial M cells overlying the gut-associated lymphoid tissues (GALT) is still largely unknown. We have prepared monoclonal antibodies (MAbs) directed against carbonate-washed plasma membranes from epithelial cells detached with EDTA from rabbit appendix, a tissue particularly rich in GALT. As determined by immunofluorescence microscopy, several MAbs specifically recognized either M cells or enterocyte-like cells of the domes from rabbit appendix, sacculus rotundus, and Peyer’s patches. M cells were identified by their large ventral pocket containing lymphoid cells and by specific labeling with antivimentin. Among various characterized MAbs, MAb 104 recognized rabbit immunoglobulins and was used as an apical marker for M cells in the rabbit appendix, MAb 58 selectively stained an integral membrane glycoprotein of greater than 205 kDa located at the apex of M cells, and MAb 214 stained a smaller soluble glycoprotein associated with the apical surfaces from neighboring enterocytes. In addition, both MAbs 58 and 214 also labeled luminal mucus and secretory granules in goblet cells. The selective association of mucin-related molecules at the surfaces of either M cells or enterocyte-like cells of the follicle-associated epithelium suggests that specific carbohydrate antigens are differentially expressed by epithelial cells and could account for the differential binding properties of pathogens.
Identification of Two Novel Genes Encoding 97- to 99-Kilodalton Outer Membrane Proteins of
Chlamydia pneumoniae
Authors: Knudsen Katrine , Madsen Anna Sofie , Mygind Per , Christiansen Gunna , Birkelund Svend
Published: December 2019

Two genes encoding 97- to 99-kDa
Chlamydia pneumoniae
VR1310 outer membrane proteins (Omp4 and Omp5) with mutual similarity were cloned and sequenced. The proteins were shown to be constituents of the
C. pneumoniae
outer membrane complex, and the deduced amino acid sequences were similar to those of putative outer membrane proteins encoded by the
Chlamydia psittaci
and
Chlamydia trachomatis
gene families. By use of a monospecific polyclonal antibody against purified recombinant Omp4, it was shown that without heating, the protein migrated at 65 to 75 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoelectron microscopy showed that epitopes of Omp4 were exposed on the surface of
C. pneumoniae
elementary bodies, reticulate bodies, and outer membrane complex. Proteins encoded by the
C. pneumoniae
gene family seem to be dominant antigens in experimentally infected mice.
Characterization ofemb, a Gene Encoding the Major Adhesin ofStreptococcus defectivus
Authors: Manganelli Riccardo , van de Rijn Ivo
Published: December 2019

Streptococcus defectivusis one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate ofS. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins.
Novel Target Antigens of the Variant-Specific Immune Response toPlasmodium falciparumIdentified by Differential Screening of an Expression Library
Authors: Le Scanf Cécile , Fandeur Thierry , Bonnefoy Serge , Guillotte Micheline , Mercereau-Puijalon Odile
Published: December 2019

A primary infection by thePlasmodium falciparumPalo Alto O and R antigenic variants induces a variant-specific immunity in theSaimiri sciureusmonkey. We have shown that these variants express distinct PfEMP1 antigens and differ in their levels of expression of additional antigens, including two conserved erythrocyte membrane-associated proteins, HRP1 and PfEMP3. To identify the antigens eliciting a variant-specific response, we conducted a differential screening of a λgt11 library with variant-specific sera. We report here the analysis of the 46 anti-R-specific clones. Two specific targets of the anti-R response were identified: (i) PfEMP3, suggesting that immunogenicity of this antigen is modulated by its relative abundance in different variants, and (ii) Asn-rich motifs. Most anti-R-specific clones, derived from so-far-undescribed genes, were detected by a cross-reaction on poly(Asn) stretches, as indicated by elimination of the signal after absorption on Asn-rich sequences. Reverse transcription-PCR (RT-PCR) showed that expression of the gene defined by clone 13 was R specific. Pepscan analysis of clone 13 identified three Asn-rich polypeptides and one unique peptide reacting specifically with antibodies eluted from the R-infected erythrocyte surface. Antisera raised to the unique peptide reacted with an R-specific protein. Attempts to demonstrate that clone 13 was derived from avargene by using PCRs combining clone 13 andvar-derived primers were unsuccessful. Thevargenes expressed by O and R parasites were identified not by this strategy but by RT-PCR withvar-specific primers. This work has provided novel insights into immunity to antigenic variants and has identified a novel gene switched on during antigenic variation.
Nucleotide Sequences of Genes Coding for Fimbrial Proteins in a Cryptic Genospecies of
Haemophilus
spp. Isolated from Neonatal and Genital Tract Infections
Authors: Gousset Nathalie , Rosenau Agnes , Sizaret Pierre-Yves , Quentin Roland
Published: December 2019

