bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of
Enterococcus faecalis fsr
Genes on Production of Gelatinase and a Serine Protease and Virulence
Authors: Qin Xiang , Singh Kavindra V. , Weinstock George M. , Murray Barbara E.
Published: July 2002

Three
agr
-like genes (
fsrA
,
fsrB
, and
fsrC
, for
Enterococcus faecalis
regulator) were found upstream of the previously reported gelatinase gene (
gelE
) and a downstream putative serine protease gene (
sprE
; accession number
Z12296
) of
Enterococcus faecalis
OG1RF. The deduced amino acid sequence of
fsrA
shows 26% identity and 38% similarity to
Staphylococcus aureus
AgrA (the response regulator of the accessory gene regulator system in the
agr
locus), FsrB shows 23% identity and 41% similarity to
S. aureus
AgrB, and FsrC shows 23% identity and 36% similarity to
S. aureus
AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that
gelE
and
sprE
are cotranscribed and that
fsrB
and
fsrC
are also cotranscribed in OG1RF. Northern blot analysis of
fsrA
,
fsrB
,
fsrC
,
gelE
, and
sprE
insertion mutants showed that
fsrB
,
fsrC
,
gelE
, and
sprE
are not expressed in
fsrA
,
fsrB
, and
fsrC
mutants, while insertion in an open reading frame further upstream of
fsrA
did not effect the expression of these genes, suggesting that
agr
-like genes may be autoregulated and that they regulate
gelE
and
sprE
expression, as further confirmed by complementation of
fsr
gene mutations with a 6-kb fragment which contains all three
fsr
genes in the shuttle vector, pAT18. Testing of 95 other isolates of
E. faecalis
showed that 62% produced gelatinase (Gel
+
), while 91% (including all Gel
+
strains) hybridized to a
gelE
probe; 71% (including all Gel
+
strains) hybridized to an
fsr
probe, corroborating the conclusion that both
gelE
and
fsr
are necessary for gelatinase production. Testing of
fsrA
,
fsrB
, and
sprE
mutants in a mouse peritonitis model showed that
sprE
and
agr
-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a
gelE
mutant. These results suggest that
sprE
and
agr
-like genes contribute to the virulence of
E. faecalis
OG1RF in this model.
Naturally Acquired and Vaccine-Elicited Antibodies Block Erythrocyte Cytoadherence of the
Plasmodium vivax
Duffy Binding Protein
Authors: Michon Pascal , Fraser Tresa , Adams John H.
Published: July 2002

Malaria merozoites require the presence of specific surface receptors on the red blood cell for invasion.
Plasmodium vivax
, requires the Duffy blood group antigen as an obligate receptor for invasion. The parasite Duffy binding protein (DBP) is the ligand involved in this process, making the DBP a potential vaccine candidate. A preliminary objective was to study whether people exposed to vivax malaria acquire antibodies that have the ability to block erythrocyte cytoadherence to the PvDBP. In comparison, we studied the immunogenicity of various recombinant DBP vaccines and investigated their potential to induct antifunctional antibodies. In order to do so, recombinant proteins to different regions of the putative ectodomain of the DBP and a DNA vaccine were used to immunize laboratory animals. An in vitro cytoadherence assay was used to investigate the presence of antifunctional antibodies in plasmas from people naturally exposed to vivax malaria, as well as in antisera obtained by animal vaccination. Our results showed that human plasma from populations naturally exposed to vivax malaria, as well as antisera obtained by vaccination using recombinant proteins, a DNA vaccine, and a synthetic peptide to DBP, inhibited in vitro binding of human erythrocytes to the DBP ligand domain (DBP
II
) in correlation to their previously measured antibody titer. Our results provide further evidence for the vaccine potential of this essential parasite adhesion molecule.
Inhibition of Staphylococcal Enterotoxin B-Induced Lymphocyte Proliferation and Tumor Necrosis Factor Alpha Secretion by MAb5, an Anti-Toxic Shock Syndrome Toxin 1 Monoclonal Antibody
Authors: Pang Liwina T. Y. , Kum Winnie W. S. , Chow Anthony W.
Published: July 2002

