bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bacterial Ghosts as an Oral Vaccine: a Single Dose of
Escherichia coli
O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge
Authors: Mayr Ulrike Beate , Haller Christoph , Haidinger Wolfgang , Atrasheuskaya Alena , Bukin Eugenij , Lubitz Werner , Ignatyev Georgy 
Published: July 2005

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.
Slow Genetic Divergence of
Helicobacter pylori
Strains during Long-Term Colonization
Authors: Lundin Annelie , Björkholm Britta , Kupershmidt Ilya , Unemo Magnus , Nilsson Peter , Andersson Dan I. , Engstrand Lars 
Published: July 2005

ABSTRACT

The genetic variability of
Helicobacter pylori
is known to be high compared to that of many other bacterial species.
H. pylori
is adapted to the human stomach, where it persists for decades, and adaptation to each host results in every individual harboring a distinctive bacterial population. Although clonal variants may exist within such a population, all isolates are generally genetically related and thus derived from a common ancestor. We sought to determine the rate of genetic change of
H. pylori
over 9 years in two asymptomatic adult patients. Arbitrary primed PCR confirmed the relatedness of individual subclones within a patient. Furthermore, sequencing of 10 loci (∼6,000 bp) in three subclones per time and patient revealed only two base pair changes among the subclones from patient I. All sequences were identical among the patient II subclones. However, PCR amplification of the highly divergent gene
amiA
revealed great variation in the size of the gene between the subclones within each patient. Thus, both patients harbored a single strain with clonal variants at both times. We also studied genetic changes in culture- and mouse-passaged strains, and under both conditions no genetic divergence was found. These results suggest that previous estimates of the rate of genetic change in
H. pylori
within an individual might be overestimates.
Distinct Defensin Profiles inNeisseria gonorrhoeaeandChlamydia trachomatisUrethritis Reveal Novel Epithelial Cell-Neutrophil Interactions
Authors: Porter Edith , Yang Huixia , Yavagal Sujata , Preza Gloria C. , Murillo Omar , Lima Heriberto , Greene Sheila , Mahoozi Laily , Klein-Patel Marcia , Diamond Gill , Gulati Sunita , Ganz Tomas , Rice Peter A. , Quayle Alison J. 
Published: July 2005

ABSTRACTDefensins are key participants in mucosal innate defense. The varied antimicrobial activity and differential distribution of defensins at mucosal sites indicate that peptide repertoires are tailored to site-specific innate defense requirements. Nonetheless, few studies have investigated changes in peptide profiles and function after in vivo pathogen challenge. Here, we determined defensin profiles in urethral secretions of healthy men and men withChlamydia trachomatis-andNeisseria gonorrhoeae-mediated urethritis by immunoblotting for the epithelial defensins HBD1, HBD2, and HD5 and the neutrophil defensins HNP1 to -3 (HNP1-3). HBD1 was not detectable in secretions, and HBD2 was only induced in a small proportion of the urethritis patients; however, HD5 and HNP1-3 were increased inC. trachomatisinfection and significantly elevated inN. gonorrhoeaeinfection. When HNP1-3 levels were low, HD5 appeared mostly as the propeptide; however, when HNP1-3 levels were >10 μg/ml, HD5 was proteolytically processed, suggesting neutrophil proteases might contribute to HD5 processing. HD5 and HNP1-3 were bactericidal againstC. trachomatisandN. gonorrhoeae, but HD5 activity was dependent upon N-terminal processing of the peptide. In vitro proteolysis of proHD5 by neutrophil proteases and analysis of urethral secretions by surface-enhanced laser desorption ionization substantiated that neutrophils contribute the key convertases for proHD5 in the urethra during these infections. This contrasts with the small intestine, where Paneth cells secrete both proHD5 and its processing enzyme, trypsin. In conclusion, we describe a unique defensin expression repertoire in response to inflammatory sexually transmitted infections and a novel host defense mechanism wherein epithelial cells collaborate with neutrophils to establish an antimicrobial barrier during infection.
Global Gene Expression and the Role of Sigma Factors in
Neisseria gonorrhoeae
in Interactions with Epithelial Cells
Authors: Du Ying , Lenz Jonathan , Arvidson Cindy Grove 
Published: July 2005

