bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Heme-Binding Proteins in Epidemic Strains of
Burkholderia cepacia
Authors: Smalley John W. , Charalabous Panagoula , Birss Andrew J. , Hart C. Anthony
Published: July 2002

A panel of 30 previously characterized strains representing five genomovars from the
Burkholderia cepacia
complex (E. Mahenthiralingam, T. Coenye, J. W. Chung, D. P. Speert, J. R. W. Govan, P. Taylor, and P. Vandamme, J. Clin. Microbiol. 38:910–913, 2000) were examined for their iron protoporphyrin IX-binding ability. These included
B. cepacia
genomovars I and III and
B. stabilis
(formerly
B. cepacia
genomovar IV),
B. multivorans
(formerly
B. cepacia
genomovar II), and
B. vietnamiensis (
formerly
B. cepacia
genomovar V
)
. Cells were exposed to μ-oxo bisheme of iron protoporphyrin IX (μ-oxo dimers) and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing, nondenaturing conditions for the presence of heme-binding proteins using tetramethylbenzidine-H
2
O
2
staining. Seven of the 30 strains, each belonging to
B. cepacia
genomovar III and designated epidemic (in possessing the
B. cepacia
epidemic strain marker), expressed a 96- to 100-kDa heme-binding protein which was located in the outer membrane. The heme-binding protein of
B. cepacia
genomovar III epidemic strain C5424 bound iron(III) protoporphyrin IX in both the monomeric and μ-oxo bisheme forms. Cells of all strains grown on Columbia agar bound iron protoporphyrin IX in the μ-oxo bisheme (dimeric) form. There were no statistical differences between the five genomovars, or those possessing the heme-binding protein, in their μ-oxo bisheme-binding ability. Possession of the outer membrane heme-binding protein may be a pathogenicity trait in enabling the bacterium to withstand oxidative stresses in inflammatory exudates in the lung and may aid identification of invasive epidemic strains of
B. cepacia
.
Role of Nijmegen Breakage Syndrome Protein in Specific T-Lymphocyte Activation Pathways
Authors: Garcı́a-Pérez Miguel Angel , Allende Luis M. , Corell Alfredo , Paz-Artal Estela , Varela Pilar , López-Goyanes Alberto , Garcı́a-Martin Francisco , Vázquez Rosario , Sotoca Amalia , Arnaiz-Villena Antonio
Published: July 2002

Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by immunodeficiency, microcephaly, and “bird-like” facies. NBS shares some clinical features with ataxia telangiectasia (AT), including increased sensitivity to ionizing radiation, increased spontaneous and induced chromosome fragility, and strong predisposition to lymphoid cancers. The mutated gene that results in NBS codes for a novel double-stranded DNA break repair protein, named nibrin. In the present work, a Spanish NBS patient was extensively characterized at the immunological and the molecular DNA levels. He showed low CD3
+
-cell numbers and an abnormal low CD4
+
naive cell/CD4
+
memory cell ratio, previously described in AT patients and also described in the present report in the NBS patient. The proliferative response of peripheral blood lymphocytes in vitro to mitogens is deficient in NBS patients, but the possible link among NBS mutations and the abnormal immune response is still unknown.
Skin Test Reactivity and Cellular Immune Responses to
Mycobacterium avium
Sensitin in AIDS Patients at Risk for Disseminated
M. avium
Infection
Authors: von Reyn C. Fordham , Williams Paige L. , Lederman Howard M. , McCutchan J. Allen , Koletar Susan L. , Murphy Robert L. , Cohn Susan E. , Evans Thomas , Heald Alison E. , Colquhoun Dodi , Bassily Ehab L. , Currier Judith S.
Published: July 2002

Skin tests and lymphocyte proliferation assays (LPA) were performed with
Mycobacterium avium
sensitin on patients with AIDS. Among 139 subjects, 13% had positive skin test results and 32% had positive LPA results. The LPA may be a more sensitive indicator of prior
M. avium
infection in this population.
B-Cell Deficiency Predisposes Mice to Disseminating Anaerobic Infections: Protection by Passive Antibody Transfer
Authors: Hou Linda , Sasakj Hajime , Stashenko Philip
Published: July 2002

