bluuebunny/crossref_metadata_2025 huggingface dataset:
Toll-Like Receptor 2- and 6-Mediated Stimulation by Macrophage-Activating Lipopeptide 2 Induces Lipopolysaccharide (LPS) Cross Tolerance in Mice, Which Results in Protection from Tumor Necrosis Factor Alpha but in Only Partial Protection from Lethal LPS Doses
Authors: Deiters Ursula , Gumenscheimer Marina , Galanos Chris , Mühlradt Peter F.
Published: July 2003

Patients or experimental animals previously exposed to lipopolysaccharide (LPS) become tolerant to further LPS challenge. We investigated the potential of the macrophage-activating lipopeptide 2 (MALP-2) to induce in vivo cross tolerance to tumor necrosis factor alpha (TNF-α) and LPS. MALP-2-induced tolerance could be of practical interest, as MALP-2 proved much less pyrogenic in rabbits than LPS. Whereas LPS signals via Toll-like receptor 4 (TLR4), MALP-2 uses TLR2 and TLR6. LPS-mediated cytokine release was studied in mice pretreated with intraperitoneal injections of MALP-2. No biologically active TNF-α could be detected in the serum of MALP-2-treated animals when challenged with LPS 24 or 72 h later, whereas suppression of LPS-dependent interleukin (IL)-6 lasted for only 24 h. Protection from lethal TNF-α shock was studied in galactosamine-treated mice. Dose dependently, MALP-2 prevented death from lethal TNF-α doses in TLR4−/−but not in TLR2−/−mice, with protection lasting from 5 to 24 h. To assay protection from LPS, mice were pretreated with MALP-2 doses of up to 10 μg. Five and 24 h later, the animals were simultaneously sensitized and challenged by intravenous coinjection of galactosamine and a lethal dose of 50 ng of LPS. There was only limited protection (four of seven mice survived) when mice were challenged 5 h after MALP-2 pretreatment, and no protection when mice were challenged at later times. The high effectiveness of MALP-2 in suppressing TNF-α, the known ways of biological inactivation, and low pyrogenicity make MALP-2 a potential candidate for clinical use.
Identification of the
agr
Locus of
Listeria monocytogenes
: Role in Bacterial Virulence
Authors: Autret Nicolas , Raynaud Catherine , Dubail Iharilalao , Berche Patrick , Charbit Alain
Published: July 2003

Listeria monocytogenes
is a gram-positive facultative intracellular food-borne pathogen that can cause severe infections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM) to identify genes involved in the virulence of
L. monocytogenes
. A new round of STM allowed us to identify a new locus encoding a protein homologous to AgrA, the well-studied response regulator of
Staphylococcus aureus
and part of a two-component system involved in bacterial virulence. The production of several secreted proteins was modified in the
agrA
mutant of
L. monocytogenes
grown in broth, indicating that the
agr
locus influenced protein secretion. Inactivation of
agrA
did not affect the ability of the pathogen to invade and multiply in cells in vitro. However, the virulence of the
agrA
mutant was attenuated in the mouse (a 10-fold increase in the 50% lethal dose by the intravenous route), demonstrating for the first time a role for the
agr
locus in the virulence of
L. monocytogenes
.
Stress-Dependent Expression of a Polymorphic, Charged Antigen in the Protozoan Parasite
Entamoeba histolytica
Authors: Satish S. , Bakre Abhijeet A. , Bhattacharya Sudha , Bhattacharya Alok
Published: July 2003

We have identified a novel stress inducible gene, Ehssp1 in
Entamoeba histolytica
, the causative agent of amebiasis. Ehssp1 belongs to a polymorphic, multigene family and is present on multiple chromosomes. No homologue of this gene was found in the NCBI database. Sequence alignment of the multiple copies, and genomic PCR data restricted the polymorphism to the central region of the gene. This region contains a polypurine stretch that encodes a domain rich in acidic and basic amino acids. Under normal culture conditions only one copy of this multigene family is expressed, as observed by Northern blot and RT-PCR analysis. The size of this copy of the gene is 1,077 nucleotides, encoding a protein of 359 amino acids. The polymorphic domain in this copy is 64 nucleotides long. However, on exposure of cells to stress conditions such as heat shock or oxidative stress, multiple polymorphic copies of the gene are expressed, suggesting a possible role of this gene in adaptation of cells to stress conditions. The gene copy expressed under normal conditions, and the expression profile of cells under heat stress was identical in two different strains of
E. histolytica
tested. Interestingly, the extent of polymorphism in this gene was very less in
E. dispar
, a nonpathogenic sibling species of
E. histolytica
. Ehssp1 was found to be antigenic in invasive amebiasis patients.
TheMycobacterium tuberculosis19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Regulated HLA-DR and FcγR1 on Human Macrophages through Toll-Like Receptor 2
Authors: Gehring Adam J. , Rojas Roxana E. , Canaday David H. , Lakey David L. , Harding Clifford V. , Boom W. Henry
Published: July 2003

