bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated Antigen Expression in Live RecombinantSalmonella entericaSerovar Typhimurium Strongly Affects Colonization Capabilities and Specific CD4+-T-Cell Responses
Authors: Bumann Dirk
Published: July 2002

Regulated antigen expression can influence the immunogenicity of live recombinantSalmonellavaccines, but a rational optimization has remained difficult since important aspects of this effect are incompletely understood. Here, attenuatedSalmonella entericaserovar Typhimurium SL3261 strains expressing the model antigen GFP_OVA were used to quantify in vivo antigen levels by flow cytometry and to simultaneously follow the crucial early steps of antigen-specific T-cell responses in mice that are transgenic for a T-cell receptor recognizing ovalbumin. Among seven tested promoters,PpagChas the highest activity in murine tissues combined with low in vitro expression, whereasPtachas a comparable in vivo and a very high in vitro activity. Both SL3261 (pPpagCGFP_OVA) and SL3261 (pPtacGFP_OVA) cells can induce potent ovalbumin-specific cellular immune responses following oral administration, but doses almost 1,000-fold lower are sufficient for the in vivo-inducible construct SL3261 (pPpagCGFP_OVA) compared to SL3261 (pPtacGFP_OVA). This efficacy difference is largely explained by impaired early colonization capabilities of SL3261 (pPtacGFP_OVA) cells. Based on the findings of this study, appropriate in vivo expression levels for any given antigen can be rationally selected from the increasing set of promoters with defined properties. This will allow the improvement of recombinantSalmonellavaccines against a wide range of pathogens.
Arginine-143 of
Yersinia enterocolitica
YopP Crucially Determines Isotype-Related NF-κB Suppression and Apoptosis Induction in Macrophages
Authors: Ruckdeschel Klaus , Richter Kathleen , Mannel Oliver , Heesemann Jürgen
Published: July 2002

Pathogenic
Yersinia
spp. counteract host defense mechanisms by modulating the cellular signal relay in response to infection. Subversion of the antiapoptotic NF-κB signaling pathway by the
Yersinia enterocolitica
virulence protein YopP crucially determines the induction of apoptosis in
Yersinia
-infected macrophages. Here, we analyzed a panel of pathogenic, phylogenetically distinct
Y. enterocolitica
serotypes for their abilities to trigger macrophage apoptosis.
Y. enterocolitica
from the highly pathogenic serogroup O8 was substantially more effective in apoptosis induction than
Yersinia
from the serogroups O3 and O9. Complementation of
yopP
-knockout mutants revealed that this effect was specifically conferred by the serogroup O8 YopP. The amino acid sequences of YopPO8 and YopPO9 share 94% identity, and both YopP isotypes were found to interact with the NF-κB-activating kinase IKKβ in macrophages. However, selectively, YopPO8 mediated efficient inhibition of IKKβ activities, which led to substantial suppression of NF-κB activation. To localize the YopPO8-related effector domain, we interchanged stretches of amino acids and single amino acid residues between YopPO8 and YopPO9. Functional characterization of the resulting mutants revealed a major role of the arginine-143 residue in determining the inhibitory impact of YopP on IKKβ activity and survival of macrophages.
Allelic Variation within
Helicobacter pylori babA
and
babB
Authors: Pride David T. , Meinersmann Richard J. , Blaser Martin J.
Published: July 2002

Helicobacter pylori
strains show both geographic and disease-associated allelic variation. We investigated the diversity present in two genes,
babA
and
babB
, which are members of a paralogous family of outer membrane proteins. Eleven family members within a single
H. pylori
strain, predicted to encode proteins with substantial N- and C-terminal similarity to each other, were classified as
babA
paralogues. In their central regions, most are less than 54% related to one another. Examining the
babA
and
babB
central regions in 42
H. pylori
strains from different geographic locales, we identified five different allele groups of
babA
(AD1 to AD5) and three different allele groups of
babB
(BD1 to BD3). Phylogenetic analysis revealed that the allelic groupings of
babA
and
babB
are independent of one another and that, for both, geographic variation is present. Analysis of synonymous and nonsynonymous substitutions in these regions showed that
babA
is more diverse, implying an earlier origin than that of the same region of
babB
, but that the
babA
diversity region may have more functional constraints. Although recombination has been central to the evolution of both genes, with
babA
and
babB
showing low mean compatibility scores and homoplasy ratios of 0.71 and 0.67, respectively, recombination is not sufficient to obscure evidence of clonal descent. Despite the involvement of
babA
in binding to the host blood group antigen Lewis B, neither the presence of different
babA
allele groups nor that of different
babB
allele groups is a determining factor in Lewis B binding of
H. pylori
strains.
Safety and Immunogenicity of ImprovedShigellaO-Specific Polysaccharide-Protein Conjugate Vaccines in Adults in Israel
Authors: Passwell Justen H. , Harlev Efrat , Ashkenazi Shai , Chu Chiayung , Miron Dan , Ramon Reut , Farzan Naheed , Shiloach Joseph , Bryla Dolores A. , Majadly Fathy , Roberson Robin , Robbins John B. , Schneerson Rachel
Published: July 2002

