bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of a Type III and a Putative Secretion System in
Edwardsiella tarda
by EsrC Is under the Control of a Two-Component System, EsrA-EsrB
Authors: Zheng J. , Tung S. L. , Leung K. Y.
Published: June 2005

Edwardsiella tarda
is a gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. A type III secretion system (TTSS) and a putative secretion system (EVP) have been found to play important roles in
E. tarda
pathogenesis. Our previous studies suggested that the TTSS and EVP gene clusters were regulated by a two-component system of EsrA-EsrB. In the present study, we characterized another regulator, EsrC, which showed significant sequence similarity to the AraC family of transcriptional regulators. Mutants with in-frame deletions of
esrC
increased the 50% lethal doses in blue gourami fish, reduced extracellular protein production, and failed to aggregate. Complementation of
esrC
restored these three phenotypes. Two-dimensional gel electrophoresis showed that EsrC regulated the expression of secreted proteins encoded by the TTSS (such as EseB and EseD) and EVP (EvpC) gene clusters. The expression of
esrC
required a functional two-component system of EsrA-EsrB. EsrC in turn regulated the expression of selected genes encoded in TTSS (such as the transcriptional unit of
orf29
and
orf30
, but not
esaC
) and genes encoded in the EVP gene cluster. The present study sheds light on the regulation of these two key virulence-associated secretion systems and provides greater insight into the pathogenic mechanisms of this bacterium.
Inactivation of the Pst System Reduces the Virulence of an Avian Pathogenic
Escherichia coli
O78 Strain
Authors: Lamarche Martin G. , Dozois Charles M. , Daigle France , Caza Mélissa , Curtiss Roy , Dubreuil J. Daniel , Harel Josée
Published: June 2005

Escherichia coli
O78 strains are frequently associated with extraintestinal diseases, such as airsacculitis and septicemia, in poultry, livestock, and humans. To understand the influence of the
pst
operon in the virulence of
E. coli
, we introduced mutations into the
pst
genes of the avian pathogenic
E. coli
(APEC) O78:K80 strain χ7122 by allelic exchange. The mutation of
pst
genes led to the constitutive expression of the Pho regulon. Furthermore, the virulence of APEC strain χ7122 in a chicken infection model was attenuated by inactivation of the Pst system. The
pst
mutant caused significantly fewer extraintestinal lesions in infected chickens, and bacterial numbers isolated from different tissues after infection were significantly lower for the mutant than for the wild-type strain. Moreover, resistance to the bactericidal effects of rabbit serum and acid shock was impaired in the
pst
mutant, in contrast to the wild-type strain. In addition, the MIC of polymyxin was twofold lower for the mutant than for the wild-type strain. Although the
pst
mutant demonstrated an increased susceptibility to rabbit serum, this strain was not killed by chicken serum, suggesting the presence of differences in host innate immune defenses and complement-mediated killing. In APEC O78 strain χ7122, a functional Pst system is required for full virulence and resistance to acid shock and polymyxin. Our results suggest that the mutation of
pst
genes induces a deregulation of phosphate sensing and changes in the cell surface composition that lead to decreased virulence, indicating the importance of the Pst system for the virulence of pathogenic
E. coli
strains from different hosts.
Impact of
Helicobacter pylori
Virulence Factors and Compounds on Activation and Maturation of Human Dendritic Cells
Authors: Kranzer Katharina , Söllner Liane , Aigner Michael , Lehn Norbert , Deml Ludwig , Rehli Michael , Schneider-Brachert Wulf
Published: June 2005

Recently, we and others have shown that
Helicobacter pylori
induces dendritic cell (DC) activation and maturation. However, the impact of virulence factors on the interplay between DCs and
H. pylori
remains elusive. Therefore, we investigated the contribution of
cag
pathogenicity island (PAI) and VacA status on cytokine release and up-regulation of costimulatory molecules in
H. pylori
-treated DCs. In addition, to characterize the stimulatory capacity of
H. pylori
compounds in more detail, we studied the effect of formalin-inactivated and sonicated
H. pylori
, as well as secreted
H. pylori
molecules, on DCs. Incubation of DCs with viable or formalin-inactivated
H. pylori
induced comparable secretion of interleukin-6 (IL-6), IL-8, IL-10, IL-12, IL-1β, and tumor necrosis factor (TNF). In contrast, IL-12 and IL-1β release was significantly reduced in DCs treated with sonicated bacteria and secreted bacterial molecules. Treatment of sonicated
H. pylori
preparations with polymyxin B resulted in a significant reduction of IL-8 and IL-6 secretion, suggesting that
H. pylori
-derived lipopolysaccharide at least partially contributes to activation of immature DCs. In addition, the capacity of
H. pylori
-pulsed DCs to activate allogeneic T cells was not affected by
cag
PAI and VacA. Pretreatment of DC with cytochalasin D significantly inhibited secretion of IL-12, IL-1β, and TNF, indicating that phagocytosis of
H. pylori
contributes to maximal activation of DCs. Taken together, our results suggest that DC activation and maturation, as well as DC-mediated T-cell activation, are independent of the
cag
PAI and VacA status of
H. pylori
.
Identification of
Mycobacterium avium
Genes That Affect Invasion of the Intestinal Epithelium
Authors: Miltner Elizabeth , Daroogheh Koorosh , Mehta Parmod K. , Cirillo Suat L. G. , Cirillo Jeffrey D. , Bermudez Luiz E.
Published: June 2005

