bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Cell-Specific Phenotype of the Polymorphic
vacA
Midregion Is Independent of the Appearance of the Cell Surface Receptor Protein Tyrosine Phosphatase β
Authors: Skibinski David A. G. , Genisset Christophe , Barone Silvia , Telford John L. 
Published: December 2005

ABSTRACT

There are two alleles, m1 and m2, of the midregion of the vacuolating cytotoxin gene (
vacA
) of
Helicobacter pylori
which code for toxins with different cell specificities. Here we describe the construction of five chimeric strains in which regions of
vacA
were exchanged between the two genotypes. By analyzing the toxicity of these strains for HeLa and RK13 cells we have confirmed that a 148-amino-acid region determines the phenotypic differences between the two forms of the protein and that this entire region is important for cytotoxicity. Furthermore, we have used our chimeric strains to investigate whether variations in the midregion of VacA have an effect on phorbol 12-myristate 13-acetate (PMA)-induced VacA sensitivity in HL-60 cells. The PMA-induced VacA sensitivity of HL-60 cells has been previously associated with the appearance of the cell surface receptor protein tyrosine phosphatase beta (RPTPβ). Our data indicate that both the m1 and m2 forms of VacA are able to utilize RPTPβ, and the cell-specific phenotype of the midregion is independent of the presence of RPTPβ. It appears that another as-yet-unidentified receptor exists in HL-60 cells that accounts for the m2 phenotype in this cell line. Also, by studying the effect of PMA on levels of RPTPβ in other cell lines and toxicity of VacA in these cell lines we have shown that RPTPβ does not play a major role in the vacuolation of HeLa cells.
IdeS, a Highly Specific Immunoglobulin G (IgG)-Cleaving Enzyme from
Streptococcus pyogenes
, Is Inhibited by Specific IgG Antibodies Generated during Infection
Authors: Åkesson Per , Moritz Linnea , Truedsson Mikael , Christensson Bertil , von Pawel-Rammingen Ulrich 
Published: December 2005

ABSTRACT

IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen
Streptococcus pyogenes
, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (
n
= 10), bacteremia (
n
= 7), and erysipelas (
n
= 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.
Urease Produced by
Coccidioides posadasii
Contributes to the Virulence of This Respiratory Pathogen
Authors: Mirbod-Donovan Fariba , Schaller Ruth , Hung Chiung-Yu , Xue Jianmin , Reichard Utz , Cole Garry T. 
Published: December 2005

ABSTRACT

Urease activity during in vitro growth in the saprobic and parasitic phases of
Coccidioides
spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of
Coccidioides posadasii
lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of
C. posadasii
is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of
C. posadasii
resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of
C. posadasii
contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.
A Recombinant Aspartyl Protease ofCoccidioides posadasiiInduces Protection against Pulmonary Coccidioidomycosis in Mice
Authors: Tarcha Eric J. , Basrur Venkatesha , Hung Chiung-Yu , Gardner Malcolm J. , Cole Garry T. 
Published: December 2005

ABSTRACTCoccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogensCoccidioidesspp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection withCoccidioides posadasii. We argue that this immunoproteomic/bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.
The UDP
N
-Acetylgalactosamine 4-Epimerase Gene Is Essential for Mesophilic
Aeromonas hydrophila
Serotype O34 Virulence
Authors: Canals Rocío , Jiménez Natalia , Vilches Silvia , Regué Miguel , Merino Susana , Tomás Juan M. 
Published: December 2005

ABSTRACT

Mesophilic
Aeromonas hydrophila
strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP
N-
acetylgalactosamine 4-epimerase (
gne
) confers the O
−
phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The
gne
gene is present in all the mesophilic
Aeromonas
strains tested. No changes were observed for the LPS core in a
gne
mutant from
A. hydrophila
strain AH-3 (serotype O34). O34 antigen LPS contains
N-
acetylgalactosamine, while no such sugar residue forms part of the LPS core from
A. hydrophila
AH-3. Some of the pathogenic features of
A. hydrophila
AH-3
gne
mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the
gne
mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic
Aeromonas
strains, possess two kinds of flagella, and the absence of O34 antigen molecules by
gne
mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type
gne
gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).
MyD88-Dependent Pathways Mediate Resistance toCryptosporidium parvumInfection in Mice
Authors: Rogers K. A. , Rogers A. B. , Leav B. A. , Sanchez A. , Vannier E. , Uematsu S. , Akira S. , Golenbock D. , Ward H. D. 
Published: December 2005