Data suggest that the O-specific polysaccharide (O-SP) domain of the lipopolysaccharide (LPS) ofShigellaspecies is both an essential virulence factor and a protective antigen and that a critical level of serum immunoglobulin G (IgG) to this antigen will confer immunity to shigellosis. Because covalent attachment of polysaccharides to proteins increases their immunogenicity, especially in infants and in young children, the O-SP ofShigellaspecies were bound to medically useful proteins, and the safety and immunogenicity of the resultant conjugates were confirmed in adults and 4- to 7-year-old children. Succinylation of the carrier protein improved the immunogenicity ofShigellaconjugates in mice and increased their yield. Based on these results, a clinical trial of O-SP conjugates ofShigella sonneiandShigella flexneri2a bound to succinylated mutantPseudomonas aeruginosaexotoxin A (rEPAsucc) or native or succinylatedCorynebacterium diphtheriaetoxin mutant (CRM9 or CRM9succ) was conducted in healthy adults. The conjugates were safe and immunogenic.S. sonnei-CRM9,S. sonnei-CRM9succ, andS. sonnei-rEPAsuccelicited significant rises of geometric mean (GM) IgG anti-LPS within 1 week of injection (P< 0.001). At 26 weeks, the GM anti-LPS levels elicited by these three conjugates were similar and higher than their prevaccination levels (P< 0.0001). GM IgG anti-LPS levels elicited byS. flexneri2a-rEPAsuccwere significantly higher than those elicited byS. flexneri2a-rCRM9succat all intervals after injection. At 26 weeks, the levels of IgG anti-LPS in vaccinees were higher than their prevaccination levels (P< 0.0001). The serum antibody responses were specific, as there was no significant rise of anti-LPS to the heterologous O-SP in any vaccinee. Both conjugates elicited statistically significant rises of serum antibodies to the injected carrier protein. At 6 months, these fiveShigellaconjugates elicited higher fold rises than similar conjugates (D. N. Taylor et al., Infect. Immun. 61:3678–3687, 1993). Based on these data, we choseS. sonnei-CRM9 andS. flexneri2a-rEPAsuccfor evaluation in children.
Cysteine Protease Secreted byParagonimus westermaniAttenuates Effector Functions of Human Eosinophils Stimulated with Immunoglobulin G
Authors: Shin Myeong Heon , Kita Hirohito , Park Hae Young , Seoh Ju Young
Published: July 2002

An immunoglobulin G (IgG)-coated surface, such as that found on helminth parasites, is one of the most effective physiologic stimuli for eosinophil activation. The cysteine proteases secreted by tissue-invasive helminth larvae play an important role in evasion of the immune response by degrading the host immunoglobulins. In this study, we investigated whether cysteine proteases in the excretory-secretory product (ESP) produced byParagonimus westermaninewly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, could modify effector functions of human eosinophils stimulated with IgG. We coated 96-well plates with human IgG in the absence or presence of the ESP produced by PwNEM. When eosinophils were incubated in the wells coated with IgG in the presence of the ESP, eosinophil degranulation and superoxide production were significantly reduced compared with results for cells incubated in wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production was dose dependent and was significantly abolished by pretreatment of the ESP with heat. These findings suggest that the cysteine proteases secreted by PwNEM attenuate both activation and degranulation of eosinophils stimulated with IgG. Thus, the cysteine proteases produced by tissue-invasive helminth larvae play crucial roles in evasion of IgG-dependent eosinophil helminthotoxicity and in reduction of eosinophil-associated tissue inflammation during the migratory period.
Two Epitopes Shared by
Taenia crassiceps
and
Taenia solium
Confer Protection against Murine
T. crassiceps
Cysticercosis along with a Prominent T1 Response
Authors: Toledo Andrea , Fragoso Gladis , Rosas Gabriela , Hernández Marisela , Gevorkian Goar , López-Casillas Fernando , Hernández Beatriz , Acero Gonzalo , Huerta Mirna , Larralde Carlos , Sciutto Edda
Published: July 2002

