bluuebunny/crossref_metadata_2025 huggingface dataset:
A NovelLeishmania infantumRecombinant Antigen Which Elicits Interleukin 10 Production by Peripheral Blood Mononuclear Cells of Patients with Visceral Leishmaniasis
Authors: Suffia Isabelle , Ferrua Bernard , Stien Xavier , Mograbi Baharia , Marty Pierre , Rousseau Deborah , Fragaki Konstantina , Kubar Joanna
Published: July 2002

We report here the characterization of a novelLeishmania infantumprotein termed papLe22 (22-kDa potentially aggravating protein ofLeishmania). A positive clone from a cDNA library was identified by serum of a visceral leishmaniasis (VL) patient. Full-length cDNA obtained using rapid amplification of cDNA ends-PCR codes for a 22-kDa protein. InL. infantumpromastigotes an endogenous nuclear protein of 14-kDa electrophoretic mobility was found by using an antiserum prepared against the fusion protein glutathioneS-transferase–papLe22. Its expression was also shown inL. infantumamastigotes and inLeishmania majorandLeishmania guyanensispromastigotes. VL patients' sera showed anti-papLe22 immunoglobulin M (IgM) and IgG reactivities, indicating that a primary response against the leishmanial protein papLe22 accompanied acute VL manifestations. Specific IgG levels were correlated with patients' clinical status. The presence of IgG1, IgG2, and IgG3 subclasses suggested a mixed Th1- and Th2-type response; there was no correlation between subclass reactivity and the disease course. The recombinant papLe22 specifically activated interleukin-10 production by VL patients' peripheral blood mononuclear cells collected at diagnosis and after treatment-induced cure, indicating its contribution to VL pathogenesis and concomitant immunosuppression and its potential role in the reactivation of latent parasites. As a dominant immunogen, papLe22 might be used as a vaccine component, provided that the vaccination protocol directs the response toward the Th1 pattern.
MTSA-10, the Product of the Rv3874 Gene of
Mycobacterium tuberculosis
, Elicits Tuberculosis-Specific, Delayed-Type Hypersensitivity in Guinea Pigs
Authors: Colangeli Roberto , Spencer John S. , Bifani Pablo , Williams Alan , Lyashchenko Konstantin , Keen Marc A. , Hill Preston J. , Belisle John , Gennaro Maria Laura
Published: July 2002

In a search for new skin test reagents specific for tuberculosis, we found that the antigen encoded by gene Rv3874 of
Mycobacterium tuberculosis
elicited delayed-type hypersensitivity in
M. tuberculosis
-infected guinea pigs but not in control animals immunized with
Mycobacterium bovis
bacillus Calmette-Guérin (BCG) or
Mycobacterium avium
. The antigen, which was named MTSA-10 (for
M. tuberculosis
-specific antigen 10), is a prime candidate for a component of a new tuberculin that will allow discrimination by a skin test of latent
M. tuberculosis
infection from vaccination with BCG or from sensitization with environmental, nontuberculous mycobacteria.
Pseudomonas aeruginosa
Cystic Fibrosis Isolates Induce Rapid, Type III Secretion-Dependent, but ExoU-Independent, Oncosis of Macrophages and Polymorphonuclear Neutrophils
Authors: Dacheux Denis , Toussaint Bertrand , Richard Marceline , Brochier Guy , Croize Jacques , Attree Ina
Published: July 2002

Pseudomonas aeruginosa
, an opportunistic pathogen responsible most notably for severe infections in cystic fibrosis (CF) patients, utilizes the type III secretion system for eukaryotic cell intoxication. The CF clinical isolate CHA shows toxicity towards human polymorphonuclear neutrophils (PMNs) which is dependent on the type III secretion system but independent of the cytotoxin ExoU. In the present study, the cytotoxicity of this strain toward human and murine macrophages was demonstrated. In low-multiplicity infections (multiplicity of infection, 10), approximately 40% of the cells die within 60 min. Analysis of CHA-infected cells by transmission electron microscopy, DNA fragmentation assay, and Hoechst staining revealed the hallmarks of oncosis: cellular and nuclear swelling, disintegration of the plasma membrane, and absence of DNA fragmentation. A panel of 29
P. aeruginosa
CF isolates was screened for type III system genotype, protein secretion profile, and cytotoxicity toward PMNs and macrophages. This study showed that six CF isolates were able to induce rapid ExoU-independent oncosis on phagocyte cells.
Evolution of Lesion Formation, Parasitic Load, Immune Response, and Reservoir Potential in C57BL/6 Mice following High- and Low-Dose Challenge withLeishmania major
Authors: Lira Rosalia , Doherty Mark , Modi Govind , Sacks David
Published: July 2002

