bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intramolecular Dimerization Is Required for the Chlamydia-Secreted Protease CPAF To Degrade Host Transcriptional Factors
Authors: Dong Feng , Sharma Jyotika , Xiao Yanming , Zhong Youmin , Zhong Guangming 
Published: June 2004

ABSTRACTWe previously identified a chlamydial protein designated CPAF (chlamydia protease/proteasome-like activity factor) that is secreted into host cell cytosol for degrading host transcription factors required for major histocompatibility complex antigen expression. Here we report that CPAF, synthesized as a 70-kDa proprotein, is processed into two fragments (designated CPAFn and CPAFc) to form intramolecular dimers that are much more stable than the naïve CPAF. Precipitation with antibodies that recognized CPAF dimers removed the proteolytic activity responsible for degrading host transcription factor RFX5 from chlamydia-infected host cell cytosol, while precipitation with antibodies that recognized free CPAF fragments alone did not remove this activity. Separation of CPAFn from CPAFc resulted in a loss of proteolytic activity. Furthermore, neither expressed full-length CPAF that was not processed nor coexpressed CPAFn and CPAFc fragments that failed to form dimers degraded RFX5. These observations demonstrate that intramolecular dimerization is required for CPAF to degrade host transcription factors, a strategy that is utilized by an obligate intracellular bacterial species to evade host defenses.
Functional Analysis of the
Streptococcus gordonii
DL1 Sialic Acid-Binding Adhesin and Its Essential Role in Bacterial Binding to Platelets
Authors: Takahashi Yukihiro , Yajima Ayako , Cisar John O. , Konishi Kiyoshi 
Published: June 2004

ABSTRACT

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The sialic acid-binding adhesin of
Streptococcus gordonii
DL1 was previously associated with the
hsa
gene encoding a 203-kDa protein. The predicted protein sequence consists of an N-terminal nonrepetitive region (NR1), including a signal sequence, a relatively short serine-rich region (SR1), a second nonrepetitive region (NR2), a long serine-rich region (SR2) containing 113 dodecapeptide repeats, and a C-terminal cell wall anchoring domain. In the present study, the contributions of SR1, NR2, and SR2 to Hsa-mediated adhesion were assessed by genetic complementation. Adhesion of an
hsa
chromosomal deletion mutant to sialic acid-containing receptors was restored by plasmids containing
hsa
constructs encoding Hsa that lacked either the N- or C-terminal portion of SR2. In contrast,
hsa
constructs that lacked the coding sequences for SR1, NR2, or the entire SR2 region failed to restore adhesion. Surface expression of recombinant Hsa was not affected by removal of SR1, NR2, or a portion of SR2 but was greatly reduced by complete removal of SR2. Wheat germ agglutinin, a probe for Hsa-specific glycosylation, reacted with recombinant Hsa lacking SR1, NR2, or SR2 but not with recombinant Hsa lacking both SR1 and SR2. Significantly, the aggregation of human platelets by
S. gordonii
DL1, an interaction implicated in the pathogenesis of infective endocarditis, required the expression of
hsa
. Moreover, neuraminidase treatment of the platelets eliminated this interaction, further supporting the hypothesis that Hsa plays an essential role in the bacterium-platelet interaction.
Identification and Characterization of a Novel Uropathogenic
Escherichia coli
-Associated Fimbrial Gene Cluster
Authors: Buckles Eric L. , Bahrani-Mougeot Farah K. , Molina Anita , Lockatell C. Virgina , Johnson David E. , Drachenberg Cinthia B. , Burland Valerie , Blattner Fred R. , Donnenberg Michael S. 
Published: June 2004

