bluuebunny/crossref_metadata_2025 huggingface dataset:
PCR-Oligonucleotide Ligation Assay for Detection of Point Mutations Associated with Quinolone Resistance in
Streptococcus pneumoniae
Authors: Bui Mai-Ha , Stone Gregory G. , Nilius Angela M. , Almer Laurel , Flamm Robert K.
Published: March 2003

We have developed a PCR-oligonucleotide ligation assay to rapidly identify base substitutions in topoisomerase genes that are associated with quinolone resistance in clinical isolates of
Streptococcus pneumoniae
. Thirty-seven strains for which the ciprofloxacin MICs were ≥4 μg/ml and 16 strains for which the MICs were ≤2 μg/ml were assayed. Compared with sequence data, the assay correctly identified the DNA bases that encoded amino acids at the four positions most commonly associated with quinolone resistance (Ser79 and Asp83 of ParC and Ser81 and Glu85 of GyrA). Therefore, this procedure can rapidly distinguish single base substitutions associated with quinolone-resistant topoisomerases in
S. pneumoniae
.
mecA
-
blaZ
Corepressors in Clinical
Staphylococcus aureus
Isolates
Authors: Rosato Adriana E. , Kreiswirth Barry N. , Craig William A. , Eisner William , Climo Michael W. , Archer Gordon L.
Published: March 2003

The presence and nucleotide sequences of the two
mecA
repressors,
mecI
and
blaI
, were assessed in 73 clinical
Staphylococcus aureus
isolates. Isolates with
mecI
mutations were grouped into unique clonal types based on their
spa
nucleotide repeat patterns. Forty-three of the 45 (96%) isolates with mutant
mecI
or with a deletion of
mecI
contained
blaI
, while
blaI
was present in only 21 of 28 (78%) isolates with wild-type
mecI
(
P
< 0.05). Among 22 additional isolates that did not contain
blaI
, all had wild-type
mecI
sequences. We conclude that oxacillin-resistant
S. aureus
must have at least one of the two functional
mecA
regulators.
Frequency of Mutation to Rifampin Resistance in
Streptococcus pneumoniae
Clinical Strains:
hexA
and
hexB
Polymorphisms Do Not Account for Hypermutation
Authors: Morosini María-Isabel , Baquero María-Rosario , Sánchez-Romero J. M. , Negri María-Cristina , Galán Juan-Carlos , del Campo Rosa , Pérez-Díaz J. C. , Baquero Fernando
Published: March 2003

The frequency of mutation to rifampin resistance of 200 clinical
Streptococcus pneumoniae
isolates was examined. Two peaks were observed in the distribution, with mode frequencies of 2.5 × 10
−7
(20% of isolates) and 2.5 × 10
−8
. The
hexA
and
hexB
gene entire sequences were analyzed in 13 isolates. Sequences from both hypermutable and “normomutable” strains were conserved relative to that of the R6
S. pneumoniae
control strain. The phenotypic Hex system proficiency, in terms of transforming efficiency, was also maintained irrespective of the variations in mutation frequency values.
Interactions of 1263W94 with Other Antiviral Agents in Inhibition of Human Cytomegalovirus Replication
Authors: Selleseth Dean W. , Talarico Christine L. , Miller Teresa , Lutz Michael W. , Biron Karen K. , Harvey Robert J.
Published: March 2003

Inhibition of human cytomegalovirus (HCMV) by 1263W94 was additive dosewise in combination with ganciclovir, acyclovir, and foscarnet. None of the commonly used anti-human immunodeficiency virus agents antagonized the inhibition of HCMV by 1263W94. The data were analyzed by a modified isobologram procedure that measures the strength and statistical significance of drug interactions.
Significance of Genomic DNA Group Delineation in Comparative Studies of Antimicrobial Susceptibility of
Acinetobacter
spp
Authors: Houang Elizabeth T. S. , Chu Yiu Wai , Chu K. Y. , Ng K. C. , Leung C. M. , Cheng A. F. B.
Published: March 2003

There were significant differences in antimicrobial susceptibilities in isolates of genomic DNA groups 2 (
Acinetobacter baumannii
), 3, and 13TU collected from the same sources, e.g., patients in intensive care units and general wards, and in isolates of the same group collected from different sources. The delineation of genomic groups is important in comparative surveillance studies of antimicrobial susceptibilities.
Pathogenic
Nocardia
,
Rhodococcus
, and Related Organisms Are Highly Susceptible to Imidazole Antifungals
Authors: Dabbs Eric R. , Naidoo Samantha , Lephoto Catherine , Nikitina Natalya
Published: March 2003