A model of cutaneous leishmaniasis using 102Leishmania majormetacyclic promastigotes inoculated into the footpads of genetically resistant C57BL/6 mice was studied in order to more accurately reproduce the evolution of lesion formation and the kinetics of parasite growth and immune response as they might occur in naturally exposed reservoirs and in human hosts. In contrast to the more conventional experimental model employing 106metacyclic promastigotes, in which the rapid development of footpad lesions was associated with an increasing number of amastigotes in the site, the low-dose model revealed a remarkably “silent” phase of parasite growth, lasting approximately 6 weeks, during which peak parasitic loads were established in the absence of any overt pathology. Footpad swelling was observed after 6 weeks, coincident with the onset of parasite clearance and with production of high levels of interleukin-12 (IL-12) and gamma interferon (IFN-γ) in draining lymph nodes. Low-dose challenge of IL-12- and IFN-γ-depleted or -deficient mice provided strong evidence that the induction or expression of cellular immunity is essentially absent during the first 6 to 8 weeks of intracellular growth, since the concentration of amastigotes in the site was not enhanced compared to that for wild-type animals during this time. By monitoring the ability of infected mice to transmit parasites to vector sand flies, it was observed that following low-dose challenge, footpads without apparent lesions provided an efficient source of parasites for exposed flies and that the low-dose challenge actually extended the duration of parasite transmissibility during the course of infection.
Cytokine Responses toPlasmodium falciparumLiver-Stage Antigen 1 Vary in Rainy and Dry Seasons in Highland Kenya
Authors: John C. C. , Sumba P. O. , Ouma J. H. , Nahlen B. L. , King C. L. , Kazura J. W.
Published: July 2002

Seasonal epidemics of malaria occur in highland areas of western Kenya where transmission intensity varies according to rainfall. This study describes the seasonal changes in cytokine responses toPlasmodium falciparumliver-stage antigen 1 (LSA-1) by children (≤17 years old) and adults (≥18 years old) living in such a highland area. Fourteen- to 24-mer peptides corresponding to the N- and C-terminal nonrepeat regions of LSA-1 stimulated production of interleukin-5 (IL-5), interleukin-10 (IL-10), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) by peripheral blood mononuclear cells (PBMC) from 17 to 73% of individuals in both age groups in both seasons. IL-10 and TNF-α responses were more frequent during the high-transmission, rainy season than during the low-transmission, dry season (73 and 67% versus 17 and 25% response rates, respectively). In contrast, there was no seasonal change in the proportion of LSA-1-driven IFN-γ and IL-5 responses. Children produced less IFN-γ than adults, but IL-5, IL-10, and TNF-α levels were similar for both age groups. Depletion of CD8+cells from PBMC decreased IFN-γ but increased IL-10 production. Individuals with LSA-1-stimulated IL-10 responses in the dry season were less likely to become reinfected in the subsequent rainy season than those without IL-10 responses (25% versus 49%;P= 0.083). These data support the notion that maintenance of LSA-1-driven IL-10 and TNF-α responses requires repeated and sustained exposure to liver-stageP. falciparum. In contrast, IFN-γ responses increase slowly with age but persist once acquired. CD8+T cells are the major source of IFN-γ but may suppress production or secretion of IL-10.
Modulation of Human Immune Response byEchinococcus granulosusAntigen B and Its Possible Role in Evading Host Defenses
Authors: Riganò R. , Profumo E. , Bruschi F. , Carulli G. , Azzarà A. , Ioppolo S. , Buttari B. , Ortona E. , Margutti P. , Teggi A. , Siracusano A.
Published: July 2002

By directly suppressing the function of certain immune cell subsets and by stimulating other cell populations related to immunopathology, parasite-derived substances play an important role in the chronic establishment of parasitic disease. Our objective was twofold: (i) to investigate further the role ofEchinococcus granulosusantigen B (AgB) in the human early inflammatory response by determining its effect on polymorphonuclear cell (PMN) random migration, chemotaxis, and oxidative metabolism and (ii) to determine its action in acquired immunity by evaluating AgB and sheep hydatid fluid (SHF)-driven Th1 (gamma interferon [IFN-γ] and interleukin 12 [IL-12]) and Th2 (IL-4 and IL-13) cytokine production by peripheral blood mononuclear cells (PBMC) from 40 patients who had cured or stable or progressive cystic echinococcosis. AgB significantly inhibited PMN recruitment but left their random migration and oxidative metabolism unchanged. Patients' PBMC stimulated with AgB produced IL-4 and IL-13 but did not produce IL-12. They also produced significantly lower IFN-γ concentrations than did PBMC stimulated with SHF (P= 10−5). AgB skewed the Th1/Th2 cytokine ratios towards a preferentially immunopathology-associated Th2 polarization, predominantly in patients with progressive disease. AgB-stimulated patients' PBMC also proliferated less than SHF-stimulated PBMC (P= 9 × 10−3). In vitro Th2 cytokine production was reflected in vivo by elevated specific immunoglobulin E (IgE) and IgG4 antibodies binding to AgB. These findings confirm that AgB plays a role in the escape from early immunity by inhibiting PMN chemotaxis. They also add new information on the host-parasite relationship, suggesting that AgB exploits the activation of T helper cells by eliciting a nonprotective Th2 cell response.
Three-Dimensional Tissue Assemblies: Novel Models for the Study ofSalmonella entericaSerovar Typhimurium Pathogenesis
Authors: Nickerson Cheryl A. , Goodwin Thomas J. , Terlonge Jacqueline , Ott C. Mark , Buchanan Kent L. , Uicker William C. , Emami Kamal , LeBlanc Carly L. , Ramamurthy Rajee , Clarke Mark S. , Vanderburg Charles R. , Hammond Timothy , Pierson Duane L.
Published: July 2002