Mycobacterium tuberculosissurvives in macrophages in the face of acquired CD4+T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-γ) has a central role in host defenses againstM. tuberculosisby activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing.M. tuberculosisinterferes with IFN-γ receptor (IFN-γR) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein fromM. tuberculosisinhibits IFN-γ-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags andM. tuberculosisbacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-γ-induced expression of FcγRI. Thus,M. tuberculosis, through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-γR signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism forM. tuberculosispersistence in macrophages.
Relative Roles of Genetic Background and Variation in PspA in the Ability of Antibodies to PspA To Protect against Capsular Type 3 and 4 Strains of
Streptococcus pneumoniae
Authors: Roche Hazeline , Ren Bing , McDaniel Larry S. , Håkansson Anders , Briles David E.
Published: July 2003

Pneumococcal surface protein A (PspA) is able to elicit antibodies in mice and humans that can protect mice against fatal infection with
Streptococcus pneumoniae
. It has been observed that immunization with a single family 1 PspA can protect mice against infections with capsular type 3 or 6B strains expressing PspA family 1 or 2. However, several studies have shown that immunity to PspA is less efficacious against several capsular type 4 strains than against strains of capsular types 3, 6A, and 6B. To determine whether the greater difficulty in protecting against capsular type 4 strains resulted from differences in their PspAs or from differences in their genetic backgrounds, we performed protection experiments using four different challenge strains: a capsular type 3 strain expressing a family 1 PspA (WU2), a capsular type 4 strain expressing a family 2 PspA (TIGR4), and genetically engineered variants of WU2 and TIGR4 expressing each other's PspAs. Prior to infection, the mice were immunized with recombinant family 1 or family 2 PspA. The results revealed that much of the difficulty in protecting against capsular type 4 strains was eliminated when mice were immunized with a homologous PspA of the same PspA family. However, regardless of which PspA the strains expressed, those on the TIGR4 background were about twice as hard to protect against as WU2 strains expressing the same PspA based on the efficacy rates seen in our experiments. These results point out the importance of including more than one PspA in any PspA vaccines developed for human use.
Induction of Specific T-Cell Responses, Opsonizing Antibodies, and Protection againstPlasmodium chabaudi adamiInfection in Mice Vaccinated with Genomic Expression Libraries Expressed in Targeted and Secretory DNA Vectors
Authors: Rainczuk A. , Scorza T. , Smooker P. M. , Spithill T. W.
Published: July 2003

It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomicPlasmodium chabaudi adamiDS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation withP. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization ofP. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge withP. chabaudi adamiDS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific toP. chabaudi adamimalaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.
Characterization of the Novel Factor Paa Involved in the Early Steps of the Adhesion Mechanism of Attaching and Effacing
Escherichia coli
Authors: Batisson Isabelle , Guimond Marie-Pierre , Girard Francis , An Hongyan , Zhu Chengru , Oswald Eric , Fairbrother John M. , Jacques Mario , Harel Josée
Published: July 2003

Nonenterotoxigenic porcine
Escherichia coli
strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic
E. coli
(PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using Tn
phoA
transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated
paa
(porcine A/E-associated gene). Sequence analysis of
paa
revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic
E. coli
O157:H7 strains (EDL933 and Sakai), PEB3 of
Campylobacter jejuni
, and AcfC of
Vibrio cholerae
, respectively. Chromosomal localization studies indicated that the region containing
paa
was inserted between the
yciD
and
yciE
genes at about 28.3 min of the
E. coli
K-12 chromosome. The presence of
paa
and
eae
sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three
eae
-positive but
paa
-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the
paa
gene in the A/E activity of O45 strains. As well, the complementation of the
paa
mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E
E. coli
virulence.
Legionella pneumophila
Catalase-Peroxidases Are Required for Proper Trafficking and Growth in Primary Macrophages
Authors: Bandyopadhyay Purnima , Byrne Brenda , Chan Yolande , Swanson Michele S. , Steinman Howard M.
Published: July 2003

