bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine Specificity of Serum Antibodies toPlasmodium falciparumMerozoite Surface Protein, PfMSP-119, Predicts Protection from Malaria Infection and High-Density Parasitemia
Authors: Okech Brenda A. , Corran Patrick H. , Todd James , Joynson-Hicks Amy , Uthaipibull Chairat , Egwang Thomas G. , Holder Anthony A. , Riley Eleanor M.
Published: February 2004

Antibodies to the C terminus of thePlasmodium falciparummerozoite surface protein, PfMSP-119, may inhibit merozoite invasion or block the effects of inhibitory antibodies. Here, using a competition enzyme-linked immunosorbent assay and antibody binding to wild-type and mutated recombinant proteins, we show that there are marked variations between individuals in the fine specificity of naturally acquired anti-MSP-119antibodies. Furthermore, although neither the prevalence nor the concentration of total anti-MSP-119antibodies was associated with resistance to malaria in African children, significant associations were observed between antibody fine specificity and subsequent risk of infection and high-density parasitemia during a follow-up period. Thus, the fine specificity of naturally acquired human anti-MSP-119antibodies is crucial in determining their function. Future field studies, including the evaluation of PfMSP-1 vaccine trials, should include assays that explore antibody fine specificity as well as titer.
A Locus Contained within a Variable Region of Pneumococcal Pathogenicity Island 1 Contributes to Virulence in Mice
Authors: Brown Jeremy S. , Gilliland Sarah M. , Spratt Brian G. , Holden David W.
Published: February 2004

We have previously described a 27-kb pathogenicity island of
Streptococcus pneumoniae
, termed pneumococcal pathogenicity island 1 (PPI1), which contains iron uptake locus
piaABCD
, required for full virulence in mice, and a further 28 previously uncharacterized genes. We have investigated one of these, Sp1051, which encodes a protein of unknown function. Disruption of Sp1051 does not affect growth in laboratory broth, serum, or blood but impairs virulence in mouse models of infection. When
S. pneumoniae
capsular serotypes were analyzed by PCR and Southern hybridization, it was found that 33% did not contain Sp1051. Analysis of other genes within PPI1 demonstrated that, compared to the serotype 4 genome published by The Institute for Genome Research (TIGR), the genomes of many strains contain deletions of a variable number of genes between Sp1046 and Sp1064, conforming to one of six different patterns. Amplification by PCR of this PPI1 variable region from a capsular serotype 17 strain and comparison of the sequence to TIGR serotype 4 strain sequence showed that Sp1051 is contained within an 11.3-kb segment of DNA flanked by 7-bp direct repeats within the serotype 4 strain which is not present in the serotype 17 strain. Further comparison of the sequences of this region between the three published
S. pneumoniae
genomes demonstrated that serotype 19F and strain R6 contain novel complements of genes not present in the serotype 4 strain. These data indicate that there is striking variation in gene content and structure of the 3′ region of PPI1 among strains and that this region includes at least one virulence determinant. Gene variation within horizontally acquired DNA such as that of PPI1 may be one factor modulating differences in virulence among strains.
Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome
Authors: Haddad Diana , Bilcikova Erika , Witney Adam A. , Carlton Jane M. , White Charles E. , Blair Peter L. , Chattopadhyay Rana , Russell Joshua , Abot Esteban , Charoenvit Yupin , Aguiar Joao C. , Carucci Daniel J. , Weiss Walter R.
Published: February 2004

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from thePlasmodium yoelii yoeliigenomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protectiveP. y. yoeliigenes can then be identified in the genomic databases ofPlasmodium falciparumandPlasmodium vivaxand investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192P. y. yoeliiexons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encodingP. y. yoeliicircumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs inP. vivaxandP. knowlesibut notP. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.
Antibodies That Inhibit Binding of
Plasmodium falciparum
-Infected Erythrocytes to Chondroitin Sulfate A and to the C Terminus of Merozoite Surface Protein 1 Correlate with Reduced Placental Malaria in Cameroonian Women
Authors: Taylor Diane Wallace , Zhou Aniong , Marsillio Lauren E. , Thuita Lucy W. , Leke Efua B. , Branch OraLee , Gowda D. Channe , Long Carole , Leke Rose F. G.
Published: February 2004