Taenia crassiceps
recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine
T. crassiceps
cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine
Taenia solium
cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal
T. crassiceps
antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in protection. The protective capacity of the peptides and their presence in all developmental stages of
T. solium
point to these two epitopes as strong candidates for inclusion in a polyepitopic synthetic vaccine against
T. solium
pig cysticercosis.
Spa33, a Cell Surface-Associated Subunit of the Mxi-Spa Type III Secretory Pathway of
Shigella flexneri
, Regulates Ipa Protein Traffic
Authors: Schuch Raymond , Maurelli Anthony T.
Published: July 2002

The Mxi-Spa type III secretion system of
Shigella flexneri
directs the host cell contact-induced secretion of a set of invasins, referred to as Ipas. In this study, we examined the role of Spa33 in Ipa secretion. A
spa33
-null mutant was both noninvasive and unable to translocate the Ipas from inner membrane to outer membrane (OM) positions of the Mxi-Spa transmembrane channel. Spa33 was found to be a Mxi-Spa substrate that is translocated to the bacterial cell surface upon the induction of Ipa secretion. This mobility may serve to drive Ipa translocation within Mxi-Spa toward OM positions. Consistent with a second distinct role in regulating Ipa traffic, the overexpression of Spa33 also blocked Ipa secretion and resulted in Ipa accumulation at the OM. Co-overexpression of Spa33 and another OM-associated element, Spa32, did not disrupt Ipa secretion, suggesting an interaction between the two proteins and an effect on the mechanism which serves to regulate Ipa release from the OM. These findings indicate that Spa33 is a mobile element within Mxi-Spa, which is required to control Ipa translocation into and out of OM positions of the secretory structure.
Induction of Cell-Associated Chemokines after Endotoxin Administration to Healthy Humans
Authors: Olszyna Dariusz P. , De Jonge Evert , Dekkers Pascale E. P. , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

Erythrocytes express the Duffy antigen receptor for chemokines. Endotoxin injection into humans induced high levels of interleukin-8 (IL-8), growth-related oncogene α, and monocyte chemoattractant protein 1 in circulating erythrocytes. IL-8 was also recovered from mononuclear and polymorphonuclear cells. Cell-associated chemokines may more accurately reflect their production than plasma concentrations.
Protection against Cutaneous Leishmaniasis Induced by Recombinant Antigens in Murine and Nonhuman Primate Models of the Human Disease
Authors: Campos-Neto Antonio , Porrozzi Renato , Greeson Kay , Coler Rhea N. , Webb John R. , Seiky Yasir A. W. , Reed Steven G. , Grimaldi Gabriel
Published: July 2002

Leishmaniasis affects approximately 2 million people each year throughout the world. This high incidence is due in part to the lack of an efficacious vaccine. We present evidence that the recombinant leishmanial antigens LmSTI1 and TSA, which we identified and characterized previously, induce excellent protection in both murine and nonhuman primate (rhesus monkey) models of human cutaneous leishmaniasis. The remarkable protection induced by LmSTI1 and TSA in an animal model that is evolutionarily close to humans qualifies this antigen combination as a promising candidate subunit vaccine against human leishmaniasis.
Decreased Apoptosis in the Ileum and Ileal Peyer's Patches: a Feature after Infection with Rabbit Enteropathogenic
Escherichia coli
O103
Authors: Heczko Ursula , Carthy Chris M. , O'Brien Bronwyn A. , Finlay B. Brett
Published: July 2002

Significant changes occur in intestinal epithelial cells after infection with enteropathogenic
Escherichia coli
(EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay;
P
= 0.01), in the tips of ileal absorptive villi (determined by H&E staining;
P
= 0.04), and in whole ileal cell lysates (determined by caspase 3 assay;
P
= 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.
Intranasal Immunization with Killed Unencapsulated Whole Cells Prevents Colonization and Invasive Disease by Capsulated Pneumococci
Authors: Malley Richard , Lipsitch Marc , Stack Anne , Saladino Richard , Fleisher Gary , Pelton Steven , Thompson Claudette , Briles David , Anderson Porter
Published: July 2002

A whole-cell killed unencapsulated pneumococcal vaccine given by the intranasal route with cholera toxin as an adjuvant was tested in two animal models. This vaccination was highly effective in preventing nasopharyngeal colonization with an encapsulated serotype 6B strain in mice and also conferred protection against illness and death in rats inoculated intrathoracically with a highly encapsulated serotype 3 strain. When the serotype 3 challenge strain was incubated in the sera of immunized rats, it was no longer virulent in an infant-rat sepsis model, indicating that the intranasal immunization elicited protective systemic antibodies. These studies suggest that killed whole-cell unencapsulated pneumococci given intranasally with an adjuvant may provide multitypic protection against capsulated pneumococci.
Neisseria meningitidisLipopolysaccharide Modulates the Specific Humoral Immune Response to Neisserial Porins but Has No Effect on Porin-Induced Upregulation of Costimulatory Ligand B7-2
Authors: Bhasin Navneet , Ho Yu , Wetzler Lee M.
Published: July 2002