Legionella pneumophila
, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of
Legionella
-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in
katA
and
katB
, encoding the two
Legionella
catalase-peroxidases, were analyzed to evaluate the hypothesis that
L. pneumophila
must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing
katA
or
katB
mutant
Legionella
colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in
dotA
and
dotB
, encoding components of the Type IV secretion system. Quantitative similarity of the
katA
/
B
phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H
2
O
2
compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that
dotA
and
dotB
mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba
Acanthamoeba castellanii
, indicating that certain
dotA
/
B
functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent
Legionella
phagosomes in macrophages.
DNA Immunization with the Cysteine-Rich Interdomain Region 1 of the
Plasmodium falciparum
Variant Antigen Elicits Limited Cross-Reactive Antibody Responses
Authors: Baruch Dror I. , Gamain Benoit , Miller Louis H.
Published: July 2003

The variant surface antigens of
Plasmodium falciparum
are an important component of naturally acquired immunity and an important vaccine target. However, these proteins appear to elicit primarily variant-specific antibodies. We tested if naked DNA immunization can elicit more cross-reactive antibody responses and allow simultaneous immunization with several variant constructs. Mice immunized with plasmid DNA expressing variant cysteine-rich interdomain region 1 (CIDR1) domains of the
P. falciparum
erythrocyte membrane protein 1 (PfEMP1) developed antibodies that were reactive to the corresponding PfEMP1s as measured by an enzyme-linked immunosorbent assay, flow cytometry, and agglutination of parasitized erythrocytes (PEs). We observed some cross-reactive immune responses; for example, sera from mice immunized with one domain agglutinated PEs of various lines and recognized heterologous domains expressed on the surface of Chinese hamster ovary (CHO) cells. We found no significant antigenic competition when animals were immunized with a mixture of plasmids or immunized sequentially with individual constructs. Moreover, mixed or sequential immunizations resulted in greater cross-reactive agglutination responses than immunization with a single domain. Recombinant protein (Sc y179) immunization after priming with DNA (prime-boost regimen) increased antibody titers to the homologous domain substantially but seemed to diminish the cross-reactive responses somewhat. The titer of agglutinating antibodies was previously shown to correlate with protection. Surprisingly, the agglutination titers of sera from DNA immunization were high, similar to those of pooled human hyperimmune sera. These sera also appeared to give limited low-titer variant transcending agglutination. Thus, DNA immunization appears to be a very useful tool for developing variant antigen vaccines.
Mycobacterium bovisBCG Scar Status and HLA Class II Alleles Influence Purified Protein Derivative-Specific T-Cell Receptor Vβ Expression in Pulmonary Tuberculosis Patients from Southern India
Authors: Shanmugalakshmi S. , Dheenadhayalan V. , Muthuveeralakshmi P. , Arivarignan G. , Pitchappan R.M.
Published: July 2003

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) Vβ expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR Vβ families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR Vβ families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR Vβ families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR Vβ families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR Vβ family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) andMycobacterium bovisBCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR Vβ families readily recalled by PPD, while the disease status of the patients was attributed to TCR Vβ downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR Vβ usage.
Shiga Toxin 2-Converting Bacteriophages Associated with Clonal Variability in
Escherichia coli
O157:H7 Strains of Human Origin Isolated from a Single Outbreak
Authors: Muniesa Maite , de Simon Mercè , Prats Guillem , Ferrer Dolors , Pañella Helena , Jofre Juan
Published: July 2003