Invasion of intestinal mucosa of the host by
Mycobacterium avium
is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an
M. avium
genomic library in
Mycobacterium smegmatis
, we have identified a number of
M. avium
genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i)
M. tuberculosis
coenzyme A carboxylase, (ii)
M. tuberculosis
membrane proteins of unknown function, (iii)
M. tuberculosis
FadE20, (iv) a
Mycobacterium paratuberculosis
surface protein, and (v)
M. tuberculosis
cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to
M. avium
the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type
M. avium
and
M. avium
containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of
M. avium
proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions.
Effects of Estradiol on Lipopolysaccharide and Pam
3
Cys Stimulation of CCL20/Macrophage Inflammatory Protein 3 Alpha and Tumor Necrosis Factor Alpha Production by Uterine Epithelial Cells in Culture
Authors: Crane-Godreau Mardi A. , Wira Charles R.
Published: June 2005

We have previously demonstrated that rat uterine epithelial cells (UEC) produce CCL20/macrophage inflammatory protein 3 alpha (MIP3α) and tumor necrosis factor alpha (TNF-α) in response to live and heat-killed
Escherichia coli
and to the pathogen-associated molecular patterns (PAMP) lipopolysaccharide (LPS) and Pam
3
Cys. To determine whether estradiol (E
2
) modulates PAMP-induced CCL20/MIP3α and TNF-α secretion, primary cultures of rat UEC were incubated with E
2
for 24 h and then treated with LPS or Pam
3
Cys or not treated for an additional 12 h. E
2
inhibited the constitutive secretion of TNF-α and CCL20/MIP3α into culture media. Interestingly, E
2
pretreatment enhanced CCL20/MIP3α secretion due to LPS and Pam
3
Cys administration. In contrast, and at the same time, E
2
lowered the TNF-α response to both PAMP. To determine whether estrogen receptors (ER) mediated the effects of E
2
, epithelial cells were incubated with E
2
and/or ICI 182,780, a known ER antagonist. ICI 182,780 had no effect on E
2
inhibition of constitutive TNF-α and CCL20/MIP3α secretion. In contrast, ICI 182,780 reversed the stimulatory effect of E
2
on LPS- and/or Pam
3
Cys-induced CCL20/MIP3α secretion as well as partially reversed the inhibitory effect of E
2
on TNF-α production by epithelial cells. Overall, these results indicate that E
2
regulates the production of TNF-α and CCL20/MIP3α by UEC in the absence as well as presence of PAMP. Since CCL20/MIP3α has antimicrobial activity and is chemotactic for immune cells, these studies suggest that regulation of CCL20/MIP3α and TNF-α by E
2
and PAMP may have profound effects on innate and adaptive immune responses to microbial challenge in the female reproductive tract.
Endocrine Perturbation Increases Susceptibility of Mice to Anthrax Lethal Toxin
Authors: Moayeri Mahtab , Webster Jeanette I. , Wiggins Jason F. , Leppla Stephen H. , Sternberg Esther M.
Published: June 2005

Bacillus anthracislethal toxin (LT) causes vascular collapse and high lethality in BALB/cJ mice, intermediate lethality in C57BL/6J mice, and no lethality in DBA/2J mice. We found that adrenalectomized (ADX) mice of all three strains had increased susceptibility to LT. The increased susceptibility of ADX-DBA/2J mice was not accompanied by changes in their macrophage sensitivity or cytokine response to LT. DBA/2J mice showed no change in serum corticosteroid levels in response to LT injection, while BALB/cJ mice showed a fivefold increase in serum corticosterone. However, LT inhibited dexamethasone (DEX)-induced glucocorticoid receptor gene activation to similar extents in all three strains. DEX treatment did not rescue ADX mice from LT-mediated mortality. Surprisingly, oral DEX treatment also sensitized adrenally intact DBA/2J mice to LT lethality at all doses tested and also exacerbated LT-mediated pathogenesis and mortality in BALB/cJ mice. Aldosterone did not protect ADX mice from toxin challenge. These results indicate that susceptibility to anthrax LT in mice depends on a fine but easily perturbed balance of endocrine functions. Thus, the potentially detrimental consequences of steroid therapy for anthrax must be considered in treatment protocols for this disease.
Innate Immune Defense against Pneumococcal Pneumonia Requires Pulmonary Complement Component C3
Authors: Kerr Alison R. , Paterson Gavin K. , Riboldi-Tunnicliffe Alan , Mitchell Tim J.
Published: June 2005

Complement is known to be involved in protection against systemic infection withStreptococcus pneumoniae. However, less is known about effects of complement within the lungs during pneumococcal pneumonia. By intranasally infecting transgenic mice unable to express complement C3, we investigated the role of complement in pulmonary defenses againstS. pneumoniae. It was demonstrated that within the lungs, there is a requirement for C3 during the initial hours of infection. It was found that within 1 h of infection, bacterial loads decreased within lung airways of control mice as C3 protein increased. The lack of C3 resulted in the inability to control growth of wild-type or attenuated pneumococci within the lungs and bloodstream, resulting in an overwhelming inflammatory response and shorter survival times. Our results show that during the initial hours of infection withS. pneumoniae, C3 is protective within the lungs and subsequently plays an important role systemically.
Alveolar Response toPseudomonas aeruginosa: Role of the Type III Secretion System
Authors: Ader F. , Le Berre R. , Faure K. , Gosset P. , Epaulard O. , Toussaint B. , Polack B. , Nowak E. , Viget N. B. , Kipnis E. , Guery B. P.
Published: June 2005