Toxic shock syndrome (TSS) is primarily caused by toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB). These toxins belong to a family of pyrogenic toxin superantigens (PTSAgs) produced byStaphylococcus aureusand exhibit several shared biological properties, including the induction of massive cytokine release and Vβ-specific T-cell proliferation. The crystal structures of most PTSAgs are now published, and they demonstrate a striking similarity in conformational architecture even though their primary protein sequences are different. Despite these structural and immunobiological similarities, no cross-reactivity between TSST-1 and other PTSAgs has been demonstrated in serological or neutralization assays. Our laboratory has developed a neutralizing murine anti-TSST-1 monoclonal antibody (MAb5) which displayed cross-reactivity with SEB by enzyme-linked immunosorbent assay. The aim of the present study was to evaluate whether MAb5 can also cross-neutralize SEB-induced superantigenic activities in vitro. MAb5 was found to partially inhibit SEB-induced T-cell mitogenesis (63%) and tumor necrosis factor alpha (TNF-α) secretion (70%) in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner, while an isotypic anti-TSST-1 monoclonal antibody showed no effect. Epitope mapping revealed that MAb5 bound to TSST-1 residues 47 to 56 (47FPSPYYSPAF56) and to SEB residues 83 to 92 (83DVFGANYYYQ92), sequences that located in different regions of these toxins and are structurally dissimilar. SEB peptide83DVFGANYYYQ92was synthesized and found to also inhibit SEB-induced mitogenesis and TNF-α secretion in human PBMC. Our results demonstrate for the first time that MAb5 binds to different epitopes on TSST-1 and SEB that appear functionally important in inducing T-cell mitogenesis and TNF-α secretion in vitro.
Toll-Like Receptors Confer Responsiveness to Lipopolysaccharide fromPorphyromonas gingivalisin Human Gingival Fibroblasts
Authors: Tabeta Kouich , Yamazaki Kazuhisa , Akashi Sachiko , Miyake Kensuke , Kumada Hidefumi , Umemoto Toshio , Yoshie Hiromasa
Published: July 2002

Gingival fibroblasts produce proinflammatory cytokines in response to lipopolysaccharide (LPS) from periodontopathic bacteria. Recently it has become evident that the human homologue ofDrosophilaToll can transduce intracellular signaling by LPS stimulation. Toll-like receptors (TLRs) have been identified in myeloid cells; however, their role in nonmyeloid cells such as gingival fibroblasts has not been fully elucidated. Here, we report that human gingival fibroblasts constitutively express TLR2 and TLR4 and that their levels of expression are increased by stimulation with LPS fromPorphyromonas gingivalis. Upregulated expression of interleukin-6 gene and protein in fibroblasts stimulated with LPS is inhibited by anti-TLR4 antibody. These findings suggest that TLRs may confer responsiveness to LPS in gingival fibroblasts.
Importance of Holotoxin Assembly in Ptl-Mediated Secretion of Pertussis Toxin from
Bordetella pertussis
Authors: Farizo Karen M. , Huang Theresa , Burns Drusilla L.
Published: July 2002

We examined the structural components of pertussis toxin that are required for efficient export from
Bordetella pertussis
via the Ptl system, a member of the type IV family of macromolecular transporters. First, we constructed a strain of
B. pertussis
that contains a functional Ptl system but does not produce pertussis toxin. Plasmids which express either the S1 subunit or the B oligomer were then introduced into this strain. We found that the B oligomer of the toxin is not secreted in the absence of the S1 subunit. Conversely, the S1 subunit is also not secreted by a Ptl-mediated mechanism in the absence of the B oligomer. Thus, an assembled holotoxin is required for Ptl-mediated export of pertussis toxin from
B. pertussis
.
Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in
Mycoplasma agalactiae
Authors: Glew M. D. , Papazisi L. , Poumarat F. , Bergonier D. , Rosengarten R. , Citti C.
Published: July 2002

A family of abundant surface proteins (Vpmas [variable proteins of
Mycoplasma agalactiae
]) undergoing phase variation in
M. agalactiae
has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple
vpma
genes which were clustered within a 14-kb
Cla
I genomic fragment. Rearrangements were found to have occurred within the
vpma
locus between clones which correlated with changes in their Vpma phenotype. Two neighboring
vpma
genes were cloned and sequenced from one
M. agalactiae
clonal variant expressing Vpma39. The two genes,
vpmaX
and
vpmaY
, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The
vpmaY
gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second
vpmaY
repeat was present, it was prematurely terminated by insertion of two nucleotides. The
vpmaX
gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of
vpmaY
. Many of the features in common between the
vpma
genes were also found to be shared by the
vsp
genes of
M. bovis
, which also undergo DNA rearrangements concomitant with phenotypic changes. Since
M. bovis
is the closest phylogenetic relative to
M. agalactiae
, the
vpma
and
vsp
gene families most probably represent homologous systems.
Human Response to
Escherichia coli
O157:H7 Infection: Antibodies to Secreted Virulence Factors
Authors: Li Yuling , Frey Elizabeth , Mackenzie Andrew M. R. , Finlay B. Brett
Published: July 2002

Vaccination has been proposed for the prevention of disease due to enterohemorrhagic
Escherichia coli
(EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized. To study this, sera were obtained from five pediatric patients with acute diarrhea caused by
E. coli
O157:H7 0, 8, and 60 days after hospitalization. These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm). The response to O157:H7 lipopolysaccharide was also examined. Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients. We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir. By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens. The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir. These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease. They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response.
Intranasal Vaccination with Pneumococcal Surface Protein A and Interleukin-12 Augments Antibody-Mediated Opsonization and Protective Immunity againstStreptococcus pneumoniaeInfection
Authors: Arulanandam Bernard P. , Lynch Joyce M. , Briles David E. , Hollingshead Susan , Metzger Dennis W.
Published: July 2002

