bluuebunny/crossref_metadata_2025 huggingface dataset:
UreR, the Transcriptional Activator of the
Proteus mirabilis
Urease Gene Cluster, Is Required for Urease Activity and Virulence in Experimental Urinary Tract Infections
Authors: Dattelbaum Jonathan D. , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T.
Published: January 2003

Proteus mirabilis
, a cause of complicated urinary tract infection, produces urease, an essential virulence factor for this species. UreR, a member of the AraC/XylS family of transcriptional regulators, positively activates expression of the
ure
gene cluster in the presence of urea. To specifically evaluate the contribution of UreR to urease activity and virulence in the urinary tract, a
ureR
mutation was introduced into
P. mirabilis
HI4320 by homologous recombination. The isogenic
ureR
::
aphA
mutant, deficient in UreR production, lacked measurable urease activity. Expression was not detected in the UreR-deficient strain by Western blotting with monoclonal antibodies raised against UreD. Urease activity and UreD expression were restored by complementation of the mutant strain with
ureR
expressed from a low-copy-number plasmid. Virulence was assessed by transurethral cochallenge of CBA mice with wild-type and mutant strains. The isogenic
ureR
::
aphA
mutant of HI4320 was outcompeted in the urine (
P
= 0.004), bladder (
P
= 0.016), and kidneys (
P
≤ 0.001) 7 days after inoculation. Thus, UreR is required for basal urease activity in the absence of urea, for induction of urease by urea, and for virulence of
P. mirabilis
in the urinary tract.
Human Eosinophil Peroxidase Induces Surface Alteration, Killing, and Lysis ofMycobacterium tuberculosis
Authors: Borelli Violetta , Vita Francesca , Shankar Sandeep , Soranzo Maria Rosa , Banfi Elena , Scialino Giuditta , Brochetta Cristiana , Zabucchi Giuliano
Published: January 2003

The antimycobacterial role of eosinophil peroxidase (EPO), one of the most abundant granule proteins in human eosinophils, was investigated. Our data indicate that purified EPO shows significant inhibitory activity towardsMycobacterium tuberculosisH37Rv. On a molar basis, this activity was similar to that exhibited by neutrophil myeloperoxidase (MPO) and was both dose and time dependent. In contrast to the activity of MPO, which requires H2O2, EPO also exhibited anti-M. tuberculosisactivity in the absence of exogenously added peroxide. Morphological evidence confirmed that the mechanism of action of EPO against mycobacteria differs from that of MPO. While MPO killsM. tuberculosisH37Rv exclusively in the presence of hydrogen peroxide, it does not induce morphological changes in the pathogen. In contrast, EPO-treated bacteria frequently had cell wall lesions and eventually underwent lysis, either in the presence or in the absence of H2O2.
Induction of Proinflammatory Cytokines in Human Lung Epithelial Cells duringChlamydia pneumoniaeInfection
Authors: Yang Jun , Hooper W. Craig , Phillips Donald J. , Tondella Maria L. , Talkington Deborah F.
Published: January 2003

Chlamydia pneumoniaeis an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established thatC. pneumoniaecan induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment ofC. pneumoniaesignificantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-α message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-β and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation ofC. pneumoniaeonly partially reduced the cytokine response, and inhibition ofC. pneumoniaeprotein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.
Clostridium sordellii
Phospholipase C: Gene Cloning and Comparison of Enzymatic and Biological Activities with Those of
Clostridium perfringens
and
Clostridium bifermentans
Phospholipase C
Authors: Karasawa Tadahiro , Wang Xingmin , Maegawa Tsuneo , Michiwa Yoshio , Kita Hiroyuki , Miwa Koichi , Nakamura Shinichi
Published: January 2003

The gene encoding
Clostridium sordellii
phospholipase C (Csp) was cloned and expressed as a histidine-tagged (His-tag) protein, and the protein was purified to compare its enzymatic and biological activities with those of
Clostridium perfringens
phospholipase C (Cpa) and
Clostridium bifermentans
phospholipase C (Cbp). Csp was found to consist of 371 amino acid residues in the mature form and to be more homologous to Cbp than to Cpa. The egg yolk phospholipid hydrolysis activity of the His-tag Csp was about one-third of that of His-tag Cpa, but the hemolytic activity was less than 1% of that of His-tag Cpa. His-tag Csp was nontoxic to mice. Immunization of mice with His-tag Cbp or His-tag Csp did not provide effective protection against the lethal activity of His-tag Cpa. These results indicate that Csp possesses similar molecular properties to Cbp and suggest that comparative analysis of toxic and nontoxic clostridial phospholipases is helpful for characterization of the toxic properties of clostridial phospholipases.
The
msbB
Mutant of
Neisseria meningitidis
Strain NMB Has a Defect in Lipooligosaccharide Assembly and Transport to the Outer Membrane
Authors: Post Deborah M. B. , Ketterer Margaret R. , Phillips Nancy J. , Gibson Bradford W. , Apicella Michael A.
Published: January 2003