Shiga toxin 2 (Stx2)-converting bacteriophages induced from 49 strains of
Escherichia coli
O157:H7 isolated during a recent outbreak of enterocolitis in Spain were examined in an attempt to identify the variability due to the
stx
2
-converting phages. The bacterial isolates were divided into low-, medium-, and high-phage-production groups on the basis of the number of phages released after mitomycin C induction. Low- and medium-phage-production isolates harbored two kinds of phages but released only one of them, whereas high-phage-production isolates harbored only one of the two phages. One of the phages, φSC370, which was detected only in the isolates with two phages, showed similarities with phage 933W. The second phage, φLC159, differed from φSC370 in morphology and DNA structure. When both phages were present in the same bacterial chromosome, as occurred in most of the isolates, only φSC370 was detected in the supernatants of the induced cultures. If φLC159 was released, its presence was masked by φSC370. When φSC370 was absent, large amounts of φLC159 were released, suggesting that there was some regulation of phage expression between the two phages. To our knowledge, this is the first description of clonal variability due to phage loss. The higher level of phage production was reflected in the larger amounts of Stx2 toxin produced by the cultures. Some relationship between phage production and the severity of symptoms was observed, and consequently these observations suggest that the virulence of the isolates studied could be related to the variability of the induced
stx
2
-converting phages.
Genome-Based Bioinformatic Selection of ChromosomalBacillus anthracisPutative Vaccine Candidates Coupled with Proteomic Identification of Surface-Associated Antigens
Authors: Ariel N. , Zvi A. , Makarova K. S. , Chitlaru T. , Elhanany E. , Velan B. , Cohen S. , Friedlander A. M. , Shafferman A.
Published: July 2003

Bacillus anthracis(Ames strain) chromosome-derived open reading frames (ORFs), predicted to code for surface exposed or virulence related proteins, were selected asB. anthracis-specific vaccine candidates by a multistep computational screen of the entire draft chromosome sequence (February 2001 version, 460 contigs, The Institute for Genomic Research, Rockville, Md.). The selection procedure combined preliminary annotation (sequence similarity searches and domain assignments), prediction of cellular localization, taxonomical and functional screen and additional filtering criteria (size, number of paralogs). The reductive strategy, combined with manual curation, resulted in selection of 240 candidate ORFs encoding proteins with putative known function, as well as 280 proteins of unknown function. Proteomic analysis of two-dimensional gels of aB. anthracismembrane fraction, verified the expression of some gene products. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses allowed identification of 38 spots cross-reacting with sera fromB. anthracisimmunized animals. These spots were found to represent eight in vivo immunogens, comprising of EA1, Sap, and 6 proteins whose expression and immunogenicity was not reported before. Five of these 8 immunogens were preselected by the bioinformatic analysis (EA1, Sap, 2 novel SLH proteins and peroxiredoxin/AhpC), as vaccine candidates. This study demonstrates that a combination of the bioinformatic and proteomic strategies may be useful in promoting the development of next generation anthrax vaccine.
Interleukin-4 and Transforming Growth Factor β Have Opposing Regulatory Effects on Gamma Interferon-Mediated Inhibition ofCryptosporidium parvumReproduction
Authors: Lean I.-Sarah , McDonald Stuart A. C. , Bajaj-Elliott Mona , Pollok Richard C. G. , Farthing Michael J. G. , McDonald Vincent
Published: July 2003

It was shown previously that enterocytes activated by gamma interferon (IFN-γ) are efficient effector cells in the killing ofCryptosporidium parvum. How this function is regulated is not clearly understood, but transforming growth factor β (TGF-β) and the Th2 regulatory cytokines may play a role. Using an in vitro cell culture system, we investigated how the key regulatory cytokines interleukin-4 (IL-4), IL-10, IL-13, and TGF-β might modulate the effect of IFN-γ in inducing resistance to infection in enterocyte cell lines. The results showed that TGF-β can abolish the inhibitory effect onC. parvumdevelopment and that neither IL-13 nor IL-10 influenced the action of IFN-γ. In contrast, IL-4 cooperated with low concentrations of IFN-γ (1 and 10 U/ml) to enhance parasite killing. One mechanism that appeared to be involved in the combined activity of IFN-γ and IL-4 was intracellular Fe2+deprivation, but induction of nitric oxide production was not involved. In one cell line, the extents and durations of phosphorylation of STAT1, a transcription factor involved in IFN-γ signaling, were similar when cells were stimulated with IFN-γ alone and with IFN-γ and IL-4γ, suggesting that the cooperative effect of the cytokines was not related to STAT1 activation. The effects of the presence of TGF-β and IL-4 on IFN-γ function did not appear to involve any alteration in the level of expression of IFN-γ receptors.
Diminished Adhesion ofAnaplasma phagocytophilum-Infected Neutrophils to Endothelial Cells Is Associated with Reduced Expression of Leukocyte Surface Selectin
Authors: Choi Kyoung-Seong , Garyu Justin , Park Jinho , Dumler J. Stephen
Published: July 2003

