bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of
Brucella suis
Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis
Authors: Foulongne Vincent , Bourg Giséle , Cazevieille Chantal , Michaux-Charachon Sylvie , O'Callaghan David 
Published: July 2002

ABSTRACT

Bacteria of the genus
Brucella
are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of
Brucella
virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially in animal models, to an in vitro human macrophage infection model. A library of 1,152
Brucella suis
1330 tagged mini-Tn
5
Km2 mutants, in 12 pools, was screened for intracellular survival and multiplication in vitamin D
3
-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells. For each avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular survival or biosynthesis of smooth lipopolysaccharide (the
virB
operon and
manB
), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of
Brucella
for the different factors identified are discussed.
Protection against Candidiasis by an Immunoglobulin G3 (IgG3) Monoclonal Antibody Specific for the Same Mannotriose as an IgM Protective Antibody
Authors: Han Yongmoon , Riesselman Marcia H. , Cutler Jim E. 
Published: July 2002

ABSTRACTWe previously reported that a liposome-mannan vaccine (L-mann) ofCandida albicansinduces production of mouse antibodies that protect against disseminated candidiasis and vaginal infection. Immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for aC. albicanscell surface β-1,2-mannotriose, protects mice against both infections. Another IgM MAb, termed B6, which is specific for a different cell surface mannan epitope, does not protect against disseminated candidiasis. The B6.1 epitope is displayed homogeneously over the entire cell surface, compared to a patchy distribution of the B6 epitope. To determine if protection is restricted to an IgM class of antibody, we tested an IgG antibody. MAb C3.1 was obtained from L-mann-immunized mice. By results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, capture enzyme-linked immunosorbent assay, and immunodiffusion tests, MAb C3.1 is an IgG3 isotype. By epitope inhibition assays, we determined that MAb C3.1 is specific for same mannotriose as MAb B6.1. As expected by the results of the inhibition assays, immunofluorescence microscopy showed that the C3.1 epitope is distributed on the yeast cell surface in a pattern identical to that of the B6.1 epitope. Kidney CFU and mean survival times of infected mice pretreated with MAb C3.1 indicated that the antibody enhanced resistance of mice against disseminated candidiasis. Mice in pseudoestrus that were given MAb C3.1 prior to vaginal infection developed fewer vaginalCandidaCFU than control animals that received buffered saline instead of the antibody. The finding that an IgG3 antibody is protective is consistent with our hypothesis that epitope specificity and complement activation are related to the ability of an antibody to protect against candidiasis.
Effects of Iron on Extracellular and Intracellular Growth of
Penicillium marneffei
Authors: Taramelli D. , Brambilla S. , Sala G. , Bruccoleri A. , Tognazioli C. , Riviera-Uzielli L. , Boelaert J. R. 
Published: July 2002

ABSTRACT

Killing of intracellular
Penicillium marneffei
conidia is demonstrated in gamma interferon-lipopolysaccharide-activated human THP1 and mouse J774 cells. Iron overload significantly reduces the antifungal activity of macrophages. Likewise, exogenous iron enhances and iron chelators inhibit the extracellular growth of
P. marneffei
. These results suggest that iron availability critically affects immunity to and the pathogenicity of
P. marneffei
.
The
sigA
Gene Which Is Borne on the
she
Pathogenicity Island of
Shigella flexneri
2a Encodes an Exported Cytopathic Protease Involved in Intestinal Fluid Accumulation
Authors: Al-Hasani Keith , Henderson Ian R. , Sakellaris Harry , Rajakumar Kumar , Grant Travis , Nataro James P. , Robins-Browne Roy , Adler Ben 
Published: July 2002

ABSTRACT

In this study, the
sigA
gene situated on the
she
pathogenicity island of
Shigella flexneri
2a was cloned and characterized. Sequence analysis showed that
sigA
encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of
Enterobacteriaceae
) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of
Shigella
infections. SigA was at least partly responsible for the ability of
S. flexneri
to stimulate fluid accumulation in ligated rabbit ileal loops.
The Flagellar Sigma Factor FliA (ς28) Regulates the Expression ofSalmonellaGenes Associated with the Centisome 63 Type III Secretion System
Authors: Eichelberg Katrin , Galán Jorge E. 
Published: July 2002