ABSTRACTCryptosporidiumspp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance toCryptosporidium parvum, we compared the course of infection in MyD88−/−mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected withC. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88−/−mice than in littermate controls. Nonetheless, both WT and MyD88−/−mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance toC. parvum. Since gamma interferon (IFN-γ) is known to mediate resistance toC. parvum, we also studied infection in MyD88−/−mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88−/−mice in which IFN-γ was neutralized than in IFN-γ-neutralized WT mice or in MyD88−/−mice in which this cytokine was active. These results suggest that MyD88 and IFN-γ had an additive effect in conferring protection fromC. parvuminfection. While this study confirms the importance of IFN-γ in conferring resistance to infection withC. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.
Iron and pH Homeostasis Intersect at the Level of Fur Regulation in the Gastric Pathogen
Helicobacter pylori
Authors: Gancz Hanan , Censini Stefano , Merrell D. Scott 
Published: December 2005

ABSTRACT

Helicobacter pylori
persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of
H. pylori
within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In
H. pylori
, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the bacterium to two conditions known to oscillate within the gastric mucosa: iron limitation and low pH. To extend our knowledge of the process of regulation and adaptation in
H. pylori
, we show that Fur is required for efficient colonization of the Mongolian gerbil: the mutant strain exhibits a 100-fold increase in the 50% infectious dose, as well as a 100-fold defect in competitive colonization, when coinfected with wild-type bacteria. Furthermore, we used DNA microarrays to identify genes whose expression was altered in a Fur-deficient strain. We show that the Fur regulon of
H. pylori
consists of approximately 30 genes, most of which have been previously annotated as acid stress associated. Finally, we investigate the role of Fur in acid-responsive modulation of gene expression and show that a large number of genes are aberrantly expressed in the Fur mutant specifically upon acid exposure. This fact likely explains the requirement for this regulator for growth and colonization in the stomach.
Virulence of Broad- and Narrow-Host-RangeSalmonella entericaSerovars in the Streptomycin-PretreatedMouse Model
Authors: Suar Mrutyunjay , Jantsch Jonathan , Hapfelmeier Siegfried , Kremer Marcus , Stallmach Thomas , Barrow Paul A. , Hardt Wolf-Dietrich 
Published: December 2005

ABSTRACTSalmonella entericasubspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a givenS. entericaserovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying otherS. entericasubspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of variousS. entericasubspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject ofSalmonellagenome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.
The Global Regulator Ler Is Necessary for Enteropathogenic
Escherichia coli
Colonization of
Caenorhabditis elegans
Authors: Mellies Jay L. , Barron Alex M. S. , Haack Kenneth R. , Korson Andrew S. , Oldridge Derek A. 
Published: December 2005

ABSTRACT

Enteropathogenic
Escherichia coli
(EPEC) is an important cause of infant diarrhea in developing countries and is useful for general investigations of the bacterial infection process. However, the study of the molecular pathogenesis of EPEC has been hampered by the lack of genetically tractable, convenient animal models. We have therefore developed the use of the nematode
Caenorhabditis elegans
as a small animal model of infection for this diarrheal pathogen. We found that nematodes died faster on nematode growth medium in the presence of EPEC pathogens than in the presence of the laboratory control strain MG1655. Increased numbers of pathogens in the gut, determined by standard plate count assays and fluorescence microscopy using green fluorescent protein-expressing bacteria, correlated with killing. Deletion of the gene encoding the global regulator Ler severely reduced the ability of EPEC to colonize the nematode gut and could be complemented by providing the
ler
gene on a multicopy plasmid in
trans
. Neither the type III secretion system nor the type IV bundle-forming pilus was required for colonization. Combined, the similarities and distinct differences between EPEC infection of nematodes and that of humans offer a unique opportunity to study several stages of the infection process, namely, attachment, colonization, and persistence, in a genetically tractable, inexpensive, and convenient in vivo system.
Invasion of the Placenta during Murine Listeriosis
Authors: Le Monnier Alban , Join-Lambert Olivier F. , Jaubert Francis , Berche Patrick , Kayal Samer 
Published: December 2005