Plasmodium falciparum
-infected erythrocytes often sequester in the placenta of pregnant women, producing placental malaria, a condition that can compromise the health of the developing fetus. Scientists are hopeful that a vaccine can be developed to prevent this condition. Immunological mechanisms responsible for eliminating parasites from the placenta remain unclear, but antibodies to the carboxyl-terminal 19-kDa segment of the merozoite surface protein 1 (MSP1-19), the ring-infected erythrocyte surface antigen (RESA), and an erythrocyte-surface ligand that binds chondroitin sulfate A (CSA-L) have been implicated. In addition, antibodies to sporozoite and liver-stage antigens could reduce initial parasite burdens. This study sought to determine if antibodies to the circumsporozoite protein (CSP), liver-stage antigen 1 (LSA1), RESA, MSP1-19, or CSA-L correlated with either the absence of placental parasites or low placental parasitemias. Using a frequency-matched case-control study design, we compared antibody levels in women (gravidity 1 to 11) with and without placental malaria. Results showed that women who were antibody negative for MSP1-19 were at a higher risk of having placental malaria than women with antibodies (
P
< 0.007). Furthermore, an association between high levels of antibodies that blocked the binding of infected erythrocytes to CSA and low placental parasitemias was observed (
P
= 0.02). On the other hand, women with high antibody levels at term to CSP, LSA1, and RESA were more likely to have placental malaria than antibody-negative women. Since antibodies to MSP1-19 and CSA-L were associated with reduced placental malaria, both antigens show promise for inclusion in a vaccine for women of child-bearing age.
Combination of the Cationic Surfactant Dimethyl Dioctadecyl Ammonium Bromide and Synthetic Mycobacterial Cord Factor as an Efficient Adjuvant for Tuberculosis Subunit Vaccines
Authors: Holten-Andersen L. , Doherty T. M. , Korsholm K. S. , Andersen P.
Published: February 2004

Recombinant, immunodominant antigens derived fromMycobacterium tuberculosiscan be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates Th1 T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of Th1 responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, againstM. tuberculosisinfection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.
Mycobacterium-InducibleNrampin Striped Bass (Morone saxatilis)
Authors: Burge Erin J. , Gauthier David T. , Ottinger Christopher A. , Van Veld Peter A.
Published: February 2004

In mammals, the natural resistance-associated macrophage protein 1 gene,Nramp1, plays a major role in resistance to mycobacterial infections. Chesapeake Bay striped bass (Morone saxatilis) is currently experiencing an epizootic of mycobacteriosis that threatens the health of this ecologically and economically important species. In the present study, we characterized anNrampgene in this species and obtained evidence that there is induction followingMycobacteriumexposure. The striped bassNrampgene (MsNramp) and a 554-amino-acid sequence contain all the signal features of theNrampfamily, including a topology of 12 transmembrane domains (TM), the transport protein-specific binding-protein-dependent transport system inner membrane component signature, three N-linked glycosylation sites between TM 7 and TM 8, sites of casein kinase and protein kinase C phosphorylation in the amino and carboxy termini, and a tyrosine kinase phosphorylation site between TM 6 and TM 7. Phylogenetic analysis most closely groupedMsNrampwith other teleostNrampgenes and revealed high sequence similarity with mammalianNramp2. MsNrampexpression was present in all tissues assayed by reverse transcription-PCR. Within 1 day of injection ofMycobacterium marinum,MsNrampexpression was highly induced (17-fold higher) in peritoneal exudate (PE) cells compared to the expression in controls. The levels ofMsNrampwere three- and sixfold higher on days 3 and 15, respectively. Injection ofMycobacterium shottsiiresulted in two-, five-, and threefold increases in gene expression in PE cells over the time course. This report is the first report of induction of anNrampgene by mycobacteria in a poikilothermic vertebrate.
Blockade of Endogenous Leukotrienes Exacerbates Pulmonary Histoplasmosis
Authors: Medeiros Alexandra I. , Sá-Nunes Anderson , Soares Edson G. , Peres Camila M. , Silva Célio L. , Faccioli Lúcia H.
Published: February 2004