Streptococcus pneumoniaeis a major pathogen in humans that enters the host primarily through the respiratory tract. Targeting mucosal surfaces directly may therefore be an optimal approach for vaccination to prevent bacterial colonization and invasive disease. We have previously demonstrated the effectiveness of interleukin-12 (IL-12) delivered intransally (i.n.) as an antiviral respiratory adjuvant. In this study, we examined the effects of i.n. IL-12 treatment on induction of protective humoral immunity againstS. pneumoniae. Immunization i.n. with pneumococcal surface protein A (PspA) and IL-12 resulted in enhanced lung IL-10 mRNA expression and marked augmentation of respiratory and systemic immunoglobulin G1 (IgG1), IgG2a, and IgA antibody levels compared to those in animals receiving PspA alone. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Flow cytometric analysis revealed a threefold increase in antibody-mediated, complement-independent opsonic activity in the sera of PspA- and IL-12-treated animals, which was mainly contributed by IgG2a and, to a lesser extent, IgA. Passive transfer of these immune sera conferred complete protection from death upon systemic pneumococcal challenge. These findings demonstrate the effectiveness of combining PspA and IL-12 at mucosal sites to achieve optimal antibody-mediated opsonization and killing ofS. pneumoniae.
Dynamic Nature of Host-Pathogen Interactions inMycobacterium marinumGranulomas
Authors: Bouley Donna M. , Ghori Nafisa , Mercer K. Lynne , Falkow Stanley , Ramakrishnan Lalita
Published: July 2002

Mycobacterium marinumcauses long-term subclinical granulomatous infection in immunocompetent leopard frogs (Rana pipiens). These granulomas, organized collections of activated macrophages, share many morphological features with persistent human tuberculous infection. We examined organs of frogs with chronicM. marinuminfection using transmission electron microscopy in conjunction with immunohistochemistry and acid phosphatase cytochemistry to better define the bacterium-host interplay during persistent infection. Bacteria were always found within macrophage phagosomes. These phagosomes were often fused to lysosomes, in sharp contrast to those formed during in vitro infection of J774 macrophage-like cells byM. marinum. The infected macrophages in frog granulomas showed various levels of activation, as evidenced by morphological changes, including epithelioid transformation, recent phagocytic events, phagolysosomal fusion, and disintegration of bacteria. Our results demonstrate that even long-term granulomas are dynamic environments with regard to the level of host cell activation and bacterial turnover and suggest a continuum between constantly replicating bacteria and phagocytic killing that maintains relatively constant bacterial numbers despite an established immune response. Infection with a mutant bacterial strain with a reduced capacity for intracellular replication shifted the balance, leading to a greatly reduced bacterial burden and inflammatory foci that differed from typical granulomas.
Trehalose 6,6′-Dimycolate (Cord Factor) ofMycobacterium tuberculosisInduces Foreign-Body- and Hypersensitivity-Type Granulomas in Mice
Authors: Yamagami Hirokazu , Matsumoto Takayuki , Fujiwara Nagatoshi , Arakawa Tetsuo , Kaneda Kenji , Yano Ikuya , Kobayashi Kazuo
Published: July 2002

Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of theMycobacterium tuberculosiscell wall participate in the pathogenesis of infection. Strains ofM. tuberculosishave cord factor (trehalose 6,6′-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.
Immune Response to Infection withMycobacterium ulcerans
Authors: Gooding Travis M. , Johnson Paul D. R. , Campbell Dianne E. , Hayman John A. , Hartland Elizabeth L. , Kemp Andrew S. , Robins-Browne Roy M.
Published: July 2002

Mycobacterium ulceransis a slow-growing, acid-fast bacillus that causes chronic necrotizing skin ulcers known as Buruli ulcers. Previously reported information on immunity to this mycobacterium is limited. We examined immune responses toM. ulceransandM. bovisBCG in patients withM. ulceransdisease and in 20 healthy control subjects (10 tuberculin test positive and 10 tuberculin test negative). Cell-mediated immunity was assessed by stimulating peripheral blood mononuclear cells (PBMC) with whole mycobacteria and then measuring PBMC proliferation and the production of gamma interferon (IFN-γ). Humoral immunity was assessed by immunoblotting. PBMC from all subjects showed significantly greater proliferation and IFN-γ production in response to stimulation with living mycobacteria compared with killed cells. However, PBMC from subjects with past or currentM. ulceransdisease showed significantly reduced proliferation and production of IFN-γ in response to stimulation with liveM. ulceransorM. bovisthan PBMC from healthy, tuberculin test-positive subjects (P< 0.001) and showed results in these assays comparable to those of tuberculin test-negative subjects (P> 0.2). Serum from 9 of 11 patients withM. ulceransdisease, but no control subject, contained antibodies toM. ulcerans. The results indicate that patients withM. ulceransinfection mount an immune response toM. ulceransas evidenced by antibody production, but they demonstrate profound systemic T-cell anergy to mycobacterial antigens. These findings may explain some of the distinct clinical and pathological features ofM. ulcerans-induced disease.
Aberrant Macrophage and Neutrophil Population Dynamics and Impaired Th1 Response toListeria monocytogenesin Colony-Stimulating Factor 1-Deficient Mice
Authors: Guleria Indira , Pollard Jeffrey W.
Published: July 2002