ABSTRACTOne of the essential features of all pathogenic strains ofSalmonella entericais the ability to enter into nonphagocytic cells. This pathogenic property is mediated by theSalmonellapathogenicity island 1 (SPI-1)-encoded type III secretion system. Expression of components and substrates of this system is subject to complex regulatory mechanisms. These mechanisms include a number of specific and global transcriptional regulatory proteins. In this study we have compared inS. entericaserovars Typhimurium and Typhi the effect of mutations in flagellar genes on the phenotypes associated with the SPI-1 type III protein secretion system. We found that serovar Typhi strains carrying a null mutation in either of the flagellar regulatory genesflhDCorfliAwere severely deficient in entry into cultured epithelial cells and macrophage cytotoxicity. This defect could not be reversed by applying a mild centrifugal force, suggesting that the effects of the mutations were not due to the absence of motility. In contrast, the same mutations had no significant effect on the ability of serovar Typhimurium to enter into cultured Henle-407 cells or to induce macrophage cell death. Consistent with these observations, we found that the mutations in the flagellar regulatory proteins significantly reduced the expression of components of the SPI-1-encoded type III system in serovar Typhi but had a marginal effect in serovar Typhimurium. Our results therefore indicate that there is an overlap between regulatory mechanisms that control flagellar and type III secretion gene expression inSalmonellaserovar Typhi.
Listeriolysin O as a Reporter To Identify Constitutive and In Vivo-Inducible Promoters in the Pathogen
Listeria monocytogenes
Authors: Dubail Iharilalao , Berche Patrick ,   The European Listeria Genome Consortium, Charbit Alain 
Published: July 2002

ABSTRACT

Listeria monocytogenes
is a facultative intracellular gram-positive bacterium capable of growing in the cytoplasm of infected host cells. Bacterial escape from the phagosomal vacuole of infected cells is mainly mediated by the pore-forming hemolysin listeriolysin O (LLO) encoded by
hly
. LLO-negative mutants of
L. monocytogenes
are avirulent in the mouse model. We have developed a genetic system with
hly
as a reporter gene allowing the identification of both constitutive and in vivo-inducible promoters of this pathogen. Genomic libraries were created by randomly inserting
L. monocytogenes
chromosomal fragments upstream of the promoterless
hly
gene cloned into gram-positive and gram-negative shuttle vectors and expressed in an LLO-negative mutant strain. With this
hly
-based promoter trap system, combined with access to the
L. monocytogenes
genome database, we identified 20 in vitro-transcribed genes, including genes encoding (i) p60, a previously known virulence gene, (ii) a putative new hemolysin, and (iii) two proteins of the general protein secretion pathway. By using the
hly
-based system as an in vivo expression technology tool, nine in vivo-induced loci of
L. monocytogenes
were identified, including genes encoding (i) the previously known in vivo-inducible phosphatidylinositol phospholipase C and (ii) a putative
N
-acetylglucosamine epimerase, possibly involved in teichoic acid biosynthesis. The use of
hly
as a reporter is a simple and powerful alternative to classical methods for transcriptional analysis to monitor promoter activity in
L. monocytogenes
.
Characterization of the
Streptococcus mutans
Pyruvate Formate-Lyase (PFL)-Activating Enzyme Gene by Complementary Reconstitution of the In Vitro PFL-Reactivating System
Authors: Yamamoto Yasuhito , Sato Yutaka , Takahashi-Abbe Shoko , Takahashi Nobuhiro , Kizaki Harutoshi 
Published: July 2002