ABSTRACT

Like many bacterial pathogens,
Neisseria gonorrhoeae
must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in
N. gonorrhoeae
in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was
rpoH
, which encodes a homolog of the heat shock sigma factor, σ
32
(RpoH), as well as genes of the RpoH regulon,
groEL
and
groES
. Attempts to construct an
rpoH
null mutant in
N. gonorrhoeae
were unsuccessful, suggesting that RpoH is essential for viability of
N. gonorrhoeae
. The extracytoplasmic sigma factor, RpoE (σ
E
), while known to regulate
rpoH
in other bacteria, was found not to be necessary for the up-regulation of
rpoH
in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an
N. gonorrhoeae
strain conditionally expressing
rpoH
was constructed. The results of our experiments showed that while induction of
rpoH
expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for
rpoH
invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that σ
32
, but not σ
E
, is important for the response of gonococci in the initial steps of an infection.
Involvement of Ganglioside GM3 in G2/M Cell Cycle Arrest of Human Monocytic Cells Induced byActinobacillus actinomycetemcomitansCytolethal Distending Toxin
Authors: Mise Koji , Akifusa Sumio , Watarai Shinobu , Ansai Toshihiro , Nishihara Tatsuji , Takehara Tadamichi 
Published: July 2005

ABSTRACTActinobacillus actinomycetemcomitansproduces a toxin called cytolethal distending toxin (CDT), which causes host cell DNA damage leading to the induction of DNA damage checkpoint pathways. CDT consists of three subunits, CdtA, CdtB, and CdtC. CdtB is the active subunit of CDT and exerts its effect as a nuclease that damages nuclear DNA, triggering cell cycle arrest. In the present study, we confirmed that the only combination of toxin proteins causing cell cycle arrest was that of all three recombinant CDT (rCDT) protein subunits. Furthermore, in order for rCDT to demonstrate toxicity, it was necessary for CdtA and CdtC to access the cell before CdtB. The coexistence of CdtA and CdtC was necessary for these subunits to bind to the cell. Cells treated with the glucosylceramide synthesis inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol showed resistance to the cytotoxicity induced by rCDT. Furthermore, LY-B cells, which are deficient in the biosynthesis of sphingolipid, also showed resistance to the cytotoxicity induced by rCDT. To evaluate the binding of each subunit for glucosylceramides, we performed thin-layer chromatography immunostaining. The results indicated that each subunit reacted with the glycosphingolipids GM1, GM2, GM3, Gb3, and Gb4. The rCDT mixture incubated with liposomes containing GM3 displayed partially reduced toxicity. These results indicate that GM3 can act as a CDT receptor.
Surfaceome of
Leptospira
spp
Authors: Cullen Paul A. , Xu Xiaoyi , Matsunaga James , Sanchez Yolanda , Ko Albert I. , Haake David A. , Adler Ben 
Published: July 2005

ABSTRACT

The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of
Leptospira
was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31
LipL45
, GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<10
7
cells) offering the potential to analyze bacterial surface expression during infection.
Expression of Arg-Gingipain RgpB Is Required for Correct Glycosylation and Stability of Monomeric Arg-Gingipain RgpA fromPorphyromonas gingivalisW50
Authors: Rangarajan Minnie , Hashim Ahmed , Aduse-Opoku Joseph , Paramonov Nikolay , Hounsell Elizabeth F. , Curtis Michael A. 
Published: July 2005

ABSTRACTArg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogenPorphyromonas gingivalisand are encoded byrgpAandrgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpAcat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPAcat), are derived fromrgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight.rgpBencodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated thatrgpBis involved in the glycosylation process, since inactivation ofrgpBresults in the loss of not only RgpB and mt-RgpB but also mt-RgpAcat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpAcatand the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivatedrgpBstrain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream ofrgpBhad no effect onrgpA-derived enzymes, suggesting that the phenotype of thergpBmutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpAcatfrom W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpAcatfrom D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived fromrgpAand that this process is required for enzyme stabilization.
Clostridium perfringens
Epsilon-Toxin Increases Permeability of Single Perfused Microvessels of Rat Mesentery
Authors: Adamson R. H. , Ly J. C. , Fernandez-Miyakawa M. , Ochi S. , Sakurai J. , Uzal F. , Curry F. E. 
Published: July 2005