Anaplasma phagocytophilumpropagates within neutrophils and causes a disease marked by inflammatory tissue injury or complicated by opportunistic infections. We hypothesized that infection withA. phagocytophilummodifies the binding of neutrophils to endothelial cells and the expression of neutrophil adhesion molecules and studied these changes in vitro. Infected dimethyl sulfoxide-differentiated HL-60 cells and neutrophils showed reduced binding to cultured brain and systemic endothelial cells and lost expression of P-selectin glycoprotein ligand 1 (PSGL-1, CD162) and L-selectin (CD62L) (to 33 and 5% of control values, respectively), at a time when the levels of β2integrin and immunoglobulin superfamily adhesion molecules and activation markers Mac-1 and intercellular adhesion molecule 1 increased (5 to 10 times that of the control). The loss of CD162 and CD62L expression was inhibited by EDTA, which suggests that neutrophil activation and sheddase cleavage occurred. The loss of selectin expression and the retained viability of the neutrophils persisted for at least 18 h withA. phagocytophiluminfection, whereasEscherichia coliandStaphylococcus aureusrapidly killed neutrophils. The adhesion defect might increase the numbers of infected cells and their persistence in the blood prior to tick bites. However, decreased CD162 expression and poor endothelial cell binding may partly explain impaired host defenses, while simultaneous neutrophil activation may aggravate inflammation. These observations may help us to understand the modified biological responses, host inflammation, and immune response that occur withA. phagocytophiluminfections.
Requirement of the
Yersinia pseudotuberculosis
Effectors YopH and YopE in Colonization and Persistence in Intestinal and Lymph Tissues
Authors: Logsdon Lauren K. , Mecsas Joan
Published: July 2003

The gram-negative enteric pathogen
Yersinia pseudotuberculosis
employs a type III secretion system and effector Yop proteins that are required for virulence. Mutations in the type III secretion-translocation apparatus have been shown to cause defects in colonization of the murine cecum, suggesting roles for one or more effector Yops in the intestinal tract. To investigate this possibility, isogenic
yop
mutant strains were tested for their ability to colonize and persist in intestinal and associated lymph tissues of the mouse following orogastric inoculation. In single-strain infections, a
yopHEMOJ
mutant strain was unable to colonize, replicate, or persist in intestinal and lymph tissues. A
yopH
mutant strain specifically fails to colonize the mesenteric lymph nodes, but
yopE
and
yopO
mutant strains showed only minor defects in persistence in intestinal and lymph tissues. While no single Yop was found to be essential for colonization or persistence in intestinal tissues in single-strain infections, the absence of both YopH and YopE together almost eliminated colonization of all tissues, indicating either that these two Yops have some redundant functions or that
Y. pseudotuberculosis
employs multiple strategies for colonization. In competition infections with wild-type
Y. pseudotuberculosis
, the presence of wild-type bacteria severely hindered the ability of the
yopH
,
yopE
, and
yopO
mutants to persist in many tissues, suggesting that the wild-type bacteria either fills colonization niches or elicits host responses that the
yop
mutants are unable to withstand.
The Absence of Linear Plasmid 25 or 28-1 ofBorrelia burgdorferiDramatically Alters the Kinetics of Experimental Infection via Distinct Mechanisms
Authors: Labandeira-Rey Maria , Seshu J. , Skare Jonathan T.
Published: July 2003