Rhodococcus equi
and species of
Nocardia
and
Gordonia
may be human opportunistic pathogens. We find that these, as well as several isolates from closely related genera, are highly susceptible to the imidazoles bifonazole, clotrimazole, econazole, and miconazole, whose MICs are ≤1 μg/ml. In liquid cultures 1 μg of the drug/ml was bacteriostatic and 10 μg/ml was bactericidal. On solid media at 10 μg of azole/ml no resistant mutants could be isolated. An MIC of 1 to 15 μg/ml was observed with ketoconazole, whereas none of these organisms was inhibited by the triazoles fluconazole and voriconazole (100 μg/ml). Imidazoles may offer the prospect of treatment of nocardioform mycetomas and may provide the basis for the development of additional antimicrobial agents to combat these pathogens.
Flagella Are Virulence Determinants of
Burkholderia pseudomallei
Authors: Chua K. L. , Chan Y. Y. , Gan Y. H.
Published: March 2003

Burkholderia pseudomallei
, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans. Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of an organism to cause disease. We used a virulent isolate of
B. pseudomallei
, KHW, to construct an isogenic deletion mutant with a mutation in the flagellin gene (
fliC
) by gene replacement transposon mutagenesis. The KHWΔ
fliC
Km mutant was aflagellate and nonmotile in semisolid agar. The isogenic KHWΔ
fliC
Km mutant was not impaired in terms of the ability to invade and replicate in cultured human lung cells compared with the wild type. It was also equally virulent in slow-killing assays involving
Caenorhabditis elegans
, but it was avirulent during intranasal infection of BALB/c mice. Very few bacteria, if any, were isolated from the lungs and spleens of KHWΔ
fliC
Km
-
infected mice. In contrast, the bacterial loads in the lungs and spleens were similar in mice infected with KHW and in mice infected with the complemented mutant, KHWΔ
fliC
Km/pUCP28T
fliC.
Unlike the Syrian hamster or diabetic rat models of infection, the
B. pseudomallei
flagellin was also a virulence factor during intraperitoneal infection of BALB/c mice. In this study, all animals infected with KHWΔ
fliC
Km remained healthy and did not succumb to disease regardless of the route of infection. The flagellum is therefore an important and necessary virulence determinant of
B. pseudomallei
during intranasal and intraperitoneal infection of mice.
Interleukin-18 Impairs the Pulmonary Host Response toPseudomonas aeruginosa
Authors: Schultz Marc J. , Knapp Sylvia , Florquin Sandrine , Pater Jennie , Takeda Kiyoshi , Akira Shizuo , van der Poll Tom
Published: March 2003

Interleukin-18 (IL-18) is a potent cytokine with many different proinflammatory activities. To study the role of IL-18 in the pathogenesis ofPseudomonaspneumonia, IL-18-deficient (IL-18−/−) and wild-type mice were intranasally inoculated withPseudomonasaeruginosa. IL-18 deficiency was associated with reduced outgrowth ofPseudomonasin the lungs and diminished dissemination of the infection. In addition, pulmonary inflammation (histopathology) and levels of tumor necrosis factor alpha, IL-6, and macrophage inflammatory protein-2 in lungs and plasma were lower inIL-18−/−mice. Consistent with results obtained forIL-18−/−mice, treatment of wild-type mice with a neutralizing IL-18 binding protein-immunoglobulin G Fc fusion construct also attenuated outgrowth ofPseudomonascompared with that for mice treated with a control protein. These results demonstrate that the presence of endogenous IL-18 activity facilitates inflammatory responses in the lung duringPseudomonaspneumonia, concurrently impairing bacterial clearance.
Prevalence and Distribution of Adhesins in Invasive Non-Type b Encapsulated
Haemophilus influenzae
Authors: Rodriguez Carina A. , Avadhanula Vasanthi , Buscher Amy , Smith Arnold L. , St. Geme Joseph W. , Adderson Elisabeth E.
Published: March 2003

Adhesion to the respiratory epithelium plays an important role in
Haemophilus influenzae
infection. The distribution of
H. influenzae
adhesins in type b and nontypeable strains has been characterized, but little is known about the prevalence of these factors in non-type b encapsulated strains. We analyzed 53 invasive type a, type e, and type f strains for the presence of
hap
,
hia
,
hmw
, and
hif
genes; Hap, Hia, and HMW1/2 adhesins; and hemagglutinating pili. The
hap
gene was ubiquitous, and homologs of
hmw
and
hia
were present in 7 of 53 (13.2%) and 45 of 53 (84.9%) strains, respectively. Hap was detected in 28 of 45 (62.2%)

hap
+

strains, HMW1/2 was detected in 5 of 7 (71.4%)

hmw
+

strains, and Hia was detected in 31 of 45 (68.8%)

hia
+

strains. The
hif
gene cluster was present in 26 of 53 strains (49.1%), and 21 of 26

hif
+

strains (80.8%) agglutinated (HA) red blood cells. Nine isolates exhibited HA but lacked the
hif
gene cluster. The distribution of adhesin genes correlated with the genetic relatedness of the strains. Strains belonging to one type a clonotype and the major type e clonotype possessed
hia
but lacked the
hif
cluster. Strains belonging to the second type a clonotype possessed both
hia
and
hif
genes. All type f strains belonging to the major type f clonotype possessed
hia
and lacked
hifB
. Although the specific complement of adhesin genes in non-type b encapsulated
H. influenzae
varies, most invasive strains express Hap and Hia, suggesting these adhesins may be especially important to the virulence of these organisms.
Effect of Interleukin-8 and Granulocyte Colony-Stimulating Factor on Priming and Activation of Bovine Neutrophils
Authors: Mitchell Gordon B. , Albright Betty N. , Caswell Jeff L.
Published: March 2003