A deletion-insertion mutation in
msbB
, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated
Neisseria meningitidis
serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galβ1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.
A/J Mice Are Susceptible and C57BL/6 Mice Are Resistant toListeria monocytogenesInfection by Intragastric Inoculation
Authors: Czuprynski Charles J. , Faith Nancy G. , Steinberg Howard
Published: January 2003

Previous studies demonstrated that the innate resistance of mice toListeria monocytogenesinfection by intravenous or intraperitoneal inoculation is regulated principally by theHclocus on mouse chromosome 2. The A/J and C57BL/6 mouse strains were identified as prototypeL. monocytogenes-susceptible and -resistant strains, respectively. In the present study, we compared the relative susceptibilities of A/J and C57BL/6 mice to intragastric (i.g.) inoculation withL. monocytogenes. The results of our study indicate that A/J mice are significantly more susceptible than C57BL/6 mice to an i.g. challenge withL. monocytogenes. This was reflected in the estimated 50% lethal doses for the two strains (106and 108CFU for A/J and C57BL/6 mice, respectively) and a more rapid and severe dissemination of the infection to the spleen and liver in A/J mice than in C57BL/6 mice. Histopathological examination of tissues from the infected mice confirmed the greater severity of disease in A/J mice. Clearance of a primary infection enhanced the resistance of both A/J and C57BL/6 mice to reinfection withL. monocytogenesvia the gastrointestinal tract. However, the relative difference in susceptibility between the two strains was evident even after immunization. The A/J mouse holds promise as a model for investigating the pathogenesis of gastrointestinal listeriosis because of its ability to develop systemic infection following challenge with numbers of organisms similar to those recovered from someL. monocytogenes-contaminated food products.
Lon, a Stress-Induced ATP-Dependent Protease, Is Critically Important for Systemic
Salmonella enterica
Serovar Typhimurium Infection of Mice
Authors: Takaya Akiko , Suzuki Masato , Matsui Hidenori , Tomoyasu Toshifumi , Sashinami Hiroshi , Nakane Akio , Yamamoto Tomoko
Published: January 2003

Studies on the pathogenesis of
Salmonella enterica
serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics—the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of
S. enterica
serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol.
184:
224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of
S. enterica
serovar Typhimurium in mice. Determination of 50% lethal doses for the
lon
disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the
lon
disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the
lon
disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with
S. enterica
serovar Typhimurium, which can be fatal. Of further interest is the finding that the
lon
disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.
Mycobacterium bovis
BCG Vaccines Exhibit Defects in Alanine and Serine Catabolism
Authors: Chen Jeffrey M. , Alexander David C. , Behr Marcel A. , Liu Jun
Published: January 2003

Mycobacterium bovis
BCG is the only accepted vaccine for the prevention of tuberculosis (TB) in humans. BCG is a live vaccine, and induction of immunity to TB requires productive infection of the host by BCG. However, BCG is not a satisfactory vaccine, because it fails to protect against pulmonary TB in adults. In this study, we found that BCG strains cannot utilize many naturally occurring amino acids as the sole nitrogen source for growth. This defect is caused, at least partially, by the lack of functional metabolic enzymes. All BCG strains are unable to catabolize
l
-alanine or
d
-alanine due to a frameshift mutation in the
l
-alanine dehydrogenase gene (
ald
). Some BCG strains, such as BCG-Pasteur and BCG-Frappier, cannot catabolize
l
-serine, apparently due to inadequate expression of
l
-serine deaminase (
sdaA
). We also found that undegraded alanine and serine inhibit the growth of BCG through blockage of glutamine synthetase. These results suggest that BCG strains are limited in nitrogen metabolic capacity and predict defects that may restrict multiplication and persistence of the live vaccine within the host.
Protective Levels of Diphtheria-Neutralizing Antibody Induced in Healthy Volunteers by Unilateral Priming-Boosting Intranasal Immunization Associated with Restricted Ipsilateral Mucosal Secretory Immunoglobulin A
Authors: Mills Kingston H. G. , Cosgrove Catherine , McNeela Edel A. , Sexton Amy , Giemza Rafaela , Jabbal-Gill Inderjit , Church Anne , Lin Wu , Illum Lisbeth , Podda Audino , Rappuoli Rino , Pizza Mariagrazia , Griffin George E. , Lewis David J. M.
Published: January 2003