ABSTRACT

Recently, we identified a fimbrial usher gene in uropathogenic
Escherichia coli
strain CFT073 that is absent from an
E. coli
laboratory strain. Analysis of the CFT073 genome indicates that this fimbrial usher gene is part of a novel fimbrial gene cluster,
aufABCDEFG
. Analysis of a collection of pathogenic and commensal strains of
E. coli
and related species revealed that the
auf
gene cluster was significantly associated with uropathogenic
E. coli
isolates. For in vitro expression analysis of the
auf
gene cluster, RNA was isolated from CFT073 bacteria grown to the exponential or stationary phase in Luria-Bertani broth and reverse transcriptase PCR (RT-PCR) with oligonucleotide primers specific to the major subunit,
aufA
, was performed. We found that
aufA
is expressed in CFT073 only during the exponential growth phase; however, no expression of AufA protein was observed by Western blotting, indicating that under these conditions, the expression of the
auf
gene cluster is low. To determine if the
auf
gene cluster is expressed in vivo, RT-PCR was performed on bacteria from urine samples of mice infected with CFT073. Out of three independent experiments, we were able to detect expression of
aufA
at least once at 4, 24, and 48 h of infection, indicating that the
auf
gene cluster is expressed in the murine urinary tract. Furthermore, antisera from mice infected with CFT073 reacted with recombinant AufA in an enzyme-linked immunosorbent assay. To identify the structure encoded by the
auf
gene cluster, a recombinant plasmid containing the
auf
gene cluster under the T7 promoter was introduced into the
E. coli
BL-21 (AI) strain. Immunogold labeling using AufA antiserum revealed the presence of amorphous material extending from the surface of BL-21 cells. No hemagglutination or cellular adherence properties were detected in association with expression of AufA. Deletion of the entire
auf
gene cluster had no effect on the ability of CFT073 to colonize the kidney, bladder, or urine of mice. In addition, no significant histological differences between the parent and
aufC
mutant strain were observed. Therefore, Auf is a uropathogenic
E. coli-
associated structure that plays an uncertain role in the pathogenesis of urinary tract infections.
Relative Importance of Heat-Labile Enterotoxin in the Causation of Severe Diarrheal Disease in the Gnotobiotic Piglet Model by a Strain of Enterotoxigenic
Escherichia coli
That Produces Multiple Enterotoxins
Authors: Berberov Emil M. , Zhou You , Francis David H. , Scott Michael A. , Kachman Stephen D. , Moxley Rodney A. 
Published: June 2004

ABSTRACT

Enterotoxigenic
Escherichia coli
(ETEC) strains that produce multiple enterotoxins are important causes of severe dehydrating diarrhea in human beings and animals, but the relative importance of these enterotoxins in the pathogenesis is poorly understood. Gnotobiotic piglets were used to study the importance of heat-labile enterotoxin (LT) in infection with an ETEC strain that produces multiple enterotoxins. LT
−
(Δ
eltAB
) and complemented mutants of an F4
+
LT
+
STb
+
EAST1
+
ETEC strain were constructed, and the virulence of these strains was compared in gnotobiotic piglets expressing receptors for F4
+
fimbria. Sixty percent of the piglets inoculated with the LT
−
mutant developed severe dehydrating diarrhea and septicemia compared to 100% of those inoculated with the nalidixic acid-resistant (Nal
r
) parent and 100% of those inoculated with the complemented mutant strain. Compared to piglets inoculated with the Nal
r
parent, the mean rate of weight loss (percent per hour) of those inoculated with the LT
−
mutant was 67% lower (
P
< 0.05) and that of those inoculated with the complemented strain was 36% higher (
P
< 0.001). Similarly, piglets inoculated with the LT
−
mutant had significant reductions in the mean bacterial colony count (CFU per gram) in the ileum; bacterial colonization scores (square millimeters) in the jejunum and ileum; and clinical pathology parameters of dehydration, electrolyte imbalance, and metabolic acidosis (
P
< 0.05). These results indicate the significance of LT to the development of severe dehydrating diarrhea and postdiarrheal septicemia in ETEC infections of swine and demonstrate that EAST1, LT, and STb may be concurrently expressed by porcine ETEC strains.
Effect of Anaerobiosis on Expression of Virulence Factors inVibrio cholerae
Authors: Krishnan H. H. , Ghosh Amalendu , Paul Kalidas , Chowdhury Rukhsana 
Published: June 2004