The 25-kb linear plasmid lp25 and one of the 28-kb linear plasmids (lp28-1) are required for experimental infection inBorrelia burgdorferi, the etiologic agent of Lyme disease. The loss of these plasmids either eliminates infectivity (lp25) or significantly increases the 50% infective dose during a 2-week infection period (lp28-1). This study assessed the kinetics of bacterial dissemination in C3H/HeN mice infected withB. burgdorferilacking either lp25 or lp28-1, as well as their wild-type parent, and tracked the development of specific borrelial antibodies over a 3-week period. The results indicated that the wild type and the lp28-1−strains were able to disseminate throughout the host, whereas the lp25−strain was cleared within 48 h of inoculation. While the wild-typeB. burgdorferipersisted in tissues for the duration of the study, the lp28-1−mutant began clearing at day 8, with no detectable bacteria present by day 18. As expected, the wild-type strain persisted in C3H/HeN mice despite a strong humoral response; however, the lp28-1−mutant was cleared coincidently with the development of a modest immunoglobulin M response. The lp28-1−mutant was able to disseminate and persist in C3H-scidmice at a level indistinguishable from that of wild-type cells, confirming that acquired immunity was required for clearance in C3H/HeN mice. Thus, within an immunocompetent host, lp28-1-encoded proteins are not required for dissemination but are essential for persistence associated with Lyme borreliosis.
Pseudomonas aeruginosaSlime Glycolipoprotein Is a Potent Stimulant of Tumor Necrosis Factor Alpha Gene Expression and Activation of Transcription Activators Nuclear Factor κB and Activator Protein 1 in Human Monocytes
Authors: Lagoumintzis George , Christofidou Myrto , Dimitracopoulos George , Paliogianni Fotini
Published: July 2003

Pseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts. Production of tumor necrosis factor alpha (TNF-α), primarily by cells of monocytic lineage, is a crucial event in the course of these infections. During in vivo infections withP. aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released. In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-α production by human monocytes. To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated withP. aeruginosaLPS or GLP. GLP was found to be a more potent stimulus for TNF-α production (threefold higher) by human monocytes than LPS. Moreover, its effect was comparable to that of viable bacteria. Quantitative mRNA analysis revealed predominantly transcriptional regulation. Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-κB and activator protein 1 (AP-1). NF-κB activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-κB through GLP. MoreoverP. aeruginosaGLP induced the formation of AP-1 complex with delayed kinetics compared with NF-κB but much more efficiently than the homologous LPS. These results identify GLP as the most important stimulant for TNF-α production by human monocytes. Activation of NF-κB and AP-1 byP. aeruginosaGLP may be involved not only in TNF-α induction but also in many of the inflammatory responses triggered in the course of infection withP. aeruginosa.
The C Terminus of YopT Is Crucial for Activity and the N Terminus Is Crucial for Substrate Binding
Authors: Sorg Isabel , Hoffmann Claudia , Dumbach Juergen , Aktories Klaus , Schmidt Gudula
Published: July 2003

Recently, it was shown thatYersiniaouter protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathioneS-transferase fusion proteins inEscherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.
Functional Selection of Vaccine Candidate Peptides from
Staphylococcus aureus
Whole-Genome Expression Libraries In Vitro
Authors: Weichhart Thomas , Horky Markus , Söllner Johannes , Gangl Susanne , Henics Tamàs , Nagy Eszter , Meinke Andreas , von Gabain Alexander , Fraser Claire M. , Gill Steve R. , Hafner Martin , von Ahsen Uwe
Published: July 2003

An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen
Staphylococcus aureus
. A library built up from genomic fragments of the virulent
S. aureus
COL strain (methicillin-resistant
S. aureus
) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against
S. aureus
were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens.
Delineation of the Molecular Mechanisms ofFrancisella tularensis-Induced Apoptosis in Murine Macrophages
Authors: Lai Xin-He , Sjöstedt Anders
Published: July 2003

Francisella tularensisis a facultative intracellular bacterium capable of inducing apoptosis in murine macrophages. Here we analyzed the pathway leading to apoptosis in the murine macrophage-like cell line J774A.1 after infection withF. tularensisstrain LVS (named LVS for live vaccine strain). We obtained evidence that the infection affected the mitochondria of the macrophages, since it induced release of the mitochondrial molecule cytochromecinto the cytosol and changed the potential over the mitochondrial membrane. Moreover, activation of caspase 9 and the executioner caspase 3 was also observed in the LVS-infected J774A.1 macrophages. The activated caspase 3 degraded poly(ADP-ribose) polymerase (PARP). All of these events were observed within 9 to 12 h after the initiation of infection, and maximum degradation of a synthetic caspase 3 substrate occurred at 18 h. The internucleosomal fragmentation and PARP degradation resulting from activation of this apoptotic pathway was prevented by the caspase 3 inhibitor Z-DEVD-fmk. No involvement of caspase 1, caspase 8, Bcl-2, or Bid was observed. Thus, theF. tularensisinfection induces macrophage apoptosis through a pathway partly resembling the intrinsic apoptotic pathway.
Stimulation of Neutrophil Granulocytes withMycobacterium bovisBacillus Calmette-Guérin Induces Changes in Phenotype and Gene Expression and Inhibits Spontaneous Apoptosis
Authors: Suttmann Henrik , Lehan Nadine , Böhle Andreas , Brandau Sven
Published: July 2003