The role of lipopolysaccharide (LPS) in the specific humoral response to meningococcal porins was investigated by measuring anti-PorA or -PorB antibody levels in mice immunized with wild-type meningococcal strain H44/76 or with its recently described LPS-negative mutant. Two murine strains were used for these immunizations: C3H/HeJ, which is LPS hyporesponsive, or C3H/HeOuJ, which is LPS responsive. A high level of anti-PorB immunoglobulin G (IgG) response was induced in both strains of mice immunized with either organism. The response induced by the wild-type strain was greater in C3H/HeOuJ mice than in C3H/HeJ mice, while the response induced by the LPS-negative mutant was similar in the two murine strains. Additionally, the anti-PorB response was similar in C3H/HeJ mice immunized with either bacterial strain. In general, the anti-PorA IgG response was lower than the anti-PorB response. These findings indicate that the presence of LPS is not essential for the induction of an antineisserial porin humoral response but can augment such a response. To determine whether LPS has any effect on the B-cell-stimulatory effect of neisserial porins (essential for the adjuvant activity of neisserial porins), B cells from both murine strains were incubated with outer membrane complexes (OMCs) prepared from strain H44/76 and its LPS-negative mutant. OMCs from either meningococcal strain were able to increase the surface expression of the costimulatory ligand B7-2 on B cells from either murine strain. Consistent with previously reported findings, LPS does not significantly affect the ability of neisserial porins to induce the costimulatory ligand B7-2.
Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells
Authors: George-Chandy Annie , Eriksson Kristina , Lebens Michael , Nordström Inger , Schön Emma , Holmgren Jan
Published: July 2002

Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.
Sensitivities of Human Monocytes and Epithelial Cells to Pneumolysin Are Different
Authors: Hirst Robert A. , Yesilkaya Hasan , Clitheroe Edwin , Rutman Andrew , Dufty Nichola , Mitchell Timothy J. , O’Callaghan Christopher , Andrew Peter W.
Published: July 2002

TheStreptococcus pneumoniaepore-forming toxin, pneumolysin, is an important virulence factor in pneumococcal pneumonia. The effect of pneumolysin on human lung epithelial and monocyte cell viability was compared. Pneumolysin caused a dose-dependent loss of viability of human lung epithelial (A549 and L132) and monocyte (U937 and THP-1) cell lines. Analysis of the dose-response curves revealed similar log 50% inhibitory concentration (pIC50) values for A549, L132, and THP-1 of 0.12± 0.1, 0.02± 0.04, and 0.12± 0.13 hemolytic units (HU), respectively, but U937 cells showed a significantly greater pIC50of 0.42± 0.12 HU. Differentiation of A549 and L132 with phorbol ester or THP-1 with gamma interferon had no effect on their sensitivity to pneumolysin. However, a significant decrease in the potency of pneumolysin against U937 cells followed gamma interferon treatment. The Hill slopes of the inhibition curves were greater than unity, indicating that pneumolysin may act with positive cooperativity. Analysis of pneumolysin-treated THP-1 cells by electron microscopy revealed membrane lesions of between 100 and 200 nm in diameter.
Replication of
Neisseria meningitidis
within Epithelial Cells Requires TonB-Dependent Acquisition of Host Cell Iron
Authors: Larson Jason A. , Higashi Dustin L. , Stojiljkovic Igor , So Magdalene
Published: July 2002

Neisseria meningitidis
(meningococcus [MC]) is able to enter and replicate within epithelial cells. Iron, an essential nutrient for nearly all organisms, is an important determinant in the ability of MC to cause disease; however, its role in MC intracellular replication has not been investigated. We analyzed the growth of MC within the A431 human epithelial cell line and the dependence of this growth on iron uptake. We present evidence here that chelation of iron from infected tissue culture cells with Desferal strongly inhibited intracellular replication of wild-type (wt) MC. We also provide genetic evidence that iron must be acquired by MC from the host cell in order for it to replicate. An
hmbR
mutant that is unable to use hemoglobin iron and could not grow in tissue culture media without iron supplementation replicated more rapidly within epithelial cells than its wt parent strain. An
fbpA
mutant that is unable to utilize human transferrin iron or lactoferrin iron replicated normally within cells. In contrast, a
tonB
mutant could not replicate intracellularly unless infected cultures were supplemented with ferric nitrate. Taken together, these findings strongly suggest that MC intracellular replication requires TonB-dependent uptake of a novel host cell iron source.
Naturally Exposed Populations Differ in Their T1 and T2 Responses to the Circumsporozoite Protein ofPlasmodium falciparum
Authors: Reece W. H. H. , Plebanski M. , Akinwunmi P. , Gothard P. , Flanagan K. L. , Lee E. A. M. , Cortina-Borja M. , Hill A. V. S. , Pinder M.
Published: July 2002