Leukotrienes are classical mediators of inflammatory response. New aspects of leukotriene function have recently been described. We examine here the previously unreported role that leukotrienes play in the regulation of cytokines in a murine model of histoplasmosis. We demonstrate that administration of MK 886, a leukotriene synthesis inhibitor, causedHistoplasma capsulatum-infected mice to die by the day 15 of infection, whereas the correlating death rate in untreated infected mice was 0%. Treating infected animals with MK 886 inhibited leukotriene synthesis but increased leukocyte recruitment to the lungs. Subsequent to this phenomenon, levels of tumor necrosis factor alpha, interleukin-1 (IL-1), IL-6, and KC chemoattractant cytokines and fungi in the lung parenchyma increased, as did inflammatory response. In contrast, IL-2, IL-5, IL-12, and gamma interferon cytokine levels actually decreased. Thus, murine response to pulmonary histoplasmosis may be leukotriene modulated. This finding may enable us to alter the course of the immune response and inflammation caused by histoplasmosis. The data from the present study suggest an important new strategy for immunologic or drug intervention in human patients.
Characterization of OralYersinia enterocoliticaInfection in Three Different Strains of Inbred Mice
Authors: Handley Scott A. , Dube Peter H. , Revell Paula A. , Miller Virginia L.
Published: February 2004

Several studies have highlighted differences in the resistances of various mouse strains to intravenous (i.v.) infection withYersinia enterocolitica. In particular, differences in resistance and immunological response between BALB/c and C57BL/6 mouse strains have been determined. Following i.v infection, C57BL/6 mice are more resistant toY. enterocoliticathan are BALB/c mice. However, becauseY. enterocoliticais typically a food-borne pathogen, the oral route of infection more accurately reflects the natural route of infection. Therefore, it was of interest to ascertain if the differences in resistance between mouse strains observed for an i.v. infection can be recapitulated following an oral infection. C57BL/6j, BALB/cj, and 129X1/Svj mouse strains presented no differences in 50% lethal dose (LD50) following oral infection withY. enterocolitica. Subsequent analysis of cytokine levels, bacterial colonization and immune cell populations following oral infection confirmed characteristics previously described following i.v.Y. enterocoliticainfection. All tissues analyzed from each mouse strain demonstrated a polarized Th1 cytokine profile and inflammatory cell influx throughout a 7-day course of infection. This immune response was present in all tissues and increased as bacterial colonization progressed. The lack of a differing LD50phenotype and common trends in immunological response among the three mouse strains tested suggests that oral infection is a useful model for studying the host response toY. enterocoliticainfection.
Relationship between Structures and Biological Activities of Mycoplasmal Diacylated Lipopeptides and Their Recognition by Toll-Like Receptors 2 and 6
Authors: Okusawa Tsugumi , Fujita Mari , Nakamura Jun-ichiro , Into Takeshi , Yasuda Motoaki , Yoshimura Atsutoshi , Hara Yoshitaka , Hasebe Akira , Golenbock Douglas T. , Morita Manabu , Kuroki Yoshio , Ogawa Tomohiko , Shibata Ken-ichiro
Published: February 2004

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam2CGDPKHPKSF] synthesized on the basis of the N-terminal structure of aMycoplasma salivariumlipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as theMycoplasma fermentans-derived lipopeptide MALP-2 (Pam2CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-κB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-κB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.
Glycolytic and Gluconeogenic Growth of
Escherichia coli
O157:H7 (EDL933) and
E. coli
K-12 (MG1655) in the Mouse Intestine
Authors: Miranda Regina L. , Conway Tyrrell , Leatham Mary P. , Chang Dong Eun , Norris Wendy E. , Allen James H. , Stevenson Sarah J. , Laux David C. , Cohen Paul S.
Published: February 2004