Subunit intranasal vaccines offer the prospect of inducing combined systemic-mucosal immunity against mucosally transmitted infections such as human immunodeficiency virus. However, although human studies have demonstrated the induction of active immunity, secretory immunoglobulin A (sIgA) responses are variable, and no study has demonstrated protection by accepted vaccine-licensing criteria as measured by direct toxin-neutralizing activity. Using the genetically inactivated mutant diphtheria toxoid CRM
197
in a bioadhesive polycationic polysaccharide chitosan delivery system, we found that a single nasal immunization was well tolerated and boosted antitoxin neutralizing activity in healthy volunteers, which could be further boosted by a second immunization. The neutralizing activity far exceeded accepted protective levels and was equivalent to that induced by standard intramuscular vaccine and significantly greater than intranasal immunization with CRM
197
in the absence of chitosan. A striking but unexpected observation was that although unilateral intranasal immunization induced circulating antitoxin antibody-secreting cells, a nasal antitoxin sIgA response was seen only after the second immunization and only in the vaccinated nostril. If these data are reproduced in larger studies, an intranasal diphtheria vaccine based on CRM
197
-chitosan could be rapidly licensed for human use. However, a restricted sIgA response suggests that care must be taken in the priming-boosting strategy and clinical sampling techniques when evaluating such vaccines for the induction of local mucosal immunity.
Role of Systemic and Mucosal Immune Responses in Reciprocal Protection against
Bordetella pertussis
and
Bordetella parapertussis
in a Murine Model of Respiratory Infection
Authors: Watanabe Mineo , Nagai Masaaki
Published: January 2003

The roles of systemic humoral immunity, cell-mediated immunity, and mucosal immunity in reciprocal protective immunity against
Bordetella pertussis
and
Bordetella parapertussis
were examined by using a murine model of respiratory infection. Passive immunization with serum from mice infected with
B. pertussis
established protective immunity against
B. pertussis
but not against
B. parapertussis
. Protection against
B. parapertussis
was induced in mice that had been injected with serum from mice infected with
B. parapertussis
but not from mice infected with
B. pertussis
. Adoptive transfer of spleen cells from mice infected with
B. pertussis
or
B. parapertussis
also failed to confer reciprocal protection. To examine the role of mucosal immunity in reciprocal protection, mice were infected with preparations of either
B. pertussis
or
B. parapertussis
, each of which had been incubated with the bronchoalveolar wash of mice that were convalescing after infection with
B. pertussis
or
B. parapertussis
. Such incubation conferred reciprocal protection against
B. pertussis
and
B. parapertussis
on infected mice. The data suggest that mucosal immunity including secreted immunoglobulin A in the lungs might play an important role in reciprocal protective immunity in this murine model of respiratory infection.
Sensitivity of Polarized Epithelial Cells to the Pore-Forming Toxin Aerolysin
Authors: Abrami Laurence , Fivaz Marc , Glauser Pierre-Etienne , Sugimoto Nakaba , Zurzolo Chiara , van der Goot F. Gisou
Published: January 2003

Aerolysin is one of the major virulence factors produced by
Aeromonas hydrophila
, a human pathogen that produces deep wound infection and gastroenteritis. The toxin interacts with target mammalian cells by binding to the glycan core of glycosylphosphatidyl inositol (GPI)-anchored proteins and subsequently forms a pore in the plasma membrane. Since epithelial cells of the intestine are the primary targets of aerolysin, we investigated its effect on three types of polarized epithelial cells: Caco-2 cells, derived from human intestine; MDCK cells, a well-characterized cell line in terms of protein targeting; and FRT cells, an unusual cell line in that it targets its GPI-anchored proteins to the basolateral plasma membrane in contrast to other epithelial cells, which target them almost exclusively to the apical surface. Surprisingly, we found that all three cell types were sensitive to the toxin from both the apical and the basolateral sides. Apical sensitivity was always higher, even for FRT cells. In contrast, FRT cells were more sensitive from the basolateral than from the apical side to the related toxin
Clostridium septicum
alpha-toxin, which also binds to GPI-anchored proteins but lacks the lectin binding domain found in aerolysin. These observations are consistent with the notion that a shuttling mechanism involving low-affinity interactions with surface sugars allows aerolysin to gradually move toward the membrane surface, where it can finally encounter the glycan cores of GPI-anchored proteins.
Saccharomyces boulardiiInterferes with EnterohemorrhagicEscherichia coli-Induced Signaling Pathways in T84 Cells
Authors: Dahan Stephanie , Dalmasso Guillaume , Imbert Veronique , Peyron Jean-Francois , Rampal Patrick , Czerucka Dorota
Published: January 2003