We have previously demonstrated that a high proportion of RAG-2 SCID knockout mice, which lack T and B cells, develop orofacial abscesses and disseminated infections following pulpal infection, whereas immunocompetent control mice do not. In the present study, we sought to identify the components of the adaptive immune response which contribute to protection against disseminating anaerobic infections and sepsis. For this purpose, various genetically engineered immunodeficient mice were employed, including RAG-2 SCID, Igh-6 (B-cell deficient), Tcrb Tcrd (T-cell deficient) and Hc0(C5 deficient). For abscess induction, the mandibular first molars were subjected to pulp exposure on day 0. Teeth were infected with a mixture of four anaerobic pathogens, includingPrevotella intermedia,Streptococcus intermedius,Fusobacterium nucleatum, andPeptostreptococcus micros, and teeth were sealed to prevent communication with the oral cavity. The findings demonstrate that both RAG-2 SCID and B-cell-deficient mice, but not T-cell- or C5-deficient mice, have increased susceptibility to the development of disseminating anaerobic infections. Abscess-susceptible RAG-2 SCID and B-cell-deficient mice also showed a significant loss of body weight, splenomegaly, and absent antibacterial antibody production. Furthermore, dissemination was significantly reduced, from 74 to 25%, in susceptible RAG-2 mice by passively transferred antibody, predominantly immunoglobulin G2b (IgG2b) and IgM, against the infecting bacterial innoculum. Fractionated IgG-enriched preparations were more efficient in transferring protection than IgM preparations. We conclude that an antibody-mediated mechanism(s), most likely bacterial opsonization, is of importance in localizing anaerobic root canal infections and in preventing their systemic spread.
Diversity of PspA: Mosaic Genes and Evidence for Past Recombination in
Streptococcus pneumoniae
Authors: Hollingshead Susan K. , Becker Robert , Briles David E.
Published: July 2002

Pneumococcal surface protein A (PspA) is a serologically variable protein of
Streptococcus pneumoniae
. Twenty-four diverse alleles of the
pspA
gene were sequenced to investigate the genetic basis for serologic diversity and to evaluate the potential of diversity to have an impact on PspA's use in human vaccination. The 24
pspA
gene sequences from unrelated strains revealed two major allelic types, termed “families,” subdivided into clades. A highly mosaic gene structure was observed in which individual mosaic sequence blocks in PspAs diverged from each other by over 20% in many cases. This level of divergence exceeds that observed for blocks in the penicillin-binding proteins of
S. pneumoniae
or in many cross-species comparisons of gene loci. Conversely, because the mosaic pattern is so complex, each pair of
pspA
genes also has numerous shared blocks, but the position of conserved blocks differs from gene pair to gene pair. A central region of
pspA
, important for eliciting protective antibodies, was found in six clades, which each diverge from the other clades by >20%. Sequence relationships among the 24 alleles analyzed over three windows were discordant, indicating that intragenic recombination has occurred within this locus. The extensive recombination which generated the mosaic pattern seen in the
pspA
locus suggests that natural selection has operated in the history of this gene locus and underscores the likelihood that PspA may be important in the interaction between the pneumococcus and its human host.
Isolation and Characterization of Mini-Tn
5
Km2 Insertion Mutants of Enterohemorrhagic
Escherichia coli
O157:H7 Deficient in Adherence to Caco-2 Cells
Authors: Tatsuno Ichiro , Kimura Hiroshi , Okutani Akiko , Kanamaru Kyoko , Abe Hiroyuki , Nagai Shinya , Makino Kozo , Shinagawa Hideo , Yoshida Mitsutaka , Sato Katsuhiro , Nakamoto Jyunichi , Tobe Toru , Sasakawa Chihiro
Published: July 2002

Adherence of enterohemorrhagic
Escherichia coli
(EHEC) to intestinal epithelium is essential for initiation of the infection. To identify genes involved in adherence, an EHEC O157:H7 strain (O157Sakai) was mutagenized by mini-Tn
5
Km2, where Km refers to kanamycin resistance, and 4,677 insertion mutants were screened for their ability to form microcolonies (MC) on Caco-2 cells. The less adherent mutants were divided into three groups: those with no adherent ability (designated as class 1 mutants,
n
= 10), those less adherent than the wild type (class 2 mutants,
n
= 16), and those unable to form MC but which adhered in a diffuse manner (class 3 mutants,
n
= 1). The sites of insertion in class 1 mutants were all found within genes of the locus for enterocyte effacement (LEE) thought to be required for type III protein secretion. Indeed, the class 1 mutants failed to secrete type III secreted proteins such as EspA and Tir into the culture medium. The insertions in class 2 mutants were outside the LEE, and all the mutants except one were able to secrete type III proteins into the culture medium. The class 3 mutant had the insertion in the
tir
gene in the LEE and was deficient in Tir and intimin expression, suggesting that in the absence of intimin-Tir, O157Sakai can still adhere to Caco-2 cells but in a diffused manner. This was confirmed by construction of a nonpolar
eae
(encoding intimin) mutant. Examination of the
eae
mutant together with O157Sakai and one of the class 1 mutants for the ability to form MC revealed that EHEC initially adhered diffusely at 1.5 h after infection. Following washing out of the nonadherent bacteria, while wild-type EHEC bacteria developed MC for another 2 to 3 h on Caco-2 cells, the
eae
mutant diffusely adhered throughout the infection without forming MC. MC with O157Sakai but not the diffusely adherent
eae
mutant could evoke F-actin condensation beneath the bacterium. Our results suggest that EHEC encodes additional adherence-associated loci and that the type III secreted proteins are involved in the initial diffuse adherence, while the intimin-Tir interaction is required for the subsequent development of MC.
Saccharomyces boulardiiPreserves the Barrier Function and Modulates the Signal Transduction Pathway Induced in EnteropathogenicEscherichia coli-Infected T84 Cells
Authors: Czerucka Dorota , Dahan Stephanie , Mograbi Baharia , Rossi Bernard , Rampal Patrick
Published: July 2002