Escherichia coli
EDL933, an O157:H7 strain, is known to colonize the streptomycin-treated CD-1 mouse intestine by growing in intestinal mucus (E. A. Wadolkowski, J. A. Burris, and A. D. O'Brien, Infect. Immun.
58:
2438-2445, 1990), but what nutrients and metabolic pathways are employed during colonization has not been determined. In this study, when the wild-type EDL933 strain was fed to mice along with an EDL933 Δ
ppsA
Δ
pckA
mutant, which is unable to utilize tricarboxylic acid cycle intermediates and gluconeogenic substrates for growth, both strains colonized the mouse intestine equally well. Therefore, EDL933 utilizes a glycolytic substrate(s) for both initial growth and maintenance when it is the only
E. coli
strain fed to the mice. However, in the presence of large numbers of MG1655, a K-12 strain, it is shown that EDL933 utilizes a glycolytic substrate(s) for initial growth in the mouse intestine but appears to utilize both glycolytic and gluconeogenic substrates in an attempt to maintain colonization. It is further shown that MG1655 predominantly utilizes glycolytic substrates for growth in the mouse intestine whether growing in the presence or absence of large numbers of EDL933. Data are presented showing that although small numbers of EDL933 grow to large numbers in the intestine in the presence of large numbers of MG1655 when both strains are fed to mice simultaneously, precolonization with MG1655 affords protection against subsequent colonization by EDL933. Moreover, in mice that are precolonized with EDL933, small numbers of MG1655 are able to grow rapidly in the intestine and EDL933 is eliminated. In situ hybridization experiments using
E. coli
-specific rRNA probes showed that while MG1655 is found only in mucus, EDL933 is found both in mucus and closely associated with intestinal epithelial cells. The data are discussed with respect to competition for nutrients and to the protection that some intestinal commensal
E. coli
strains might afford against infection by O157:H7 strains.
Immunization with a DNA Vaccine Cocktail Protects Mice Lacking CD4 Cells against an Aerogenic Infection withMycobacterium tuberculosis
Authors: Derrick Steven C. , Repique Charlene , Snoy Philip , Yang Amy Li , Morris Sheldon
Published: February 2004

Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV). Effective vaccination against TB in persons with HIV has been considered unlikely because of the central role that CD4 cells play in controlling tuberculous infections. Here we show that the vaccination of CD8−/−mice with a TB DNA vaccine cocktail did not significantly enhance protective responses to aMycobacterium tuberculosisinfection. In contrast, immunization with a DNA vaccine cocktail or with the current TB vaccine,Mycobacterium bovisBCG, induced considerable antituberculosis protective immunity in immune-deficient mice lacking CD4 cells. In vaccinated CD4−/−animals, substantially reduced bacterial burdens in organs and much improved lung pathology were seen 1 month after an aerogenicM. tuberculosischallenge. Importantly, the postchallenge mean times to death of vaccinated CD4−/−mice were significantly extended (mean with DNA cocktail, 172 ± 7 days; mean with BCG, 156 ± 22 days) compared to that of naïve CD4−/−mice (33 ± 6 days). Furthermore, the treatment of DNA-vaccinated CD4−/−mice with an anti-CD8 or anti-gamma interferon (IFN-γ) antibody significantly reduced the effect of immunization, and neither IFN-γ−/−nor tumor necrosis factor receptor-deficient mice were protected by DNA immunization; therefore, the primary vaccine-induced protective mechanism in these immune-deficient mice likely involves the secretion of cytokines from activated CD8 cells. The substantial CD8-mediated protective immunity that was generated in the absence of CD4 cells suggests that it may be possible to develop effective TB vaccines for use in HIV-infected populations.
Susceptibility to Tuberculosis: Clues from Studies with Inbred and Outbred New Zealand White Rabbits
Authors: Dorman Susan E. , Hatem Christine L. , Tyagi Sandeep , Aird Katherine , Lopez-Molina Javier , Pitt M. Louise M. , Zook Bernard C. , Dannenberg Arthur M. , Bishai William R. , Manabe Yukari C.
Published: February 2004

The rabbit model of tuberculosis (TB) is important because rabbits develop a disease that is similar to TB in humans, namely, granulomas with caseous necrosis, liquefaction, and cavities. We describe here a comparison of inbred and outbred New Zealand White rabbits infected by aerosol with eitherMycobacterium tuberculosisErdman or H37Rv strain. Five weeks after infection with either bacillary strain, the inbred rabbits had significantly larger pulmonary tubercles than did outbred rabbits (2.7 versus 1.4 mm in diameter;P< 0.01). After infection with H37Rv, the inbred rabbits had significantly more pulmonary tubercles than did the outbred rabbits (98 ± 12 versus 33 ± 13;P< 0.01), with more mycobacterial CFU per tubercle (809 ± 210 versus 215 ± 115;P= 0.027) (means ± standard errors of the means). Compared with histologic examination of lung granulomas from outbred rabbits, histologic examination of those from inbred rabbits showed more caseous necrosis, more visible bacilli, and fewer mature epithelioid cells. The delayed-type hypersensitivity (DTH) responses to intradermal tuberculin were significantly lower, and peritoneal macrophages from uninfected inbred rabbits produced significantly less tumor necrosis factor alpha after lipopolysaccharide (LPS) stimulation in vitro than those from the outbred rabbits (2,413 ± 1,154 versus 8,879 ± 966 pg/ml). We conclude that these inbred rabbits were more susceptible to TB than their outbred counterparts and had an impaired ability to contain disease, resulting in more grossly visible tubercles that were larger than those observed in outbred rabbits. Preliminary evidence is presented for a cell-mediated immune defect with lower DTH responses and macrophages that have a decreased ability to respond to in vitro stimulation with LPS orM. tuberculosisinfection.
Porphyromonas gingivalisInduces Receptor Activator of NF-κB Ligand Expression in Osteoblasts through the Activator Protein 1 Pathway
Authors: Okahashi Nobuo , Inaba Hiroaki , Nakagawa Ichiro , Yamamura Taihei , Kuboniwa Masae , Nakayama Koji , Hamada Shigeyuki , Amano Atsuo
Published: February 2004

Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viableP. gingivalison cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection withP. gingivalisinduced receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-κB) did not prevent the RANKL expression induced byP. gingivalis. Degradation of inhibitor of NF-κB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced byP. gingivalisinfection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection withP. gingivalisKDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest thatP. gingivalisinfection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.
FimbriatedPorphyromonas gingivalisIs More Efficient than Fimbria-DeficientP. gingivalisin Entering Human Dendritic Cells In Vitro and Induces an Inflammatory Th1 Effector Response
Authors: Jotwani Ravi , Cutler Christopher W.
Published: February 2004

Porphyromonas gingivalisis a fimbriated mucosal pathogen implicated in chronic periodontitis (CP). The fimbriae are required for invasion of the gingival mucosa and for induction of CP in animal models of periodontitis. CP is associated with infection of immature dendritic cells (DCs) byP. gingivalisin situ and with increased numbers of dermal DCs (DDCs) and mature DCs in the lamina propria. The role of fimbriae in gaining entry into human DCs and how this modulates the inflammatory and effector immune responses, however, have not been explored. To address this, we generated monocyte-derived DCs (MDDCs) in vitro which phenotypically and functionally resemble DDCs. We show here that virulent fimbriatedP. gingivalis381, in contrast to its fimbria-deficient mutant,P. gingivalisDPG3, efficiently gains entry to MDDCs in a manner dependent on active cell metabolism and cytoskeletal rearrangement. In addition, uptake of 381, unlike DPG3, induces DCs to undergo maturation, upregulate costimulatory molecules, and secrete inflammation cytokines interleukin-1β (IL-1β), IL-6, tumor necrosis factor alpha, IL-10, and IL-12. Moreover, MDDCs pulsed with 381 also stimulated a higher autologous mixed lymphocyte reaction and induced a Th1-type response, with gamma interferon (IFN-γ) being the main cytokine. Monocytes used as controls demonstrated fimbria-dependent uptake of 381 as well but produced low levels of inflammatory cytokines compared to MDDCs. When MDDCs were pulsed with recombinant fimbrillin ofP. gingivalis(10 μg/ml), maturation of MDDCs was also induced; moreover, matured MDDCs induced proliferation of autologous CD4+T cells and release of IFN-γ. Thus, these results establish the significance ofP. gingivalisfimbriae in the uptake ofP. gingivalisby MDDCs and in induction of immunostimulatory Th1 responses.
Fusarium oxysporum
as a Multihost Model for the Genetic Dissection of Fungal Virulence in Plants and Mammals
Authors: Ortoneda Montserrat , Guarro Josep , Madrid Marta P. , Caracuel Zaira , Roncero M. Isabel G. , Mayayo Emilio , Di Pietro Antonio
Published: February 2004

Fungal pathogens cause disease in plant and animal hosts. The extent to which infection mechanisms are conserved between both classes of hosts is unknown. We present a dual plant-animal infection system based on a single strain of
Fusarium oxysporum
, the causal agent of vascular wilt disease in plants and an emerging opportunistic human pathogen. Injection of microconidia of a well-characterized tomato pathogenic isolate (isolate 4287) into the lateral tail vein of immunodepressed mice resulted in disseminated infection of multiple organs and death of the animals. Knockout mutants in genes encoding a mitogen-activated protein kinase, a pH response transcription factor, or a class V chitin synthase previously shown to be implicated in virulence on tomato plants were tested in the mouse model. The results indicate that some of these virulence factors play functionally distinct roles during the infection of tomato plants and mice. Thus, a single
F. oxysporum
strain can be used to study fungal virulence mechanisms in plant and mammalian pathogenesis.
C3 Promotes Clearance ofKlebsiella pneumoniaeby A549 Epithelial Cells
Authors: de Astorza Beatriz , Cortés Guadalupe , Crespí Catalina , Saus Carles , Rojo José María , Albertí Sebastián
Published: February 2004