EnterohemorrhagicEscherichia coli(EHEC) infections are associated with the modification of tight-junction permeability and synthesis of proinflammatory cytokine interleukin-8 (IL-8). In a previous study, it was demonstrated that EHEC-induced IL-8 secretion is due to the involvement of the mitogen-activated protein kinase (MAPK), AP-1, and NF-κB pathways. In this study, we investigated the effect of the yeastSaccharomyces boulardiion EHEC infection in T84 cells. For this purpose, cells were (i) incubated with bacteria and yeast at the same time or (ii) incubated overnight with yeast cells that were maintained during infection or eliminated by several washes before infection. Coincubation is sufficient to maintain the transmonolayer electrical resistance (TER) of EHEC-infected cells, whereas the preincubation of cells with the yeast without elimination of the yeast during infection is necessary to significantly decrease IL-8 secretion. We thus analyzed the mechanisms ofS. boulardiiaction. We showed thatS. boulardiihas no effect on EHEC growth or on EHEC adhesion. Kinetics studies revealed that EHEC-induced myosin light chain (MLC) phosphorylation precedes the decrease of TER. ML-7, an MLC kinase inhibitor, abolishes the EHEC-induced MLC phosphorylation and decrease of TER. Studies show thatS. boulardiialso abolishes EHEC-induced MLC phosphorylation. We demonstrated that the preincubation of cells withS. boulardiiwithout washes before EHEC infection inhibits NF-κB DNA binding activity, phosphorylation and degradation of IκB-α, and activation of the three members of a MAPK group (extracellular signal-regulated protein kinases 1 and 2, p38, and c-jun N-terminal kinase). These findings demonstrate thatS. boulardiiexerts a preventive effect on EHEC infection by (i) interfering with one of the transduction pathways implicated in the control of tight-junction structure and (ii) decreasing IL-8 proinflammatory secretion via inhibition of the NF-κB and MAPK signaling pathways in infected T84 cells.
Streptococcus pyogenesGlycoprotein-Binding Strepadhesin Activity Is Mediated by a Surface-Associated Carbohydrate-Degrading Enzyme, Pullulanase
Authors: Hytönen Jukka , Haataja Sauli , Finne Jukka
Published: January 2003

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel antiadhesive drugs and vaccines. We have previously identified strepadhesin, a novel glycoprotein-binding activity inStreptococcus pyogenesbinding to thyroglobulin, submaxillar mucin, fetuin, and asialofetuin. The activity is known to be regulated by Mga, a regulator of streptococcal virulence factors, and is carried by the surface-associated streptococcal cysteine protease, SpeB. In the present study, we focused on the high strepadhesin activity in anS. pyogenesstrain (NZ131rgg) lacking SpeB expression. By extracting surface proteins from the bacteria, a new strepadhesin protein was identified, and mass spectrometric analysis and database search identified it as a putative pullulanase. The gene was cloned, and the recombinant pullulanase (PulA) exhibited pullulanase and starch hydrolyzing activity, as well as strepadhesin activity. Sequencing of thepulAgene revealed an open reading frame with 3,498 bp encoding a protein of 1,165 amino acids with a predicted molecular mass of 129 kDa. PulA exhibited properties typical for a gram-positive surface protein with a putative signal sequence and LPKTGE cell wall anchoring motif and contained the four highly conserved regions common to pullulanases. Mutant bacteria deficient in PulA expression showed diminished strepadhesin activity on bacterial dot blot assay and reduced adherence to thyroglobulin immobilized on microtiter plates. Thus,S. pyogenesstrepadhesin activity is carried by a surface-bound pullulanase, which combines glycoprotein-binding and carbohydrate-degrading activities in the same molecule.
Five-Member Gene Family of
Bartonella quintana
Authors: Minnick Michael F. , Sappington Kate N. , Smitherman Laura S. , Andersson Siv G. E. , Karlberg Olof , Carroll James A.
Published: January 2003

Bartonella quintana
, the agent of trench fever and an etiologic agent of bacillary angiomatosis, has an extraordinarily high hemin requirement for growth compared to other bacterial pathogens. We previously identified the major hemin receptor of the pathogen as a 30-kDa surface protein, termed HbpA. This report describes four additional homologues that share approximately 48% amino acid sequence identity with
hbpA
. Three of the genes form a paralagous cluster, termed
hbpCAB
, whereas the other members,
hbpD
and
hbpE
, are unlinked. Secondary structure predictions and other evidence suggest that Hbp family members are β-barrels located in the outer membrane and contain eight transmembrane domains plus four extracellular loops. Homologs from a variety of gram-negative pathogens were identified, including
Bartonella henselae
Pap31,
Brucella
Omp31,
Agrobacterium tumefaciens
Omp25, and neisserial opacity proteins (Opa). Family members expressed in vitro-synthesized proteins ranging from ca. 26.5 to 35.1 kDa, with the exception of HbpB, an ∼55.9-kDa protein whose respective gene has been disrupted by a ∼510 GC-rich element containing variable-number tandem repeats. Transcription analysis by quantitative reverse transcriptase-PCR (RT-PCR) indicates that all family members are expressed under normal culture conditions, with
hbpD
and
hbpB
transcripts being the most abundant and the rarest, respectively. Mutagenesis of
hbpA
by allelic exchange produced a strain that exhibited an enhanced hemin-binding phenotype relative to the parental strain, and analysis by quantitative RT-PCR showed elevated transcript levels for the other
hbp
family members, suggesting that compensatory expression occurs.
Induction by Sphingomyelinase of Shiga Toxin Receptor and Shiga Toxin 2 Sensitivity in Human Microvascular Endothelial Cells
Authors: Obrig T. G. , Seaner R. M. , Bentz M. , Lingwood C. A. , Boyd B. , Smith A. , Narrow W.
Published: January 2003