Use of the nonpathogenic yeastSaccharomyces boulardiiin the treatment of infectious diarrhea has attracted growing interest. The present study designed to investigate the effect of this yeast on enteropathogenicEscherichia coli(EPEC)-associated disease demonstrates thatS. boulardiiabrogated the alterations induced by an EPEC strain on transepithelial resistance, [3H]inulin flux, and ZO-1 distribution in T84 cells. Moreover, EPEC-mediated apoptosis of epithelial cells was delayed in the presence ofS. boulardii. The yeast did not modify the number of adherent bacteria but lowered by 50% the number of intracellular bacteria. Infection by EPEC induced tyrosine phosphorylation of several proteins in T84 cells, including p46 and p52 SHC isoforms, that was attenuated in the presence ofS. boulardii. Similarly, EPEC-induced activation of the ERK1/2 mitogen-activated protein (MAP) kinase pathway was diminished in the presence of the yeast. Interestingly, inhibition of the ERK1/2 pathway with the specific inhibitor PD 98059 decreased EPEC internalization, suggesting that modulation of the ERK1/2 MAP pathway might account for the lowering of the number of intracellular bacteria observed in the presence ofS. boulardii. Altogether, this study demonstrated thatS. boulardiiexerts a protective effect on epithelial cells after EPEC adhesion by modulating the signaling pathway induced by bacterial infection.
Purification and Characterization of a Cytotonic Protein Expressed In Vitro by the Live Cholera Vaccine Candidate CVD 103-HgR
Authors: Sathyamoorthy Venugopal , Hall Robert H. , McCardell Barbara A. , Kothary Mahendra H. , Ahn Susie J. , Ratnayake Shashikala
Published: July 2002

Cholera vaccines developed by the deletion of CTX genes from
Vibrio cholerae
induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467–470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an
M
r
of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor
V. cholerae.
Differential Effects of Virulent versus Avirulent
Legionella pneumophila
on Chemokine Gene Expression in Murine Alveolar Macrophages Determined by cDNA Expression Array Technique
Authors: Nakachi Noriya , Matsunaga Kazuto , Klein Thomas W. , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

The cDNA expression array technique is a powerful tool to determine, at one time from many genes, specific gene messages modulated by infection. In the present study, we identified genes modulated in response to virulent versus avirulent
Legionella pneumophila
infection of the alveolar macrophage cell line MH-S by the cDNA expression array technique. Many macrophage genes were found to be modulated after 5 h of in vitro infection with
L. pneumophila
. In particular, it was found that the monocyte chemotactic protein 3 (MCP-3) gene expression was significantly induced by infection with virulent
L. pneumophila
but not with avirulent
L. pneumophila
. In contrast, other chemokine genes, such as macrophage inflammatory protein (MIP) 1α, were induced by both virulent and avirulent
L. pneumophila
. Reverse transcription (RT)-PCR assay of total RNA isolated from macrophages infected with the bacteria for 5 or 24 h confirmed the differential induction of the chemokine genes by virulent versus avirulent
L. pneumophila
. Thus, the cDNA expression array technique readily revealed differential induction by
L. pneumophila
infection of select chemokine genes of macrophages from more than 1,100 genes. These results also indicate that certain chemokine genes may be selectively induced by virulent bacteria.
B Cells Are Essential for Vaccination-Induced Resistance to VirulentToxoplasma gondii
Authors: Sayles Peter C. , Gibson George W. , Johnson Lawrence L.
Published: July 2002

T lymphocytes and gamma interferon (IFN-γ) are known mediators of immune resistance toToxoplasma gondiiinfection, but whether B cells also play an important role is not clear. We have investigated this issue using B-cell-deficient (μMT) mice. If vaccinated with attenuatedT. gondiitachyzoites, μMT mice are susceptible to a challenge intraperitoneal infection with highly virulent tachyzoites that similarly vaccinated B-cell-sufficient mice resist. Susceptibility is evidenced by increased numbers of parasites at the challenge infection site and by extensive mortality. The susceptibility of B-cell-deficient mice does not appear to be caused by deficient T-cell functions or diminished capacity of vaccinated and challenged B-cell-deficient mice to produce IFN-γ. Administration ofToxoplasma-immune serum, but not nonimmune serum, to vaccinated B-cell-deficient mice significantly prolongs their survival after challenge with virulent tachyzoites. Vaccinated mice lacking Fc receptors or the fifth component of complement resist a challenge infection, suggesting that neither Fc-receptor-dependent phagocytosis of antibody-coated tachyzoites nor antibody-dependent cellular cytotoxicity nor antibody-and-complement-dependent lysis of tachyzoites is a crucial mechanism of resistance. However,Toxoplasma-immune serum effectively inhibits the infection of host cells by tachyzoites in vitro. Together, the results support the hypothesis that B cells are required for vaccination-induced resistance to virulent tachyzoites in order to produce antibodies and that antibodies may function protectively in vivo by blocking infection of host cells by tachyzoites.
Salmonella entericaSerovar TyphimuriumsurAMutants Are Attenuated and Effective Live Oral Vaccines
Authors: Sydenham Mark , Douce Gillian , Bowe Frances , Ahmed Saddif , Chatfield Steve , Dougan Gordon
Published: July 2002