Neutrophils are important effector cells in innate and acquired immunity, but the magnitude and character of their phagocytic and bactericidal responses depend on cues derived from mediators in the local microenvironment. This study investigated the effect of bovine interleukin-8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) on priming and activation of bovine neutrophils in vitro and in vivo. Neutrophils were isolated from blood and cultured for up to 18 h, with or without cytokines, and thenMannheimia haemolytica-induced oxidative burst and phagocytosis ofStaphylococcus aureuswere measured by flow cytometry. Neither IL-8 nor G-CSF directly triggered an oxidative burst, but incubation with these cytokines for 18 h primed neutrophils for a greater oxidative burst triggered byM. haemolyticaand for enhanced uptake ofS. aureus. The maximal response was observed when neutrophils were incubated with both cytokines together, at concentrations of 200 ng/ml for G-CSF and 400 ng/ml for IL-8. The IL-8-induced priming effect was reduced by treatment with a neutralizing antibody to IL-8, and was not attributed to endotoxin contamination. Instillation of IL-8 into the lung using a bronchoscope induced neutrophil recruitment within 18 h. Neutrophils from IL-8-treated lung showed dose-dependent enhancement of the oxidative burst triggered byM. haemolytica. Histologically, neutrophils filled alveoli and bronchioles, and scattered macrophages contained neutrophils with morphological features of apoptosis. Thus, prolonged in vitro or in vivo exposure to IL-8 and/or G-CSF enhances the subsequent oxidative burst and phagocytic responses of bovine neutrophils.
Cross-Reactivity of Antibodies against PorA after Vaccination with a Meningococcal B Outer Membrane Vesicle Vaccine
Authors: Vermont C. L. , van Dijken H. H. , Kuipers A. J. , van Limpt C. J. P. , Keijzers W. C. M. , van der Ende A. , de Groot R. , van Alphen L. , van den Dobbelsteen G. P. J. M.
Published: March 2003

The cross-reactivity of PorA-specific antibodies induced by a monovalent P1.7-2,4 (MonoMen) and/or a hexavalent (HexaMen) meningococcal B outer membrane vesicle vaccine (OMV) in toddlers and school children was studied by serum bactericidal assays (SBA). First, isogenic vaccine strains and PorA-identical patient isolates were compared as a target in SBA, to ensure that the vaccine strains are representative for patient isolates. Geometric mean titers (GMTs) in SBA against patient isolates with subtypes P1.5-2,10 and P1.5-1,2-2 after vaccination with HexaMen were generally lower than those against vaccine strains with the same subtype, although the percentage of vaccine responders (≥4-fold increase in SBA after vaccination) was not affected. Using various P1.7-2,4 patient isolates, GMTs as well as the number of vaccine responders were higher than for the P1.7-2,4 vaccine strain, indicating that the use of the P1.7-2,4 vaccine strain may have underestimated the immunogenicity of this subtype in HexaMen. Secondly, the cross-reactivity of antibodies induced by MonoMen and HexaMen was studied using several patient isolates that differed from the vaccine subtypes by having minor antigenic variants of one variable region (VR), by having a completely different VR or by having a different combination of VRs. MonoMen induced P1.4-specific antibodies that were cross-reactive with P1.4 variants P1.4-1 and P1.4-3. HexaMen induced a broader cross-reactive antibody response against various patient isolates with one VR identical to a vaccine subtype or a combination of VRs included in HexaMen. Cross-reactivity, measured by a fourfold increase in SBA after vaccination, against these strains ranged from 23 to 92% depending on the subtype of the tested strain and was directed against both VR1 and VR2. The extended cross-reactivity of vaccinee sera induced by HexaMen against antigenic variants has important favorable implications for meningococcal B OMV vaccine coverage.
Virulence, Immunogenicity, and Protective Efficacy of Two RecombinantMycobacterium bovisBacillus Calmette-Guérin Strains Expressing the Antigen ESAT-6 fromMycobacterium tuberculosis
Authors: Bao Lang , Chen Wei , Zhang Huidong , Wang Xiaoying
Published: March 2003

