bluuebunny/crossref_metadata_2025 huggingface dataset:
A Recombinant β-1,3-Glucanosyltransferase Homolog of
Coccidioides posadasii
Protects Mice against Coccidioidomycosis
Authors: Delgado Nelson , Xue Jianmin , Yu Jieh-Juen , Hung Chiung-Yu , Cole Garry T.
Published: May 2003

Coccidioides posadasii
is a fungal respiratory pathogen which is responsible for recurrent epidemics of San Joaquin Valley fever (coccidioidomycosis) in desert regions of the southwestern United States. Numerous studies have revealed that the cell wall of the parasitic phase of the fungus is a reservoir of immunoreactive macromolecules and a potential source of a vaccine against this mycosis. A 495-bp fragment of a
C. posadasii
gene which encodes a putative wall-associated, glycosylphosphatidylinositol (GPI)-anchored β-1,3-glucanosyltransferase was identified by computational analysis of the partially sequenced genome of this pathogen. The translated, full-length gene (
GEL1
) showed high sequence homology to a reported β-1,3-glucanosyltransferase of
Aspergillus fumigatus
(70% identity, 90% similarity) and was selected for further study. The
GEL1
mRNA of
C. posadasii
was detected at the highest level during the endosporulation stage of the parasitic cycle, and the mature protein was immunolocalized to the surface of endospores. BALB/c or C57BL/6 mice were immunized subcutaneously with the bacterium-expressed recombinant protein (rGel1p) to evaluate its protective efficacy against a lethal challenge of
C. posadasii
by either the intraperitoneal or intranasal route. In both cases, rGel1p-immune mice infected with the pathogen showed a significant reduction in fungal burden and increased survival compared to nonimmune mice. The recombinant β-1,3-glucanosyltransferase is a valuable addition to an arsenal of immunoreactive proteins which could be incorporated into a human vaccine against coccidioidomycosis.
Isolation and Characterization of Mini-Tn
5
Km2 Insertion Mutants of
Brucella abortus
Deficient in Internalization and Intracellular Growth in HeLa Cells
Authors: Kim Suk , Watarai Masahisa , Kondo Yuki , Erdenebaatar Janchivdorj , Makino Sou-ichi , Shirahata Toshikazu
Published: May 2003

Brucella
spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells,
Brucella abortus
was mutagenized by mini-Tn
5
Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including
virB
genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.
A Fluoroquinolone Induces a Novel Mitogen-Encoding Bacteriophage inStreptococcus canis
Authors: Ingrey Keely T. , Ren Jun , Prescott John F.
Published: May 2003

This study investigated whether the recently recognized emergence of canine streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) might be partly attributed to the use of fluoroquinolones to treatStreptococcus canisinfections in dogs. Both mitomycin and the fluoroquinolone enrofloxacin caused bacteriophage-induced lysis ofS. canisstrain 34, an isolate from a case of canine STSS and NF. Fluoroquinolone-evoked, bacteriophage-induced lysis occurred over a range of concentrations similar to those that would occur after treatment of dogs with these agents. To search for a possible bacteriophage-encoded streptococcal superantigen gene(s), a library of the 36.5 (±1.1)-kb bacteriophage, designated φsc1, was made by ligating 3- to 7-kbTsp5091-digested φsc1 fragments into anEcoRI-digested λZapII vector. Recombinants were screened for mitogenic activity by using canine peripheral blood lymphocytes. Of 800 recombinants screened, 11 recombinants with mitogenic effects were identified, and their inserts were sequenced. The highest homology of 11.6 kb of sequenced φsc1 DNA was to the completely sequencedStreptococcus pneumoniaebacteriophage MM1. Seven of the 11 φsc1 sequenced inserts contained a 552-bp open reading frame,scm, with 27% amino acid similarity to pokeweed (Phytolacca americana) mitogen. PCR showed this gene to be present in 22 of 23S. canisisolates tested. Quantitative reverse transcription-PCR showed that bacteriophage induction was associated with a 58-fold enhancement of expression of this gene relative to that in a noninduced culture of a similar age. The presence of this gene on a fluoroquinolone-induced bacteriophage may explain the association observed between fluoroquinolone use in dogs and the development of canine STTS and NF.
Killing of
Aspergillus fumigatus
by Alveolar Macrophages Is Mediated by Reactive Oxidant Intermediates
Authors: Philippe B. , Ibrahim-Granet O. , Prévost M. C. , Gougerot-Pocidalo M. A. , Sanchez Perez M. , Van der Meeren A. , Latgé J. P.
Published: May 2003