A previously described attenuated TnphoAmutant (BRD441) ofSalmonella entericaserovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758–2763, 1989) was characterized, and the transposon was shown to be inserted insurA, a gene which encodes a peptidylprolyl-cis,trans-isomerase. A definedsurAdeletion mutation was introduced intoS. entericaserovar Typhimurium C5 and the mutant strain, namedS. entericaserovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo.S. entericaserovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore,S. entericaserovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intactsurAgene almost completely restored the virulence of BRD1115. In addition,S. entericaserovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge withS. entericaserovar Typhimurium C5.S. entericaserovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.
Cytokines inMycoplasma hyorhinis-Induced Arthritis in Pigs Bred Selectively for High and Low Immune Responses
Authors: Jayagopala Reddy N. R. , Wilkie Bruce N. , Borgs Peter , Mallard Bonnie A.
Published: July 2002

Yorkshire pigs were bred selectively for high and low immune responses (H and L pigs, respectively) based on multiple antibody (Ab) and cell-mediated immune response traits. In a previous experiment, generation 4 (G4) pigs of each line were infected withMycoplasma hyorhinis. High responders had a more rapid and higher Ab response and less polyserositis, but arthritis was more severe in H pigs than in L pigs. To test the hypothesis that line differences were attributable to differential expression of cytokines,M. hyorhinisinfection was induced in pigs of G8. Arthritis was more severe clinically (P, ≤0.05) and postmortem (P, ≤0.001) whenM. hyorhinisCFU were more numerous in synovial fluid (SF) of H pigs than of L pigs (P, ≤0.03). In H pigs but not L pigs, CFU and lesion scores were correlated positively. In H pigs, infection increased the frequency of expression of mRNAs for interleukin-8 (IL-8), IL-10, and tumor necrosis factor alpha (TNF-α) in mononuclear cells from synovial membranes (SM). In L pigs, IL-1α, IL-6, IL-10, and TNF-α mRNAs were increased in frequency of expression. The quantity of the cytokine message for IL-6 was increased in infected H pigs. For L pigs, infection increased the cytokine message for IL-1α, IL-6, IL-10, and TNF-α. IL-6 in SM and gamma interferon (IFN-γ) in SF were produced at a higher copy number in H pigs than in L pigs after infection. For H pigs, there were no positive rank correlations between lesion or CFU scores and cytokines. For L pigs, IL-1α, IL-8, IL-10, and TNF-α in SM correlated with CFU, while IL-6, TNF-β, and IFN-γ in SF correlated with CFU. Lesion score in L pigs correlated with IL-1α in SF. While these results indicate that H and L pigs differ in the cytokine response toM. hyorhinisinfection, they do not confirm a characteristic cytokine response in association with the relative susceptibility to infection and arthritis observed in H pigs.
Phospholipid Synthesis by
Staphylococcus aureus
during (Sub)Lethal Attack by Mammalian 14-Kilodalton Group IIA Phospholipase A2
Authors: Foreman-Wykert Amy K. , Weiss Jerrold , Elsbach Peter
Published: July 2002

Killing of gram-positive bacteria by mammalian group IIA phospholipases A2 (PLA2) requires the catalytic activity of the enzyme. However, nearly complete degradation of the phospholipids can occur with little effect on bacterial viability, suggesting that PLA2-treated bacteria can biosynthetically replace phospholipids that are lost due to PLA2 action. In the presence of albumin, phospholipid degradation products are quantitatively sequestered extracellularly. In the absence of albumin, the bacteria retain and substantially reutilize the phospholipid breakdown products and survive an otherwise lethal dose of PLA2. PLA2-treated bacteria also continue to incorporate sodium [2-
14
C]acetate into phospholipids, suggesting that the bacteria are attempting to repair the damaged membranes by de novo synthesis of phospholipids. To determine whether PLA2 action also triggers activation of bacterial lipolytic enzymes, the effects of nisin and PLA2 on the degradation of
S. aureus
lipids were compared. In contrast to nisin treatment, PLA2 treatment does not stimulate endogenous phospholipase activity in
S. aureus
. These findings show that
S. aureus
responds to PLA2 attack by continued phospholipid (re)synthesis by both de novo and salvage pathways. The fate of PLA2-treated
S. aureus
therefore appears to depend on the relative rates of phospholipid degradation and synthesis.
Molecular and Evolutionary Characterization of the cp32/18 Family of Supercoiled Plasmids in
Borrelia burgdorferi
297
Authors: Caimano Melissa J. , Yang Xiaofeng , Popova Taissia G. , Clawson Michael L. , Akins Darrin R. , Norgard Michael V. , Radolf Justin D.
Published: July 2002