The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen,Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulatedK. pneumoniaeclinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonizeK. pneumoniae. CD46 could competitively inhibit the internalization ofK. pneumoniaeby the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonizedK. pneumoniaeto these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.
Identification and Molecular Characterization of EatA, an Autotransporter Protein of Enterotoxigenic
Escherichia coli
Authors: Patel Seema K. , Dotson Jimmie , Allen Kenneth P. , Fleckenstein James M.
Published: February 2004

Enterotoxigenic
Escherichia coli
(ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed Tn
phoA
mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant Tn
phoA
.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal
E. coli
K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the
Enterobacteriaceae
proteins. The gene identified in H10407,
eatA
(ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by
Shigella flexneri
. DNA hybridization and PCR demonstrated that
eatA
resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the Tn
phoA
.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H
134
, D
162
, and S
267
. Finally, interruption of the
eatA
gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.
Inhibitory Effect of Antiserum to Surface Antigen P50 of
Babesia gibsoni
on Growth of Parasites in Severe Combined Immunodeficiency Mice Given Canine Red Blood Cells
Authors: Fukumoto Shinya , Xuan Xuenan , Takabatake Noriyuki , Igarashi Ikuo , Sugimoto Chihiro , Fujisaki Kozo , Nagasawa Hideyuki , Mikami Takeshi , Suzuki Hiroshi
Published: February 2004

The inhibitory effect of an antiserum to surface protein P50 of
Babesia gibsoni
on the growth of the parasite was determined with severe combined immunodeficiency mice given canine red blood cells. The antiserum to the recombinant P50 protein significantly inhibited the parasite growth, indicating that P50 might be a useful vaccine candidate.
Identification of
srv
, a PrfA-Like Regulator of Group A
Streptococcus
That Influences Virulence
Authors: Reid Sean D. , Montgomery Alison G. , Musser James M.
Published: February 2004

We have identified a Crp/Fnr-like transcriptional regulator of
Streptococcus pyogenes
that when inactivated attenuates virulence. The gene, named
srv
for
s
treptococcal regulator of virulence, encodes a 240-amino-acid protein with 53% amino acid similarity to PrfA, a transcriptional activator of virulence in
Listeria monocytogenes
.
Characterization of Cytolethal Distending Toxin Genes and Expression in Shiga Toxin-Producing
Escherichia coli
Strains of Non-O157 Serogroups
Authors: Bielaszewska Martina , Fell Marina , Greune Lilo , Prager Rita , Fruth Angelika , Tschäpe Helmut , Schmidt M. Alexander , Karch Helge
Published: February 2004

We identified cytolethal distending toxin and its gene (
cdt
) in 17 of 340 non-O157 Shiga toxin-producing
Escherichia coli
(STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were
eae
negative.
cdt
is either chromosomal and homologous to
cdt-V
(serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to
cdt-III
(serotype O153:H18). Among
eae
-negative STEC,
cdt
was associated with disease (
P
= 0.003).
Inclusion of CpG Adjuvant with Plasmid DNA Coding for NcGRA7 Improves Protection against Congenital Neosporosis
Authors: Jenkins Mark , Parker Carolyn , Tuo Wenbin , Vinyard Bryan , Dubey J. P.
Published: February 2004

The present study showed that incorporation of CpG adjuvant into plasmid DNA coding for NcGRA7 antigen resulted in a twofold increase in the level of protection against congenital transfer of
Neospora caninum
. The level of protection was considerably higher than that observed in pups born from dams immunized with nonrecombinant plasmid.
Heterozygous Toll-Like Receptor 2 Polymorphism Does Not Affect Lipoteichoic Acid-Induced Chemokine and Inflammatory Responses
Authors: von Aulock Sonja , Schröder Nicolas W. J. , Traub Stephanie , Gueinzius Katja , Lorenz Eva , Hartung Thomas , Schumann Ralf R. , Hermann Corinna
Published: February 2004