We constructed two recombinantMycobacterium bovisBCG (rBCG) strains expressing ESAT-6 ofMycobacterium tuberculosis, named rBCG-1 and rBCG-2. rBCG-1 contained the ESAT-6 gene linked to BCGhsp60and expressed a fusion protein, while rBCG-2, with a secretory sequence, could secret ESAT-6 into the culture medium. There was no evidence for increased virulence of the two rBCG strains when we made a comparison between them and BCG with regard to organ bacterial loads, lung histology, and survival time. rBCG-1 induced significantly higher specific antibody titers and stronger cellular immune response than BCG, whereas rBCG-2 had immunogenicity similar to that of the parental BCG strain. Both rBCG-1 and rBCG-2 conferred marked protection againstM. tuberculosisinfection, yet in terms of protective efficacy, they showed no significant improvements upon conventional BCG vaccine.
Surfactant Protein D-Mediated Aggregation ofPneumocystis cariniiImpairs Phagocytosis by Alveolar Macrophages
Authors: Yong Suk-Joong , Vuk-Pavlovic Zvezdana , Standing Joseph E. , Crouch Erika C. , Limper Andrew H.
Published: March 2003

Pneumocystis cariniiremains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces duringP. cariniipneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with activeP. cariniipneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients withP. cariniipneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation ofP. cariniiin vitro by SP-D.P. cariniiorganisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium.P. cariniiincubated with natural SP-D (10 μg/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-strippedP. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid.P. cariniiaggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers—but not trimeric subunits—mediate optimal aggregation ofP. carinii. Aggregation ofP. cariniiby SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation ofP. cariniimay represent one means by which the organism avoids elimination by the host.
A New Vaccine against Tuberculosis Affords Greater Survival after Challenge than the Current Vaccine in the Guinea Pig Model of Pulmonary Tuberculosis
Authors: Horwitz Marcus A. , Harth Günter
Published: March 2003

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine,MycobacteriumbovisBCG, is urgently needed. We describe a recombinant BCG vaccine (rBCG30) expressing and secreting the 30-kDa major secretory protein ofMycobacterium tuberculosis, the primary causative agent of TB, that affords greater survival after challenge than parental BCG in the highly demanding guinea pig model of pulmonary TB. Animals immunized with rBCG30 and then challenged by aerosol with a highly virulent strain ofM. tuberculosissurvived significantly longer than animals immunized with conventional BCG. The parental and recombinant vaccine strains are comparably avirulent in guinea pigs, as they display a similar pattern of growth and clearance in the lung, spleen, and regional lymph nodes. The pMTB30 plasmid encoding the 30-kDa protein is neither self-transmissible nor mobilizable to other bacteria, including mycobacteria. The pMTB30 plasmid can be stably maintained inEscherichia colibut is expressed only in mycobacteria. The recombinant and parental strains are sensitive to the same antimycobacterial antibiotics. rBCG30, the first vaccine against TB more potent than nearly century-old BCG, is being readied for human clinical trials.
Cloning and Expression of theEscherichia coliK1 Outer Membrane Protein A Receptor, a gp96 Homologue
Authors: Prasadarao Nemani V. , Srivastava Pramod K. , Rudrabhatla Rajyalakshmi S. , Kim Kwang Sik , Huang Sheng-he , Sukumaran Sunil K.
Published: March 2003

Escherichia coliis one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) ofE. coliinteracts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5′ rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increasesE. colibinding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blockedE. coliinvasion efficiently by binding directly to the bacteria. These results suggest that OmpA ofE. coliK1 interacts with a gp96-like molecule on HBMECs for invasion.
Profiling of Temperature-Induced Changes inBorrelia burgdorferiGene Expression by Using Whole Genome Arrays
Authors: Ojaimi Caroline , Brooks Chad , Casjens Sherwood , Rosa Patricia , Elias Abdallah , Barbour Alan , Jasinskas Algis , Benach Jorge , Katona Laura , Radolf Justin , Caimano Melissa , Skare Jon , Swingle Kristen , Akins Darrin , Schwartz Ira
Published: March 2003

Borrelia burgdorferiis the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affectB. burgdorferigene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putativeB. burgdorferiopen reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression inB. burgdorferiB31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment ofB. burgdorferito growth under diverse environmental conditions.
Expression of Multiple Outer Membrane Protein Sequence Variants from a Single Genomic Locus of
Anaplasma phagocytophilum
Authors: Barbet A. F. , Meeus P. F. M. , Bélanger M. , Bowie M. V. , Yi J. , Lundgren A. M. , Alleman A. R. , Wong S. J. , Chu F. K. , Munderloh U. G. , Jauron S. D.
Published: March 2003

Anaplasma phagocytophilum
is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of
A. phagocytophilum
within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism
Anaplasma marginale
, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of
A. phagocytophilum
, MSP2(P44), is homologous to MSP2 of
A. marginale
, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in
A. phagocytophilum
. As in
A. marginale
, the
msp2
(
p44
) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain
msp2
(
p44
) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in
msp2
(
p44
) mRNA. These data suggest that, similarly to
A. marginale
,
A. phagocytophilum
uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.
Role of Antibodies in Immunity to
Bordetella
Infections
Authors: Kirimanjeswara Girish S. , Mann Paul B. , Harvill Eric T.
Published: March 2003