The type III secretion system (TTSS) is a specialized cytotoxin-translocating apparatus of gram-negative bacteria which is involved in lung injury, septic shock, and a poor patient outcome. Recent studies have attributed these effects mainly to the ExoU effector protein. However, few studies have focused on the ExoU-independent pathogenicity of the TTSS. For the present study, we compared the pathogenicities of two strains ofPseudomonas aeruginosain a murine model of acute lung injury. We compared the CHA strain, which has a functional TTSS producing ExoS and ExoT but not ExoU, to an isogenic mutant with an inactivatedexsAgene, CHA-D1, which does not express the TTSS at all. Rats challenged with CHA had significantly increased lung injury, as assessed by the wet/dry weight ratio for the lungs and the protein level in bronchoalveolar lavage fluid (BALF) at 12 h, compared to those challenged with CHA-D1. Consistent with these findings, the CHA strain was associated with increased in vitro cytotoxicity on A549 cells, as assessed by the release of lactate dehydrogenase. CHA was also associated at 12 h with a major decrease in polymorphonuclear neutrophils in BALF, with a proinflammatory response, as assessed by the amounts of tumor necrosis factor alpha and interleukin-1β, and with decreased bacterial clearance from the lungs, ultimately leading to an increased mortality rate. These results demonstrate that the TTSS has a major role inP. aeruginosapathogenicity independent of the role of ExoU. This report underscores the crucial roles of ExoS and ExoT or other TTSS-related virulence factors in addition to ExoU.
Identification of a Novel Adhesion Molecule Involved in the Virulence ofLegionella pneumophila
Authors: Chang Bin , Kura Fumiaki , Amemura-Maekawa Junko , Koizumi Nobuo , Watanabe Haruo
Published: June 2005

Legionella pneumophilais an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of anL. pneumophilastrain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue ofSaccharomyces cerevisiae, was namedL. pneumophilaadhesion molecule homologous with integrin analogue ofS. cerevisiae(LaiA). ThelaiAgene product was involved inL. pneumophilaadhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication ofL. pneumophilawithin a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, thelaiAmutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that thelaiAgene encodes a virulence factor that is involved in transmission ofL. pneumophila80-045 and may play a role in Legionnaires' disease in humans.
Production of Nontypeable
Haemophilus influenzae
HtrA by Recombinant
Bordetella pertussis
with the Use of Filamentous Hemagglutinin as a Carrier
Authors: Alonso Sylvie , Willery Eve , Renauld-Mongénie Genevieve , Locht Camille
Published: June 2005

Bordetella pertussis
, the etiologic agent of whooping cough, is a highly infectious human pathogen capable of inducing mucosal and systemic immune responses upon a single intranasal administration. In an attenuated, pertussis toxin (PTX)-deficient recombinant form, it may therefore constitute an efficient bacterial vector that is particularly well adapted for the delivery of heterologous antigens to the respiratory mucosa. Filamentous hemagglutinin (FHA) has been used as a carrier to present foreign antigens at the bacterial surface, thereby inducing local, systemic, and protective immune responses to these antigens in mice. Both full-length and truncated (Fha44) forms of FHA have been used for antigen presentation. To investigate the effect of the carrier (FHA or Fha44) on antibody responses to passenger antigens, we genetically fused the HtrA protein of nontypeable
Haemophilus influenzae
to either FHA form. The
fha-htrA
and Fha44 gene
-htrA
hybrids were expressed as single copies inserted into the chromosome of PTX-deficient
B. pertussis
. Both chimeras were secreted into the culture supernatants of the recombinant strains and were recognized by anti-FHA and anti-HtrA antibodies. Intranasal infection with the strain producing the FHA-HtrA hybrid led to significantly higher anti-HtrA and anti-FHA antibody titers than those obtained in mice infected with the Fha44-HtrA-producing strain. Interestingly, the
B. pertussis
strain producing the Fha44-HtrA chimera colonized the mouse lungs more efficiently than the parental, Fha44-producing strain and gave rise to higher anti-FHA antibody titers than those induced by the parental strain.
Virulence Attenuation and Live Vaccine Potential of
aroA
,
crp cdt cya
, and Plasmid-Cured Mutants of
Salmonella enterica
Serovar Abortusovis in Mice and Sheep
Authors: Uzzau Sergio , Marogna Gavino , Leori Guido Sisinnio , Curtiss Roy , Schianchi Giuseppe , Stocker Bruce A. D. , Rubino Salvatore
Published: June 2005