Listeria monocytogenes, a facultative intracellular bacterium, has been used extensively to study innate immune responses. Macrophages act as hosts for this bacterium as well as a major defense against it. Using mice homozygous for a null mutation (Csf1op) in the gene for the mononuclear phagocytic growth factor colony-stimulating factor 1 (CSF-1), we have demonstrated that CSF-1-regulated macrophages were essential to defend against a listerial infection. In the absence of CSF-1, monocytes were not recruited to the sites of infection due to the lack of synthesis of the macrophage chemoattractant chemokine MCP-1. In addition, there was no burst of interleukin-10 (IL-10) synthesis that has been shown to result in the egress of neutrophils from sites of infection. Consequently, neutrophils were not replaced by macrophages, and numerous neutrophil-filled microabscesses developed, followed by tissue destruction and death of the mice. In the CSF-1 nullizygous mice compared to wild-type mice, there was also a very low synthesis of gamma interferon (IFN-γ), resulting in reduced macrophage activation. However, the concentrations of the IFN-γ-inducing cytokines IL-12 and IL-18 at this bacterial load were similar in these mutant mice. In contrast, IL-6 concentrations were dramatically reduced. Administration of IL-6 toCsf1op/Csf1opmice significantly increased the synthesis of IFN-γ and reduced the bacterial burden to a greater extent than treatment with IFN-γ alone. These data indicate that IL-6 occupies a central role in the CSF-1-regulated macrophage response toL. monocytogenes.
Recognition of Multiple Antibody Epitopes throughout
Borrelia burgdorferi
p66, a Candidate Adhesin, in Patients with Early or Late Manifestations of Lyme Disease
Authors: Ntchobo Hyacinthe , Rothermel Holly , Chege Wambui , Steere Allen C. , Coburn Jenifer
Published: July 2002

Antibody responses to p66, a candidate integrin ligand of
Borrelia burgdorferi
, were studied in 79 patients with early or late manifestations of Lyme disease. The central portion of p66 was previously shown to contain all of the information required for specific recognition of β
3
-chain integrins, but work by others had suggested that the C-terminal portion of the protein contains a single surface-exposed, immunodominant loop. In examining antibody responses to full-length p66 and to three overlapping fragments of the protein, we found that the majority of Lyme disease patients had immunoglobulin M (IgM) and/or IgG responses to p66 and that, particularly early in the disease, epitopes throughout p66 were recognized. Among patients with later manifestations of the illness, antibody responses to the C-terminal portion of the protein were more prominent. These results demonstrate that Lyme disease patient sera recognize epitopes throughout p66.
Clostridium perfringens
Iota-Toxin: Mapping of Receptor Binding and Ia Docking Domains on Ib
Authors: Marvaud Jean-Christophe , Smith Theresa , Hale Martha L. , Popoff Michel R. , Smith Leonard A. , Stiles Bradley G.
Published: July 2002

Clostridium perfringens
iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing ≥200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice.
Trypanosome-Derived Oligopeptidase B Is Released into the Plasma of Infected Rodents, Where It Persists and Retains Full Catalytic Activity
Authors: Morty Rory E. , Lonsdale-Eccles John D. , Mentele Reinhardt , Auerswald Ennes A. , Coetzer Theresa H. T.
Published: July 2002

A trypsin-like serine peptidase activity, levels of which correlate with blood parasitemia levels, is present in the plasma of rats acutely infected with
Trypanosoma brucei brucei
. Antibodies to a trypanosome peptidase with a trypsin-like substrate specificity (oligopeptidase B [OP-Tb]) cross-reacted with a protein in the plasma of trypanosome-infected rats on a Western blot. These antibodies also abolished 80% of the activity in the plasma of trypanosome-infected rats, suggesting that the activity may be attributable to a parasite-derived peptidase. We purified the enzyme responsible for the bulk of this activity from parasite-free
T. b. brucei
-infected rat plasma and confirmed its identity by protein sequencing. We show that live trypanosomes do not release OP-Tb in vitro and propose that disrupted parasites release it into the host circulation, where it is unregulated and retains full catalytic activity and may thus play a role in the pathogenesis of African trypanosomiasis.
Characterization of Binding of Human Lactoferrin to Pneumococcal Surface Protein A
Authors: Håkansson Anders , Roche Hazeline , Mirza Shaper , McDaniel Larry S. , Brooks-Walter Alexis , Briles David E.
Published: July 2002