The persistence of
Bordetella
pertussis
and
B
.
parapertussis
within vaccinated populations and the reemergence of associated disease highlight the need to better understand protective immunity. The present study examined host immunity to bordetellae and addressed potential concerns about the mouse model by using a comparative approach including the closely related mouse pathogen
B
.
bronchiseptica
. As previously observed with
B
.
pertussis
, all three organisms persisted throughout the respiratory tracts of B-cell-deficient mice, indicating that B cells are required for bacterial clearance. However, adoptively transferred antibodies rapidly cleared
B
.
bronchiseptica
but not human pathogens. These results obtained with the mouse model are consistent with human clinical observations, including the lack of correlation between antibody titers and protection, as well as the limited efficacy of intravenous immunoglobulin treatments against human disease. Together, this evidence suggests that the mouse model accurately reflects substantial differences between immunities to these organisms. Although both
B
.
pertussis
and
B
.
parapertussis
are more closely related to
B
.
bronchiseptica
than they are to each other, they share the ability to resist rapid clearance from the lower respiratory tract by adoptively transferred antibodies, an adaptation that correlates with their emergence as human pathogens that circulate within vaccinated populations.
Differential Modulation by Ca
2+
of Type III Secretion of Diffusely Adhering Enteropathogenic
Escherichia coli
Authors: Ide Tina , Michgehl Silke , Knappstein Sabine , Heusipp Gerhard , Schmidt M. Alexander
Published: March 2003

Enteropathogenic
Escherichia coli
(EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca
2+
is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca
2+
. While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca
2+
-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.
Modification in the
ppk
Gene of
Helicobacter
pylori
during Single and Multiple Experimental Murine Infections
Authors: Ayraud Sarah , Janvier Blandine , Salaun Laurence , Fauchère Jean-Louis
Published: March 2003

The bacterial pathogen
Helicobacter
pylori
is highly adapted to the human stomach, and a high level of polymorphism is observed among clinical isolates. This polymorphism may be the consequence of adaptive changes during colonization, making a strain better able to survive, to evade the immune system, and to provoke a chronic infection. To investigate the mechanisms involved in the acquisition of diversity in
H
.
pylori
, mouse models of single infections, coinfections, and superinfections were developed. These experimental infections were conducted with strain SS1, well known to be mouse adapted, and with two strains freshly isolated from infected patients: Hp141 and Hp145. Genetic modifications occurring in these strains were studied over time by comparing randomly selected colonies of the emerging strains to those of the infecting strains by using randomly amplified polymorphic DNA fingerprinting with six different primers and by using PCR to amplify the
vacA
and
cagA
genes. We showed that, regardless of the number of infecting strains, only one emerged from the animals and that the establishment of a first strain thwarted the implantation of a second strain. During both a single infection and a coinfection with SS1, Hp141 was replaced by a genetic variant (Hp141v) that overcame SS1 in coinfection experiments. Hp141v exhibited a deletion of a 102-bp repeated sequence within the
ppk
gene, which encodes polyphosphate kinase (PPK), an enzyme involved in the physiological adaptation of the microbial cell to nutritional and environmental stresses. The deletion led to higher enzymatic activity of PPK, and the variant exhibited a better capacity to colonize mice. Considering that the modified gene is known to be involved in adaptation to a new environment, our results are consistent with an adaptive change in strain Hp141 and suggest that PPK is an important virulence factor in
H
.
pylori
.
Cholera Toxin and Its B Subunit Promote Dendritic Cell Vaccination with Different Influences on Th1 and Th2 Development
Authors: Eriksson Kristina , Fredriksson Margareta , Nordström Inger , Holmgren Jan
Published: March 2003

Cholera toxin (CT) is a strong mucosal adjuvant for codelivered antigens, whereas its nontoxic B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. We investigated the effects of CT and CTB on the immunogenicity of in vitro-treated antigen-pulsed dendritic cells (DC) following intravenous injection into mice. Prior to infusion, DC were pulsed for 90 min with either free ovalbumin (OVA), OVA mixed with CT or CTB, or chemical conjugates of OVA with CT and CTB (OVA-CT and OVA-CTB). DC pulsed with OVA or with OVA and CTB gave rise to modest antibody and T-cell responses. Conjugation of OVA with CTB enhanced both the subsequent B-cell and T-cell responses to OVA and preferentially induced Th2 responses. CT was shown to be a strong adjuvant when it was coadministered to DC with OVA and was even stronger when it was coadministered with OVA-CTB and primed for a mixed Th1-Th2 response. The antibody and T-cell responses were further enhanced if OVA was coupled to CT, implying that CT can utilize a combined carrier and adjuvant function vis-a-vis linked antigens for DC vaccination. The immunopotentiating capacity of CT- and CTB-linked antigen was associated with both upregulated secretion of interleukin-1β by the pulsed DC and increased expression of CD80 and CD86 on the DC surface. These results imply that CT and CTB can be used to both markedly increase and partially direct the DC vaccine-induced immune response with respect to Th1 and Th2 responses, which has obvious implications for DC-based vaccine development.
Induction of Protective Immunity toListeria monocytogeneswith Dendritic Cells Retrovirally Transduced with a Cytotoxic T Lymphocyte Epitope Minigene
Authors: Nakamura Yutaro , Suda Takafumi , Nagata Toshi , Aoshi Taiki , Uchijima Masato , Yoshida Atsushi , Chida Kingo , Koide Yukio , Nakamura Hirotoshi
Published: March 2003