ABSTRACT

Epsilon-toxin, the primary virulence factor of
Clostridium perfringens
type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (

L
p

) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 μg ml
−1
the

L
p

increased irreversibly to more than 15 times the control value by 10 min. At 0.3 μg ml
−1
no increase in

L
p

was observed for up to 90 min. The toxin-induced increase in

L
p

was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.
Comparison ofHelicobacter pyloriVirulence Gene Expression In Vitro and in the Rhesus Macaque
Authors: Boonjakuakul Jenni K. , Canfield Don R. , Solnick Jay V. 
Published: July 2005

ABSTRACTWe used a quantitative real-time reverse transcriptase PCR assay to measure the transcript abundance of 46 known and putativeHelicobacter pylorivirulence genes, including 24 genes on the Cag pathogenicity island. The expression profile ofH. pyloricells grown in vitro was also compared to expression in vivo after experimental infection of rhesus macaques. Transcript abundance in vitro (mid-log phase) ranged from about 0.004 (feoBandhpaA) to 20 (ureAB,napA, andcag25) copies/cell. Expression of most genes was repressed during the transition from logarithmic- to stationary-phase growth, but several well-characterizedH. pylorivirulence genes (katA,napA,vacA, andcagA) were induced. Comparison of results in the rhesus macaque with similar data from humans showed a strong correlation (r= 0.89). The relative in vivo expression in the rhesus monkey was highly correlated with in vitro expression during mid-log (r= 0.89)- and stationary (r= 0.88)-phase growth. Transcript abundance was on average three- to fourfold reduced in vivo compared to in vitro during mid-log phase. However, when compared to stationary phase, increased expression in vivo was observed for 6 of 7 genes on a contiguous portion of the pathogenicity island, several of which are thought to encode theH. pyloritype IV structural pilus and its accessory proteins. These results suggest the possibility that some genes encoding theH. pyloritype IV structural pilus and accessory proteins may form an operon that is induced during growth in vivo.
Divergence of Protection Induced by Bacterial Products and Sepsis-Induced Immune Suppression
Authors: Sterns Theo , Pollak Nils , Echtenacher Bernd , Männel Daniela N. 
Published: July 2005

ABSTRACTSusceptibility to bacterial infections after a primary immune stimulation differs drastically depending on the presensitization of the innate immune system. To determine the conditions that either induce protection or enhanced susceptibility to infection withSalmonella entericaserovar Typhimurium, we pretreated mice either with tumor necrosis factor (TNF), whole killed bacteria, or sublethal cecal ligation and puncture (CLP) as a mouse model for septic peritonitis. Impaired production of the cytokines TNF, interleukin-6 (IL-6), and IL-10 was induced by these pretreatment schedules, with TNF-signaling not being essential for this effect. Injection of TNF or killed bacteria enhanced survival of mice infected subsequently with serovar Typhimurium. In contrast, sepsis such as that induced by CLP only protected from shock induced byd-galactosamine and lipopolysaccharide or by a high dose of bacteria but sensitized to a secondary bacterial infection. Such sepsis-induced enhanced susceptibility to infection was critically dependent on TNF function.
NK Cells Help To Induce CD8+-T-Cell Immunity againstToxoplasma gondiiin the Absence of CD4+T Cells
Authors: Combe Crescent L. , Curiel Tyler J. , Moretto Magali M. , Khan Imtiaz A. 
Published: July 2005