Nineteen isolates belonging to a cryptic genospecies of
Haemophilus
(referred to here as genital strains) isolated from genital tract infections (6 strains) and from neonatal infections (13 strains) were studied for fimbrial genes. Sixteen strains exhibit peritrichous fimbriae observed by electron microscopy. By PCR with primers corresponding to the extreme ends of the
Haemophilus influenzae
type b (Hib)
hifA
and
hifD
genes and Southern blotting, a
hifA
-like gene (named
ghfA
) and a
hifD
-like gene (named
ghfD
) were identified in 6 of the 19 strains. Five of these six strains were from the genital tracts of adults, and one was from a neonate. For each gene, the nucleotide sequence was identical for the six strains. A
hifE
-like gene (named
ghfE
) was amplified from only one of the 19 genital strains of
Haemophilus
, but the
ghfE
probe gave a signal in Southern hybridization with the five other strains positive for
ghfA
and
ghfD
. Therefore, these strains may carry a
ghfE
-like gene. The Hib fimbrial gene cluster is located between the
purE
and
pepN
genes as previously described. For the 13 genital
Haemophilus
strains that lack fimbrial genes, this region corresponds to a noncoding sequence. Another major fimbrial gene designated the fimbrin gene was previously identified in a nontypeable
H. influenzae
strain. A fimbrin-like gene was identified for all of our 19 genital strains. This gene is similar to the
ompP5
gene of many
Haemophilus
strains. Therefore, other, unidentified genes may explain the piliation observed in electron microscopy on genital
Haemophilus
strains which do not possess LKP-like fimbrial genes. Fimbrial genes were significantly associated with strains isolated from the genital tract. They may confer on the strain the ability to survive in the genital tract.
Cloning and Sequencing of the Genes Downstream of thewbfGene Cluster ofVibrio choleraeSerogroup O139 and Analysis of the Junction Genes in Other Serogroups
Authors: Sozhamannan Shanmuga , Deng Ying Kang , Li Manrong , Sulakvelidze Alexander , Kaper James B. , Johnson Judith A. , Nair G. Balkrish , Morris J. Glenn
Published: December 2019

The DNA sequence of the O-antigen biosynthesis cluster (wbf) of a recently emergent pathogen,Vibrio choleraeserogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139wbfXgene and analysis of the genes flanking thewbfgene cluster in other serogroups. The gene downstream ofwbfX, designatedrjg(right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of therjggene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106V. choleraestrains showed the presence of the left and right junction genes,gmhDandrjg, respectively, in all strains. Further, these genes mapped to a single I-CeuI fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS1358, found in both O1 and O139wb* regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to thewb* region. These results indicated a cassette-like organization of thewb* region, with the conserved genes (gmhDandrjg) flanking the divergent, serogroup-specificwb* genes and IS1358. A similar organization of thewb* region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes.
Use of In Vivo-Regulated Promoters To Deliver Antigens from Attenuated
Salmonella enterica
var. Typhimurium
Authors: Dunstan Sarah J. , Simmons Cameron P. , Strugnell Richard A.
Published: December 2019

This study describes the construction and analysis of three in vivo-inducible promoter expression plasmids, containing p
nirB
, p
pagC
, and p
katG
, for the delivery of foreign antigens in the Δ
aroAD
mutant of
Salmonella enterica
var. Typhimurium (hereafter referred to as
S. typhimurium
). The reporter genes encoding β-galactosidase and firefly luciferase were used to assess the comparative levels of promoter activity in
S. typhimurium
in vitro in response to different induction stimuli and in vivo in immunized mice. It was determined that the p
pagC
construct directed the expression of more β-galactosidase and luciferase in
S. typhimurium
than the p
nirB
and p
katG
constructs, both in vitro and in vivo. The gene encoding the C fragment of tetanus toxin was expressed in the
aroAD
mutant of
S. typhimurium
(BRD509) under the control of the three promoters. Mice orally immunized with attenuated
S. typhimurium
expressing C fragment under control of the
pagC
promoter [BRD509(pKK/p
pagC
/C frag)] mounted the highest tetanus toxoid-specific serum antibody response. Levels of luciferase expression in vivo and C-fragment expression in vitro from the
pagC
promoter appeared to be equivalent to if not lower than the levels of expression detected with the constitutive
trc
promoter. However, mice immunized with BRD509(pKK/p
pagC
/C frag) induced significantly higher levels of tetanus toxoid-specific antibody than BRD509(pKK/C frag)-immunized mice, suggesting that the specific location of foreign antigen expression may be important for immunogenicity. Mutagenesis of the ribosome binding sites (RBS) in the three promoter/C fragment expression plasmids was also performed. Despite optimization of the RBS in the three different promoter elements, the expression levels in vivo and overall immunogenicity of C fragment when delivered to mice by attenuated
S. typhimurium
were not affected. These studies suggest that in vivo-inducible promoters may give rise to enhanced immunogenicity and increase the efficacy of
S. typhimurium
as a vaccine vector.
Dual Role of Interleukin-10 in Murine Lyme Disease: Regulation of Arthritis Severity and Host Defense
Authors: Brown Jeanette P. , Zachary James F. , Teuscher Cory , Weis Janis J. , Wooten R. Mark
Published: December 2019