In the present study, we developed a cytotoxic T lymphocyte (CTL) epitope minigene-transduced dendritic cell (DC)-based vaccine againstListeria monocytogenes. Murine bone marrow-derived DCs were retrovirally transduced with a minigene for listeriolysin O (LLO) 91-99, a dominant CTL epitope ofL. monocytogenes, and were injected into BALB/c mice intravenously. We found that the DC vaccine was capable of generating peptide-specific CD8+T cells exhibiting LLO 91-99-specific cytotoxic activity and gamma interferon production, leading to induction of protective immunity to the bacterium. Furthermore, we demonstrated that the retrovirally transduced DC vaccine was more effective than a CTL epitope peptide-pulsed DC vaccine and a minigene DNA vaccine for eliciting antilisterial immunity. These results provide an alternative strategy in which retrovirally transduced DCs are used to design vaccines against intracellular pathogens.
Helicobacter
pylori
-
Specific CD4
+
CD25
high
Regulatory T Cells Suppress Memory T-Cell Responses to
H
.
pylori
in Infected Individuals
Authors: Lundgren Anna , Suri-Payer Elisabeth , Enarsson Karin , Svennerholm Ann-Mari , Lundin B. Samuel
Published: March 2003

Helicobacter
pylori
colonizes the gastric and duodenal mucosa. The infection normally persists for life and causes peptic ulcers and gastric cancer in a subset of infected individuals. We hypothesized that the inability to clear the infection may be a consequence of
H
.
pylori
-specific regulatory T cells that actively suppress T-cell responses. Therefore, we characterized the T-cell responses to
H
.
pylori
in
H
.
pylori
-infected individuals without any subjective symptoms and in uninfected control subjects and investigated the role of regulatory CD4
+
CD25
high
T cells during infection. The stimulation of CD4
+
peripheral blood T cells with monocyte-derived dendritic cells pulsed with a membrane preparation of
H
.
pylori
resulted in proliferation and gamma interferon production in both infected and uninfected individuals. Sorted memory cells from infected individuals responded less than cells from uninfected subjects, and the unresponsiveness could be abolished by depletion of CD4
+
CD25
high
regulatory T cells or the addition of interleukin 2. Furthermore, CD4
+
CD25
high
T cells suppressed
H
.
pylori
-induced responses in cocultures with CD25
low/−
cells. Tetanus toxoid induced comparable responses in memory cells from infected and uninfected individuals in both the presence and the absence of regulatory T cells, suggesting that the suppression was
H
.
pylori
specific. In conclusion, we have shown that
H
.
pylori
-infected individuals have impaired memory CD4
+
T-cell responses to
H
.
pylori
that are linked to the presence of
H
.
pylori
-specific regulatory T cells that actively suppress the responses.
Differential Effects of Control and Antigen-Specific T Cells on Intracellular Mycobacterial Growth
Authors: Worku S. , Hoft D. F.
Published: March 2003