T-cell responses directed against the circumsporozoite protein (CS) ofPlasmodium falciparumcan mediate protection against malaria. We determined the frequency of T cells reactive to different regions of the CS in the blood of donors naturally exposed toP. falciparumby examining T1 (gamma interferon [IFN-γ] ELISPOT assay), T2 (interleukin 4 [IL-4] ELISPOT assay), and proliferative T-cell responses. The proliferative responses were weak, which confirmed previous observations. The responses to the CS in the IL-4 and IFN-γ ELISPOT assays were also weak (<40 responding cells per 106cells), much weaker than the response to the purified protein derivative ofMycobacterium tuberculosisin the same donors. Moreover, a response in one assay could not be used to predict a response in either of the other assays, suggesting that although these assays may measure different responding cells, all of the responses are weakly induced by natural exposure. Interestingly, the two different study populations used had significantly different T1 and T2 biases in their responses in the C terminus of the protein, suggesting that the extent ofP. falciparumexposure can affect regulation of the immune system.
Receptor-Based Antidote for Diphtheria
Authors: Cha Jeong-Heon , Brooke Joanna S. , Chang Mee Young , Eidels Leon
Published: July 2002

Although equine diphtheria antitoxin may be an effective therapy for human diphtheria, its use often induces serum sickness. We describe here a strategy for developing an alternative treatment based on the human diphtheria toxin (DT) receptor/heparin-binding epidermal growth factor-like growth factor (HB-EGF) precursor. Recombinant mature human HB-EGF acts as a soluble receptor analog, binding radioiodinated DT and preventing its binding to the cellular DT receptor/HB-EGF precursor. However, the possibility existed that radioiodinated DT-HB-EGF complexes associate with cells due to the binding of the heparin-binding domain of recombinant HB-EGF to cell surface heparan sulfate proteoglycans. This possibility was confirmed by performing DT binding studies in the presence of heparin. A recombinant truncated HB-EGF (residues 106 to 149), which lacks most of the heparin-binding domain, showed an essentially heparin-independent binding of radioiodinated DT to cells. Furthermore, it was a more effective inhibitor of DT binding than was recombinant mature HB-EGF. Since mature HB-EGF is a known ligand for the EGF receptor and is thus highly mitogenic (tumorigenic), we then changed amino acid residues in the EGF-like domain of the recombinant truncated HB-EGF and demonstrated that this DT receptor analog (I117A/L148A) displayed a low mitogenic effect. The truncated (I117A/L148A) HB-EGF protein retained high DT binding affinity, as confirmed by using surface plasmon resonance. Our results suggest that the truncated (I117A/L148A) HB-EGF protein could be an effective, safe antidote for human diphtheria.
Immunoglobulin G Monoclonal Antibodies toCryptococcus neoformansProtect Mice Deficient in Complement Component C3
Authors: Shapiro Scott , Beenhouwer David O. , Feldmesser Marta , Taborda Carlos , Carroll Michael C. , Casadevall Arturo , Scharff Matthew D.
Published: July 2002

Passive administration of monoclonal antibodies (MAbs) to the capsular polysaccharide ofCryptococcus neoformanscan alter the course of infection in mice. In a murine model of cryptococcal infection, immunoglobulin G1 (IgG1), IgG2a, and IgG2b switch variants of the anti-capsular 3E5 MAb prolong the survival of lethally infected mice, whereas the 3E5 IgG3 MAb does not protect and in some cases enhances infection, shortening the life spans of infected mice. We examined the role of complement component C3 in Ab-mediated protection by determining the efficacy of the four mouse IgG subclasses againstC. neoformansin mice genetically deficient in factor C3 as well as mice acutely depleted of C3. Similar to other complement-deficient animal models, C3−/−mice and mice depleted of C3 by cobra venom factor were more susceptible toC. neoformansinfection than control mice, providing further evidence that complement is important in the host defense against the fungus. In the absence of C3, all IgG isotypes prolonged the lives of mice infected withC. neoformans, indicating that protection by IgG does not require the complement pathways. Furthermore, we observed protection with IgG3 in the complement-deficient mice, suggesting that complement is involved in the lack of protection observed with IgG3 in other mouse models.
Lipopolysaccharide Down Regulates Both Scavenger Receptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells
Authors: Baranova Irina , Vishnyakova Tatyana , Bocharov Alexander , Chen Zhigang , Remaley Alan T. , Stonik John , Eggerman Thomas L. , Patterson Amy P.
Published: July 2002