ABSTRACTInVibrio cholerae, the transmembrane DNA binding proteins, ToxR and TcpP, activate expression of the regulatory genetoxTin response to specific environmental signals. The resulting enhanced level of ToxT leads to a coordinated increase in the production of a subset of virulence factors, including cholera toxin (CT) and toxin-coregulated pilus (TCP). The effect of anaerobiosis on expression of theV. choleraevirulence regulatory cascade was examined. The expression of the major regulatory genes,tcpP,toxR, andtoxT, in anaerobically grownV. choleraewas comparable to that in cells grown under aerobic conditions, and no significant difference in the ToxT-dependent expression oftcpAwas detected when aerobic and anaerobic cultures were compared. However, in spite of the presence of functional ToxT,ctxABexpression was drastically reduced, and practically no CT was detected in cells grown under anaerobic conditions. In aV. cholerae hnsmutant, however, high levels ofctxABexpression occurred even under anaerobic conditions. Also, deletion of the H-NS binding site from thectxABpromoter eliminated anaerobic repression ofctxABexpression. These results suggest that H-NS directly repressesctxABexpression under anaerobic growth conditions. It has been reported that in the first stage of infection of infant miceby V. cholerae,tcpAis expressed butctxABexpression is shut off (S. H. Lee, D. L. Hava, M. K. Waldor, and A. Camilli, Cell99:625-634, 1999). This pattern is similar to the pattern in anaerobic cultures ofV. cholerae. Under all other in vitro conditions,ctxABandtcpAare known to be coordinately expressed.
Identification of theEntamoeba histolyticaGalactose-Inhibitable Lectin Epitopes Recognized by Human Immunoglobulin A Antibodies following Cure of Amebic Liver Abscess
Authors: Abd-Alla Mohamed D. , Jackson Terry F. G. H. , Soong Ginny C. , Mazanec Mary , Ravdin Jonathan I. 
Published: June 2004

ABSTRACTImmunity toEntamoebaspecies intestinal infection is associated with the presence of intestinal IgA antibodies against the parasite's galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectin's carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrentEntamoeba histolyticaintestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected withE. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence ofE. histolyticatrophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P< 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.
Characterization of a Complement-Binding Protein, DRS, from Strains of
Streptococcus pyogenes
Containing the
emm12
and
emm55
Genes
Authors: Binks Michael , Sriprakash K. S. 
Published: June 2004

ABSTRACT

An extracellular protein of
Streptococcus pyogenes
, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some
S. pyogenes
strains. SIC from type 1 (M1) isolates of
S. pyogenes
interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that
S. pyogenes
strains carrying
emm12
and
emm55
(the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.
Yersinia enterocolitica
Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB
Authors: Wilharm Gottfried , Lehmann Verena , Krauss Kristina , Lehnert Beatrix , Richter Susanna , Ruckdeschel Klaus , Heesemann Jürgen , Trülzsch Konrad 
Published: June 2004

ABSTRACT

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In
Yersinia enterocolitica
, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide
m
-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted
motAB
, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the
motAB
deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.
Disruption of the Phagosomal Membrane and Egress ofLegionella pneumophilainto the Cytoplasm during the Last Stages of Intracellular Infection of Macrophages andAcanthamoeba polyphaga
Authors: Molmeret Maëlle , Bitar Dina M. , Han Lihui , Kwaik Yousef Abu 
Published: June 2004

ABSTRACTAlthough the early stages of intracellular infection byLegionella pneumophilaare well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of theL. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages andAcanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages andA. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infectedA. polyphagaamoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages andA. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that anlspGmutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease,p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore,L. pneumophiladisrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages andA. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages andA. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion system.
Protection Afforded by Heat Shock Protein 60 fromFrancisella tularensisIs Due to Copurified Lipopolysaccharide
Authors: Hartley M. G. , Green M. , Choules G. , Rogers D. , Rees D. G. C. , Newstead S. , Sjostedt A. , Titball R. W. 
Published: June 2004

ABSTRACTHeat shock proteins (Hsps) have attracted significant attention as protective antigens against a range of diseases caused by bacterial pathogens. However, more recently there have been suggestions that the protective response is due to the presence of peptide components other than Hsps. We have shown that mice that had been immunized with purified heat shock protein 60 (Hsp60) isolated fromFrancisella tularensiswere protected against a subsequent challenge with some strains of the bacterium. However, this protection appeared to be due to trace amounts of lipopolysaccharide, which were too low to be detected by using theLimulusamoebocyte lysate assay. This finding raises the possibility that the protection afforded by other bacterial Hsp60 proteins may be due to trace quantities of polysaccharide antigens carried by and acting in conjunction with the Hsps.
Toxoplasma gondiiAntigen-Pulsed-Dendritic Cell-Derived Exosomes Induce a Protective Immune Response againstT. gondiiInfection
Authors: Aline Fleur , Bout Daniel , Amigorena Sébastian , Roingeard Philippe , Dimier-Poisson Isabelle 
Published: June 2004