Phagocytosis and mechanisms of killing of
Aspergillus fumigatus
conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of
A. fumigatus
conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47

phox
−/−

129Sv, and wild-type and congenic

iNOS
−/−

C57BL/6 mice. AM from p47

phox
−/−

mice were unable to kill
A. fumigatus
conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of
A. fumigatus
without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of
A. fumigatus
conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.
Comparison of
bipA
Alleles within and across
Bordetella
Species
Authors: Fuchslocher Bryna , Millar Laura L. , Cotter Peggy A.
Published: May 2003

The
Bordetella
BvgAS signal transduction system controls the expression of at least three phenotypic phases, the Bvg
+
or virulent phase, the Bvg
−
or avirulent phase, and the Bvg
i
or Bvg intermediate phase, which has been hypothesized to be important for transmission.
bipA
, the first identified Bvg
i
-phase gene, encodes a protein with similarity to the well-characterized bacterial adhesins intimin and invasin. Proteins encoded by the
bipA
genes present in
Bordetella pertussis
Tohama I and
Bordetella bronchiseptica
RB50 differ in the number of 90-amino-acid repeats which they possess and in the sequence of the C-terminal domain. To investigate the possibility that
bipA
alleles segregate according to host specificity and to gain insight into the role of BipA and the Bvg
i
phase in the
Bordetella
infectious cycle, we compared
bipA
alleles across members of the
B. bronchiseptica
cluster, which includes both human-infective (
B. pertussis
and
B. parapertussis
hu
) and non-human-infective (
B. bronchiseptica
and
B. parapertussis
ov
) strains.
bipA
genes were present in most, but not all, strains. All
bipA
genes present in
B. bronchiseptica
strains were identical to
bipA
of RB50 (at least with regard to the DNA sequence of the 3′ C-terminal-domain-encoding region, the number of 90-amino-acid repeats encoded, and expression patterns). Although all
bipA
genes present in the other
Bordetella
strains were identical in the 3′ C-terminal-domain-encoding region to
bipA
of
B. pertussis
Tohama I, they varied in the number of 90-amino-acid repeats that they encoded and in expression level. Notably, the genes present in
B. parapertussis
hu
strains were pseudogenes, and the genes present in
B. parapertussis
ov
strains were expressed at significantly reduced levels compared with the levels in
B. pertussis
and
B. bronchiseptica
strains. Our results indicate that there is a correlation between specific
bipA
alleles and specific hosts. They also support the hypothesis that both horizontal gene transfer and fine-tuning of gene expression patterns contribute to the evolution of host adaptation in lineages of the
B. bronchiseptica
cluster.
Involvement of Beta Interferon in Enhancing Inducible Nitric Oxide Synthase Production and Antimicrobial Activity of
Burkholderia
pseudomallei-
Infected Macrophages
Authors: Utaisincharoen P. , Anuntagool N. , Limposuwan K. , Chaisuriya P. , Sirisinha S.
Published: May 2003

Burkholderia
pseudomallei
is the causative agent of melioidosis, a life-threatening disease that affects both humans and animals. This bacterium is able to survive and multiply inside both phagocytic and nonphagocytic cells. We recently reported that mouse macrophages infected with
B
.
pseudomallei
fail to produce a significant level of inducible nitric oxide synthase (iNOS), a crucial enzyme needed for the cells to control the intracellular growth of this bacterium. In the present study, we extended our investigation to demonstrate that, unlike other gram-negative bacteria that have been investigated,
B
.
pseudomallei
only minimally activates beta interferon (IFN-β) production; this minimal activation leads to a low level of interferon regulating factor 1 (IRF-1) in the macrophages, in parallel with poor iNOS expression. Adding exogenous IFN-β to the system could upregulate IRF-1 production, which in turn could enhance iNOS expression in the
B
.
pseudomallei
-infected macrophages and lead to suppression of the intracellular growth of this bacterium. Taken together, these results imply that the failure of macrophages to successfully control the growth and survival of intracellular
B
.
pseudomallei
is related, at least in part, to the defective production of IFN-β, which modulates the ability of macrophages to synthesize iNOS.
Stimulation of Hematopoiesis by the Fms-Like Tyrosine Kinase 3 Ligand Restores Bacterial Induction of Th1 Cytokines in Thermally Injured Mice
Authors: Toliver-Kinsky Tracy E. , Lin Cheng Y. , Herndon David N. , Sherwood Edward R.
Published: May 2003