Three live vaccine candidates of
Salmonella enterica
subspecies I serotype Abortusovis (
aroA
,
cya crp cdt
, and plasmid-cured strains) have been developed, and their efficacies in inducing humoral antibodies and protecting against abortion after challenge with wild-type strain SS44 were evaluated in sheep. Following estrus synchronization, animals were immunized 3 weeks after fertilization and boosted once 3 weeks later. Following challenge with wild-type SS44, pregnancy failure of vaccinated ewes was reduced compared to that of nonimmunized controls. Attenuation of each vaccine was also assessed in challenge experiments with nonimmunized pregnant ewes and in BALB/c mice. All three vaccine candidates appear to be safe for use in sheep and provide a model for the development of live vaccine candidates against naturally occurring ovine salmonellosis.
Bovine Intestinal Alkaline Phosphatase Attenuates the Inflammatory Response in Secondary Peritonitis in Mice
Authors: van Veen Suzanne Q. , van Vliet Arlène K. , Wulferink Marty , Brands Ruud , Boermeester Marja A. , van Gulik Thomas M.
Published: June 2005

Lipopolysaccharide (LPS) contributes importantly to morbidity and mortality in sepsis. Bovine intestinal alkaline phosphatase (BIAP) was demonstrated to detoxify LPS through dephosphorylation. LPS injection combined with BIAP reduced inflammation and improved survival in various experimental settings. In this study, single-dose intravenous administration of BIAP (0.15 IU/g) was applied in a murine cecal ligation and puncture (CLP) model of polymicrobial sepsis. Saline was given as control (S group). Treatment with BIAP prior to CLP (prophylaxis; BIAP-P group) or shortly after (early treatment; BIAP-ET group) reduced cytokine concentrations in plasma and peritoneal lavage fluid (PLF). Tumor necrosis factor-alpha peak levels decreased from 170 pg/ml (S) to 57.5 (BIAP-P) and 82.5 (BIAP-ET) in plasma and in PLF from 57.5 pg/ml (S) to 35.3 (BIAP-P) and 16.8 (BIAP-ET) (all,P< 0.05). Peak interleukin-6 levels in plasma decreased from 19.3 ng/ml (S) to 3.4 (BIAP-P) and 11.5 (BIAP-ET) and in PLF from 32.6 ng/ml (S) to 13.4 (BIAP-P) and 10.9 (BIAP-ET) (all,P< 0.05). Macrophage chemoattractant protein 1 peak levels in plasma decreased from 2.0 ng/ml (S) to 1.0 (BIAP-P) and 0.7 (BIAP-ET) and in PLF from 6.4 (S) to 2.3 (BIAP-P) and 1.3 ng/ml (BIAP-ET) (all,P< 0.05). BIAP-treated groups showed decreased transaminase activity in plasma and decreased myeloperoxidase activity in the lung, indicating reduced associated hepatocellular and pulmonary damage. Survival was not significantly altered by BIAP in this single-dose regimen. In polymicrobial secondary peritonitis, both prophylactic and early BIAP treatment attenuates the inflammatory response both locally and systemically and reduces associated liver and lung damage.
Effect of
Chlamydia pneumoniae
on Cellular ATP Content in Mouse Macrophages: Role of Toll-Like Receptor 2
Authors: Yaraei Kambiz , Campbell Lee Ann , Zhu Xiaodong , Liles W. Conrad , Kuo Cho-chou , Rosenfeld Michael E.
Published: June 2005

Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live
C. pneumoniae
and inactivated
C. pneumoniae
induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of
C. pneumoniae
on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated
C. pneumoniae
cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated
C. pneumoniae
increased the ATP content of the cells. In cells infected with live
C. pneumoniae
, the increase was inversely proportional to the MOI. In cells treated with inactivated
C. pneumoniae
, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of
C. pneumoniae
, while sustained induction required live organisms. The capacity of
C. pneumoniae
to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.
sciS
, an
icmF
Homolog in
Salmonella enterica
Serovar Typhimurium, Limits Intracellular Replication and Decreases Virulence
Authors: Parsons Duncan A. , Heffron Fred
Published: June 2005

Salmonella enterica
serovar Typhimurium utilizes macrophages to disseminate from the intestine to deeper tissues within the body. While
S. enterica
serovar Typhimurium has been shown to kill its host macrophage, it can persist intracellularly beyond 18 h postinfection. To identify factors involved in late stages of infection, we screened a transposon library made in
S. enterica
serovar Typhimurium for the ability to persist in J774 macrophages at 24 h postinfection. Through this screen, we identified a gene,
sciS
, found to be homologous to
icmF
in
Legionella pneumophila. icmF
, which is required for intracellular multiplication, is conserved in several gram-negative pathogens, and its homolog appears to have been acquired horizontally in
S. enterica
serovar Typhimurium. We found that an
sciS
mutant displayed increased intracellular numbers in J774 macrophages when compared to the wild-type strain at 24 h postinfection.
sciS
was maximally transcribed at 27 h postinfection and is repressed by SsrB, an activator of genes required for promoting intracellular survival. Finally, we demonstrate that an
sciS
mutant is hypervirulent in mice when administered intragastrically. Taken together, these data indicate a role for SciS in controlling intracellular bacterial levels at later stages of infection and attenuating virulence in a murine host
Quantitative Dynamics of
Plasmodium yoelii
Sporozoite Transmission by Infected Anopheline Mosquitoes
Authors: Medica Darcy L. , Sinnis Photini
Published: June 2005