Lipopolysaccharide (LPS) has recently been shown to facilitate macrophage foam cell formation and has been suggested to be a proatherogenic factor. The mechanism of LPS induced cholesterol accumulation, however, is unclear. In this report, using the macrophage-like RAW 264.7 cell line, we provide experimental evidence that LPS's proatherogenic effects may at least in part reflect altered cholesterol metabolism. Data presented demonstrate that in a dose-dependent manner, LPS is able to down regulate the mRNA expression of the two primary high-density lipoprotein (HDL) receptors, scavenger receptor B1 (SR-B1) and ATP binding cassette A1 (ABCA1), with a 50% inhibitory concentration of less than 0.2 ng/ml, as well as to decrease SR-B1 protein expression by 80%. We also found that LPS treatment resulted in a significant decrease (to 20% of the control level) of the specific125I-HDL binding as well as in 50% inhibition of the HDL-mediated cholesterol efflux compared to untreated cells. In addition, we compared the potencies of various modified LPS preparations and demonstrated that the phosphorylated lipid A portion of LPS, which is highly conserved among gram-negative microorganisms, includingChlamydia, is primarily responsible for the effects of LPS on SR-B1 and ABCA1 expression. Inhibitors of NF-κB activation were observed to efficiently block the suppressive effect of LPS on SR-B1 and ABCA1, suggesting a mechanism involving NF-κB. These data indicate that the LPS effects on cholesterol metabolism may contribute to the proatherogenic properties of LPS.
Protein Disulfide Isomerase, a Component of the Estrogen Receptor Complex, Is Associated withChlamydia trachomatisSerovar E Attached to Human Endometrial Epithelial Cells
Authors: Davis C. H. , Raulston J. E. , Wyrick P. B.
Published: July 2002

Chlamydia trachomatisserovar E, the leading bacterial agent responsible for sexually transmitted diseases, is required to invade genital epithelial cells for its growth and survival, yet little is known about the adhesin-receptor interactions promoting its entry. In contrast, much has been published on the heparan sulfate receptor for bindingC. trachomatisL2 elementary bodies (EBs) prior to entry into HeLa cells. Using a different experimental approach in which a biotinylated apical membrane protein receptor(s) attached to EB at 4°C was stripped off the surface of polarized HEC-1B cells and immunoprecipitated with polyclonal anti-EB antibodies, an ∼55-kDa protein was reproducibly detected by enhanced chemiluminescence and two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization mass-spectrometry sequence analysis revealed the 55-kDa protein to be protein disulfide isomerase (PDI), a member of the estrogen receptor complex which carries out thiol-disulfide exchange reactions at infected host cell surfaces. Exposure of HEC-1B cells during EB attachment (1.5 to 2 h) to three different inhibitors of PDI reductive reactions—(i) the thiol-alkylating reagent DTNB (5,5′-dithiobis[2-nitrobenzoic acid]), (ii) bacitracin, and (iii) anti-PDI antibodies—resulted in reduced chlamydial infectivity. Since (i)C. trachomatisserovar E attachment to estrogen-dominant primary human endometrial epithelial cells is dramatically enhanced and (ii) productive entry into and infectivity of EB in host cells is dependent on reduction of EB cross-linked outer membrane proteins at the host cell surface, these data provide some preliminary evidence for an intriguing new potential receptor candidate for further analysis of luminalC. trachomatisserovar E entry.
A Plasmid Partition System of the P1-P7
par
Family from the pMT1 Virulence Plasmid of
Yersinia pestis
Authors: Youngren Brenda , Radnedge Lyndsay , Hu Ping , Garcia Emilio , Austin Stuart
Published: July 2002