Patients with large burn injuries are susceptible to opportunistic infections due to impaired functions of multiple effector cells of innate immunity and acquired immunity, including macrophages, dendritic cells (DC), natural killer (NK) cells, and T cells. The ability of a host to produce Th1 cytokines, such as gamma interferon (IFN-γ) and interleukin-12 (IL-12), upon infectious challenge is also impaired after burn injury. Stimulation of hematopoiesis, to regenerate new immune cells, may be an effective strategy for improving resistance to infections after severe burn trauma. Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that stimulates the expansion and differentiation of NK cells and DC. Using a mouse model, we tested the hypothesis that Flt3L treatments after burn injury stimulate the production of functional effector cells of innate immunity and restore appropriate Th1 cytokine responses toPseudomonas aeruginosa, a common source of pneumonia and wound infections in burn victims. Flt3L increased splenic cellularity in sham (uninjured) and burned mice and increased the numbers of NK cells (DX5+) and DC (CD11c+). In response toP. aeruginosa, significant increases in the serum IFN-γ levels and the numbers of splenic IFN-γ-producing DC, NK cells, and T cells were observed in Flt3L-treated burned mice compared to the values obtained for untreated burned mice. The splenic levels of IL-12 and IL-15 mRNAs and the IL-12 and IL-15 receptors were also increased. In addition, Flt3L treatment restored the ability of splenic cultures prepared from burned mice to produce IFN-γ and IL-12 after in vitro challenge withP. aeruginosa. Flt3L may have potential for restoring NK cell and DC functions and improving immunity after burn injury.
Role of Phosphoglucomutase of
Stenotrophomonas
maltophilia
in Lipopolysaccharide Biosynthesis, Virulence, and Antibiotic Resistance
Authors: McKay Geoffrey A. , Woods Donald E. , MacDonald Kelly L. , Poole Keith
Published: May 2003

A homologue of the
algC
gene, responsible for the production of a phosphoglucomutase (PGM) associated with LPS and alginate biosynthesis in
Pseudomonas
aeruginosa
,
spgM
, was cloned from
Stenotrophomonas
maltophilia
. The
spgM
gene was shown to encode a bifunctional enzyme with both PGM and phosphomannomutase activities. Mutants lacking
spgM
produced less LPS than the SpgM
+
parent strain and had a tendency for shorter O polysaccharide chains. No changes in LPS chemistry were obvious as a result of the loss of
spgM
. Significantly, however,
spgM
mutants displayed a modest increase in susceptibility to several antimicrobial agents and were completely avirulent in an animal model of infection. The latter finding may relate to the resultant serum sensitivity of
spgM
mutants which, unlike the wild-type parent strain, were rapidly killed by human serum. These data highlight the contribution made by LPS to the antimicrobial resistance and virulence of
S
.
maltophilia
.
T-Cell Responses to CD1-Presented Lipid Antigens in Humans with
Mycobacterium tuberculosis
Infection
Authors: Ulrichs Timo , Moody D. Branch , Grant Ethan , Kaufmann Stefan H. E. , Porcelli Steven A.
Published: May 2003

CD1-restricted presentation of lipid or glycolipid antigens derived from
Mycobacterium tuberculosis
has been demonstrated by in vitro experiments using cultured T-cell lines. In the present work, the frequency of T-cell responses to natural mycobacterial lipids was analyzed in ex vivo studies of peripheral blood lymphocytes from human patients with pulmonary tuberculosis, from asymptomatic individuals with known contact with
M. tuberculosis
documented by conversion of their tuberculin skin tests, and from healthy tuberculin skin test-negative individuals or individuals vaccinated with
Mycobacterium bovis
BCG. Proliferation and gamma interferon enzyme-linked immunospot assays using peripheral blood lymphocytes and autologous CD1
+
immature dendritic cells revealed that T cells from asymptomatic
M. tuberculosis-
infected donors responded with significantly greater magnitude and frequency to mycobacterial lipid antigen preparations than lymphocytes from uninfected healthy donors. By use of these methods, lipid-antigen-specific proliferative responses were minimally detectable or absent in blood samples from patients with active tuberculosis prior to chemotherapy but became detectable in blood samples drawn 2 weeks after the start of treatment. Lipid antigen-reactive T cells were detected predominantly in the CD4-enriched T-cell fractions of circulating lymphocytes, and anti-CD1 antibody blocking experiments confirmed the CD1 restriction of these T-cell responses. Our results provide further support for the hypothesis that lipid antigens serve as targets of the recall response to
M. tuberculosis
, and they indicate that CD1-restricted T cells responding to these antigens comprise a significant portion of the circulating pool of
M. tuberculosis-
reactive T cells in healthy individuals with previous exposure to
M. tuberculosis
.
degS
Is Necessary for Virulence and Is among Extraintestinal
Escherichia coli
Genes Induced in Murine Peritonitis
Authors: Redford Peter , Roesch Paula L. , Welch Rodney A.
Published: May 2003