Malaria transmission begins with the injection of
Plasmodium
sporozoites into the skin of a vertebrate host by infected anopheline mosquitoes. Although the size of the sporozoite inoculum likely affects the course of the disease, the number of sporozoites injected by infected mosquitoes has not been determined in vivo. Using a quantitative PCR assay, we determined the number of sporozoites injected into mice by single mosquitoes. Analysis of 59 mosquito feedings showed that a single infected mosquito injected between 0 and 1,297 sporozoites, with a mean of 123 and a median of 18. Twenty-two percent of infected mosquitoes injected no sporozoites. The number of sporozoites injected was only weakly correlated to the salivary gland load. To better understand the large variability in sporozoite injection among mosquitoes, we quantified the sporozoites injected by individual mosquitoes on three different days. Approximately 20% of moderately to heavily infected mosquitoes injected few to no sporozoites on all 3 days, suggesting that some mosquitoes are poor transmitters of sporozoites. Other mosquitoes injected high numbers of sporozoites on at least one of the days observed and minimal numbers on the other day(s), supporting the hypothesis that sporozoite injection is discontinuous, a pattern that may aid in the establishment of malaria infection.
Enteropathogenic
Escherichia coli
Type III Effectors EspG and EspG2 Disrupt the Microtubule Network of Intestinal Epithelial Cells
Authors: Shaw Robert K. , Smollett Katherine , Cleary Jennifer , Garmendia Junkal , Straatman-Iwanowska Ania , Frankel Gad , Knutton Stuart
Published: June 2005

Enteropathogenic
Escherichia coli
infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.
KatA, the Major Catalase, Is Critical for Osmoprotection and Virulence inPseudomonas aeruginosaPA14
Authors: Lee Ji-Sun , Heo Yun-Jeong , Lee Jeong K. , Cho You-Hee
Published: June 2005

We demonstrate that among the three monofunctional catalases ofPseudomonas aeruginosaPA14, KatA and, to a lesser extent, KatB, but not KatE, are required for resistance to peroxide and osmotic stresses. KatA is crucial for adaptation to H2O2stress and full virulence in bothDrosophila melanogasterand mice. This dismantling of catalase roles represents a specialized catalytic system primarily involving KatA in responses to adverse environmental conditions.
Adherence to and Invasion of Human Brain Microvascular Endothelial Cells Are Promoted by Fibrinogen-Binding Protein FbsA ofStreptococcus agalactiae
Authors: Tenenbaum Tobias , Bloier Christiane , Adam Rüdiger , Reinscheid Dieter J. , Schroten Horst
Published: June 2005

Streptococcus agalactiaeis a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection,S. agalactiaecolonizes and invades a number of host compartments, thereby interacting with different host tissues. Deletion of thefbsAgene, encoding the fibrinogen protein FbsA, significantly impaired the adherence and invasion of human brain microvascular endothelial cells (HBMEC) byS. agalactiae. The adherence and invasiveness of anfbsAdeletion mutant were restored by reintroducing thefbsAgene on an expression vector. Heterologous expression offbsAinLactococcus lactisenabled this bacterium to adhere to but not to invade HBMEC, suggesting that FbsA is a streptococcal adhesin. Finally, host cell adherence and invasion were significantly blocked in competition experiments with either purified FbsA fusion protein or a monoclonal antibody directed against the fibrinogen-binding epitope of FbsA. TheS. agalactiae fbsAmutant induced a release of the neutrophil chemoattractant interleukin-8 (IL-8) equal to that induced by the wild type. Taken together, our studies demonstrate that FbsA promotes the adherence ofS. agalactiaeto HBMEC but that FbsA neither mediates the bacterial invasion into host cells nor plays a role in IL-8 release for HBMEC.
Inhibition of Proteolytic Activity of a Chlamydial Proteasome/Protease-Like Activity Factor by Antibodies from Humans Infected with
Chlamydia trachomatis
Authors: Sharma Jyotika , Dong Feng , Pirbhai Mustak , Zhong Guangming
Published: June 2005

We have previously shown that individuals infected with
Chlamydia trachomatis
can develop a robust antibody response to a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor [CPAF]). We now report that sera collected from these infected individuals neutralized the proteolytic activity of CPAF. Depletion of the serum sample with CPAF proteins to remove the CPAF-specific antibodies effectively blocked the neutralization, whereas similar depletion with the HSP60 proteins failed to do so. We further demonstrated that the CPAF central region covering residues 200 to 338 was predominantly recognized by the human neutralization antibodies. The significance of the CPAF neutralization antibodies generated in chlamydia-infected individuals is discussed.
Complement Depletion Renders C57BL/6 Mice Sensitive to the
Bacillus anthracis
Sterne Strain
Authors: Harvill Eric T. , Lee Gloria , Grippe Vanessa K. , Merkel Tod J.
Published: June 2005

Concerns regarding safety and control of virulent
Bacillus anthracis
have created substantial hurdles to the study of anthrax. The Sterne strain is considered relatively safe to study, but this acapsular strain has a defect in normal mice and is often studied in A/J mice. A/J mice are highly susceptible to the Sterne strain, due to a defect in the Hc locus, which encodes complement factor 5 (C5). Here we show that normally resistant C57BL/6 mice become highly susceptible to the Sterne strain upon complement depletion with cobra venom factor. This generalizable approach should allow the virulence of anthrax to be studied under relatively safe conditions and using a wide variety of mouse strains.
Endogenous CD4+CD25+Regulatory T Cells Play No Apparent Role in the Acute Humoral Response to IntactStreptococcus pneumoniae
Authors: Lee Katherine S. , Sen Goutam , Snapper Clifford M.
Published: June 2005