The complete sequence of the virulence plasmid pMT1 of
Yersinia pestis
KIM5 revealed a region homologous to the plasmid partition (
par
) region of the P7 plasmid prophage of
Escherichia coli
. The essential genes
parA
and
parB
and the downstream partition site gene,
parS
, are highly conserved in sequence and organization. The pMT1
parS
site and the
parA-parB
operon were separately inserted into vectors that could be maintained in
E. coli
. A mini-P1 vector containing pMT1
parS
was stably maintained when the pMT1 ParA and ParB proteins were supplied in
trans
, showing that the pMT1
par
system is fully functional for plasmid partition in
E. coli
. The pMT1
par
system exerted a plasmid silencing activity similar to, but weaker than those of P7
par
and P1
par
. In spite of the high degree of similarity, especially to P7
par
, it showed unique specificities with respect to the interactions of key components. Neither the P7 nor P1 Par proteins could support partition via the pMT1
parS
site, and the pMT1 Par proteins failed to support partition with P1
parS
or P7
parS
. Typical of other partition sites, supernumerary copies of pMT1
parS
exerted incompatibility toward plasmids supported by pMT1
par
. However, no interspecies incompatibility effect was observed between pMT1par, P7
par
, and P1
par
.
Effects of Organic Antagonists of Ca
2+
, Na
+
, and K
+
on Chemotaxis and Motility of
Escherichia coli
Authors: Tisa Louis S. , Sekelsky Jeff J. , Adler Julius
Published: July 2002

Various Ca
2+
antagonists used in animal research, many of them known to be Ca
2+
channel blockers, inhibited
Escherichia coli
chemotaxis (measured as entry of cells into a capillary containing attractant). The most effective of these, acting in the nanomolar range, was ω-conotoxin GVIA. The next most effective were gallopamil and verapamil. At concentrations around 100-fold higher than that needed for inhibition of chemotaxis, each of these antagonists inhibited motility (measured as entry of cells into a capillary lacking attractant). Various other Ca
2+
antagonists were less effective, though chemotaxis was almost always more sensitive to inhibition than was motility. Cells treated with each of these Ca
2+
antagonists swam with a running bias, i.e., tumbling was inhibited. Similarly, some Na
+
antagonists used in animal research inhibited bacterial chemotaxis.
E. coli
chemotaxis was inhibited by saxitoxin at concentrations above 10
−7
M, while more than 10
−4
M was needed to inhibit motility. Cells treated with saxitoxin swam with a tumbling bias. In the case of other Na
+
antagonists in animals, aconitine inhibited bacterial chemotaxis 10 times more effectively than it inhibited motility, and two others inhibited chemotaxis and motility at about the same concentration. In the case of K
+
antagonists used in animal research, 4-aminopyridine blocked
E. coli
chemotaxis between 10
−3
M and, totally, 10
−2
M, while motility was not affected at 10
−2
M; on the other hand, tetraethylammonium chloride failed to inhibit either chemotaxis or motility at 10
−2
M.
Molecular Cloning and Characterization of Two Genes for the Biotin Carboxylase and Carboxyltransferase Subunits of Acetyl Coenzyme A Carboxylase in
Myxococcus xanthus
Authors: Kimura Yoshio , Miyake Rina , Tokumasu Yushi , Sato Masayuki
Published: July 2002

We have cloned a DNA fragment from a genomic library of
Myxococcus xanthus
using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the
accB
and
accA
genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the
accA
gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO
2
fixation, and biotin binding were found in AccA. The
accB
gene was located upstream of the
accA
gene, and they formed a two-gene operon. The protein (AccB) encoded by the
accB
gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An
accA
disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the
accA
gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this
M. xanthus
acetyl-CoA carboxylase consists of two subunits, which are encoded by the
accB
and
accA
genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.
Developmental Aggregation of
Myxococcus xanthus
Requires
frgA
, an
frz
-Related Gene
Authors: Cho Kyungyun , Treuner-Lange Anke , O'Connor Kathleen A. , Zusman David R.
Published: July 2002

Myxococcus xanthus
is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the
frz
genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called
frgA
(for
frz
-related gene), which confers a subset of the frizzy phenotype when mutated. The
frgA
null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of
frz
mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the
frgA
mutant could be complemented extracellularly by wild-type cells or strains carrying non-
frz
mutations. The phenotype of the
frgA
mutant is similar to that of the
abcA
mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The
frgA
gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The
frgA
gene is part of an operon which includes two additional genes,
frgB
and
frgC
. The
frgB
gene encodes a putative histidine protein kinase, and the
frgC
gene encodes a putative response regulator. The
frgB
and
frgC
null mutants, however, formed wild-type fruiting bodies.
Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, fromThermotoga maritima
Authors: Yamanaka Kunitoshi , Hwang Jihwan , Inouye Masayori
Published: July 2002