Shiga toxin-producing enterohemorrhagic
Escherichia coli
is the major cause of acute renal failure in young children. The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome. Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins. In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2. Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours. Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2. A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway. The product of CGT (glucosylceramide) was also increased. In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC. These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.
Protection of Chickens againstEscherichia coliInfections by DNA Containing CpG Motifs
Authors: Gomis Susantha , Babiuk Lorne , Godson Dale L. , Allan Brenda , Thrush Tannis , Townsend Hugh , Willson Philip , Waters Edwin , Hecker Rolf , Potter Andrew
Published: January 2003

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate ofEscherichia coliwas applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after theE. colichallenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN(2007)had a survival rate of 15%. In contrast, groups that received CpG-ODN(2007), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P< 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN(2007)by the subcutaneous route (P< 0.01). A dose of as little as 3.16 μg of CpG-ODN(2007), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds againstE. coliinfection (P< 0.01). This study demonstrates that CpG-ODN(2007)has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN(2007)has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.
Disruption of Epithelial Barrier Integrity bySalmonella entericaSerovar Typhimurium Requires Geranylgeranylated Proteins
Authors: Tafazoli Farideh , Magnusson Karl-Eric , Zheng Limin
Published: January 2003

Epithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such asSalmonellaandYersiniaspp., have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasiveSalmonella entericaserovar Typhimurium SL1344 exhibited marked changes in F-actin organization, an increase in the paracellular flux of dextran, and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with an isogenic noninvasive mutant (hilA) increased the TER in these cells. Pretreating MDCK-1 cells with the inhibitors for tyrosine kinase (genistein) or phosphatidylinositol 3-kinase (wortmannin) did not affect invasion and subsequent perturbation of the epithelial barrier by serovar Typhimurium. Instead, the geranylgeranyltransferase 1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of serovar Typhimurium to disrupt the epithelial barrier. The substrates for GGTI-298 include Rho family GTPases, as indicated by inhibiting prenylation of Rac1 and Cdc42. Infection with wild-type serovar Typhimurium increased the level of activated Rac1 and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. ThisSalmonella-induced accumulation of Rac1 and Cdc42 and alteration of the junction-associated proteins ZO-1, occludin, and E-cadherin in MDCK-1 cells were markedly inhibited by GGTI-298. These results suggest that activation of geranylgeranylated proteins, including Rac1 and Cdc42, is critical for disruption of barrier integrity by serovar Typhimurium in polarized MDCK-1 cells.
Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model
Authors: Kadurugamuwa Jagath L. , Sin Lin , Albert Eddie , Yu Jun , Francis Kevin , DeBoer Monica , Rubin Michael , Bellinger-Kawahara Carole , Parr, T. R. , Contag Pamela R.
Published: January 2003

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens,
Staphylococcus aureus
and
Pseudomonas aeruginosa
, were made bioluminescent by insertion of a complete
lux
operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (
S. aureus r
= 0.98;
P. aeruginosa r
= 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10
3
to 10
5
CFU/catheter for
S. aureus
and
P. aeruginosa
resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the
lux
constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.
Colonial Morphology of
Burkholderia cepacia
Complex Genomovar III: Implications in Exopolysaccharide Production, Pilus Expression, and Persistence in the Mouse
Authors: Chung Jacqueline W. , Altman Eleonora , Beveridge Terry J. , Speert David P.
Published: January 2003

The purpose of this study was to determine the role of colonial morphology of
Burkholderia cepacia
complex (BCC) organisms in pathogenicity in a mouse model of pulmonary infection. BCC strain C1394 was rapidly cleared by leukopenic mice after intranasal challenge, whereas a spontaneous variant (C1394mp2) that was indistinguishable from the parent strain by genetic typing persisted in the lungs and differed in colonial morphology. The parent strain had a matte colonial phenotype, made scant exopolysaccharide (EPS), and was lightly piliated. The variant had a shiny phenotype, produced abundant EPS, and was heavily piliated. Matte to shiny colonial transformation was induced by growth at 42°C. Colonial morphology in the BCC strain variant was associated with persistence after pulmonary challenge and appeared to be correlated with the elaboration of putative virulence determinants.
Vaccine-Induced Reduction ofHelicobacter pyloriColonization in Mice Is Interleukin-12 Dependent but Gamma Interferon and Inducible Nitric Oxide Synthase Independent
Authors: Garhart Christine A. , Heinzel Frederick P. , Czinn Steven J. , Nedrud John G.
Published: January 2003