Human lactoferrin is an iron-binding glycoprotein that is particularly prominent in exocrine secretions and leukocytes and is also found in serum, especially during inflammation. It is able to sequester iron from microbes and has immunomodulatory functions, including inhibition of both complement activation and cytokine production. This study used mutants lacking pneumococcal surface protein A (PspA) and PspC to demonstrate that the binding of human lactoferrin to the surface ofStreptococcus pneumoniaewas entirely dependent on PspA. Lactoferrin bound both family 1 and family 2 PspAs. Binding of lactoferrin to PspA was shown by surface colocalization with PspA and was verified by the lack of binding to PspA-negative mutants. Lactoferrin was expressed on the body of the cells but was largely absent from the poles. PspC showed exactly the same distribution on the pneumococcal surface as PspA but did not bind lactoferrin. PspA's binding site for lactoferrin was mapped using recombinant fragments of PspA of families 1 and 2. Binding of human lactoferrin was detected primarily in the C-terminal half of the α-helical domain of PspA (amino acids 167 to 288 of PspA/Rx1), with no binding to the N-terminal 115 amino acids in either strain. The interaction was highly specific. As observed previously, bovine lactoferrin bound poorly to PspA. Human transferrin did not bind PspA at all. The binding of lactoferrin toS. pneumoniaemight provide a way for the bacteria to interfere with host immune functions or to aid in the acquisition of iron at the site of infection.
Early Local Cytokine Profiles in Strains of Mice with Different Outcomes from Chlamydial Genital Tract Infection
Authors: Darville Toni , Andrews Charles W. , Sikes James D. , Fraley Patrick L. , Rank Roger. G.
Published: July 2002

In this study, we expand on the examination of genetically determined differences in host responses that correlate with clearance ofChlamydia trachomatisfrom the genital tract. We infected C57BL/6, BALB/c, and C3H/HeN mice with the mouse pneumonitis agent ofC. trachomatis(MoPn). C57BL/6 mice had the shortest course of infection (22 days) and the lowest incidence of severe hydrosalpinx. BALB/c mice also had a short course of infection (25 days), but all developed hydrosalpinx. C3H/HeN mice had the longest course of infection (38 days), and all developed severe hydrosalpinx. Determination of local cytokine responses by enzyme-linked immunosorbent assay (ELISA) of genital tract secretions revealed that the levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were significantly increased in the C57BL/6 and BALB/c strains compared to those in the C3H/HeN strain whereas the level of IL-6 was not different. The level of the neutrophil chemokine macrophage inflammatory protein 2 (MIP-2) was increased during the first week of infection in all three strains but was significantly higher in the BALB/c strain, the strain with the most rapid influx of neutrophils into the genital tract. Prolonged detection of MIP-2 in C3H/HeN mice was associated with a protracted presence of neutrophils in the genital tract. Early increases in the levels of the proinflammatory cytokines TNF-α and IL-1β are associated with earlier eradication of infection in the C57BL/6 and BALB/c strains than in the C3H/HeN strain. Increased levels of MIP-2 and neutrophils in BALB/c and C3H/HeN mice relative to C57BL/6 mice suggest that these responses may contribute to pathology.
Selective Enhancement of Systemic Th1 Immunity in Immunologically Immature Rats with an Orally Administered Bacterial Extract
Authors: Bowman L. M. , Holt P. G.
Published: July 2002

Infant rats primed during the first week of life with soluble antigen displayed adult-equivalent levels of T-helper 2 (Th2)-dependent immunological memory development as revealed by production of secondary immunoglobulin G1 (IgG1) antibody responses to subsequent challenge, but in contrast to adults failed to prime for Th1-dependent IgG2b responses. We demonstrate that this Th2 bias in immune function can be redressed by oral administration to neonates of a bacterial extract (Broncho-Vaxom OM-85) comprising lyophilized fractions of several common respiratory tract bacterial pathogens. Animals given OM-85 displayed a selective upregulation in primary and secondary IgG2b responses, accompanied by increased gamma interferon and decreased interleukin-4 production (both antigen specific and polyclonal), and increased capacity for development of Th1-dependent delayed hypersensitivity to the challenge antigen. We hypothesize that the bacterial extract functions via enhancement of the process of postnatal maturation of Th1 function, which is normally driven by stimuli from the gastrointestinal commensal microflora.
Gene Fragments Distinguishing an Epidemic-Associated Strain from a Virulent Prototype Strain of
Listeria monocytogenes
Belong to a Distinct Functional Subset of Genes and Partially Cross-Hybridize with Other
Listeria
Species
Authors: Herd Martin , Kocks Christine
Published: July 2002

Most major food-borne outbreaks of listeriosis in Europe and in the United States have been caused by genetically closely related
Listeria monocytogenes
strains of serotype 4b. In order to assess whether genomic loci exist that could underlie this increased epidemic potential, we subtracted the genome of the virulent prototype
L. monocytogenes
strain EGD from a prototype epidemic strain. A total of 39 DNA fragments corresponding to 20% of an estimated total of 150 to 190 kb of differential genome material were isolated. For 21 of these fragments, no function on the basis of homology could be predicted. Of the remaining 18 fragments, 15 had homologies to bacterial surface proteins, some of which have been implicated in virulence mechanisms such as cell invasion, adhesion, or immune escape. Southern hybridization of arrays containing the epidemic-clone-specific DNA segments with genomic DNA of different
L. monocytogenes
strains was consistent with the current lineage division. Surprisingly, however, some of the fragments hybridized in a mosaic-like fashion to genomes of two other
Listeria
species, the animal pathogen
L. ivanovii
and the nonpathogen
L. innocua
. Taken together, our results provide a starting point for the identification of epidemic-trait-associated genes.
Motility and the Polar Flagellum Are Required for
Aeromonas caviae
Adherence to HEp-2 Cells
Authors: Rabaan Ali A. , Gryllos Ioannis , Tomás Juan M. , Shaw Jonathan G.
Published: July 2002