In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in
Borrelia burgdorferi
297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526–1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.
Monoclonal Antibody-Mediated Modulation of the Humoral Immune Response against Mucosally AppliedStreptococcus mutans
Authors: Brady L. Jeannine , van Tilburg Marloes L. J. A. , Alford Connie E. , McArthur William P.
Published: July 2002

Systemic immunization with antigen coupled to monoclonal antibody (MAb) has been used by several investigators to increase the number of MAb-producing hybridomas against an antigen and to elicit antibodies specific for poorly immunogenic epitopes. This strategy has implications for vaccine design in that protective immunity is not necessarily directed at immunodominant epitopes of pathogens and may be improved by deliberately shifting the immune response toward subdominant epitopes. To our knowledge, no studies to date have addressed the potential for immunomodulatory activity mediated by MAbs bound to mucosally applied antigen. To test whether administration of an exogenous MAb directed against a streptococcal surface protein could influence the humoral immune response, BALB/c mice were immunized orally by gastric intubation or intranasally withStreptococcus mutansalone orS. mutanscomplexed with a MAb directed against the major surface protein P1. Significant changes in the subclass distribution, as well as the specificity, of anti-P1 serum immunoglobulin G antibodies were demonstrated in groups of mice which receivedS. mutanscoated with the anti-P1 MAb versus those which receivedS. mutansalone. Alterations in the humoral immune response were dependent on the amount of anti-P1 MAb used to coat the bacteria. In addition, differences in the anti-P1 immune responses were observed between groups of mice immunized via oral versus intranasal routes. In summary, an exogenous MAb complexed with a streptococcal antigen prior to mucosal immunization can influence the immunoglobulin isotype and specificity of the host humoral immune response against the antigen.
Biochemical and Genetic Evidence for Phospholipase C Activity inMycobacterium ulcerans
Authors: Gomez Arley , Mve-Obiang Armand , Vray Bernard , Remacle José , Chemlal Karim , Meyers Wayne M. , Portaels Françoise , Fonteyne Pierre-Alain
Published: July 2002

This study reports the existence of phospholipase C and D enzymatic activities inMycobacterium ulceranscultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine.M. ulceransDNA sequences homologous to the genes encoding phospholipase C inMycobacterium tuberculosisandPseudomonas aeruginosawere identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes ofM. ulceransplays a role in the pathogenesis of the disease needs further investigation.
Ineffective Cellular Immune Response Associated with T-Cell Apoptosis in SusceptibleMycobacterium bovisBCG-Infected Mice
Authors: Kremer Laurent , Estaquier Jérôme , Wolowczuk Isabelle , Biet Franck , Ameisen Jean-Claude , Locht Camille
Published: July 2002

It has previously been reported that inhibition of delayed-type hypersensitivity-mediating functions of T cells during mycobacterial infection in mice is haplotype dependent. In the present study, we show thatMycobacterium bovisBCG infection induced, in susceptible C57BL/6 and BALB/c mice but not in resistant C3H/HeJ and DBA/2 mice, an important splenomegaly. An in vitro defect in T-cell proliferation in response to T-cell receptor (TCR) stimulation with mitogens or anti-CD3 antibodies was associated with enhanced levels of CD4+and CD8+T-cell apoptosis in susceptible but not in resistant mice 2 weeks after infection. Further investigations of C57BL/6 and C3H/HeJ mice revealed that in vivo splenomegaly was associated with destruction of the lymphoid tissue architecture, liver cellular infiltrates, and increased numbers of apoptotic cells in both spleen and liver tissue sections. Infection of C57BL/6 mice but not of C3H/HeJ mice induced massive production of tumor necrosis factor alpha (TNF-α) in serum, as well as an increase in Fas and Fas ligand (FasL) expression in T cells. In vitro addition of neutralizing anti-TNF-α antibodies led to a significant reduction in CD3-induced T-cell apoptosis of both CD4+and CD8+T cells of C57BL/6 mice, while the blockade of Fas-FasL interactions reduced apoptosis only in CD4+but not in CD8+T cells. Together, these results suggest that TNF-α and Fas-FasL interactions play a role in the activation-induced cell death (AICD) process associated with a defect in T-cell proliferation of the susceptible C57BL/6 mice. T-cell death by apoptosis may represent one of the important components of the ineffective immune response against mycobacterium-induced immunopathology in susceptible hosts.
Does Inhibition of Tumor Necrosis Factor Alpha Affect Chlamydial Genital Tract Infection in Mice and Guinea Pigs?
Authors: Darville Toni , Andrews C. W. , Rank R. G.
Published: July 2002