Extraintestinal
Escherichia coli
strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal
E. coli
strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless
gfp
vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes
metA
,
gltB
, and
sucA
, and another was linked to
iha
, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with
degS
, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073
degS
was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073
degS
shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σ
E
activity, our findings implicate σ
E
and its regulon in
E. coli
extraintestinal pathogenesis.
T Cells Are Required for Host Protection againstBrugia malayibut Need Not Produce or Respond to Interleukin-4
Authors: Spencer L. , Shultz L. , Rajan T. V.
Published: May 2003

T cells are known to be required for host protection in mouse models ofBrugia malayiinfection. Several independent studies in murine models have also highlighted the rapid induction of Th2-like responses after infection withB. malayiorB. pahangi. Previous data from our laboratory have described a significant increase in permissiveness in the absence of interleukin-4 (IL-4), the “prototypical” Th2 cytokine, involved in both the induction and maintenance of Th2 responses. These observations led to our hypothesis that T cells involved in murine host protection would respond to IL-4 signaling and differentiate into cells of the “type 2” phenotype. As such, these cells would presumably also act as major sources of IL-4. To investigate these hypotheses, we performed several adoptive transfers in which we controlled the cell population(s) able to produce or respond to IL-4. We show here that, in contrast to our original hypotheses, IL-4 production and IL-4 receptor expression by T cells are both dispensable for T-cell-mediated host protection. Instead, our data imply that T cells may be required for eosinophil accumulation at the site of infection.
Nonpathogenic
Escherichia coli
Can Contribute to the Production of Shiga Toxin
Authors: Gamage Shantini D. , Strasser Jane E. , Chalk Claudia L. , Weiss Alison A.
Published: May 2003

The food-borne pathogen,
Escherichia coli
O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in
E. coli
O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic
E. coli
could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with
E. coli
that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal
E. coli
isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant
E. coli
was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.
Relationship of
plcR
-Regulated Factors to
Bacillus
Endophthalmitis Virulence
Authors: Callegan Michelle C. , Kane Scott T. , Cochran D. Clay , Gilmore Michael S. , Gominet Myriam , Lereclus Didier
Published: May 2003

The explosive, destructive course of
Bacillus
endophthalmitis has been attributed to the production of toxins during infection. In this study we analyzed the contribution of toxins controlled by the global regulator
plcR
to the pathogenesis of experimental
Bacillus
endophthalmitis. Isogenic
plcR-
deficient mutants of
Bacillus cereus
and
Bacillus thuringiensis
were constructed by insertional inactivation of
plcR
by the kanamycin resistance cassette,
aphA3
. Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type
B. cereus
or
B. thuringiensis
or a
plcR
-deficient mutant. The evolution of endophthalmitis resulting from each
plcR
-deficient mutant was considerably slower than that caused by each wild-type strain. Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the
plcR
-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h. The intraocular inflammatory cell influx and retinal destruction in
plcR
-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection. Gross and histological examinations of eyes infected with
plcR
mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types. The loss of
plcR
-regulated factors significantly attenuated the severity of
Bacillus
endophthalmitis. The results therefore suggest that
plcR
may represent a target for which adjunct therapies could be designed for the prevention of blindness during
Bacillus
endophthalmitis.
Stx2-Specific Human Monoclonal Antibodies Protect Mice against Lethal Infection with
Escherichia coli
Expressing Stx2 Variants
Authors: Sheoran Abhineet S. , Chapman Susan , Singh Pradeep , Donohue-Rolfe Arthur , Tzipori Saul
Published: May 2003