Aeromonas caviae
is increasingly being recognized as a cause of gastroenteritis, especially among the young. The adherence of aeromonads to human epithelial cells in vitro has been correlated with enteropathogenicity, but the mechanism is far from well understood. Initial investigations demonstrated that adherence of
A. caviae
to HEp-2 cells was significantly reduced by either pretreating bacterial cells with an antipolar flagellin antibody or by pretreating HEp-2 cells with partially purified flagella. To precisely define the role of the polar flagellum in aeromonad adherence, we isolated the
A. caviae
polar flagellin locus and identified five polar flagellar genes, in the order
flaA, flaB, flaG, flaH
, and
flaJ
. Each gene was inactivated using a kanamycin resistance cartridge that ensures the transcription of downstream genes, and the resulting mutants were tested for motility, flagellin expression, and adherence to HEp-2 cells. N-terminal amino acid sequencing, mutant analysis, and Western blotting demonstrated that
A. caviae
has a complex flagellum filament composed of two flagellin subunits encoded by
flaA
and
flaB
. The predicted molecular mass of both flagellins was ∼31,700 Da; however, their molecular mass estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was ∼35,500 Da. This aberrant migration was thought to be due to their glycosylation, since the proteins were reactive in glycosyl group detection assays. Single mutations in either
flaA
or
flaB
did not result in loss of flagella but did result in decreased motility and adherence by approximately 50%. Mutation of
flaH, flaJ
, or both flagellin genes resulted in the complete loss of motility, flagellin expression, and adherence. However, mutation of
flaG
did not affect motility but did significantly reduce the level of adherence. Centrifugation of the flagellate mutants (
flaA, flaB
, and
flaG
) onto the cell monolayers did not increase adherence, whereas centrifugation of the aflagellate mutants (
flaH, flaJ
, and
flaA flaB
) increased adherence slightly. We conclude that maximum adherence of
A. caviae
to human epithelial cells in vitro requires motility and optimal flagellar function.
Type IHelicobacter pyloriLipopolysaccharide Stimulates Toll-Like Receptor 4 and Activates Mitogen Oxidase 1 in Gastric Pit Cells
Authors: Kawahara Tsukasa , Teshima Shigetada , Oka Ayuko , Sugiyama Toshiro , Kishi Kyoichi , Rokutan Kazuhito
Published: July 2002

Guinea pig gastric pit cells express an isozyme of gp91-phox, mitogen oxidase 1 (Mox1), and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phox). Helicobacter pylorilipopolysaccharide (LPS) andEscherichia coliLPS have been shown to function as potent activators for the Mox1 oxidase. These cells spontaneously secreted about 10 nmol of superoxide anion (O2−)/mg of protein/h under LPS-free conditions. They expressed the mRNA and protein of Toll-like receptor 4 (TLR4) but not those of TLR2. LPS from type IH. pyloriat 2.1 endotoxin units/ml or higher stimulated TLR4-mediated phosphorylations of transforming growth factor β-activated kinase 1 and its binding protein 1 induced TLR4 and p67-phoxand up-regulated O2−production 10-fold. In contrast, none of these events occurred withH. pyloriLPS from complete or partial deletion mutants of thecagpathogenicity island. Lipid A was confirmed to be a bioactive component for the priming effects, while removal of bisphosphates from lipid A completely eliminated the effects, suggesting the importance of the phosphorylation pattern besides the acylation pattern for the bioactivity.H. pyloriLPS is generally accepted as having low toxicity; however, our results suggest that type IH. pylorilipid A may be a potent stimulator for innate immune responses of gastric mucosa by stimulating the TLR4 cascade and Mox1 oxidase in pit cells.
Safety and Immunogenicity of a Proteosome-
Shigella flexneri
2a Lipopolysaccharide Vaccine Administered Intranasally to Healthy Adults
Authors: Fries Louis F. , Montemarano Andrew D. , Mallett Corey P. , Taylor David N. , Hale Thomas L. , Lowell George H.
Published: July 2002

We studied the safety and immunogenicity of a
Shigella flexneri
2a vaccine comprising native
S. flexneri
2a lipopolysaccharide (LPS) complexed to meningococcal outer membrane proteins—proteosomes—in normal, healthy adults. A two-dose series of immunizations was given by intranasal spray, and doses of 0.1, 0.4, 1.0, and 1.5 mg (based on protein) were studied in a dose-escalating design. The vaccine was generally well tolerated. The most common reactions included rhinorrhea and nasal stuffiness, which were clearly dose related (
P
≤ 0.05). These reactions were self-limited and generally mild. The vaccine elicited
S. flexneri
2a LPS-specific immunoglobulin A (IgA), IgG, and IgM antibody-secreting cells (ASCs) in a dose-responsive manner. At doses of 1.0 or 1.5 mg, highly significant (
P
< 0.001) increases in ASCs of all antibody isotypes occurred and 95% of subjects had an ASC response in at least one antibody isotype. Dose-related serum antibody responses were observed, with geometric mean two- to fivefold rises in specific serum IgA and IgG titers and two- to threefold rises in IgM in the 1.0- and 1.5-mg-dose groups (
P
< 0.0001 for each isotype). Elevated serum antibody levels persisted through day 70. Increases in fecal IgG and IgA and also in urinary IgA specific for
S. flexneri
2a LPS were demonstrated. These were most consistent and approached statistical significance (
P
= 0.02 to 0.12 for various measures) on day 70 after the first dose. The magnitude of immune responses to intranasally administered proteosome-
S. flexneri
2a LPS vaccine is similar to those reported for live vaccine candidates associated with protective efficacy in human challenge models, and further evaluation of this product is warranted.
Brain Granulomas in Neurocysticercosis Patients Are Associated with a Th1 and Th2 Profile
Authors: Restrepo Blanca I. , Alvarez Jorge I. , Castaño Jorge A. , Arias Luis F. , Restrepo Margarita , Trujillo Judith , Colegial Carlos H. , Teale Judy M.
Published: July 2002