We investigated the effects of peripheral blood mononuclear cells expanded with irrelevant control and mycobacterial antigens on the intracellular growth ofMycobacterium bovisbacillus Calmette-Guérin (BCG) in human macrophages. More than 90% of the cells present after 1 week of in vitro expansion were CD3+. T cells were expanded from purified protein derivative-negative controls, persons with latent tuberculosis, and BCG-vaccinated individuals. T cells expanded with nonmycobacterial antigens enhanced the intracellular growth of BCG in suboptimal cultures of macrophages. T cells expanded with live BCG or lysates ofMycobacterium tuberculosisdirectly inhibited intracellular BCG. Recent intradermal BCG vaccination significantly enhanced the inhibitory activity of T cells expanded with mycobacterial antigens (P< 0.02), consistent with the induction of memory-immune inhibitory T-cell responses. Selected mycobacterial antigens (Mtb41 > lipoarabinomannan > 38kd > Ag85B > Mtb39) expanded inhibitory T cells, demonstrating the involvement of antigen-specific T cells in intracellular BCG inhibition. We studied the T-cell subsets and molecular mechanisms involved in the memory-immune inhibition of intracellular BCG. Mycobacteria-specific γδ T cells were the most potent inhibitors of intracellular BCG growth. Direct contact between T cells and macrophages was necessary for the BCG growth-enhancing and inhibitory activities mediated by control and mycobacteria-specific T cells, respectively. Increases in tumor necrosis factor alpha, interleukin-6, transforming growth factor β, and vascular endothelial growth factor mRNA expression were associated with the enhancement of intracellular BCG growth. Increases in gamma interferon, FAS, FAS ligand, perforin, granzyme, and granulysin mRNA expression were associated with intracellular BCG inhibition. These culture systems provide in vitro models for studying the opposing T-cell mechanisms involved in mycobacterial survival and protective host immunity.
Afa/Dr Diffusely AdheringEscherichia coliInfection in T84 Cell Monolayers Induces Increased Neutrophil Transepithelial Migration, Which in Turn Promotes Cytokine-Dependent Upregulation of Decay-Accelerating Factor (CD55), the Receptor for Afa/Dr Adhesins
Authors: Bétis Fréderic , Brest Patrick , Hofman Véronique , Guignot Julie , Kansau Imad , Rossi Bernard , Servin Alain , Hofman Paul
Published: March 2003

Ulcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains ofEscherichia coli. We report here that two wild-type Afa/Dr diffusely adheringE. coli(DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and theE. colilaboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis of tumor necrosis factor alpha and IL-1β, which in turn promoted the upregulation of DAF.
Expression of
Clostridium difficile
Toxins A and B and Their Sigma Factor TcdD Is Controlled by Temperature
Authors: Karlsson Sture , Dupuy Bruno , Mukherjee Kakoli , Norin Elisabeth , Burman Lars G. , Åkerlund Thomas
Published: March 2003

Growth temperature was found to control the expression of toxins A and B in
Clostridium difficile
VPI 10463
,
with a maximum at 37°C and low levels at 22 and 42°C in both peptone yeast (PY) and defined media. The up-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37°C followed the same kinetics, showing that temperature control occurred at the level of transcription. Experiments with
Clostridium perfringens
using
gusA
as a reporter gene demonstrated that both toxin gene promoters were temperature controlled and that their high activity at 37°C was dependent on the alternative sigma factor TcdD. Furthermore,
tcdD
was found to be autoinduced at 37°C. Glucose down-regulated all these responses in the
C. perfringens
constructs, similar to its impact on toxin production in
C. difficile
PY broth cultures.
C. difficile
proteins induced at 37°C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved in butyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxin production in
C. difficile
by temperature is a novel finding apparently reflecting an adaptation of the expression of its virulence to mammalian hosts.
Genetic Organization and Iron-Responsive Regulation of theBrucella abortus2,3-Dihydroxybenzoic Acid Biosynthesis Operon, a Cluster of Genes Required for Wild-Type Virulence in Pregnant Cattle
Authors: Bellaire Bryan H. , Elzer Philip H. , Hagius Sue , Walker Joel , Baldwin Cynthia L. , Roop R. Martin
Published: March 2003

Brucella abortusreportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulentB. abortus2308 and genetic complementation of definedEscherichia colimutants were used to identify theB. abortusgenes (designateddhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA withdhb-specific primers demonstrated thatdhbC, -B, and -Aare transcribed as components of an operon, together withdhbE, a functional homolog of theEscherichia coli entEgene. Homologs of theE. coli entDandVibrio cholerae vibHgenes were also detected in the flanking regions immediately adjacent to theB. abortus dhbCEBAoperon, suggesting thatB. abortushas the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of theB. abortus dhbCEBAoperon originates from two iron-regulated promoters located upstream ofdhbC. Consistent with their iron-dependent regulation, both of thedhbCEBApromoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection byB. abortusin mice, experimental infection of pregnant cattle with theB. abortus dhbCmutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.
AttenuatedYersiniaenterocoliticaMutant Strains Exhibit Differential Virulence in Cytokine-Deficient Mice: Implications for the Development of Novel Live Carrier Vaccines
Authors: Di Genaro María S. , Waidmann Marc , Kramer Uwe , Hitziger Niclas , Bohn Erwin , Autenrieth Ingo B.
Published: March 2003