The lack of readily available experimental systems has limited knowledge pertaining to the development ofSalmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity ofSalmonella entericaserovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed thatSalmonellaestablished infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection withSalmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore,Salmonellaexhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection withSalmonellarevealed significant differences in expression of interleukin 1α (IL-1α), IL-1β, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor β1 mRNA and prostaglandin E2than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.
Distribution and Genetic Diversity of Suilysin in
Streptococcus suis
Isolated from Different Diseases of Pigs and Characterization of the Genetic Basis of Suilysin Absence
Authors: King Samantha J. , Heath Peter J. , Luque Inmaculada , Tarradas Carmen , Dowson Christopher G. , Whatmore Adrian M.
Published: July 2002

Streptococcus suis
is an economically important pathogen of pigs responsible for a variety of diseases including meningitis, septicemia, arthritis, and pneumonia, although little is known about the mechanisms of pathogenesis or virulence factors associated with this organism. Here, we report on the distribution and genetic diversity of the putative virulence factor suilysin, a member of the thiol-activated toxin family of gram-positive bacteria. On the basis of PCR analysis of over 300 isolates of
S. suis
, the suilysin-encoding gene,
sly
, was detected in 69.4% of isolates. However,
sly
was present in a considerably higher proportion of isolates obtained from cases of meningitis, septicemia, and arthritis (>80%) and isolates obtained from asymptomatic tonsillar carriage (>90%) than lung isolates associated with pneumonia (44%). With the exception of serotypes 1, 14, and 1/14, there was no strong correlation between the presence of suilysin and serotype. Analysis of the genetic diversity of suilysin by restriction fragment length polymorphism and sequence analysis found that the suilysin gene, where present, is highly conserved with a maximum of 1.79% diversity at the nucleotide level seen between
sly
alleles. Assays of hemolytic activity and hybridization analysis provided no evidence for a second member of the thiol-activated toxin family in
S. suis
. Inverse PCR was used to characterize regions flanking
sly
, which in turn allowed the first characterization of the equivalent region in a strain lacking
sly
. Sequence comparison of these regions from
sly
-positive (P1/7) and
sly
-negative (DH5) strains indicated that two alternative arrangements are both flanked by genes with highest similarity to haloacid dehalogenase-like hydrolases (5′ end) and putative
N
-acetylmannosamine-6-phosphate epimerases (3′ end). However,
sly
appears to be completely absent from the alternative arrangement, and a gene of unknown function is located in the equivalent position. Finally, PCR analysis of multiple
sly
-positive and -negative strains indicated that these two alternative genetic arrangements are conserved among many
S. suis
isolates.
Mycobacterium tuberculosis19-Kilodalton Lipoprotein InhibitsMycobacterium smegmatis-Induced Cytokine Production by Human Macrophages In Vitro
Authors: Post Frank A. , Manca Claudia , Neyrolles Olivier , Ryffel Bernhard , Young Douglas B. , Kaplan Gilla
Published: July 2002

Vaccination of mice withMycobacterium vaccaeorM. smegmatisinduces some protection againstM. tuberculosischallenge. The 19-kDa lipoprotein ofM. tuberculosis, expressed inM. vaccaeorM. smegmatis(M. smeg19kDa), abrogates this protective immunity. To investigate the mechanism of this suppression of immunity, human monocyte-derived macrophages (MDM) were infected withM. smeg19kDa. Infection resulted in reduced production of tumor necrosis factor alpha (TNF-α) (P< 0.01), interleukin-12 (IL-12) (P< 0.05), IL-6 (P< 0.05), and IL-10 (P< 0.05), compared to infection withM. smegmatisvector (M. smegV). Infection withM. smeg19kDa and withM. smegV had no differential effect on expression of costimulatory molecules on MDM, nor did it affect the proliferation of presensitized T cells cocultured with infected MDM. When MDM were infected withM. smegmatisexpressing mutated forms of the 19-kDa lipoprotein, including non-O-glycosylated (M. smeg19NOG), nonsecreted (M. smeg19NS), and nonacylated (M. smeg19NA) variants, the reduced production of TNF-α or IL-12 was not observed. When the purified 19-kDa lipoprotein was added directly to cultures of infected monocytes, there was little effect on either induction of cytokine production or its inhibition. Thus, the immunosuppressive effect is dependent on glycosylated and acylated 19-kDa lipoprotein present in the phagosome containing the mycobacterium. These results suggest that the diminished protection against challenge withM. tuberculosisseen in mice vaccinated withM. smegmatisexpressing the 19-kDa lipoprotein is the result of reduced TNF-α and IL-12 production, possibly leading to reduced induction of T-cell activation.
Staphylococcus aureusandSalmonella entericaSerovar Dublin Induce Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Expression by Normal Mouse and Human Osteoblasts
Authors: Alexander Emily H. , Bento Jennifer L. , Hughes Francis M. , Marriott Ian , Hudson Michael C. , Bost Kenneth L.
Published: July 2002