Immunoglobulin G (IgG) antiprotein and antipolysaccharide responses to intactStreptococcus pneumoniaeare CD4+-T-cell dependent and therefore might be under the negative control of CD4+CD25+regulatory T cells. Injection of anti-interleukin 2 receptor α (anti-IL-2Rα) MAb to deplete regulatory T cells, injection of agonistic MAb against glucocorticoid-induced tumor necrosis factor receptor family-related protein to inhibit regulatory-T-cell function, and adoptive transfer of regulatory-T-cell-depleted CD4+T cells into athymic nude mice each had no effect on either the primary or secondary protein- or polysaccharide-specific IgG response to intactS. pneumoniae. Surprisingly, anti-IL-2Rα MAb also had no effect on the IgG response to intactS. pneumoniaein MyD88−/−mice or to a soluble protein-polysaccharide conjugate injected into wild-type mice in the absence of adjuvant. Collectively, these data are the first to suggest that, in contrast to their role in limiting chronic cell-mediated immunity, regulatory T cells may play no significant role in an acute humoral immune response to an intact extracellular bacterial pathogen.
Protective Immunization of Mice with an Active-Site Mutant of Subtilase Cytotoxin of Shiga Toxin-Producing
Escherichia coli
Authors: Talbot Ursula M. , Paton James C. , Paton Adrienne W.
Published: June 2005

We have recently described a novel AB
5
cytotoxin produced by certain Shiga toxin-producing
Escherichia coli
strains. The A subunit of this toxin is a subtilase-like serine protease, while the B pentamer mediates binding to host cell glycolipid receptors. The subtilase cytotoxin is lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidneys, and liver. In the present study, we have immunized mice with a purified derivative of the toxin with a Ser
272
→ Ala mutation in the A subunit which abolishes cytotoxicity. This elicited strong antibody responses, as judged by enzyme-linked immunosorbent assay, which conferred protection against intraperitoneal challenge with purified toxin. Immunized mice were also protected from weight loss resulting from oral challenge with an
E. coli
K-12 clone expressing the active toxin.
The Order of Prime-Boost Vaccination of Neonatal Calves with
Mycobacterium bovis
BCG and a DNA Vaccine Encoding Mycobacterial Proteins Hsp65, Hsp70, and Apa Is Not Critical for Enhancing Protection against Bovine Tuberculosis
Authors: Skinner Margot A. , Wedlock D. Neil , de Lisle Geoffrey W. , Cooke Michèle M. , Tascon Ricardo E. , Ferraz Jose C. , Lowrie Douglas B. , Vordermeier H. Martin , Hewinson R. Glyn , Buddle Bryce M.
Published: June 2005

Priming neonatal calves at birth with a
Mycobacterium bovis
bacillus Calmette-Guérin (BCG) vaccine and boosting with a DNA vaccine consisting of plasmids encoding mycobacterial antigens Hsp65, Hsp70, and Apa or the reverse prime-boost sequence induced similar levels of protection against experimental challenge with
Mycobacterium bovis
. When
M. bovis
was isolated from a thoracic lymph node following challenge, the two groups of calves given the prime-boost regimen had significantly lower numbers of
M. bovis
isolates than those vaccinated with BCG alone. These observations suggest that the exact sequence of administration of a prime-boost vaccination regimen in a neonatal animal model is not critical to the development of immunity.
DIVISIBILITY OF DEDEKIND FINITE SETS
Authors: BLAIR DAVID , BLASS ANDREAS , HOWARD PAUL
Published: June 2005

A Dedekind-finite set is said to be divisible by a natural number n if it can be partitioned into pieces of size n. We study several aspects of this notion, as well as the stronger notion of being partitionable into n pieces of equal size. Among our results are that the divisors of a Dedekind-finite set can consistently be any set of natural numbers (containing 1 but not 0), that a Dedekind-finite power of 2 cannot be divisible by 3, and that a Dedekind-finite set can be congruent modulo 3, to all of 0, 1, and 2 simultaneously. (In these results, 2 and 3 serve as typical examples; the full results are more general.)
THE CLUB GUESSING IDEAL: COMMENTARY ON A THEOREM OF GITIK AND SHELAH
Authors: FOREMAN MATTHEW , KOMJATH PETER
Published: June 2005

It is shown in this paper that it is consistent (relative to almost huge cardinals) for various club guessing ideals to be saturated.
MULTIBUMP SOLUTIONS OF NONLINEAR PERIODIC SCHRÖDINGER EQUATIONS IN A DEGENERATE SETTING
Authors: ACKERMANN NILS , WETH TOBIAS
Published: June 2005

We prove the existence of infinitely many geometrically distinct two bump solutions of periodic superlinear Schrödinger equations of the type -Δu + V(x)u = f(x,u), where x ∈ ℝN and lim |x| → ∞u(x) = 0. The solutions we construct change sign and have exactly two nodal domains. The usual multibump constructions for these equations rely on strong non-degeneracy assumptions. We present a new approach that only requires a weak splitting condition. In the second part of the paper we exhibit classes of potentials V for which this splitting condition holds.
COMPLEX FOLIATED TORI AND THEIR MODULI SPACES
Authors: CARESSA PAOLO , TOMASSINI ADRIANO
Published: June 2005