Yersiniaenterocoliticamutant strains, including mutants deficient in the chaperone SycH resulting in a functional deficiency in tyrosine phosphatase (YopH), Mn-cofactored superoxide dismutase (SodA), iron-repressive protein 1 (IRP-1), andYersiniaadhesin A (YadA), were demonstrated to be highly attenuated in wild-type C57BL/6 mice. TNFRp55−/−, IL-12p40−/−, and IL-18−/−mutant mice, in which theYersiniawild-type strain causes severe systemic infections, were used to investigate whether theseYersiniamutant strains would be attenuated in immunodeficient hosts. A plasmid-curedYersiniamutant strain was unable to colonize any of the mutant mice tested. A SycH-deficient mutant strain colonized intestinal tissues of these mice but was attenuated for systemic infection in all of the mutant mice. Both YadA- and Irp-1-deficientYersiniamutants were still attenuated in IL-12−/−and IL-18−/−mice but were pathogenic in TNFRp55−/−mice. By contrast, aYersiniasodAmutant was highly pathogenic for TNFRp55−/−and IL-12p40−/−mice while interleukin-18 (IL-18) was dispensable. This finding demonstrates that certain virulence factors enable yersiniae to compete with distinct cytokine-dependent host defense mechanisms. Moreover, while gamma interferon mRNA expression did not reflect protective host responses in cytokine-deficient mice, IL-10 expression coincided with a heavy splenic bacterial load and was associated with progressive infection courses. We can thus segregate minor (SodA), intermediate (YadA and IRP-1), and major (YopH) virulence factors ofY.enterocolitica. Finally, we demonstrate that, even in immunocompromised hosts,YersiniasycHand, with some restrictions,irp-1mutants may be suitable for use as live carrier vaccines.
Pneumococcal Behavior and Host Responses during Bronchopneumonia Are Affected Differently by the Cytolytic and Complement-Activating Activities of Pneumolysin
Authors: Jounblat Rania , Kadioglu Aras , Mitchell Tim J. , Andrew Peter W.
Published: March 2003

Pneumolysin, a multifunctional toxin produced by all clinical isolates of
Streptococcus pneumoniae
, is strongly implicated in the pathogenesis of pneumococcal bronchopneumonia and septicemia. Using isogenic mutant strains, we examined the effect of deletion of the cytotoxic activity or complement-activating activity of pneumolysin on bacterial growth in lungs and blood, histological changes in infected lung tissue, and the pattern of inflammatory cell recruitment. Both of the activities of pneumolysin contributed to the pathology in the lungs, as well as the timing of the onset of bacteremia. Histological changes in the lungs were delayed after infection with either mutant compared to the changes seen after infection with the wild-type pneumococcus. The complement-activating activity of pneumolysin affected the accumulation of T cells, whereas the toxin's cytolytic activity influenced neutrophil recruitment into lung tissue.
Protective Efficacy of Anti-HelicobacterpyloriImmunity following Systemic Immunization of Neonatal Mice
Authors: Eisenberg Julia C. , Czinn Steven J. , Garhart Christine A. , Redline Raymond W. , Bartholomae Wolf C. , Gottwein Judith M. , Nedrud John G. , Emancipator Steven E. , Boehm Bernhard B. , Lehmann Paul V. , Blanchard Thomas G.
Published: March 2003

Helicobacterpyloriinfection of the gastric mucosa is a significant cause of morbidity and mortality because of its etiologic role in symptomatic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Infection occurs in young children; therefore, a prophylactic vaccine would have to be administered within the first year of life, a period thought to be immunologically privileged. We investigated vaccine formulations administered by different routes to confer protective anti-H.pyloriimmunity in neonatal mice. Neonatal mice immunized with a single dose of vaccine in complete Freund's adjuvant (CFA) generated antigen-specific gamma interferon-, interleukin-2 (IL-2)-, IL-4-, and IL-5-secreting T cells in numbers similar to those in immunized adult mice, while vaccine administered to neonates in incomplete Freund's adjuvant (IFA) induced such cells in reduced numbers compared to those in adult mice. Both IFA and CFA, however, provided partial protection from a challenge with infectiousH. pyloriwhen the vaccine was administered subcutaneously. Neonatal immunized mice also had reduced bacterial loads when immunized intraperitoneally with CFA. In all cases, protection was equivalent to that achieved when adult counterparts were immunized. These studies suggest that an efficacious vaccine might be successfully administered to very young children to prevent perinatal infection ofH.pylori.
Characterization of
Cryptosporidium meleagridis
of Human Origin Passaged through Different Host Species
Authors: Akiyoshi Donna E. , Dilo Julia , Pearson Christine , Chapman Susan , Tumwine James , Tzipori Saul
Published: March 2003

Cryptosporidium meleagridis
, a protozoon first observed in turkeys, has been linked by several investigators to cryptosporidiosis in humans.
C. meleagridis
is the only known
Cryptosporidium
species that infects both avian and mammalian species. We describe the successful propagation of
C. meleagridis
(isolate TU1867), originally purified from a patient with diarrhea, in laboratory animals including chickens, mice, piglets, and calves. TU1867 was readily transmitted from one animal host to another, maintaining genetic homogeneity and stability. The rate of infectivity and virulence of TU1867 for the mammalian species were similar to those of
Cryptosporidium parvum
. Laboratory propagation of genetically and phenotypically stable and well-characterized reference isolates, representing various
Cryptosporidium
species, particularly those infectious to humans, will improve considerably the spectrum and quality of laboratory and field investigations on this medically important protozoa.