Neurocysticercosis (NCC) is a common central nervous system (CNS) infection caused byTaenia soliummetacestodes. Despite the well-documented importance of the granulomatous response in the pathogenesis of this infection, there is limited information about the types of cells and cytokines involved. In fact, there has been limited characterization of human brain granulomas with any infectious agent. In the present study a detailed histological and immunohistochemical analysis of the immune response was performed on eight craniotomy specimens where a granuloma surrounded eachT. soliummetacestode. The results indicated that in all the specimens there was a dying parasite surrounded by a mature granuloma with associated fibrosis, angiogenesis, and an inflammatory infiltrate. The most abundant cell types were plasma cells, B and T lymphocytes, macrophages, and mast cells. Th1 cytokines were prevalent and included gamma interferon, interleukin-18 (IL-18), and the immunosuppressive, fibrosis-promoting cytokine transforming growth factor β. The Th2 cytokines IL-4, IL-13, and IL-10 were also present. These observations indicate that a chronic immune response is elicited in the CNS environment with multiple cell types that together secrete inflammatory and anti-inflammatory cytokines. In addition, both collagen type I and type III deposits were evident and could contribute to irreversible nervous tissue damage in NCC patients.
Porphyromonas gingivalisFimbriae Inhibit Caspase-3-Mediated Apoptosis of Monocytic THP-1 Cells under Growth Factor Deprivation via Extracellular Signal-Regulated Kinase-Dependent Expression of p21 Cip/WAF1
Authors: Ozaki Ken , Hanazawa Shigemasa
Published: July 2002

Apoptotic regulation of monocytes/macrophages appears to be closely associated with chronic inflammatory reactions. Since it was demonstrated earlier that certain bacterial cell components are involved in apoptotic regulation of these cells, in the present study, we investigated whether the bacterial fimbria, an important cell structure involved in bacterial adherence to host cells, regulates apoptosis of human monocytic THP-1 cells induced under growth factor deprivation. To investigate this point, we used fimbriae ofPorphyromonas gingivalis,a pathogen causing periodontal disease, which is a chronic inflammatory disease. The fimbriae inhibited apoptosis of the cells under growth factor deprivation. This inhibitory action of the fimbriae was completely neutralized by anti-fimbrial antibody. The fimbriae stimulated activation of extracellular signal-regulated kinase (ERK) and expression of cyclin-dependent kinase inhibitor p21 Cip/WAF1 (p21) in the cells. The stimulatory effect of the fimbriae on the expression of the p21 protein was inhibited by treatment with PD98059, a specific inhibitor of ERK. The cell apoptosis was inhibited by treatment with Ac-DEVD-CHO, an inhibitor of caspase-3. The fimbriae inhibited the serum withdrawal-induced cleavage of the caspase-3 proform and poly(ADP-ribose) polymerase, one of the caspase-3 substrates. Furthermore, PD98059 and antisense p21 oligonucleotide blocked the fimbrial inhibition of apoptosis and caspase-3 activation of the cells induced by serum withdrawal. These results show that the bacterial fimbriae inhibited apoptosis of THP-1 cells induced under growth factor deprivation via ERK-dependent expression of p21. The present study suggests that bacterial fimbriae act as potent regulators of chronic inflammatory disease, e.g., periodontal disease, through blocking apoptosis of monocytes/macrophages.
Monocytic Cell Activation by Nonendotoxic Glycoprotein fromPrevotella intermediaATCC 25611 Is Mediated by Toll-Like Receptor 2
Authors: Sugawara Shunji , Yang Shuhua , Iki Koichi , Hatakeyama Junko , Tamai Riyoko , Takeuchi Osamu , Akashi Sachiko , Espevik Terje , Akira Shizuo , Takada Haruhiko
Published: July 2002

Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such asPorphyromonas gingivalisandPrevotella intermediaactivate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract ofP. intermediaATCC 25611 and designatedPrevotellaglycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-1 cells compared withSalmonellaLPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IVA. These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.
Purification, Characterization, and Immunogenicity of a Disulfide Cross-Linked
Plasmodium vivax
Vaccine Candidate Antigen, Merozoite Surface Protein 1, Expressed in
Escherichia coli
Authors: Dutta Sheetij , Ware Lisa A. , Barbosa Arnoldo , Ockenhouse Christian F. , Lanar David E.
Published: July 2002