Polymorphonuclear neutrophil granulocytes (PMN) have been implicated in the early inflammatory response against mycobacteria besides monocytes/macrophages. Yet, little is known about the interaction of mycobacteria with PMN. We investigated the potential ofMycobacterium bovisbacillus Calmette-Guérin (BCG) to stimulate and influence PMN phenotype, gene expression profile and spontaneous apoptosis. Flow cytometric analyses revealed an upregulation of the function-associated molecules Fcγ receptor III (FcγR III) and II (CD16 and CD32) as well as MAC-1 (CD11b and CD18) on BCG-stimulated PMN. As determined by cDNA microarrays and multiplex reverse transcriptase PCR, stimulation with BCG alters the expression of various genes for proinflammatory cytokines/chemokines or receptors in PMN. We detected an upregulation or de novo synthesis of interleukin 1α (IL-1α), IL-1β, IL-8, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, GRO-α, transforming growth factor β, MCP-1, IL-2 receptor γ (IL-2Rγ), IL-10Rα, and IL-6R. Genes for IL-9, IL-12α, IL-15, IL-5Rα, and IL-13Rα1were found to be downregulated or switched off. Furthermore, Giemsa and annexin V-propidium iodide double staining demonstrated an inhibition of spontaneous PMN apoptosis following BCG stimulation. Changes in phenotype and inhibition of apoptosis did not depend on direct mycobacterial stimulation alone, but were a result of an autocrine-paracrine stimulation mechanism. Our findings support the hypothesis that PMN become activated at the site of mycobacterial infections and that this activation might set the stage for a subsequent antimycobacterial immune response.
Binding of
Actinobacillus pleuropneumoniae
to Phosphatidylethanolamine
Authors: Jeannotte Marie-Eve , Abul-Milh Maan , Dubreuil J. Daniel , Jacques Mario
Published: July 2003

The gram-negative bacterium
Actinobacillus pleuropneumoniae
is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, a disease that causes important economic losses to the swine industry worldwide. In general, the initial step of bacterial colonization is attachment to host cells. The purpose of the present study was to evaluate the binding of
A. pleuropneumoniae
serotype 1 to phospholipids, which are the major constituents of biological membranes. Phospholipids serve as receptors for several bacteria, including respiratory pathogens. To study this effect, we used thin-layer chromatography overlay binding assays to test commercial phospholipids such as phosphatidic acid, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylethanolamine (PE). Our results indicate that
A. pleuropneumoniae
serotype 1 binds to PE but not to the other phospholipids tested. Serotypes 5b and 7, which, along with serotype 1, are the most prevalent serotypes of
A. pleuropneumoniae
in North America, share the ability to bind PE. Inhibition of binding with a monoclonal antibody against
A. pleuropneumoniae
serotype 1 O antigen and the use of isogenic lipopolysaccharide (LPS) mutants of
A. pleuropneumoniae
serotype 1 showed that the O antigen seems to be implicated in the binding to PE, at least for
A. pleuropneumoniae
serotype 1.
A. pleuropneumoniae
was also shown to bind to a phospholipid extracted from swine lungs by using the method of Folch. Chemical staining with molybdenum blue and ninhydrin, migration with neutral, acidic, and basic solvent systems, and mass spectrometry analysis all indicated that this lipid is PE. This study is, to the best of our knowledge, the first description of
A. pleuropneumoniae
binding to phospholipids. Our data also suggest that LPS O antigens could be involved in binding to PE.
Construction, Characterization, and Immunogenicity of an AttenuatedSalmonella entericaSerovar TyphimuriumpgtEVaccine Expressing Fimbriae with Integrated Viral Epitopes from thespiCPromoter
Authors: Chen Huaiqing , Schifferli Dieter M.
Published: July 2003