ABSTRACTFeto-placental infections due toListeria monocytogenesrepresent a major threat during pregnancy, and the underlying mechanisms of placental invasion remain poorly understood. Here we used a murine model of listeriosis (pregnant mice, infected at day 14 of gestation) to investigate how this pathogen invades and grows within the placenta to ultimately infect the fetus. WhenL. monocytogenesis injected intravenously, the invasion of the placenta occurs early after the initial bacteremia, allowing the placental growth of the bacteria, which is an absolute requirement for vertical transmission to the fetus. Kinetically, bacteria first target the cells lining the central arterial canal of the placenta, which stain positively with cytokeratin, demonstrating their fetal trophoblast origin. Bacteria then disseminate rapidly to the other trophoblastic structures, like syncytiotrophoblast cells lining the villous core in the labyrinthine zone of placenta. Additionally, we found that an inflammatory reaction predominantly constituted of polymorphonuclear cells occurs in the villous placenta and participates in the control of infection. Altogether, our results suggest that the infection of murine placenta is dependent, at the early phase, on circulating bacteria and their interaction with endovascular trophoblastic cells. Subsequently, the bacteria spread to the other trophoblastic cells before crossing the placental barrier.
Genome Engineering in
Bacillus anthracis
Using Cre Recombinase
Authors: Pomerantsev Andrei P. , Sitaraman Ramakrishnan , Galloway Craig R. , Kivovich Violetta , Leppla Stephen H. 
Published: December 2005

ABSTRACT

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of
Bacillus anthracis
, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1
loxP
sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the
loxP
sites excised the spectinomycin marker, leaving a single
loxP
site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes:
pepM
and
spo0A
, and
mcrB
and
mrr
. Furthermore,
loxP
sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the
capBCAD
region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the
mcrB
and
mrr
genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth.
A Bacterial Flagellin,
Vibrio vulnificus
FlaB, Has a Strong Mucosal Adjuvant Activity To Induce Protective Immunity
Authors: Lee Shee Eun , Kim Soo Young , Jeong Byung Chul , Kim Young Ran , Bae Soo Jang , Ahn Ouk Seon , Lee Je-Jung , Song Ho-Chun , Kim Jung Mogg , Choy Hyon E. , Chung Sun Sik , Kweon Mi-Na , Rhee Joon Haeng 
Published: December 2005

ABSTRACT

Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a
Vibrio vulnificus
major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200× minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-κB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.
Intercellular Spreading of
Porphyromonas gingivalis
Infection in Primary Gingival Epithelial Cells
Authors: Yilmaz Özlem , Verbeke Philippe , Lamont Richard J. , Ojcius David M. 
Published: December 2005

ABSTRACT

Porphyromonas gingivalis
, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether
P. gingivalis
resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that
P. gingivalis
spreads cell to cell and that the amount of spreading increases gradually over time. There was a very low level of propagation of bacteria to uninfected cells early in the infection (3 h postinfection), but there were 20-fold and 45-fold increases in the propagation rate after 24 h and 48 h, respectively, of infection. Immunofluorescence microscopy of infected cells suggested that intercellular translocation of
P. gingivalis
may be mediated through actin-based membrane protrusions, bypassing the need for release of bacteria into extracellular medium. Consistent with these observations, cytochalasin D treatment of infected cells resulted in significant inhibition of bacterial spreading. This study shows for the first time that
P. gingivalis
disseminates from cell to cell without passing through the extracellular space. This mechanism of spreading may allow
P. gingivalis
to colonize oral tissues without exposure to the humoral immune response.