The
Plasmodium vivax
merozoite surface protein 1 (MSP-1) 42-kDa fragment (PvMSP-1 p42) is a promising vaccine candidate antigen against the blood stage of the malarial parasite. We have developed a process for the production of this vaccine target, keeping in mind its use in human volunteers. A novel strain, Origami(DE3), of
Escherichia coli
with mutations in the glutathione and thioredoxin reductase genes yielded 60% more soluble PvMSP-1 p42 than the conventional
E. coli
BL21(DE3) strain. Recombinant PvMSP-1 p42 was purified to ≥99% purity with a rapid two-step protocol designed for easy scaling up. The final product had a low endotoxin content and was stable in its lyophilized form. PvMSP-1 p42 was found to have the predicted primary and tertiary structures and consisted of a single conformer containing one free cysteine, as predicted. The product was recognized by conformational monoclonal antibodies against
P. vivax
MSP-1. Immunogenicity studies of PvMSP-1 p42 were carried out with two strains of mice and the adjuvants Montanide ISA51 and Montanide ISA720. Both formulations were found to induce high levels of immunoglobulin G1 (IgG1), IgG2b, and IgG2a antibodies along with low levels of IgG3. Lymphocytes from animals in all the PvMSP-1 p42-immunized groups showed proliferative responses upon stimulation with PvMSP-1 p42; the cytokines interleukin 2 (IL-2), gamma interferon, IL-4, and IL-10 were detected in the culture supernatants. These results indicate that PvMSP-1 p42 in combination with both of the adjuvants elicited cellular and humoral responses in mice.
pH-Enhanced Cytopathic Effects of
Clostridium sordellii
Lethal Toxin
Authors: Qa'Dan Maen , Spyres Lea M. , Ballard Jimmy D.
Published: July 2002

Clostridium sordellii
lethal toxin (TcsL) is a large clostridial toxin (LCT) that glucosylates Ras, Rac, and Ral. TcsL differs from other LCTs because it modifies Ras, which does not cycle from cytosol to membrane. By using a suite of inhibitors, steps in cell entry by TcsL were dissected, and entry appears to be dependent on endosomal acidification. However, in contrast to TcdB, TcsL was substantially slower in its time course of entry. TcsL cytopathic effects (CPE) were blocked by bafilomycin A1 and neutralized by antiserum up to 2 h following treatment of cells with the toxin. The slow time course of intoxication and relatively high cytopathic dose were alleviated by exposing TcsL to acid pH, resulting in a time course similar to that of TcdB. The optimal pH range for activation was 4.0 to 5.0, which increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, as shown by differential glucosylation. Fluorescence analysis of TcsL with 2-(
p
-toluidinyl) naphthalene-6-sulfonic acid as a probe suggested the acid pH stimulated a hydrophobic transition in the protein, a likely prelude to membrane insertion. Finally, acid entry by TcsL caused TcdB-like morphological changes in CHO cells, which suggestings that acid activation may impact substrate recognition profiles for TcsL.
Mutualism versus Independence: Strategies of Mixed-Species Oral Biofilms In Vitro Using Saliva as the Sole Nutrient Source
Authors: Palmer Robert J. , Kazmerzak Karen , Hansen Martin C. , Kolenbrander Paul E.
Published: July 2002

During initial dental plaque formation, the ability of a species to grow when others cannot would be advantageous, and enhanced growth through interspecies and intergeneric cooperation could be critical. These characteristics were investigated in three coaggregating early colonizers of the tooth surface (
Streptococcus gordonii
DL1,
Streptococcus oralis
34, and
Actinomyces naeslundii
T14V). Area coverage and cell cluster size measurements showed that attachment of
A. naeslundii
and of
S. gordonii
to glass flowcells was enhanced by a salivary conditioning film, whereas attachment of
S. oralis
was hindered. Growth experiments using saliva as the sole carbon and nitrogen source showed that
A. naeslundii
was unable to grow either in planktonic culture or as a biofilm, whereas
S. gordonii
grew under both conditions.
S. oralis
grew planktonically, but to a much lower maximum cell density than did
S. gordonii
;
S. oralis
did not grow reproducibly as a biofilm. Thus, only
S. gordonii
possessed all traits advantageous for growth as a solitary and independent resident of the tooth. Two-species biofilm experiments analyzed by laser confocal microscopy showed that neither
S. oralis
nor
A. naeslundii
grew when coaggregated pairwise with
S. gordonii
. However, both
S. oralis
and
A. naeslundii
showed luxuriant, interdigitated growth when paired together in coaggregated microcolonies. Thus, the
S. oralis-A. naeslundii
pair formed a mutualistic relationship, potentially contact dependent, that allows each to grow where neither could survive alone.
S. gordonii
, in contrast, neither was hindered by nor benefited from the presence of either of the other strains. The formation of mutually beneficial interactions within the developing biofilm may be essential for certain initial colonizers to be retained during early plaque development, whereas other initial colonizers may be unaffected by neighboring cells on the substratum.