ABSTRACT

The
act
gene was identified and an
act
mutant as well as the
pfl
mutant was constructed in
Streptococcus mutans
. Pyruvate formate-lyase (PFL) activity was regenerated with the mixture of the respective cell extracts from these mutants by complementary reconstitution of the in vitro reactivating system. The
S. mutans act
gene encoded the sole enzyme able to activate the PFL protein in this organism.
Shiga Toxin-Induced Tumor Necrosis Factor Alpha Expression: Requirement for Toxin Enzymatic Activity and Monocyte Protein Kinase C and Protein Tyrosine Kinases
Authors: Foster Gregory H. , Armstrong Cassandra S. , Sakiri Ramesh , Tesh Vernon L. 
Published: July 2002

ABSTRACTInfections with Shiga toxin (Stx)-producing bacteria cause bloody diarrhea which may progress to life-threatening complications, including acute renal failure and neurological abnormalities. The precise mechanism of disease progression is unclear, although evidence suggests that the localized production of the host proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 may exacerbate toxin-mediated vascular damage. Purified Stxs have been demonstrated to elicit proinflammatory cytokine synthesis from human peripheral blood mononuclear cells and monocytic cell lines in vitro. To understand toxin-monocyte interactions required for cytokine synthesis, we have treated differentiated THP-1 cells with purified wild-type toxins, enzymatic mutants, or B subunits and measured TNF-α production. Our data suggest that A subunit enzymatic activity is essential for cytokine production. THP-1 cells were treated with a series of protein kinase C (PKC), PKA, and protein tyrosine kinase inhibitors to examine the role of intracellular signaling molecules in Stx-mediated cytokine production. Treatment of cells with PKC and tyrosine kinase inhibitors blocked TNF-α secretion by Stx-stimulated THP-1 cells. Stx treatment directly activated PKC, which occurred at a point upstream of transcriptional activation of the gene encoding TNF-α.
Susceptibility to SecondaryFrancisella tularensisLive Vaccine Strain Infection in B-Cell-Deficient Mice Is Associated with Neutrophilia but Not with Defects in Specific T-Cell-Mediated Immunity
Authors: Bosio Catharine M. , Elkins Karen L. 
Published: July 2002

ABSTRACTPrevious studies have demonstrated a role for B cells, not associated with antibody production, in protection against lethal secondary infection of mice withFrancisella tularensislive vaccine strain (LVS). However, the mechanism by which B cells contribute to this protection is not known. To study the specific role of B cells during secondary LVS infection, we developed an in vitro culture system that mimics many of the same characteristics of in vivo infection. Using this culture system, we showed that B cells do not directly control LVS infection but that control of LVS growth is mediated primarily by LVS-primed T cells. Importantly, B cells were not required for the generation of effective memory T cells since LVS-primed, B-cell-deficient (BKO) mice generated CD4+and CD8+T cells that controlled LVS infection similarly to LVS-primed CD4+and CD8+T cells from wild-type mice. The control of LVS growth appeared to depend primarily on gamma interferon and nitric oxide and was similar in wild-type and BKO mice. Rather, the inability of BKO mice to survive secondary LVS infection was associated with marked neutrophil influx into the spleen very early after challenge. The neutrophilia was directly associated with B cells, since BKO mice reconstituted with naive B cells prior to a secondary challenge with LVS had decreased bacterial loads and neutrophils in the spleen and survived.
vimA
Gene Downstream of
recA
Is Involved in Virulence Modulation in
Porphyromonas gingivalis
W83
Authors: Abaibou Hafid , Chen Zhuo , Olango G. Jon , Liu Yi , Edwards Jessica , Fletcher Hansel M. 
Published: July 2002