We consider complex foliated tori, their periods and polarizations on them. We define the moduli space of polarized complex foliated tori and show that it is a normal analytic complex space. Finally we discuss some examples.
On the suppression of plasma nonesterified fatty acids by insulin during enhanced intravascular lipolysis in humans
Authors: Carpentier André C. , Frisch Frédérique , Cyr Denis , Généreux Philippe , Patterson Bruce W. , Giguère Robert , Baillargeon Jean-Patrice
Published: June 2005

During the fasting state, insulin reduces nonesterified fatty acid (NEFA) appearance in the systemic circulation mostly by suppressing intracellular lipolysis in the adipose tissue. In the postprandial state, insulin may also control NEFA appearance through enhanced trapping into the adipose tissue of NEFA derived from intravascular triglyceride lipolysis. To determine the contribution of suppression of intracellular lipolysis in the modulation of plasma NEFA metabolism by insulin during enhanced intravascular triglyceride lipolysis, 10 healthy nonobese subjects underwent pancreatic clamps at fasting vs. high physiological insulin level with intravenous infusion of heparin plus Intralipid. Nicotinic acid was administered orally during the last 2 h of each 4-h clamp to inhibit intracellular lipolysis and assess insulin’s effect on plasma NEFA metabolism independently of its effect on intracellular lipolysis. Stable isotope tracers of palmitate, acetate, and glycerol were used to assess plasma NEFA metabolism and total triglyceride lipolysis in each participant. The glycerol appearance rate was similar during fasting vs. high insulin level, but plasma NEFA levels were significantly lowered by insulin. Nicotinic acid significantly blunted the insulin-mediated suppression of plasma palmitate appearance and oxidation rates by ∼60 and ∼70%, respectively. In contrast, nicotinic acid did not affect the marked stimulation of palmitate clearance by insulin. Thus most of the insulin-mediated reduction of plasma NEFA appearance and oxidation can be explained by suppression of intracellular lipolysis during enhanced intravascular triglyceride lipolysis in healthy humans. Our results also suggest that insulin may affect plasma NEFA clearance independently of the suppression of intracellular lipolysis.
Collagen synthesis in human musculoskeletal tissues and skin
Authors: Babraj J. A. , Cuthbertson D. J. R. , Smith K. , Langberg H. , Miller B. , Krogsgaard M. R. , Kjaer M. , Rennie M. J.
Published: June 2005

We have developed a direct method for the measurement of human musculoskeletal collagen synthesis on the basis of the incorporation of stable isotope-labeled proline or leucine into protein and have used it to measure the rate of synthesis of collagen in tendon, ligament, muscle, and skin. In postabsorptive, healthy young men (28 ± 6 yr) synthetic rates for tendon, ligament, muscle, and skin collagen were 0.046 ± 0.005, 0.040 ± 0.006, 0.016 ± 0.002, and 0.037 ± 0.003%/h, respectively (means ± SD). In postabsorptive, healthy elderly men (70 ± 6 yr) the rate of skeletal muscle collagen synthesis is greater than in the young (0.023 ± 0.002%/h, P < 0.05 vs. young). The rates of synthesis of tendon and ligament collagen are similar to those of mixed skeletal muscle protein in the postabsorptive state, whereas the rate for muscle collagen synthesis is much lower in both young and elderly men. After nutritient provision, collagen synthesis was unaltered in tendon and skeletal muscle, remaining at postabsorptive values (young: tendon, 0.045 ± 0.008%/h; muscle, 0.016 ± 0.003%/h; elderly: muscle, 0.024 ± 0.003%/h). These results demonstrate that the rate of human musculoskeletal tissue collagen synthesis can be directly and robustly measured using stable isotope methodology.
PKA-dependent ENaC trafficking requires the SNARE-binding protein complexin
Authors: Butterworth M. B. , Frizzell R. A. , Johnson J. P. , Peters K. W. , Edinger R. S.
Published: June 2005

Acute regulation of epithelial sodium channel (ENaC) function at the apical surface of polarized kidney cortical collecting duct (CCD) epithelial cells occurs in large part by changes in channel number, mediated by membrane vesicle trafficking. Several soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNARE) have been implicated in this process. A novel SNARE-binding protein, complexin, has been identified in nervous tissue which specifically binds to and stabilizes SNARE complexes at synaptic membranes to promote vesicle fusion. To test whether this protein is present in mouse CCD (mCCD) cells and its possible involvement in acute ENaC regulation, we cloned complexin (isoform II) from a mouse kidney cDNA library. Complexin II mRNA coexpressed with α-, β-, and γ-ENaC subunits in Xenopus laevis oocytes reduced sodium currents to 16 ± 3% ( n = 19) of control values. Short-circuit current ( Isc) measurements on mCCD cell lines stably over- or underexpressing complexin produced similar results. Basal Isc was reduced from 12.0 ± 1.0 ( n = 15) to 2.0 ± 0.4 ( n = 15) and 1.8 ± 0.3 ( n = 17) μA/cm2, respectively. Similarly forskolin-stimulated Isc was reduced from control values of 20.0 ± 2 to 2.7 ± 0.5 and 2.3 ± 0.4 μA/cm2 by either increasing or decreasing complexin expression. Surface biotinylation demonstrated that the complexin-induced reduction in basal Iscwas due to a reduction in apical membrane-resident ENaC and the inhibition in forskolin stimulation was due to the lack of ENaC insertion into the apical membrane to increase surface channel number. Immunofluorescent localization of SNARE proteins in polarized mCCD epithelia detected the presence of syntaxins 1 and 3 and synaptosomal-associated protein of 23 kDa (SNAP-23) at the apical membrane, and vesicle-associated membrane protein (VAMP2) was localized to intracellular compartments. These findings identify SNAREs that may mediate ENaC-containing vesicle insertion in mCCD epithelia and suggest that stabilization of SNARE interactions by complexin is an essential aspect of the regulated trafficking events that increase apical membrane ENaC density either by constitutive or regulated trafficking pathways.
A different analysis applied to a mathematical model on output of exhaled nitric oxide
Authors: Rottier Bart L. , Cohen Judith , van der Mark Thom W. , Douma W. Rob , Duiverman Eric J. , ten Hacken Nick H. T.
Published: June 2005