Previous studies with mice have shown that major histocompatibility complex class II (MHC-II) is required for protection fromHelicobacter pylori, while MHC-I and antibodies are not. Thus, CD4+T cells are presumed to play an essential role in protective immunity via secretion of cytokines. To determine which cytokines are associated with a reduction of bacterial load in immunized mice, gastric cytokine expression was examined by semiquantitative reverse transcription-PCR in protected (defined as ≥2-log-unit decrease in bacterial load) and unprotected mice 4 weeks after challenge. Elevated levels of mRNA for interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) were associated with protection in immunized-challenged (I/C) mice, but Th2 cytokine (IL-4, IL-5, IL-10, and IL-13) and chemokine (KC, MIP-2, and MCP-1) expression was not associated with protection. Despite the association of IFN-γ and iNOS message with protection, I/C mice genetically lacking either of these products were able to reduce the bacterial load as well as the wild-type I/C controls. The I/C mice lacking IL-12p40 were not protected compared to unimmunized-challenged mice. All I/C groups developed gastritis. We conclude that neither IFN-γ nor iNOS is essential for vaccine-induced protection fromH. pyloriinfection. The p40 subunit of IL-12, which is a component of both IL-12 and IL-23, is necessary for protection in immunized mice. These findings suggest a novel IFN-γ-independent function of IL-12p40 in effective mucosal immunization againstH. pylori.
Phagocytic Activity and Monocyte Chemotactic Protein Expression by Pulmonary Macrophages in Persistent Pulmonary Cryptococcosis
Authors: He Wu , Casadevall Arturo , Lee Sunhee C. , Goldman David L.
Published: January 2003

The mechanisms by whichCryptococcus neoformanspersists in an immunocompetent host are not well understood. Using a rat model of persistent infection, we investigated the ability of pulmonary macrophages (PuM) to phagocytizeC. neoformansand produce monocyte chemotactic protein 1 (MCP-1) as a function of the length of time of infection and opsonin. The ability of macrophages to affect serum-mediated phagocytosis varied over the course of infection and was dependent on CD11b/c and CD18 expression. Infection resulted in increased MCP-1 levels within the lung, though the actual amounts varied over the course of infection. Immunohistochemical studies localized MCP-1 expression to macrophages and epithelioid cells. Enhanced production of MCP-1 by PuM from infected rats was confirmed by ex vivo studies. Induction of MCP-1 following serum-mediated phagocytosis was observed for PuM from both infected and noninfected rats and depended on the interaction ofC. neoformanswith CD11b/c and CD18. Specific antibody was more efficient than serum in promoting phagocytosis and consistently elicited more MCP-1. The relative amount of MCP-1 produced in association with phagocytosis was similar for PuM at all lengths of time of infection. Decreased MCP-1 production was observed for PuM obtained from older rats, including long-term (8 to 10 months)-infected and age-matched controls, suggesting that aging may affect the production of MCP-1 by PuM in response to cryptococcal infection. In summary, our results show that macrophages are an important source of MCP-1 during pulmonary cryptococcosis and that MCP-1 production is actively regulated during infection. Furthermore, we find that phagocytosis ofC. neoformanscan serve as an important stimulus for MCP-1 production by PuM, though the efficiency of this process is dependent on the opsonin type and may be affected by aging.
Infection byStreptococcus pyogenesInduces the Receptor Activator of NF-κB Ligand Expression in Mouse Osteoblastic Cells
Authors: Okahashi Nobuo , Sakurai Atsuo , Nakagawa Ichiro , Fujiwara Taku , Kawabata Shigetada , Amano Atsuo , Hamada Shigeyuki
Published: January 2003