A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned fromThermotoga maritima, a hyperthermophilic bacterium.T. maritimaTrmE was overexpressed inEscherichia coliand purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP.Kmandkcatat 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.
Molecular, Antigenic, and Functional Analyses of Omp2b Porin Size Variants of
Brucella
spp
Authors: Paquet Jean-Yves , Diaz Maria A. , Genevrois Stephanie , Grayon Maggy , Verger Jean-Michel , De Bolle Xavier , Lakey Jeremy H. , Letesson Jean-Jacques , Cloeckaert Axel
Published: July 2002

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus
Brucella
, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the
Brucella
genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various
Brucella
species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of
Brucella melitensis
reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus
Brucella
seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of
Brucella suis
biovar 3), the porin variability does not result in major antigenic variability of the
Brucella
surface that could help the bacteria during the reinfection of a host. Porin variation in
Brucella
seems to result mainly in porin conductivity modifications.
Control of a Family of Phosphatase Regulatory Genes (
phr
) by the Alternate Sigma Factor Sigma-H of
Bacillus subtilis
Authors: McQuade Ryan S. , Comella Natalia , Grossman Alan D.
Published: July 2002

A family of 11 phosphatases can help to modulate the activity of response regulator proteins in
Bacillus subtilis
. Downstream of seven of the
rap
(phosphatase) genes are
phr
genes, encoding secreted peptides that function as phosphatase regulators. By using fusions to
lacZ
and primer extension analysis, we found that six of the seven
phr
genes are controlled by the alternate sigma factor sigma-H. These results expand the potential of sigma-H to contribute to the output of several response regulators by controlling expression of inhibitors of phosphatases.
Molecular Evidence for Independent Occurrence of IS
6110
Insertions at the Same Sites of the Genome of
Mycobacterium tuberculosis
in Different Clinical Isolates
Authors: Fang Z. , Kenna D. T. , Doig C. , Smittipat D. N. , Palittapongarnpim P. , Watt B. , Forbes K. J.
Published: July 2002

Several characteristics of
Mycobacterium tuberculosis
(e.g., conserved genome and low growth rate) have severely restricted the study of the microorganism. The discovery of IS
6110
raised hopes of overcoming these obstacles. However, our knowledge of this IS element is relatively limited; even its two basic characteristics (transposition mechanism and target site selection) are far from well understood. In this study, IS
6110
insertions in
ipl
loci (
iplA
and
iplB
) in two collections of clinical isolates of
M. tuberculosis
from different geographic locations, one from Scotland and the other from Thailand, were investigated. Five different IS
6110
insertions in the loci were identified:
ipl-4
::IS
6110
,
ipl-5
::IS
6110
,
ipl-11
::IS
6110
,
ipl-12
::IS
6110
, and
ipl-13
::IS
6110
. An attempt to establish the phylogenetic relationship of the isolates containing these insertions was unsuccessful, suggesting that some of these insertions may have arisen from more than one event. This possibility is further supported by the observation that IS
6110
copies existed in the same site but with different orientations in different isolates, and the insertion site of
ipl-1
::IS
6110
harbored IS
6110
copies in both
iplA
and
iplB
in different strains. All these suggest the independent occurrence of IS
6110
insertions at the same sites of the genome of
M. tuberculosis
in different clinical isolates. The implications of this finding are discussed.
Directed Evolution of Biphenyl Dioxygenase: Emergence of Enhanced Degradation Capacity for Benzene, Toluene, and Alkylbenzenes
Authors: Suenaga Hikaru , Mitsuoka Mariko , Ura Yuko , Watanabe Takahito , Furukawa Kensuke
Published: July 2002

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the
bphA1
gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of
bphA1
of
Pseudomonas pseudoalcaligenes
KF707 and
Burkholderia cepacia
LB400, a number of evolved Bph Dox enzymes were created. Among them, an
Escherichia coli
clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.
Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Authors: Adams Michael W. W. , Holden James F. , Menon Angeli Lal , Schut Gerrit J. , Grunden Amy M. , Hou Chun , Hutchins Andrea M. , Jenney Francis E. , Kim Chulhwan , Ma Kesen , Pan Guangliang , Roy Roopali , Sapra Rajat , Story Sherry V. , Verhagen Marc F. J. M.
Published: July 2002

The hyperthermophilic archaeon
Pyrococcus furiosus
grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S
0
). Growth rates were highest on media containing peptides and S
0
, with or without maltose. Growth did not occur on the peptide medium without S
0
. S
0
had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S
0
with or without maltose were the same, suggesting that S
0
is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of
P. furiosus
were shown to be regulated under the five different growth conditions studied. The presence of S
0
in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S
0
-reducing enzyme in this organism and the mechanism of the S
0
dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in
P. furiosus
and a significant regulatory role for elemental sulfur or its metabolites.