The role of tumor necrosis factor alpha (TNF-α) in host defense against chlamydial infection remains unclear. In order to further evaluate the relevance of TNF-α to host resistance in chlamydial genital tract infection, we examined the effect of local inhibition of the TNF-α response in normal C57 mice and in interferon gamma gene-deficient C57 mice infected intravaginally with the mouse pneumonitis agent ofChlamydia trachomatis. Since the guinea pig model of female genital tract infection more closely approximates the human in terms of ascending infection and development of pathology, we also examined the effect of local inhibition of the TNF-α response in guinea pigs infected intravaginally with the guinea pig strain ofChlamydia psittaci. We successfully blocked the early TNF-α response in the respective animal models. This blockade had no effect on the numbers of organisms isolated from the genital tract during the time of TNF-α inhibition in mice or guinea pigs. Analysis of interleukin-1β, macrophage inflammatory protein-2, and granulocyte macrophage-colony stimulating factor in the mouse model revealed that blockade of the TNF-α response did not alter the release of these proinflammatory proteins. Yet, in TNF-α-depleted mice, increased numbers of neutrophils were detected in the genital tract, and, in TNF-α-depleted guinea pigs, increased numbers of neutrophils as well as infiltrating lymphocytes were seen in the endocervix. Blockade of TNF-α does not affect the level of infection in mice or guinea pigs, but it may decrease TNF-α-induced apoptosis of infiltrating inflammatory cells.
Novel Group of Virulence Activators within the AraC Family That Are Not Restricted to Upstream Binding Sites
Authors: Munson George P. , Holcomb Lisa G. , Scott June R.
Published: July 2002

Several regulators within the AraC family control the expression of genes known or thought to be required for virulence of bacterial pathogens. One of these, Rns, activates transcription from an unprecedented variety of binding-site locations. Although nearly all prokaryotic activators bind within a small region upstream and adjacent to the promoter that they regulate, Rns does not bind within this region to activate its own promoter, P
rns
. Instead, to activate P
rns
, Rns requires one binding site 224.5 bp upstream and one downstream of the transcription start site. We show in this study that several other AraC family activators recognize the same binding sites as Rns and share with it the ability to utilize a downstream binding site. Like Rns, other members of this group of activators positively regulate the expression of virulence factors in pathogenic bacteria. These regulators are also able to activate transcription from promoter-proximal upstream binding sites since they are able to substitute for Rns at P
coo
, a promoter with only upstream binding sites. Thus, Rns is the prototype for a group of regulators, which include CfaR, VirF, AggR, and CsvR and which activate transcription from locations that are more diverse than those of any other known activator.
Group BStreptococcusCapsular Polysaccharide-Cholera Toxin B Subunit Conjugate Vaccines Prepared by Different Methods for Intranasal Immunization
Authors: Shen Xuzhuang , Lagergård Teresa , Yang Yonghong , Lindblad Marianne , Fredriksson Margareta , Holmgren Jan
Published: July 2002

Group BStreptococcus(GBS) type III capsular polysaccharide (CPS III) was conjugated to recombinant cholera toxin B subunit (rCTB) using three different methods which employed (i) cystamine andN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), (ii) carbodiimide with adipic acid dihydrazide (ADH) as a spacer, or (iii) reductive amination (RA). The CPS III-rCTB conjugates were divided into large- and small-molecular-weight (Mr) fractions, and the immunogenicities of the different preparations after intranasal (i.n.) immunization were studied in mice. Both large- and small-Mrconjugates of CPS III-rCTBRAor CPS III-rCTBADHinduced high, almost comparable levels of CPS-specific immunoglobulin G (IgG) in serum, lungs, and vagina that were generally superior to those obtained with CPS III-rCTBSPDPconjugates or a CPS III and rCTB mixture. However, the smaller-Mrconjugates of CPS III-rCTBRAor CPS III-rCTBADHin most cases elicited a lower anti-CPS IgA immune response than the large-Mrconjugates, and the highest anti-CPS IgA titers in both tissues and serum were obtained with the large-MrCPS III-rCTBRAconjugate. Serum IgG anti-CPS titers induced by the CPS III-rCTBRAconjugate had high levels of specific IgG1, IgG2a, IgG2b, and IgG3 antibodies. Based on the effectiveness of RA for coupling CPS III to rCTB, RA was also tested for conjugating GBS CPS Ia with rCTB. As for the CPS III-rCTB conjugates, the immunogenicity of CPS Ia was greatly increased by conjugation to rCTB. Intranasal immunization with a combination of CPS Ia-rCTB and CPS III-rCTB conjugates was shown to induce anti-CPS Ia and III immune responses in serum and lungs that were fully comparable with the responses to immunization with the monovalent CPS Ia-rCTB or CPS III-rCTB conjugates. These results suggest that the GBS CPS III-rCTB and CPS Ia-rCTB conjugates prepared by the RA method may be used in bivalent and possibly also in multivalent mucosal GBS conjugate vaccines.
Severity of Group B Streptococcal Arthritis in Selected Strains of Laboratory Mice
Authors: Puliti Manuela , Bistoni Francesco , von Hunolstein Christina , Orefici Graziella , Tissi Luciana
Published: July 2002