The following is the abstract of the article discussed in the following letter:

The relatively recent detection of nitric oxide (NO) in the exhaled breath has prompted a great deal of experimentation in an effort to understand the pulmonary exchange dynamics. There has been very little progress in theoretical studies to assist in the interpretation of the experimental results. We have developed a two-compartment model of the lungs in an effort to explain several fundamental experimental observations. The model consists of a nonexpansile compartment representing the conducting airways and an expansile compartment representing the alveolar region of the lungs. Each compartment is surrounded by a layer of tissue that is capable of producing and consuming NO. Beyond the tissue barrier in each compartment is a layer of blood representing the bronchial circulation or the pulmonary circulation, which are both considered an infinite sink for NO. All parameters were estimated from data in the literature, including the production rates of NO in the tissue layers, which were estimated from experimental plots of the elimination rate of NO at end exhalation (ENO) vs. the exhalation flow rate (V̇e). The model is able to simulate the shape of the NO exhalation profile and to successfully simulate the following experimental features of endogenous NO exchange: 1) an inverse relationship between exhaled NO concentration and V̇E, 2) the dynamic relationship between the phase III slope and V̇E, and 3) the positive relationship between ENO and V̇E. The model predicts that these relationships can be explained by significant contributions of NO in the exhaled breath from the nonexpansile airways and the expansile alveoli. In addition, the model predicts that the relationship between ENO and V̇E can be used as an index of the relative contributions of the airways and the alveoli to exhaled NO.
Energy cost and muscular activity required for leg swing during walking
Authors: Gottschall Jinger S. , Kram Rodger
Published: June 2005

To investigate the metabolic cost and muscular actions required for the initiation and propagation of leg swing, we applied a novel combination of external forces to subjects walking on a treadmill. We applied a forward pulling force at each foot to assist leg swing, a constant forward pulling force at the waist to provide center of mass propulsion, and a combination of these foot and waist forces to evaluate leg swing. When the metabolic cost and muscle actions were at a minimum, the condition was considered optimal. We reasoned that the difference in energy consumption between the optimal combined waist and foot force trial and the optimal waist force-only trial would reflect the metabolic cost of initiating and propagating leg swing during normal walking. We also reasoned that a lower muscle activity with these assisting forces would indicate which muscles are normally responsible for initiating and propagating leg swing. With a propulsive force at the waist of 10% body weight (BW), the net metabolic cost of walking decreased to 58% of normal walking. With the optimal combination, a propulsive force at the waist of 10% BW plus a pulling force at the feet of 3% BW the net metabolic cost of walking further decreased to 48% of normal walking. With the same combination, the muscle activity of the iliopsoas and rectus femoris muscles during the swing phase was 27 and 60% lower, respectively, but the activity of the medial gastrocnemius and soleus before swing did not change. Thus our data indicate that ∼10% of the net metabolic cost of walking is required to initiate and propagate leg swing. Additionally, the hip flexor muscles contribute to the initiation and propagation leg swing.
Initial (Latent) Polycythemia vera with Thrombocytosis Mimicking Essential Thrombocythemia
Authors: Thiele Jürgen , Kvasnicka Hans Michael , Diehl Volker
Published: June 2005

Patients have previously been described who showed clinical signs and symptoms suggesting essential thrombocythemia (ET), but later transformed to polycythemia vera (PV). From a series of 344 patients with a sustained borderline to moderate erythrocytosis, 44 failed to conform initially with the diagnostic criteria of the WHO for PV, because of their low hemoglobin level. Twenty-three patients of this group presented with a thrombocytosis exceeding 600 × 109/l and therefore suggested ET, but later developed full-blown PV. For comparison we investigated also 164 patients with manifest PV, 90 patients with ET and 22 patients with reactive thrombocytosis (Th). The histopathology of initial PV was evaluated by stepwise discriminant analysis of 17 standardized features. Quantity and left shifting of erythro- and granulopoiesis, giant forms and naked nuclei of megakaryocytes, cellularity and reticulin fibers proved to exert a significant relevance concerning differentiation from true ET and Th. In conclusion, initial PV with thrombocytosis is characterized by a special pattern of BM histopathology. Therefore, so-called masked PV in patients with ET or simultaneous PVR-1 gene expression and endogeneous erythroid colony growth in ET patients are probably in keeping with initial PV mimicking ET.