In the murine model of Lyme disease, C3H/He mice exhibit severe arthritis while C57BL/6N mice exhibit mild lesions when infected withBorrelia burgdorferi. Joint tissues from these two strains of mice harbor similar concentrations ofB. burgdorferi, suggesting that the difference in disease severity reflects differences in the magnitude of the inflammatory response toB. burgdorferilipoproteins. Stimulation of bone marrow macrophages from C3H/HeN mice with theB. burgdorferilipoprotein OspA resulted in higher-level production of the inflammatory mediators tumor necrosis factor alpha, nitric oxide, and interleukin-6 (IL-6) than that of macrophages from C57BL/6N mice. In contrast, macrophages from C57BL/6N mice consistently produced larger amounts of the anti-inflammatory cytokine IL-10 than did C3H/HeN macrophages. Addition of recombinant IL-10 suppressed the production of inflammatory mediators by macrophages from both strains. IL-10 was found to modulateB. burgdorferi-induced inflammation in vivo, since C57BL/6J mice deficient in IL-10 (IL-10−/−) developed more severe arthritis than wild-type C57BL/6J mice. The increase in arthritis severity was associated with a 10-fold decrease in the number ofB. burgdorferiorganisms present in ankle tissues from IL-10−/−mice. These findings suggest that in C57BL/6 mice, IL-10-dependent regulation of arthritis severity occurs at the expense of effective control of bacterial numbers.
Purification and Characterization of Novel Toxin Produced byVibrio choleraeO1
Authors: Walia Kamini , Ghosh Sujata , Singh Harminder , Nair G. B. , Ghosh Amit , Sahni Girish , Vohra Harpreet , Ganguly Nirmal Kumar
Published: December 2019

Vibrio choleraeWO7 (serogroup O1) isolated from patients with diarrhea produces an extracellular toxin despite the absence ofctx,zot, andacegenes from its genome. The toxin elongates Chinese hamster ovary cells, produces fluid accumulation in ligated rabbit ileal loops, and agglutinates freshly isolated rabbit erythrocytes. Maximal production of this toxin (WO7 toxin) was seen in AKI medium with the pH adjusted to 8.5 at 37°C under shaking conditions. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, affinity chromatography using a fetuin-Sepharose CL-4B column, and gel filtration chromatography, which increased the specific activity of the toxin by 1.6 × 106-fold. The toxin is heat labile and sensitive to proteases and has a subunit structure consisting of two subunits with molecular masses of about 58 and 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Agglutination of GM1-coated sheep erythrocytes by toxin suggests that GM1 might be the physiologic receptor for WO7 toxin on the enterocytes. An immunodiffusion test between the antiserum raised against the purified WO7 toxin and the purified toxin gave a well-defined precipitation band. In the immunoblot assay, two bands were observed in the 58- and 40-kDa region. At the same time, antiserum against WO7 toxin failed to show any cross-reactivity with cholera toxin orEscherichia coliheat-labile toxin (LT1) in an immunodiffusion test or immunoblot assay. The enterotoxic activity of WO7 toxin could be inhibited by antiserum against purified WO7 toxin. Our results indicate that WO7 toxin is structurally and functionally distinct from other cholera toxins and that the enterotoxic activities expressed by WO7 toxin appear to contribute to the pathogenesis of disease associated withV. choleraeO1 strains.
Expression of the
soxR
Gene of
Pseudomonas aeruginosa
Is Inducible during Infection of Burn Wounds in Mice and Is Required To Cause Efficient Bacteremia
Authors: Ha Unhwan , Jin Shouguang
Published: December 2019

Burn wounds are prone to infection by
Pseudomonas aeruginosa
, which is an opportunistic pathogen causing various human diseases. During infection, the bacterium senses environmental changes and regulates the expression of genes appropriate for survival. A purine-auxotrophic mutant of
P. aeruginosa
was unable to replicate efficiently on burn wounds, suggesting that burn wounds are purine-deficient environments. An in vivo expression technology based on
purEK
gene expression was applied to the burned mouse infection model to isolate
P. aeruginosa
genes that are specifically induced during infection. Four such in vivo-inducible (
ivi
) genetic loci were identified, including the gene for a superoxide response regulator (
soxR
), the gene for a malate synthase G homologue (
glcG
), an antisense transcript of a putative regulator responding to copper (
copR
), and an uncharacterized genetic locus. SoxR of
Escherichia coli
is known to regulate genes involved in protecting the bacterium against oxidative stress. The expression of
soxR
was proven to be highly inducible during the infection of burned mice and also inducible by treatment with paraquat, which is a redox-cycling reagent generating intracellular superoxide. The SoxR protein functions as an autorepressor in the absence of paraquat, whereas in the presence of paraquat, this autorepression is diminished. Furthermore, a
soxR
null mutant was shown to be much more sensitive than wild-type
P. aeruginosa
to macrophage-mediated killing. In support of this observation, a
soxR
null mutant exhibited a significant delay in causing systemic infections in the burned mice. Since most mortality in burn patients is caused by systemic infection, the defect in the ability to cause efficient bacteremia in burned mice suggests an important role of the
soxR
gene in the infection of burn wounds.