Shiga toxin-producing
Escherichia coli
(STEC) strains are responsible for causing hemolytic-uremic syndrome (HUS), and systemic administration of Shiga toxin (Stx)-specific human monoclonal antibodies (HuMAbs) is considered a promising approach for prevention or treatment of the disease in children. The goal of the present study was to investigate the ability of Stx2-specific HuMAbs to protect against infections with STEC strains that produce Stx2 variants. Dose-response studies on five HuMAbs, using the mouse toxicity model, revealed that only the three directed against the A subunit were protective against Stx2 variants, and 5C12 was the most effective among the three tested. Two HuMAbs directed against the B subunit, while highly effective against Stx2, were ineffective against Stx2 variants. In a streptomycin-treated mouse model, parenteral administration of 5C12 significantly protected mice up to 48 h after oral bacterial challenge. We conclude that 5C12, reactive against the Stx2 A subunit, is an excellent candidate for immunotherapy against HUS and that antibodies directed against the A subunit of Stx2 have broad-spectrum activity that includes Stx2 variants, compared with those directed against the B subunit.
Critical Contribution of CD28-CD80/CD86 Costimulatory Pathway to Protection fromTrypanosoma cruziInfection
Authors: Miyahira Yasushi , Katae Masaharu , Kobayashi Seiki , Takeuchi Tsutomu , Fukuchi Yoshinosuke , Abe Ryo , Okumura Ko , Yagita Hideo , Aoki Takashi
Published: May 2003

The CD28-CD80/CD86-mediated T-cell costimulatory pathway has been variably implicated in infectious immunity. In this study, we investigated the role of this costimulatory pathway in resistance toTrypanosoma cruziinfection by using CD28-deficient mice and blocking antibodies against CD80 and CD86. CD28-deficient mice exhibited markedly exacerbatedT. cruziinfection, as evidenced by unrelenting parasitemia and 100% mortality after infection with doses that are nonlethal in wild-type mice. The blockade of both CD80 and CD86 by administering specific monoclonal antibodies also exacerbatedT. cruziinfection in wild-type mice. Splenocytes fromT. cruzi-infected, CD28-deficient mice exhibited greatly impaired gamma interferon production in response toT. cruziantigen stimulation in vitro compared to those from infected wild-type mice. The induction ofT. cruziantigen-specific CD8+T cells was also impaired inT. cruzi-infected, CD28-deficient mice. In addition to these defects in natural protection againstT. cruziinfection, CD28-deficient mice were also defective in the induction of CD8+-T-cell-mediated protective immunity againstT. cruziinfection by DNA vaccination. These results demonstrate, for the first time, a critical contribution of the CD28-CD80/CD86 costimulatory pathway not only to natural protection against primaryT. cruziinfection but also to DNA vaccine-induced protective immunity to Chagas' disease.
Plasmid Stability during In Vitro Propagation of
Borrelia burgdorferi
Assessed at a Clonal Level
Authors: Grimm Dorothee , Elias Abdallah F. , Tilly Kit , Rosa Patricia A.
Published: May 2003

Borrelia burgdorferi
causes Lyme disease in humans. The genome of the sequenced type strain B31 MI consists of a linear chromosome, 12 linear plasmids, and 9 circular plasmids. Previous studies by other investigators indicated that some of these plasmids are essential for the survival of the spirochetes in vivo but not in vitro. We have studied plasmid stability during in vitro growth at 23 and 35°C, conditions that approximate the temperatures of the tick vector and the mammalian host, respectively. Starting with two clones that have all 21 plasmids, we investigated plasmid maintenance within the population and on a clonal level. After three passages (27 generations), the cultures were no longer homogeneous and some derivative clones had already lost multiple plasmids. Despite this, one of six clones analyzed after 25 passages (225 generations) retained all but one plasmid (cp9) and was able to complete the mouse-tick-mouse infectious cycle. We analyzed protein composition and regulation of gene expression of clones differing in plasmid content after serial passages. All clones tested exhibited temperature-regulated expression of several proteins, including OspC. In addition, analysis of cultures inoculated from frozen stocks suggests that freezing and/or thawing contributes to heterogeneity in the outgrowth population with respect to plasmid content. Our investigations show that in vitro propagation of a clone leads to a heterogeneous population but that virulent clones can persist through extended passage. We therefore conclude that isogenicity of clones must be confirmed irrespective of their in vitro passage history.
TheMycobacterium tuberculosisRecombinant 27-Kilodalton Lipoprotein Induces a Strong Th1-Type Immune Response Deleterious to Protection
Authors: Hovav Avi-Hai , Mullerad Jacob , Davidovitch Liuba , Fishman Yolanta , Bigi Fabiana , Cataldi Angel , Bercovier Herve
Published: May 2003

Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDaMycobacterium tuberculosislipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged byM. tuberculosisH37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.
Optimal Tumor Necrosis Factor Induction byPlasmodium falciparumRequires the Highly Localized Release of Parasite Products
Authors: O'Dea Kieran P. , Pasvol Geoffrey
Published: May 2003

Overproduction of tumor necrosis factor (TNF) has been linked with the pathogenesis ofPlasmodium falciparummalaria. Here, we examined why the high levels of TNF-inducing activity associated withP. falciparum-parasitized erythrocytes (PE) appear to be lost after cell lysis. Static coculture of PE and peripheral blood mononuclear cells (PBMC), with or without separation by porous membranes, demonstrated that rupture of live PE in the presence of responder cells was required for optimal TNF induction. Although the insoluble fraction of lysed PE was found to partially inhibit TNF responses, supernatants prepared from large numbers of lysed PE still contained only low levels of TNF-inducing activity, which showed no evidence of instability. A dramatic reduction in TNF levels resulted when noncytoadherent PE lines were maintained under low-cell-proximity conditions by suspension coculture. This reduction was much less marked with PE capable of adhering to PBMC, despite the fact that cytoadherent and noncytoadherent parasite lines induced comparable levels of TNF in high-cell-proximity, static coculture. These results suggest that rupture of PE in a highly localized setting, facilitated by either static coculture or the more biologically relevant phenomenon of cytoadherence to PBMC, can result in considerable enhancement of theP. falciparum-induced TNF response.
Humoral and Cellular Immune Responses toTrypanosoma cruzi-Derived Paraflagellar Rod Proteins in Patients with Chagas' Disease
Authors: Michailowsky Vladimir , Luhrs Keith , Rocha Manoel Otávio C. , Fouts David , Gazzinelli Ricardo T. , Manning Jerry E.
Published: May 2003

Sera and peripheral blood mononuclear cells (PBMC) from patients displaying different clinical symptoms as well as from normal uninfected individuals (NI) were used to evaluate the humoral and cellular responses of Chagas' disease patients toTrypanosoma cruzi-derived paraflagellar rod proteins (PFR). Our results show that sera from both asymptomatic Chagas' disease patients (ACP) and cardiac Chagas' disease patients (CCP) have higher levels of antibodies to PFR than sera from NI. Immunoglobulin G1 (IgG1) and IgG3 were the main Ig isotypes that recognized PFR. We also tested three recombinant forms of PFR, named rPAR-1, rPAR-2, and rPAR-3, by Western blot analysis. Sera from seven out of eight patients with Chagas' disease recognized one of the three rPAR forms. Sera from 75, 50, and 37.5% of Chagas' disease patients tested recognized rPAR-3, rPAR-2, and rPAR-1, respectively. PFR induced proliferation of 100 and 70% of PBMC from ACP and CCP, respectively. Further, stimulation of cells from Chagas' disease patients with PFR enhanced the frequencies of both small and large CD4+CD25+and CD4+CD69+lymphocytes, as well as that of small CD8+CD25+lymphocytes. Finally, we evaluated the ability of PFR to elicit the production of gamma interferon (IFN-γ) by PBMC from patients with Chagas' disease. Fifty percent of the PBMC from ACP as well as CCP produced IFN-γ upon stimulation with PFR. PFR enhanced the percentages of IFN-γ-producing cells in both CD3+and CD3−populations. Within the T-cell population, large CD4+T lymphocytes were the main source of IFN-γ.
Perforin and Gamma Interferon Are Critical CD8+T-Cell-Mediated Responses in Vaccine-Induced Immunity againstLeishmania amazonensisInfection
Authors: Colmenares María , Kima Peter E. , Samoff Erika , Soong Lynn , McMahon-Pratt Diane
Published: May 2003

Previous studies have demonstrated that protection against New World leishmaniasis caused byLeishmania amazonensiscan be elicited by immunization with the developmentally regulatedLeishmaniaamastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8+(as well as CD4+) effector responses is required for protection. Further, mice lacking β2-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8+T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8+T cells, secreting gamma interferon (IFN-γ) and expressing perforin, was observed at the site of infection; subsequently, activated CD4+T cells producing IFN-γ were primarily found. As protection correlated with the ratio of total IFN-γ-producing cells (CD4+and CD8+T cells) to macrophages found at the site of infection, a role for IFN-γ was evident; in addition, vaccination of IFN-γ-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8+T cell effector mechanisms (perforin, IFN-γ) are clearly required in the protective immune response againstL. amazonensisinfection in vaccinated mice.
Characterization of Anthrolysin O, theBacillus anthracisCholesterol-Dependent Cytolysin
Authors: Shannon Jeffrey G. , Ross Cana L. , Koehler Theresa M. , Rest Richard F.
Published: May 2003

