bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins
Authors: Cai Fang , Adrion Carrie B. , Keller James E. 
Published: September 2006

ABSTRACTLevels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+withN,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+is not required for toxin entry into neurons, and removal of Zn2+from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.
Opsonic Antibodies to
Enterococcus faecalis
Strain 12030 Are Directed against Lipoteichoic Acid
Authors: Theilacker Christian , Kaczynski Zbigniew , Kropec Andrea , Fabretti Francesca , Sange Tatjana , Holst Otto , Huebner Johannes 
Published: September 2006

ABSTRACT

A teichoic acid (TA)-like polysaccharide in
Enterococcus faecalis
has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from
E. faecalis
strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with
d
-Ala and kojibiose. We also detected a novel substituent at position C-2, [
d
-Ala→6]-α-
d
-Glc
p
-(1→2-[
d
-Ala→6]-α-
d
-Glc
p
-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the
E. faecalis
strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to
E. faecalis
12030 are directed predominantly to nonalanylated epitopes on the LTA molecule.
Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of
Cryptococcus neoformans
Authors: Petzold Elizabeth Wills , Himmelreich Uwe , Mylonakis Eleftherios , Rude Thomas , Toffaletti Dena , Cox Gary M. , Miller Jackie L. , Perfect John R. 
Published: September 2006

ABSTRACT

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of
Cryptococcus neoformans
identified the presence of a functioning trehalose pathway during infection and suggested its importance for
C. neoformans
survival in the host. Therefore, in
C. neoformans
we created null mutants of the trehalose-6-phosphate (T6P) synthase (
TPS1
), trehalose-6-phophate phosphatase (
TPS2
), and neutral trehalase (
NTH1
) genes. We found that both
TPS1
and
TPS2
are required for high-temperature (37°C) growth and glycolysis but that the block at
TPS2
results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the
tps1
and
tps2
mutants at 37°C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in
C. neoformans
. The essential nature of this pathway for disease was confirmed when a
tps1
mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate
C. elegans
, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the
tps1
mutant, and this supports the hypothesis that the trehalose pathway in
C. neoformans
is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in
C. neoformans
and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.
Interleukin-10 Anti-Inflammatory Response toBorrelia burgdorferi, the Agent of Lyme Disease: a Possible Role for Suppressors of Cytokine Signaling 1 and 3
Authors: Dennis Vida A. , Jefferson Ayanna , Singh Shree R. , Ganapamo Frédéric , Philipp Mario T. 
Published: September 2006

ABSTRACTIt has been established that interleukin-10 (IL-10) inhibits inflammatory cytokines produced by macrophages in response toBorrelia burgdorferior its lipoproteins. The mechanism by which IL-10 exerts this anti-inflammatory effect is still unknown. Recent findings indicate that suppressors of cytokine signaling (SOCS) proteins are induced by cytokines and Toll-like receptor (TLR)-mediated stimuli, and in turn they can down-regulate cytokine and TLR signaling in macrophages. Because it is known that SOCS are induced by IL-10 and thatB. burgdorferiand its lipoproteins most likely interact via TLR2 or the heterodimers TLR2/1 and/or TLR2/6, we hypothesized that SOCS are induced by IL-10 andB. burgdorferiand its lipoproteins in macrophages and that SOCS may mediate the inhibition by IL-10 of concomitantly elicited cytokines. We report here that mouse J774 macrophages incubated with IL-10 and addedB. burgdorferispirochetes (freeze-thawed, live, or sonicated) or lipidated outer surface protein A (L-OspA) augmented their SOCS1/SOCS3 mRNA and protein expression, with SOCS3 being more abundant. Pam3Cys, a synthetic lipopeptide, also induced SOCS1/SOCS3 expression under these conditions, but unlipidated OspA was ineffective. Neither endogenous IL-10 nor the translation inhibitor cycloheximide blocked SOCS1/SOCS3 induction byB. burgdorferiand its lipoproteins, indicating that the expression of other genes is not required. This temporally correlated with the IL-10-mediated inhibition of the inflammatory cytokines IL-1β, IL-6, IL-12p40, IL-18, and tumor necrosis factor α. Our data are evidence to suggest that expression of SOCS is part of the mechanism of IL-10-mediated inhibition of inflammatory cytokines elicited byB. burgdorferiand its lipoproteins.
Cord Blood Dendritic Cell Subsets in African Newborns Exposed toPlasmodium falciparumIn Utero
Authors: Breitling Lutz P. , Fendel Rolf , Mordmueller Benjamin , Adegnika Ayola A. , Kremsner Peter G. , Luty Adrian J. F. 
Published: September 2006