The susceptibilities of C3H/HeN, BALB/c, and C57BL/6N mouse strains to group B streptococci (GBS) infection were evaluated. C3H/HeN mice developed severe polyarthitis; mild lesions and no lesions were observed in BALB/c and C57BL/6N mice, respectively. A correlation between the severity of arthritis, the number of GBS in the joints, and local interleukin-6 and interleukin-1β production was evident.
Cytotoxic Necrotizing Factor from
Escherichia coli
Induces RhoA-Dependent Expression of the Cyclooxygenase-2 Gene
Authors: Thomas Warren , Ascott Zoe K. , Harmey Dympna , Slice Lee W. , Rozengurt Enrique , Lax Alistair J.
Published: July 2002

Cytotoxic necrotizing factor 1 (CNF) is a toxin produced by some isolates of
Escherichia coli
that cause extraintestinal infections. CNF can initiate signaling pathways that are mediated by the Rho family of small GTPases through a covalent modification that results in constitutive activation. In addition to regulating the assembly of actin stress fibers and focal adhesion complexes, RhoA can also regulate gene expression at the level of transcription. Here we demonstrate for the first time, by using a luciferase-based reporter system, that the transcription of cyclooxygenase-2 (COX-2) is strongly upregulated in NIH 3T3 fibroblasts treated with CNF and that this effect is dependent upon the activation of RhoA by the toxin. Subsequent protein tyrosine phosphorylation events modulate the induction, but the transcription signal is not mediated by Rho-associated kinase (p160/ROCK) and so must rely upon another effector that is activated by RhoA. CNF therefore induces COX-2 expression via a RhoA-dependent signaling pathway that diverges from the pathway that regulates cytoskeletal rearrangements in response to RhoA activation.
Effect of SpeB and EndoS from
Streptococcus pyogenes
on Human Immunoglobulins
Authors: Collin Mattias , Olsén Arne
Published: July 2002

Streptococcus pyogenes
secretes a specific immunoglobulin G (IgG)-protease, SpeB, as well as the IgG glycan-hydrolyzing enzyme EndoS. Here we show that SpeB also degrades IgA, IgM, IgD, and IgE. We also show that EndoS only hydrolyzes the glycan moiety on native but not denatured IgG. Thus, SpeB has a broad immunoglobulin-degrading activity, while EndoS is highly specific for IgG.
Glycocalyx on Rabbit Intestinal M Cells Displays Carbohydrate Epitopes from Muc2
Authors: Lelouard Hugues , Reggio Hubert , Roy Christian , Sahuquet Alain , Mangeat Paul , Montcourrier Philippe
Published: July 2002

It is essential to investigate the apical surface properties of both M cells and dome enterocytes to understand the mechanisms involved in the binding of pathogens to M cells. In rabbit appendix tissue, monoclonal antibodies (MAbs) highlight differences between M cells (MAb 58) and dome enterocytes (MAb 214). Such antibodies ultimately recognized intestinal mucin-related epitopes. To further characterize these differences, the labeling patterns obtained with these MAbs were compared to those obtained with other antibodies to intestinal mucins on dissected domes from all gut-associated lymphoid tissues. A glycoprotein recognized by MAb 58 was purified on a CsCl isopycnic density gradient and microsequenced, and its mRNA expression was localized by in situ hybridization. It was identified as the rabbit homologue of human Muc2, i.e., the major mucin secreted in intestine tissue. Two other Muc2 carbohydrate epitopes were also expressed on M cells, although Muc2 mRNA was not detected. All results indicated that M cells express, on their apical membrane, glycoconjugates bearing at least three glycosidic epitopes from Muc2. MAb 214 and MAb 6G2, which recognized a partially characterized mucin expressed on dome enterocytes, were negative markers for M cells in rabbit gut-associated lymphoid tissues. We propose that the presence, on the surface of M cells, of carbohydrates also expressed on Muc2, together with the absence of an enterocyte-associated mucin, could favor pathogen attachment and accessibility to the M-cell luminal membrane.
The Common Vaccine Adjuvant Aluminum Hydroxide Up-Regulates Accessory Properties of Human Monocytes via an Interleukin-4-Dependent Mechanism
Authors: Ulanova Marina , Tarkowski Andrej , Hahn-Zoric Mirjana , Hanson Lars Å.
Published: July 2002