We characterized the expression of a putative toxin ofBacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). AlthoughB. anthracisexpresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR ofalomRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which thealogene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented intranswith nativealoon a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain ofB. anthracisin rich media in vitro. To further support thealogene product being a hemolysin, recombinantB. anthracisALO (rALO) purified fromEscherichia coliwas extremely active against washed human erythrocytes, with complete hemolysis detected at ∼30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.
Expression of MultipleCPBGenes Encoding Cysteine Proteases Is Required forLeishmania mexicanaVirulence In Vivo
Authors: Denise Hubert , McNeil Kathryn , Brooks Darren R. , Alexander James , Coombs Graham H. , Mottram Jeremy C.
Published: May 2003

Leishmania mexicanamutants deficient in the multicopyCPBgene array have reduced virulence, demonstrated by poor lesion growth in BALB/c mice and induction of a protective Th1 response. Reinsertion of the amastigote-specificCPB2.8or metacyclic stage-specificCPB2gene into aCPB-deficient mutantL. mexicanafailed to restore either a Th2 response or sustained virulence. However, reexpression of multipleCPBgenes from a cosmid significantly restored virulence. This was characterized by increased lesion and parasite growth and the acquisition of a Th2 response, as determined by measuring interleukin-4 production and immunoglobulin G1 (IgG1) and IgE levels. These studies confirm thatL. mexicanacysteine proteases are important virulence factors and provide an explanation for the presence inL. mexicanaof a multicopy tandem array ofCPBgenes.
Modulation of Virulence by Two Acidified Nitrite-Responsive Loci ofSalmonella entericaSerovar Typhimurium
Authors: Kim Charles C. , Monack Denise , Falkow Stanley
Published: May 2003

Two acidified nitrite-inducible genes ofSalmonella entericaserovar Typhimurium were identified with a green fluorescent protein-based promoter-trap screen. The nitrite-inducible promoters were located upstream of loci that we designatednipABandnipC, which correspond tohcp-hcr(hybrid cluster protein) ofEscherichia coliandnorAofAlcaligenes eutrophus, respectively. Maximal induction of the promoters by nitrite was dependent on pH. ThenipABpromoter was regulated by oxygen in an Fnr-dependent manner. ThenipCpromoter was also regulated by oxygen but in an Fnr-independent manner. The promoters were upregulated in activated RAW264.7 macrophage-like cells, which produce NO via the inducible nitric oxide synthase (iNOS), and the induction was inhibited by aminoguanidine, an inhibitor of iNOS. Although thenipABandnipCmutants displayed no defects under a variety of in vitro conditions or in tissue culture infections, they exhibited lower oral 50% lethal doses (LD50s) than did the wild type in C57BL/6J mouse infections. The lower LD50s reflected an unexpected increased ability of small inoculating doses of the mutant bacteria to cause lethal infection 2 to 3 weeks after challenge, compared to a similar challenge dose of wild-type bacteria. We conclude that these genes are regulated by physiological nitrogen oxides and that the absence of these bacterial genes in some way diminishes the ability of mice to clear a low dose infection.
Mutation ofluxSofStreptococcus pneumoniaeAffects Virulence in a Mouse Model
Authors: Stroeher Uwe H. , Paton Adrienne W. , Ogunniyi A. David , Paton James C.
Published: May 2003