ABSTRACTCD8+T-cell immunity plays an important role in protection against intracellular infections. Earlier studies have shown that CD4+T-cell help was needed for launching in vivo CD8+T-cell activity against these pathogens and tumors. However, recently CD4+T-cell-independent CD8 responses during several microbial infections including those withToxoplasma gondiihave been described, although the mechanism is not understood. We now demonstrate that, in the absence of CD4+T cells,T. gondii-infected mice exhibit an extended NK cell response, which is mediated by continued interleukin-12 (IL-12) secretion. This prolonged NK cell response is critical for priming parasite-specific CD8+T-cell immunity. Depletion of NK cells inhibited the generation of CD8+T-cell immunity in CD4−/−mice. Similarly neutralization of IL-12 reduces NK cell numbers in infected animals and leads to the down-regulation of CD8+T-cell immunity againstT. gondii. Adoptive transfer of NK cells into the IL-12-depleted animals restored their CD8+T-cell immune response, and animals exhibited reduced mortality. NK cell gamma interferon was essential for cytotoxic T-lymphocyte priming. Our studies for the first time demonstrate that, in the absence of CD4+T cells, NK cells can play an important role in induction of primary CD8+T-cell immunity against an intracellular infection. These observations have therapeutic implications for immunocompromised individuals, including those with human immunodeficiency virus infection.
Cryptococcus neoformans
α Strains Preferentially Disseminate to the Central Nervous System during Coinfection
Authors: Nielsen Kirsten , Cox Gary M. , Litvintseva Anastasia P. , Mylonakis Eleftherios , Malliaris Stephanie D. , Benjamin Daniel K. , Giles Steven S. , Mitchell Thomas G. , Casadevall Arturo , Perfect John R. , Heitman Joseph 
Published: July 2005

ABSTRACT

Cryptococcus neoformans
is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (
gattii
,
grubii
, and
neoformans
). Each variety manifests differences in epidemiology and disease, and var.
grubii
strains are responsible for the vast majority of human disease. In previous studies, α strains were more virulent than congenic
a
strains in var.
neoformans
, whereas var.
grubii
congenic
a
and α strains exhibited equivalent levels of virulence. Here the role of mating type in the virulence of var.
grubii
was further characterized in a panel of model systems. Congenic var.
grubii
a
and α strains had equivalent survival rates when cultured with amoebae, nematodes, and macrophages. No difference in virulence was observed between
a
and α congenic strains in multiple inbred-mouse genetic backgrounds, and there was no difference in accumulations in the central nervous system (CNS) late in infection. In contrast, during coinfections,
a
and α strains are equivalent in peripheral tissues but α cells have an enhanced predilection to penetrate the CNS. These studies reveal the first virulence difference between congenic
a
and α strains in the most common pathogenic variety and suggest an explanation for the prevalence of α strains in clinical isolates.
Type 1 Immunity Provides Both Optimal Mucosal and Systemic Protection against a Mucosally Invasive, Intracellular Pathogen
Authors: Hoft Daniel F. , Eickhoff Chris S. 
Published: July 2005

ABSTRACTIt has been hypothesized that optimal vaccine immunity against mucosally invasive, intracellular pathogens may require the induction of different types of immune responses in mucosal and systemic lymphoid tissues. Mucosal type 2/3 responses (producing interleukin-4 [IL-4], IL-6 and/or transforming growth factor β) could be necessary for optimal induction of protective secretory immunoglobulin A responses. On the other hand, systemic type 1 responses (including gamma interferon [IFN-γ], tumor necrosis factor alpha, and optimal cytotoxic T-cell responses) are likely to be critical for protection against the disseminated intracellular replication that occurs after mucosal invasion. Despite these predictions, we recently found that vaccines inducing highly polarized type 1 immunity in both mucosal and systemic tissues provided optimal mucosal and systemic protection against the protozoan pathogenTrypanosoma cruzi. To further address this important question in a second model system, we now have studied the capacity of knockout mice to develop protective immune memory.T. cruziinfection followed by nifurtimox treatment rescue was used to immunize CD4, CD8, β2-microglobulin, inducible nitric oxide synthase (iNOS), IL-12, IFN-γ, and IL-4 knockout mice. Despite the previously demonstrated importance of CD4+T cells, CD8+T cells, and nitric oxide forT. cruziimmunity, CD4, CD8, and iNOS knockout mice developed mucosal and systemic protective immunity. However, IL-12, IFN-γ, and β2-microglobulin-deficient mice failed to develop mucosal or systemic protection. In contrast, IL-4 knockout mice developed maximal levels of both mucosal and systemic immune protection. These results strongly confirm our earlier conclusion from studies with polarizing vaccination protocols that type 1 immunity provides optimal mucosal and systemic protection against a mucosally invasive, intracellular pathogen.