ABSTRACTPlacentalPlasmodium falciparuminfection affects birth outcomes and sensitizes fetal lymphocytes to parasite antigens. We assessed the influence of maternalP. falciparuminfection on fetal myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC), analyzing the cord blood of offspring of Gabonese mothers with different infection histories. Cord blood from newborns of mothers with malarial infection at delivery had significantly more mDC than that from nonexposed newborns (P= 0.028) but mDC and pDC HLA-DR expression was unrelated to maternal infection history. Independently of these findings, cord blood mDC and pDC numbers declined significantly as a function of increasing maternal age (P= 0.029 andP= 0.033, respectively). The inducible antigen-specific interleukin-10-producing regulatory-type T-cell population that we have previously detected in cord blood of newborns with prolonged in utero exposure toP. falciparummay directly reflect the altered DC numbers in such neonates, while the maintenance of cord blood DC HLA-DR expression contrasts with that of DC fromP. falciparummalaria patients.
A New Immunoglobulin-Binding Protein, EibG, Is Responsible for the Chain-Like Adhesion Phenotype of Locus of Enterocyte Effacement-Negative, Shiga Toxin-Producing
Escherichia coli
Authors: Lu Yan , Iyoda Sunao , Satou Hiromi , Satou Hitomi , Itoh Kenichiro , Saitoh Takehito , Watanabe Haruo 
Published: September 2006

ABSTRACT

Shiga toxin-producing
Escherichia coli
(STEC) are important enteropathogens causing severe diseases such as hemorrhagic colitis and hemolytic-uremic syndrome in humans. The majority of STEC strains of serogroups O157, O26, or O111 associated with severe cases of these diseases possess a pathogenicity island termed the locus of enterocyte effacement (LEE). LEE, which is responsible for the formation of attaching-and-effacing lesions on intestinal epithelial cells, is important for the full virulence of STEC. Nonetheless, LEE-negative STEC strains have repeatedly been reported to be associated with severe diseases in humans. In this study, we characterized adhesion to cultured epithelial cells of certain LEE-negative STEC isolated from humans with or without bloody diarrhea. Several LEE-negative STEC belonging to serogroup O91 showed an unusual, chain-like adhesion pattern to HEp-2 cells. Using Tn
5
-based transposon mutagenesis, we identified the gene essential for the chain-like adhesion phenotype of this O91 STEC strain. Sequence analysis of the Tn
5
-inserted allele identified a novel chromosomal open reading frame (ORF) encoding a polypeptide with a high degree of similarity to the
E. coli
immunoglobulin-binding (Eib) proteins EibA, -C, -D, -E, and -F. Therefore, the ORF was designated EibG. Laboratory
E. coli
strain MC4100 transformed with a multicopy plasmid carrying
eibG
showed chain-like adhesion to HEp-2 cells, and whole-cell lysates of the strain bound to human-derived immunoglobulin G (IgG) Fc and IgA. These results indicate that EibG acts as an IgG Fc- and IgA-binding protein, as well as an adhesin of LEE-negative STEC.
Effects of Temperature on Gene Expression Patterns in
Leptospira interrogans
Serovar Lai as Assessed by Whole-Genome Microarrays
Authors: Lo Miranda , Bulach Dieter M. , Powell David R. , Haake David A. , Matsunaga James , Paustian Michael L. , Zuerner Richard L. , Adler Ben 
Published: September 2006

ABSTRACT

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic
Leptospira
spp. from infected animals or contaminated water or soil. The availability of genome sequences for
Leptospira interrogans
, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression.
Leptospira
spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20°C, 30°C, 37°C, and 39°C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.
The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System
Authors: Shea John M. , Kechichian Talar B. , Luberto Chiara , Del Poeta Maurizio 
Published: September 2006

ABSTRACT

In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (
ISC1
) gene from the fungal pathogen
Cryptococcus neoformans
and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects
C. neoformans
from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the
C. neoformans Δisc1
mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the
C. neoformans Δisc1
strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting
C. neoformans
from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the
Δisc1
strain to the central nervous system and the development of meningoencephalitis.
Growth Phase- and Nutrient Limitation-Associated Transcript Abundance Regulation inBordetella pertussis
Authors: Nakamura Mari M. , Liew Sin-Yee , Cummings Craig A. , Brinig Mary M. , Dieterich Christine , Relman David A. 
Published: September 2006