The LuxS protein is required for the biosynthesis of the type 2 autoinducer (AI-2), which is involved in quorum sensing in a wide range of bacterial species. We have determined the effects of a definedluxSmutation on the virulence ofStreptococcus pneumoniae. Although theluxSmutant displayed reduced virulence relative to its wild-type parent, the type 2 strain D39, it was by no means avirulent in a mouse model. After intranasal administration, theluxSmutant was able to colonize the nasopharynx of the mouse as efficiently as the wild type. However, it was less able to spread from the nasopharynx to the lungs or the blood. Intraperitoneal coadministration studies indicated that theluxSmutant was less fit and was readily outcompeted by wild-type D39. However, when administered on its own by this route, the mutant was able to proliferate and cause fatal systemic disease, albeit at a lower rate than the wild type. Western blot analysis of whole-cell lysates of the mutant and its parent did not reveal any differences in the levels of several well-characterized virulence proteins. However, analysis of Coomassie blue-stained protein profiles after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that mutation ofluxShad pleiotropic effects on protein expression in all cellular compartments. This is consistent with the product ofluxShaving a regulatory role inS.pneumoniae. This is the first report of a direct role forluxS(and by inference, AI-2) in the virulence of a gram-positive pathogen. However, the fact that mutagenesis ofluxSdoes not completely attenuateS.pneumoniaehas implications for the possible use of AI-2 antagonists for treatment of pneumococcal infections.
Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis
Authors: Kothary Mahendra H. , Lowman Heather , McCardell Barbara A. , Tall Ben D.
Published: May 2003

The halophilic bacteriumVibrio fluvialisis an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature- and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-Leu-Pro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin ofVibrio choleraeand the heat-labile hemolysin ofVibrio mimicus. This homology was further confirmed by PCR analysis using a 5′ primer derived from the amino-terminal sequence of the hemolysin and a 3′ primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1V. cholerae.
Lipopolysaccharide-Mimetic Activities of a Toll-Like Receptor 2-Stimulatory Substance(s) in Enterobacterial Lipopolysaccharide Preparations
Authors: Muroi Masashi , Ohnishi Takahiro , Azumi-Mayuzumi Satoko , Tanamoto Ken-ichi
Published: May 2003

Lipopolysaccharide (LPS) preparations are known to often contain substances which activate cells through Toll-like receptor 2 (TLR2), and it is suspected that bacterial lipoproteins are responsible for this activation. We compared the mode of action of the TLR2-stimulatory substances with that of a synthetic bacterial lipopeptide (tripalmitoyl-Cys-Ser-Ser-Asn-Ala [Pam3CSSNA]), as well as with that of peptidoglycan. Six out of eight LPS preparations tested induced NF-κB-dependent reporter activity in 293 cells expressing CD14 and TLR2. Phenol extract (PEX) prepared fromEscherichia coliLPS by modified phenol extraction induced reporter activity in 293 cells expressing TLR2, and this activity was enhanced by coexpression of CD14, whereas the activity of Pam3CSSNA was not dependent on CD14. The activity of PEX, but not that of Pam3CSSNA or peptidoglycan, was also enhanced by LPS binding protein or serum and blocked by polymyxin B. In addition, the activity of PEX was inhibited by a lipid A precursor (compound 406) in 293 cells expressing CD14 and TLR2. These results indicate thatE. coliLPS preparations contain LPS-mimetic TLR2-stimulatory substances which differ from bacterial lipopeptides or peptidoglycan.
The Secreted Aspartyl Proteinases Sap1 and Sap2 Cause Tissue Damage in an In Vitro Model of Vaginal Candidiasis Based on Reconstituted Human Vaginal Epithelium
Authors: Schaller Martin , Bein Matthias , Korting Hans C. , Baur Stefan , Hamm Gerald , Monod Michel , Beinhauer Sabine , Hube Bernhard
Published: May 2003

Secreted aspartyl proteinases (Saps) contribute to the ability of
Candida albicans
to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these
C. albicans
proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by
C. albicans
SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of
SAP2
,
SAP9
, and
SAP10
was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of
SAP1
,
SAP4
, and
SAP5
. Additional transcripts of
SAP6
and
SAP7
were detected at a later stage of the artificial infection (24 h). Similar
SAP
expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by
C. albicans
was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by
C. albicans
cells. Furthermore,
SAP
null mutants lacking either
SAP1
or
SAP2
had a drastically reduced potential to cause tissue damage even though
SAP3
,
SAP4
, and
SAP7
were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking
SAP3
or
SAP4
to
SAP6
was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in
C. albicans
vaginal infections.
Protection against Shiga Toxin 1 Challenge by Immunization of Mice with Purified Mutant Shiga Toxin 1
Authors: Ishikawa Satoshi , Kawahara Kazuyoshi , Kagami Yutaka , Isshiki Yasunori , Kaneko Aki , Matsui Hidenori , Okada Nobuhiko , Danbara Hirofumi
Published: May 2003

Shiga toxin 1 (Stx1) of enterohemorrhagic
Escherichia coli
O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing
E. coli.