Group AStreptococcus pyogenesis known to induce nongonococcal septic arthritis in addition to pharyngitis, scarlet fever, and poststreptococcal sequelae. However, little is known about the interaction betweenS. pyogenesand bone cells. We report here thatS. pyogenesstrain JRS4 (M6) attached to and invaded mouse primary osteoblasts. Reverse transcription-PCR demonstrated thatS. pyogenesinfection of osteoblasts stimulated expression of mRNA for the receptor activator of NF-κB ligand (RANKL). Western blot analysis followed by ligand precipitation with the receptor activator of NF-κB receptor showed that there was an increase in RANKL protein in infected osteoblasts. Production of interleukin-6 was also stimulated, but no production of interleukin-1β or tumor necrosis factor alpha was observed. Stimulation of RANKL production was not observed in osteoblasts stimulated with heat-inactivatedS. pyogenes, suggesting that an active interaction ofS. pyogeneswith osteoblasts is essential for this phenomenon. A Western blot analysis performed with antibodies specific for phosphorylated signal transduction proteins demonstrated thatS. pyogenesinfection induces phosphorylation of p38 mitogen-activated protein kinase. A specific inhibitor of this kinase, SB203580, inhibited RANKL production by infected osteoblasts. These results suggest that infection of osteoblasts byS. pyogenesstimulates RANKL production and may trigger bone destruction in infected bone tissue.
Immunohistochemical Evaluation of T Cells in Oral Lesions from Human Immunodeficiency Virus-Positive Persons with Oropharyngeal Candidiasis
Authors: Myers Tammy A. , Leigh Janet E. , Arribas Alfredo R. , Hager Shannon , Clark Rebecca , Lilly Elizabeth , Fidel, Paul L.
Published: January 2003

Oropharyngeal candidiasis (OPC), caused byCandida albicans, is the most frequent opportunistic fungal infection in human immunodeficiency virus (HIV)-positive persons. Although Th1-type CD4+T cells are considered important for host defense against mucosalC. albicansinfections, there is a paucity of information regarding the presence and/or role of T cells in OPC lesions. In pursuit of this, initial chromophore immunohistochemical studies showed a majority of CD8+rather than CD4+cells equally distributed throughout the buccal mucosa of OPC−persons (HIV−or HIV+), irrespective of blood CD4+cell numbers. In contrast, CD8+cells in lesions from HIV+OPC+persons were in significantly higher numbers and concentrated at the lamina propria-epithelium interface, a considerable distance from theCandidaat the outer epithelium. Dual fluorescence and confocal microscopy confirmed that the majority of CD8+, but not CD4+, cells were T cells by the presence or absence, respectively, of CD3 on each cell type. These results suggest that CD8+T cells may be important for oral host defense against OPC, especially when CD4 cell numbers are reduced, with a potential CD8 cell-specific dysfunction associated with susceptibility to OPC.
Apoptotic Killing and Phagocytosis of Host Cells by the ParasiteEntamoeba histolytica
Authors: Huston Christopher D. , Boettner Douglas R. , Miller-Sims Vanessa , Petri, William A.
Published: January 2003

The ability ofEntamoeba histolyticato kill and phagocytose host cells correlates with parasite virulence. This study addressed the role of apoptotic cell killing and host cell phosphatidylserine exposure in the subsequent phagocytosis of Jurkat T cells byE. histolytica. Ingested host cells were apoptotic, as evidenced by the activation of caspase 3 in 88% ± 3% (mean and standard deviation [SD] of the mean) of Jurkat cells engulfed byE. histolytica; ingested cells without detectable active caspase 3 were already disrupted and partially digested. That apoptotic cell killing preceded phagocytosis was supported by the demonstration that a higher percentage of amebae ingested apoptotic cells than ingested healthy cells (62% ± 7% versus 30% ± 9%, respectively [mean and SD]) (P= 0.008).E. histolyticaalso ingested apoptotic Jurkat cells more rapidly than necrotic control cells (8.5% ± 0.4% versus 3.5% ± 0.7%, respectively [mean and SD]) (P< 0.001). The inhibition of amebic cytotoxicity withd-galactose (which blocks the amebic Gal/GalNAc lectin) blocked the phagocytosis of healthy cells by greater than 80%, providing further evidence that apoptosis preceded engulfment. In contrast,d-galactose blocked the phagocytosis of already apoptotic cells by only 40%, implicating an additional host ligand (besidesd-galactose) in amebic engulfment of apoptotic cells. The most characteristic surface change on apoptotic cells is phosphatidylserine exposure. Consistent with a role for host cell phosphatidylserine exposure in amebic ingestion of killed cells, Jurkat cell phosphatidylserine was exposed during incubation withE. histolytica(27% ± 1% [mean and SD] specific increase at 30 min) (thePvalue versus the control was 0.0003). Approximately 50% more amebae ingested viable Jurkat cells expressing phosphatidylserine on the outer leaflet of the plasma membrane than ingested control cells (30.3% ± 2.2% versus 19.8% ± 1.9%, respectively [mean and SD]) (P= 0.003). By analogy with phagocytic clearance during apoptosis in metazoans, amebic apoptotic host cell killing followed by phagocytosis may limit inflammation and enable amebae to evade the host immune response.
Restricted Fusion of
Chlamydia trachomatis
Vesicles with Endocytic Compartments during the Initial Stages of Infection
Authors: Scidmore Marci A. , Fischer Elizabeth R. , Hackstadt Ted
Published: January 2003