Aluminum adjuvants are widely used in human vaccines based on their ability to enhance antibody production. However, the mechanisms underlying these effects remain unknown. In the present study we assessed the direct in vitro effect of aluminum hydroxide on human peripheral blood monocytes, specifically with regard to its impact on the phenotype and functional properties of this cell population. Our results revealed significant changes in the accessory properties of monocytes following short-term exposure of cultured cells to aluminum hydroxide. Thus, flow cytometry analyses showed an increase in the expression of major histocompatibility complex (MHC) class II, CD40, CD54, CD58, CD83, and CD86 molecules on the monocytes. In addition, many cells in the cultures containing aluminum hydroxide acquired typical dendritic morphology. Increased synthesis of interleukin-4 (IL-4) mRNA, but not gamma interferon mRNA, was also noted after exposure to aluminum hydroxide. The increase in cell surface expression of MHC class II did not occur in the presence of neutralizing IL-4 antibody or in cultures of highly purified monocytes or CD4-depleted mononuclear cells. Our findings suggest that aluminum hydroxide directly stimulates monocytes to produce proinflammatory cytokines activating T cells. Activated Th2 cells release IL-4, which in turn can induce an increase in the expression of MHC class II molecules on monocytes. The increase in the expression of antigen-presenting and costimulatory molecules leads to enhanced accessory functions of monocytes. These properties of aluminum hydroxide observed in vitro may explain its potent in vivo adjuvant effect.
Comparison of theexoSGene and Protein Expression in Soil and Clinical Isolates ofPseudomonas aeruginosa
Authors: Ferguson Michael W. , Maxwell Jill A. , Vincent Timothy S. , da Silva Jack , Olson Joan C.
Published: July 2002

Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis ofPseudomonas aeruginosa. To gain further understanding of how ExoS might contribute toP. aeruginosasurvival and virulence, ExoS expression and the structural gene sequence were determined inP. aeruginosasoil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity.ExoSstructural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced byP. aeruginosaor residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral toP. aeruginosasurvival in the soil and suggest that, in the transition ofP. aeruginosafrom the soil to certain clinical settings, the loss of ExoS expression is favored.
Involvement of HxuC Outer Membrane Protein in Utilization of Hemoglobin by
Haemophilus influenzae
Authors: Cope Leslie D. , Love Robert P. , Guinn Sarah E. , Gilep Andrei , Usanov Sergei , Estabrook Ronald W. , Hrkal Zbynek , Hansen Eric J.
Published: July 2002

Haemophilus influenzae
can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable
Haemophilus influenzae
(NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic
hgbA hgbB hgbC
mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a
tonB
mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the
hxuC
gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by
H. influenzae
.
Delineation of
Borrelia burgdorferi
p66 Sequences Required for Integrin α
IIb
β
3
Recognition
Authors: Defoe Gillianne , Coburn Jenifer
Published: July 2002

The outer membrane protein p66 of the Lyme disease agent,
Borrelia burgdorferi
, has been identified as a candidate ligand for β
3
-chain integrins. To identify portions of p66 required for integrin recognition, fusions of maltose-binding protein to fragments of p66 were tested for binding to integrin α
IIb
β
3
, and synthetic peptides derived from the p66 amino acid sequence were tested for the ability to inhibit
B. burgdorferi
attachment to the same integrin. The data identify two noncontiguous segments of p66 that are important for α
IIb
β
3
recognition, suggesting that, as is true for other integrin ligands, the tertiary structure of p66 is important for receptor recognition.
Construction, Genotypic and Phenotypic Characterization, and Immunogenicity of Attenuated ΔguaBA Salmonella entericaSerovar Typhi Strain CVD 915
Authors: Wang Jin Yuang , Pasetti Marcela F. , Noriega Fernando R. , Anderson Richard J. , Wasserman Steven S. , Galen James E. , Sztein Marcelo B. , Levine Myron M.
Published: July 2002

A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in theguaBAlocus of pathogenicSalmonella entericaserovar Typhi strain Ty2. The resultant ΔguaBAmutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence withinguaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA(harboring mutations inaroC, aroD, andhtrA). CVD 915 was less invasive than CVD 908-htrAin tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 × 107CFU) was significantly higher than that of wild-type Ty2 (1.4 × 102CFU) and was only slightly lower than that of Ty21a (1.9 × 108CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915.
Demonstration of the Genetic Stability and Temporal Expression of Select Members of the Lyme Disease Spirochete OspF Protein Family during Infection in Mice
Authors: McDowell John V. , Sung Shian Ying , Price Gregory , Marconi Richard T.
Published: July 2002

Infection with Lyme disease spirochetes can be chronic. This suggests that the spirochetes are capable of immune evasion. In a previous study we demonstrated that the
ospE
gene family, which is one of three gene families whose members are flanked at their 5′ end by the highly conserved upstream homology box (UHB) element, undergoes mutation and rearrangement during infection. This results in the generation of antigenically distinct variants that may contribute to immune evasion. In this study we have assessed the genetic stability of the UHB-flanked
ospF
gene family during infection in mice. Using postinfection clonal populations of
Borrelia burgdorferi
B31MI, PCR amplicons were generated for three members of the
ospF
gene family after a 3-month infection time frame. The amplicons were analyzed by single-nucleotide polymorphism pattern analysis and DNA sequencing. Members of the
ospF
gene family were found to be stable during infection, as no mutations or rearrangements were detected. An analysis of the humoral immune response to these proteins during infection revealed that the immune response to each is specific and that there is a delayed humoral immune response to some OspF protein family members. These analyses suggest that there is a temporal component to the expression of these genes during infection. In addition to a possible contribution to immune evasion, members of the OspF protein family may play specific roles at different stages of infection.