ABSTRACT

A 0.9-kb open reading frame encoding a unique 32-kDa protein was identified downstream of the
recA
gene of
Porphyromonas gingivalis
. Reverse transcription-PCR and Northern blot analysis showed that both the
recA
gene and this open reading frame are part of the same transcriptional unit. This cloned fragment was insertionally inactivated using the
ermF-ermAM
antibiotic resistance cassette to create a defective mutant by allelic exchange. When plated on
Brucella
blood agar, the mutant strain, designated
P. gingivalis
FLL92, was non-black pigmented and showed significant reduction in beta-hemolysis compared with the parent strain,
P. gingivalis
W83. Arginine- and lysine-specific cysteine protease activities, which were mostly soluble, were approximately 90% lower than that of the parent strain. Expression of the
rgpA
,
rgpB
, and
kgp
protease genes was the same in
P. gingivalis
FLL92 as in the wild-type strain. In contrast to the parent strain,
P. gingivalis
FLL92 showed increased autoaggregration in addition to a significant reduction in hemagglutinating and hemolysin activities. In in vivo experiments using a mouse model,
P. gingivalis
FLL92 was dramatically less virulent than the parent strain. A molecular survey of this mutant and the parent strain using all known
P. gingivalis
insertion sequence elements as probes suggested that no intragenomic changes due to the movement of these elements have occurred in
P. gingivalis
FLL92. Taken together, these results suggest that the
recA
downstream gene, designated
vimA
(virulence-modulating gene), plays an important role in virulence modulation in
P. gingivalis
W83, possibly representing a novel posttranscriptional or translational regulation of virulence factors in
P. gingivalis
.
Microtubule- and Dynein-Mediated Movement of
Orientia tsutsugamushi
to the Microtubule Organizing Center
Authors: Kim Sang-Wook , Ihn Kyung-Soo , Han Seung-Hoon , Seong Seung-Yong , Kim Ik-Sang , Choi Myung-Sik 
Published: July 2002

ABSTRACT

The host cell microfilaments and microtubules (MTs) are known to play a critical role in the life cycles of several pathogenic intracellular microbes by providing for successful invasion and promoting movement of the pathogen once inside the host cell cytoplasm.
Orientia tsutsugamushi
, an obligate intracellular bacterium, enters host cells by induced phagocytosis, escapes to the cytosol, and then replicates in the cytosol. ECV304 cells infected with
O. tsutsugamushi
revealed the colocalization of the MT organizing center (MTOC) and cytosolic orientiae by indirect immunofluorescence assay. Using immunofluorescence microscopy in the presence and absence of MT-depolymerizing agents (colchicine and nocodazole), it was shown that the cytosolic oriential movement was mediated by MTs. By transfection study (overexpression of dynamitin [also called p50], which is known to associate with dynein-dependent movement), the movement of
O. tsutsugamushi
to the MTOC was also mediated by dynein, the minus-end-directed MT-related motor. Although the significance of this movement in the life cycle of
O. tsutsugamushi
was not proven, we propose that the cytosolic
O. tsutsugamushi
bacteria use MTs and dyneins to propel themselves from the cell periphery to the MTOC.
Resistance of T-Cell Receptor δ-Chain-Deficient Mice to ExperimentalCandidaalbicansVaginitis
Authors: Wormley Floyd L. , Steele Chad , Wozniak Karen , Fujihashi Kohtaro , McGhee Jerry R. , Fidel Paul L. 
Published: July 2002

ABSTRACTConditions consistent with tolerance or immunoregulation have been observed in experimentalCandida albicansvaginal infections. The present study investigated the role of γ/δ T cells in experimental vaginal candidiasis. Results showed that T-cell receptor δ-chain-knockout mice had significantly less vaginal fungal burden when compared to wild-type mice, suggesting an immunoregulatory role for γ/δ T cells inCandidavaginitis.
Interleukin-4 Receptor–Stat6 Signaling in Murine Infections with a Tissue-Dwelling Nematode Parasite
Authors: Spencer L. , Shultz L. , Rajan T. V. 
Published: July 2002