ABSTRACTTo survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation inBordetella pertussishas been reported previously. We characterized changes in genome-wide transcript abundance ofB. pertussisas a function of growth phase and availability of glutamate, a key nutrient for this organism. Using aBordetellaDNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by whichB. pertussisregulates virulence according to the stage of infection or the location within the human airway.
Characterization of
igaB
, a Second Immunoglobulin A1 Protease Gene in Nontypeable
Haemophilus influenzae
Authors: Fernaays Matthew M. , Lesse Alan J. , Cai Xueya , Murphy Timothy F. 
Published: September 2006

ABSTRACT

Nontypeable
Haemophilus influenzae
is an important respiratory pathogen, causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Immunoglobulin A1 (IgA1) protease is a well-described protein and potential virulence factor in this organism as well as other respiratory pathogens. IgA1 proteases cleave human IgA1, are involved in invasion, and display immunomodulatory effects. We have identified a second IgA1 protease gene,
igaB
, in
H. influenzae
that is present in addition to the previously described IgA1 protease gene,
iga
. Reverse transcriptase PCR and IgA1 protease assays indicated that the gene is transcribed, expressed, and enzymatically active in
H. influenzae
. The product of this gene is a type 2 IgA1 protease with homology to the
iga
gene of
Neisseria
species. Mutants that were deficient in
iga
,
igaB
, and both genes were constructed in
H. influenzae
strain 11P6H, a strain isolated from a patient with COPD who was experiencing an exacerbation. Analysis of these mutants indicated that
igaB
is the primary mediator of IgA1 protease activity in this strain. IgA1 protease activity assays on 20 clinical isolates indicated that the
igaB
gene is associated with increased levels of IgA1 protease activity. Approximately one-third of 297 strains of
H. influenzae
of diverse clinical and geographic origin contained
igaB
. Significant differences in the prevalence of
igaB
were observed among isolates from different sites of isolation (sputum > middle ear > nasopharynx). These data support the hypothesis that the newly discovered
igaB
gene is a potential virulence factor in nontypeable
H. influenzae
.
Porphyromonas gingivalis
Minor Fimbriae Are Required for Cell-Cell Interactions
Authors: Lin Xinghua , Wu Jie , Xie Hua 
Published: September 2006

ABSTRACT

Two distinctive types of fimbriae have been identified in
Porphyromonas gingivalis
. In this report, we demonstrate that minor fimbriae are involved in
P. gingivalis
autoaggregation and colonization. A mutant with a deficiency in minor fimbriae can bind to a saliva-coated surface but does not form microcolonies as the wild-type strain does.
The
mgtC
Gene of
Burkholderia cenocepacia
Is Required for Growth under Magnesium Limitation Conditions and Intracellular Survival in Macrophages
Authors: Maloney Kendra E. , Valvano Miguel A. 
Published: September 2006

ABSTRACT

Burkholderia cenocepacia
, a bacterium commonly found in the environment, is an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known about the mechanisms by which
B. cenocepacia
causes disease, but chronic infection of the airways in CF patients may be associated, at least in part, with the ability of this bacterium to survive within epithelial cells and macrophages. Survival in macrophages occurs in a membrane-bound compartment that is distinct from the lysosome, suggesting that
B. cenocepacia
prevents phagolysosomal fusion. In a previous study, we employed signature-tagged mutagenesis and an agar bead model of chronic pulmonary infection in rats to identify
B. cenocepacia
genes that are required for bacterial survival in vivo. One of the most significantly attenuated mutants had an insertion in the
mgtC
gene. Here, we show that
mgtC
is also needed for growth of
B. cenocepacia
in magnesium-depleted medium and for bacterial survival within murine macrophages. Using fluorescence microscopy, we demonstrated that
B. cenocepacia mgtC
mutants, unlike the parental isolate, colocalize with the fluorescent acidotropic probe LysoTracker Red. At 4 h postinfection,
mgtC
mutants expressing monomeric red fluorescent protein cannot retain this protein within the bacterial cytoplasm. Together, these results demonstrate that, unlike the parental strain, an
mgtC
mutant does not induce a delay in phagolysosomal fusion and the bacterium-containing vacuoles are rapidly targeted to the lysosome, where bacteria are destroyed.