The chlamydial inclusion occupies a unique niche within the eukaryotic cell that does not interact with endocytic compartments but instead is fusogenic with a subset of sphingomyelin-containing exocytic vesicles. The
Chlamydia trachomatis
inclusion acquires these distinctive properties by as early as 2 h postinfection as demonstrated by the ability to acquire sphingomyelin, endogenously synthesized from 6{
N
-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproylsphingosine} (C
6
-NBD-ceramide). The molecular mechanisms involved in transformation of the properties and cellular interactions of the inclusion are unknown except that they require early chlamydial transcription and translation. Although the properties of the inclusion are established by 2 h postinfection, the degree of interaction with endocytic pathways during the brief interval before fusogenicity with an exocytic pathway is established is unknown. Using a combination of confocal and electron microscopy to localize endocytic and lysosomal markers in
C. trachomatis
infected cells during the early stages of infection, we demonstrate a lack of these markers within the inclusion membrane or lumen of the inclusion to conclude that the nascent chlamydial inclusion is minimally interactive with endosomal compartments during this interval early in infection. Even when prevented from modifying the properties of the inclusion by incubation in the presence of protein synthesis inhibitors, vesicles containing elementary bodies are very slow to acquire lysosomal characteristics. These results imply a two-stage mechanism for chlamydial avoidance of lysosomal fusion: (i) an initial phase of delayed maturation to lysosomes due to an intrinsic property of elementary bodies and (ii) an active modification of the vesicular interactions of the inclusion requiring chlamydial protein synthesis.
DEVIATION ALGORITHMS FOR RANKING SHORTEST PATHS
Authors: DE QUEIRÓS VIEIRA MARTINS ERNESTO , PASCOAL MARTA MARGARIDA BRAZ , SANTOS JOSÉ LUIS ESTEVES DOS
Published: January 2003

The shortest path problem is a classical network problem that has been extensively studied. The problem of determining not only the shortest path, but also listing the K shortest paths (for a given integer K>1) is also a classical one but has not been studied so intensively, despite its obvious practical interest. Two different types of problems are usually considered: the unconstrained and the constrained K shortest paths problem. While in the former no restriction in considered in the definition of a path, in the constrained K shortest paths problem all the paths have to satisfy some condition – for example, to be loopless.

In this paper new algorithms are proposed for the uncontrained problem, which compute a super set of the K shortest paths. It is also shown that ranking loopless paths does not hold in general the Optimality Principle and how the proposed algorithms for the unconstrained problem can be adapted for ranking loopless paths.
SELF-SPECIFYING MACHINES
Authors: HEMASPAANDRA LANE A. , HEMPEL HARALD , WECHSUNG GERD
Published: January 2003

We study the computational power of machines that specify their own acceptance types, and show that they accept exactly the languages that [Formula: see text]-reduce to NP sets. A natural variant accepts exactly the languages that [Formula: see text]-reduce to P sets. We show that these two classes coincide if and only if [Formula: see text], where the latter class denotes the sets acceptable via at most one question to #P followed by at most a constant number of questions to NP.
OPTIMAL LAYOUT OF TRIVALENT CAYLEY INTERCONNECTION NETWORKS
Authors: CALAMONERI TIZIANA , PETRESCHI ROSSELLA
Published: January 2003

In this paper we deal with the layout of Trivalent Cayley Interconnection Networks. Namely, we prove that a lower bound on their layout area is Ω(2n-1× 2n-1) and we exhibit some methods to lay these networks out in O(2n-1× 2n-1).

We piont out that these layout methods work for other networks decomposable into equal length cycles connected by following fixed rules.
THE ISOPERIMETRIC NUMBER OF d–DIMENSIONAL k–ARY ARRAYS
Authors: AZIZOĞLU M. CEMIL , EĞECIOĞLU ÖMER
Published: January 2003

The d–dimensional k-ary array [Formula: see text] is the d–fold Cartesian product graph of the path graph Pk with k vertices. We show that the (edge) isoperimetric number [Formula: see text] of [Formula: see text] is given by [Formula: see text] and identify the cardinalities and the structure of the isoperimetric sets. For odd k, the cardinalities of isoperimetric sets in [Formula: see text] are [Formula: see text], whereas every isoperimetric set for k even has cardinality [Formula: see text].
ON GROUPING IN RELATIONAL ALGEBRA
Authors: LARSEN KIM S.
Published: January 2003

The concept of grouping in relational algebra is well-known from its connection to aggregation. In this paper we generalize the grouping notion by defining a simultaneous grouping of more than one relation, and we discuss the application of operations on grouping elements other than just arithmetic aggregation. Finally we show that this grouping mechanism can be added to relational algebra without increasing its computational power.