ABSTRACTInterleukin-4 (IL-4) has been shown to be crucial in parasite expulsion in several gastrointestinal nematode infection models. Data from both epidemiological studies with humans and experimental infections in animals imply a critical role for the type II helper response, dominated by IL-4, in host protection. Here we utilized inbred mice on two distinct backgrounds to document the involvement of IL-4 in the clearance of a primary infection ofBrugiafrom the murine host. Our data from infections of IL-4 receptor−/−and Stat6−/−mice further indicate that IL-4 exerts its effects by activating the Stat6 molecule in host target cells, a finding which links clearance requirements of a gastrointestinal tract-dwelling nematode with those of a tissue-dwelling nematode. Additionally, we show that the requirements for IL-4 receptor binding and Stat6 activation extend to accelerated clearance of a secondary infection as well. The data shown here, including analysis of cell populations at the site of infection and infection of immunoglobulin E (IgE)−/−mice, lead us to suggest that deficiencies in eosinophil recruitment and isotype switching to IgE production may be at least partially responsible for slower parasite clearance in the absence of IL-4.
Pretreatment with Recombinant Flt3 Ligand Partially Protects against Progressive Cutaneous Leishmaniasis in Susceptible BALB/c Mice
Authors: Kremer Inger B. , Gould Meetha P. , Cooper Kevin D. , Heinzel Frederick P. 
Published: July 2002

ABSTRACTDendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-γ)-producing Th1-type CD4+T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneousLeishmania majorinfection. Pretreatment of disease-susceptible BALB/c mice with 10 μg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alterL. major-induced IFN-γ and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection withL. majorreduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.
Systemic and Mucosal Immunizations with Fibronectin-Binding Protein FBP54 Induce Protective Immune Responses againstStreptococcus pyogenesChallenge in Mice
Authors: Kawabata Shigetada , Kunitomo Eiji , Terao Yutaka , Nakagawa Ichiro , Kikuchi Ken , Totsuka Kyo-ichi , Hamada Shigeyuki 
Published: July 2002

ABSTRACTThe purpose of this study was to examine the suitability of fibronectin-binding protein FBP54 as a putative vaccine forStreptococcus pyogenesinfections. When the distribution of thefbp54gene among the clinical isolates representing various M serotypes was tested by PCR and Southern blot assays, it was found that all of the strains possess this gene. Furthermore, a significant increase in immunoglobulin G (IgG) antibody titers against FBP54 was observed in sera from patients withS. pyogenesinfections compared with those from healthy volunteers (P< 0.005). Mice were immunized with FBP54 subcutaneously, orally, or nasally. An enzyme-linked immunosorbent assay revealed that antigen-specific IgG antibodies were induced in the sera of immunized mice, while high salivary levels of IgA antibodies were detected after oral and nasal immunizations. Mice subcutaneously or orally immunized with FBP54 survived significantly longer following the challenge withS. pyogenesthan did nonimmunized mice (P< 0.001). These results indicate that FBP54 is a promising vaccine for the prevention ofS. pyogenesinfections.
Neutralization of Shiga Toxins Stx1, Stx2c, and Stx2e by Recombinant Bacteria Expressing Mimics of Globotriose and Globotetraose
Authors: Paton Adrienne W. , Morona Renato , Paton James C. 
Published: July 2002

ABSTRACT

Strains of
Escherichia coli
producing Shiga toxins Stx1, Stx2, Stx2c, and Stx2d cause gastrointestinal disease and the hemolytic-uremic syndrome in humans. We have recently constructed a recombinant bacterium which displays globotriose (the receptor for these toxins) on its surface and adsorbs and neutralizes these Shiga toxins with very high efficiency. This agent has great potential for the treatment of humans with such infections.
E. coli
strains which cause edema disease in pigs produce a variant toxin, Stx2e, which has a different receptor specificity from that for the other members of the Stx family. We have now modified the globotriose-expressing bacterium such that it expresses globotetraose (the preferred receptor for Stx2e) by introducing additional genes encoding a
N
-acetylgalactosamine transferase and a UDP-
N
-acetylgalactosamine-4-epimerase. This bacterium had a reduced capacity to neutralize Stx1 and Stx2c in vitro, but remarkably, its capacity to bind Stx2e was similar to that of the globotriose-expressing construct; both constructs neutralized 98.4% of the cytotoxicity in lysates of
E. coli
JM109 expressing cloned
stx
2e
. These data suggest that either globotriose- or globotetraose-expressing constructs may be suitable for treatment and/or prevention of edema disease in pigs.