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that causes diarrhea, leading to near 100% mortality in neonatal piglets with corresponding devastating economic consequences. For the protection of neonatal and older animals, oral live vaccines present the attractive property of inducing desired mucosal immune responses, including colostral antibodies in sows—an effective means to passively protect suckling piglets. Newly attenuatedSalmonellavaccine constructs expressing TGEV S protein epitopes were studied and evaluated for improved humoral immune response to TGEV. The macrophage-inducibleSalmonella ssaHandspiC/ssaBpromoters were compared for their ability to express the TGEV C and A epitopes in the context of the heterologous 987P fimbriae onSalmonellavaccines. Compared to thessaHpromoter, theSalmonella cya crpvector elicited significantly higher levels of mucosal and systemic antibodies in orally immunized mice when the chimeric fimbriae were expressed from thespiCpromoter. TheSalmonella spiCpromoter construct induced the highest level of chimeric fimbriae after being taken up by the J774A.1 macrophagelike cells. TheSalmonella cya crpvaccine vector was shown to incorporate into 987P partially degraded chimeric subunits lacking the TGEV epitopes. In contrast, its isogenicpgtEmutant produced fimbriae consisting exclusively of intact chimeric subunits. Mice immunized orally with theSalmonella pgtEvaccine expressing chimeric fimbriae from thespiCpromoter elicited significantly higher systemic and mucosal antibody titers against the TGEV epitopes compared to the parental vaccine. This study indicates that theSalmonella cya crp pgtEvector and thespiCpromoter can be used successfully to improve immune responses toward heterologous antigens.
Comparative Genomic Indexing Reveals the Phylogenomics of
Escherichia coli
Pathogens
Authors: Anjum Muna F. , Lucchini Sacha , Thompson Arthur , Hinton Jay C. D. , Woodward Martin J.
Published: July 2003

The
Escherichia coli
O26 serogroup includes important food-borne pathogens associated with human and animal diarrheal disease. Current typing methods have revealed great genetic heterogeneity within the O26 group; the data are often inconsistent and focus only on verotoxin (VT)-positive O26 isolates. To improve current understanding of diversity within this serogroup, the genomic relatedness of VT-positive and -negative O26 strains was assessed by comparative genomic indexing. Our results clearly demonstrate that irrespective of virulence characteristics and pathotype designation, the O26 strains show greater genomic similarity to each other than to any other strain included in this study. Our data suggest that enteropathogenic and VT-expressing
E. coli
O26 strains represent the same clonal lineage and that VT-expressing
E. coli
O26 strains have gained additional virulence characteristics. Using this approach, we established the core genes which are central to the
E. coli
species and identified regions of variation from the
E. coli
K-12 chromosomal backbone.
Sulfolipid Deficiency Does Not Affect the Virulence ofMycobacterium tuberculosisH37Rv in Mice and Guinea Pigs
Authors: Rousseau Cécile , Turner Oliver C. , Rush Erik , Bordat Yann , Sirakova Tatiana D. , Kolattukudy Pappachan E. , Ritter Shannon , Orme Ian M. , Gicquel Brigitte , Jackson Mary
Published: July 2003

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival ofMycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant ofM. tuberculosisH37Rv (Sirakova et al., J. Biol. Chem.276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity ofM. tuberculosisH37Rv in mice and guinea pigs or in cultured macrophages.
Efficacy of the 26-Kilodalton Outer Membrane Protein and Two P5 Fimbrin-Derived Immunogens To Induce Clearance of Nontypeable
Haemophilus influenzae
from the Rat Middle Ear and Lungs as Well as from the Chinchilla Middle Ear and Nasopharynx
Authors: Kyd Jennelle M. , Cripps Allan W. , Novotny Laura A. , Bakaletz Lauren O.
Published: July 2003

The rat middle ear and lung clearance model has been used to show that the nontypeable
Haemophilus influenzae
26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)
2,1,3
, are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable
H. influenzae
upon challenge of these sites. Here, these three nontypeable
H. influenzae
immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)
2,1,3
were assayed in the rat host with a mucosal immunization route and clearance of nontypeable
H. influenzae
from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable
H. influenzae
.