While transfection of
tlr2
conveyed responsiveness to lipoteichoic acid (LTA), the Arg753Gln polymorphic gene could not. LTA induced a stronger chemokine and anti-inflammatory response than lipopolysaccharides did. Blood from heterozygous polymorphic and wild-type donors reacted uniformly to LTA and
Staphylococcus aureus.
Thus, one functional allele for Toll-like receptor 2 suffices for full cytokine response.
Shiga Toxin 1 Targets Bovine Leukemia Virus-Expressing Cells
Authors: Ferens Witold A. , Grauke Luke J. , Hovde Carolyn J.
Published: February 2004

Direct evidence that
Escherichia coli
Shiga toxin (Stx) acts against bovine leukemia virus (BLV)-expressing cells was obtained. The active A subunit of Stx type 1 (StxA1) targeted a selected population of permeable cells expressing BLV and inhibited BLV replication in a culture of bovine peripheral blood mononuclear cells. Cells were cultured with and without StxA1, and at various times cells expressing BLV were identified by being stained with MW1 monoclonal antibody specific for the BLV protein gp51. Before culture, permeable cells were tagged by uptake of one of the following: acetoxymethyl of 2′,7′-
bis
-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF), BCECF conjugated to 70-kDa dextran, or 70-kDa dextran conjugated to fluorescein. The tagged cells costaining with anti-gp51 were selectively eliminated in StxA1-treated cultures. Electron microscopy analysis of purified B lymphocytes showed sharply reduced numbers of BLV particles in StxA1-treated cultures.
Role of Class A Penicillin-Binding Proteins in PBP5-Mediated β-Lactam Resistance inEnterococcus faecalis
Authors: Arbeloa Ana , Segal Heidi , Hugonnet Jean-Emmanuel , Josseaume Nathalie , Dubost Lionnel , Brouard Jean-Paul , Gutmann Laurent , Mengin-Lecreulx Dominique , Arthur Michel
Published: February 2004

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. InEnterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes ofE. faecalisJH2-2 (ponA,pbpF, andpbpZ). Among mutants with single or double deletions, only JH2-2 ΔponAΔpbpFwas susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression ofpbpFfromEnterococcus faeciumbut not bymgtencoding the monofunctional glycosyltransferase ofStaphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs ofE. faecalis, and heterospecific complementation was observed with an ortholog fromE. faecium. Site-directed mutagenesis ofpbpFconfirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As theE. faecalischromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.
Plasmid-Dependent Methylotrophy in Thermotolerant
Bacillus methanolicus
Authors: Brautaset Trygve , Jakobsen Øyvind M. , Flickinger Michael C. , Valla Svein , Ellingsen Trond E.
Published: February 2004

Bacillus methanolicus
can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50°C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from
B. methanolicus
MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene,
mdh
, which is crucial for methanol consumption in this bacterium. In addition, five genes (
pfk
, encoding phosphofructokinase;
rpe
, encoding ribulose-5-phosphate 3-epimerase;
tkt
, encoding transketolase;
glpX
, encoding fructose-1,6-bisphosphatase; and
fba
, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (
repB
and
ori
) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete
mdh
gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant
B. methanolicus
wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.
Identification and Functional Characterization of the
Lactococcus lactis rfb
Operon, Required for dTDP-Rhamnose Biosynthesis
Authors: Boels Ingeborg C. , Beerthuyzen Marke M. , Kosters Marit H. W. , Van Kaauwen Martijn P. W. , Kleerebezem Michiel , de Vos Willem M.
Published: February 2004

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in
Lactococcus lactis
. We cloned the
rfbACBD
operon from
L. lactis
MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in
Escherichia coli
, the lactococcal
rfbACBD
genes could sustain heterologous production of the
Shigella flexneri
O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in
L. lactis
resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the
rfb
genes have an essential role in
L. lactis
. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Capsule Shields the Function of Short Bacterial Adhesins
Authors: Schembri Mark A. , Dalsgaard Dorte , Klemm Per
Published: February 2004