ABSTRACTIt was previously demonstrated that immunizing mice with spleen dendritic cells (DCs) that had been pulsed ex vivo withToxoplasma gondiiantigens triggers a systemic Th1-biased specific immune response and induces protection against infection.T. gondiican cause severe sequelae in the fetuses of mothers who acquire the infection during pregnancy, as well as life-threatening neuropathy in immunocompromised patients, in particular those with AIDS. Here, we investigate the efficacy of a novel cell-free vaccine composed of DC exosomes, which are secreted antigen-presenting vesicles that express functional major histocompatibility complex class I and II and T-cell-costimulatory molecules. They have already been shown to induce potent antitumor immune responses. We investigated the potential of DC2.4 cell line-derived exosomes to induce protective immunity against toxoplasmosis. Our data show that most adoptively transferredT. gondii-pulsed DC-derived exosomes were transferred to the spleen, elicited a strong systemic Th1-modulatedToxoplasma-specific immune response in vivo, and conferred good protection against infection. These findings support the possibility that DC-derived exosomes can be used forT. gondiiimmunoprophylaxis and for immunoprophylaxis against many other pathogens.
Mycobacterium bovisBCG Urease Attenuates Major Histocompatibility Complex Class II Trafficking to the Macrophage Cell Surface
Authors: Sendide Khalid , Deghmane Ala-Eddine , Reyrat Jean-Marc , Talal Amina , Hmama Zakaria 
Published: June 2004

ABSTRACTWe have previously shown thatMycobacterium tuberculosisattenuates cell surface expression of major histocompatibility complex class II molecules in response to gamma interferon (IFN-γ) by a mechanism dependent on intracellular sequestration of α,β dimers. In this study we examined whether intracellular alkalinization due to mycobacterial urease could account for the defect in intracellular trafficking of class II molecules. Phagocytosis of wild-typeMycobacterium bovisBCG was associated with secretion of ammonia intracellularly, which increased substantially upon addition of exogenous urea to the culture medium. Increased intracellular ammonia, due to urea degradation by the bacterium, correlated with inhibition of class II surface expression. Conversely, no ammonia was detected in cells infected with a urease-negative mutant strain ofM. bovisBCG, which also displayed a reduced effect on surface expression of class II molecules. A direct cause-effect relationship between urease and class II molecule trafficking was established with experiments where cells ingesting beads coated with purified urease showed an increased ammonia level and decreased surface expression of class II in response to IFN-γ. In contrast to BCG, infection of macrophages withMycobacterium smegmatis, which expresses relatively greater urease activity in cell-free culture, had a marginal effect on both the intracellular level of ammonia and class II expression. The limited effect ofM. smegmatiswas consistent with a failure to resist intracellular killing, suggesting that urease alone is not sufficient to resist macrophage microbicidal mechanisms and that this is required for a more distal effect on cell regulation. Our results demonstrate that alkalinization of critical intracellular organelles by pathogenic mycobacteria expressing urease contributes significantly to the intracellular retention of class II dimers.
NadA Diversity and Carriage in
Neisseria meningitidis
Authors: Comanducci Maurizio , Bambini Stefania , Caugant Dominique A. , Mora Marirosa , Brunelli Brunella , Capecchi Barbara , Ciucchi Laura , Rappuoli Rino , Pizza Mariagrazia 
Published: June 2004

ABSTRACT

NadA is a novel vaccine candidate recently identified in
Neisseria meningitidis
and involved in adhesion to host tissues. The
nadA
gene has been found in approximately 50% of the strains isolated from patients and in three of the four hypervirulent lineages of non-serogroup A strains. Here we investigated the presence of the
nadA
gene in 154 meningococcal strains isolated from healthy people (carrier strains). Only 25 (16.2%) of the 154 carrier isolates harbored the
nadA
gene. The commensal species
Neisseria lactamica
was also found not to have the
nadA
gene. Eighteen of the carrier strains belonged to the ET-5 and ET-37 hypervirulent clusters, indicating that only the 5.1% of the genuine carrier population actually harbored
nadA
(7 of 136 strains). Five of the seven strains harbored a novel allele of the
nadA
gene that was designated
nadA4
. The NadA4 protein was present on the bacterial surface as heat-stable high-molecular-weight oligomers. Antibodies against the recombinant NadA4 protein were bactericidal against homologous strains, whereas the activity against other NadA alleles was weak. In conclusion, the
nadA
gene segregates differently in the population of strains isolated from healthy individuals and in the population of strains isolated from patients. The presence of NadA can therefore be used as a tool to study the dynamics of meningococcal infections and understand why this bacterium, which is mostly a commensal, can become a severe pathogen.