Bacterial surface structures such as capsules and adhesins are generally regarded as important virulence factors. Here we demonstrate that capsules block the function of the self-recognizing protein antigen 43 through physical shielding. The phenomenon is not restricted to
Escherichia coli
but can occur in other gram-negative bacteria. Likewise, we show that other short adhesins exemplified by the AIDA-I protein are blocked by the presence of a capsule. The results support the notion that capsule polysaccharides sterically prevent receptor-target recognition of short bacterial adhesins. This negative interference has important biological consequences, such as affecting the ability of bacteria to form biofilms.
Differential Impact of MexB Mutations on Substrate Selectivity of the MexAB-OprM Multidrug Efflux Pump of
Pseudomonas aeruginosa
Authors: Middlemiss Jocelyn K. , Poole Keith
Published: February 2004

The integral inner membrane resistance-nodulation-division (RND) components of three-component RND-membrane fusion protein-outer membrane factor multidrug efflux systems define the substrate selectivity of these efflux systems. To gain a better understanding of what regions of these proteins are important for substrate recognition, a plasmid-borne
mexB
gene encoding the RND component of the MexAB-OprM multidrug efflux system of
Pseudomonas aeruginosa
was mutagenized in vitro by using hydroxylamine and mutations compromising the MexB contribution to antibiotic resistance identified in a Δ
mexB
strain. Of 100 mutants that expressed wild-type levels of MexB and showed increased susceptibility to one or more of carbenicillin, chloramphenicol, nalidixic acid, and novobiocin, the
mexB
genes of a representative 46 were sequenced, and 19 unique single mutations were identified. While the majority of mutations occurred within the large periplasmic loops between transmembrane segment 1 (TMS-1) and TMS-2 and between TMS-7 and TMS-8 of MexB, mutations were seen in the TMSs and in other periplasmic as well as cytoplasmic loops. By threading the MexB amino acid sequence through the crystal structure of the homologous RND transporter from
Escherichia coli
, AcrB, a three-dimensional model of a MexB trimer was obtained and the mutations were mapped to it. Unexpectedly, most mutations mapped to regions of MexB predicted to be involved in trimerization or interaction with MexA rather than to regions expected to contribute to substrate recognition. Intragenic second-site suppressor mutations that restored the activity of the G220S mutant version of MexB, which was compromised for resistance to all tested MexAB-OprM antimicrobial substrates, were recovered and mapped to the apparently distal portion of MexB that is implicated in OprM interaction. As the G220S mutation likely impacted trimerization, it appears that either proper assembly of the MexB trimer is necessary for OprM interaction or OprM association with an unstable MexB trimer might stabilize it, thereby restoring activity.
The PhoP-PhoQ Two-Component Regulatory System of
Photorhabdus luminescens
Is Essential for Virulence in Insects
Authors: Derzelle Sylviane , Turlin Evelyne , Duchaud Eric , Pages Sylvie , Kunst Frank , Givaudan Alain , Danchin Antoine
Published: February 2004

Photorhabdus luminescens
is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (
phoP
) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the
phoP
bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg
2+
-dependent modifications in lipopolysaccharides and controlled a locus (
pbgPE
) required for incorporation of 4-aminoarabinose into lipid A. Mg
2+
-regulated gene expression of
pbgP1
was absent in the mutant and was restored when
phoPQ
was complemented in
trans
. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.
Evidence for a Symbiosis Island Involved in Horizontal Acquisition of Pederin Biosynthetic Capabilities by the Bacterial Symbiont of
Paederus fuscipes
Beetles
Authors: Piel Jörn , Höfer Ivonne , Hui Dequan
Published: February 2004

Pederin belongs to a group of antitumor compounds found in terrestrial beetles and marine sponges. It is used by apparently all members of the rove beetle genera
Paederus
and
Paederidus
as a chemical defense against predators. However, a recent analysis of the putative pederin biosynthesis (
ped
) gene cluster strongly suggests that pederin is produced by bacterial symbionts. We have sequenced an extended region of the symbiont genome to gain further insight into the biology of this as-yet-unculturable bacterium and the evolution of pederin symbiosis. Our data indicate that the symbiont is a very close relative of
Pseudomonas aeruginosa
that has acquired several foreign genetic elements by horizontal gene transfer. Besides one functional tellurite resistance operon, the region contains a genomic island spanning 71.6 kb that harbors the putative pederin biosynthetic genes. Several decayed insertion sequence elements and the mosaic-like appearance of the island suggest that the acquisition of the
ped
symbiosis genes was followed by further insertions and rearrangements. A horizontal transfer of genes for the biosynthesis of protective substances could explain the widespread occurrence of pederin-type compounds in unrelated animals from diverse habitats.