Staphylococcus aureusandSalmonella entericaserovar Dublin invade osteoblasts and are causative agents of human bone disease. In the present study, we examined the ability ofS. aureusandSalmonellaserovar Dublin to induce the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by normal osteoblasts. Normal mouse and human osteoblasts were cocultured withS. aureusorSalmonellaserovar Dublin at different multiplicities of infection. Following initial incubation and examination of TRAIL expression, extracellular bacteria were killed by the addition of media containing the antibiotic gentamicin. Lysates and conditioned media from osteoblast cultures were then collected at various times following invasion and analyzed. The results demonstrated thatS. aureusandSalmonellaserovar Dublin are potent inducers of TRAIL expression by osteoblasts. Mouse and human TRAIL mRNA expression was induced by bacterial infection and demonstrated a dose-dependent response. Analysis of kinetics suggested that TRAIL mRNA was induced within 30 min after exposure to bacteria and that its level of expression remained relatively constant over the time period examined. mRNA molecules encoding TRAIL receptors were constitutively expressed by osteoblasts. Furthermore, TRAIL protein was detected as early as 45 min and up to 24 h following infection. The quantity of TRAIL protein produced also increased in a dose-dependent manner. Collectively, these findings suggest a mechanism whereby bacterial pathogens mediate bone destruction via osteoblast apoptosis.
Characterization of a Cell Surface Protein of
Clostridium difficile
with Adhesive Properties
Authors: Waligora Anne-Judith , Hennequin Claire , Mullany Peter , Bourlioux Pierre , Collignon Anne , Karjalainen Tuomo
Published: July 2002

Our laboratory has previously shown that
Clostridium difficile
adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The
cwp66
gene, encoding a cell surface-associated protein of
C. difficile
79-685, was isolated by immunoscreening of a
C. difficile
gene library with polyclonal antibodies against
C. difficile
heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of
Bacillus subtilis
. A survey of 36 strains of
C. difficile
representing 11 serogroups showed that the 3′ portion of the
cwp66
gene is variable; this was confirmed by sequencing of
cwp66
from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione
S
-transferase in
Escherichia coli
and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of
C. difficile
to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia.
Staphylococcus aureusagrandsarAFunctions Are Required for Invasive Infection but Not Inflammatory Responses in the Lung
Authors: Heyer Geoffrey , Saba Shahryar , Adamo Robert , Rush William , Soong Grace , Cheung Ambrose , Prince Alice
Published: July 2002

Staphylococcus aureusstrains lackingagr- andsarA-dependent gene products or specific MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesins were compared for the ability to activate inflammatory responses in the lung. The mutants were evaluated for virulence in a mouse model of pneumonia and by quantifying their ability to stimulate interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in respiratory epithelial cells. In a neonatal mouse, only strains with intactagrandsarAloci were consistently associated with invasive, fatal pulmonary infection (P< 0.001) andsarA was specifically required to cause bacteremia (P< 0.001). Theagrand/orsarAmutants were, nonetheless, fully capable of producing pneumonia and were as proficient as the wild-type strain in stimulating epithelial IL-8 expression, a polymorphonuclear leukocyte chemokine, in airway cells. In contrast,agrand especiallysarAmutants induced less epithelial GM-CSF expression, and MSCRAMM mutants lacking fibronectin binding proteins or clumping factor A, a ligand for fibrinogen, were unable to stimulate epithelial GM-CSF production. The ability to induce IL-8 expression was independent of the adherence properties of intact bacteria, indicating that shed and/or secreted bacterial components activate epithelial responses. While conserved staphylococcal components such as peptidoglycan are sufficient to evoke inflammation and cause pneumonia, theagrandsarAloci ofS. aureusare critical for the coordination of invasive infection of the lungs.
Antigenic Specificity of theMycobacterium lepraeHomologue of ESAT-6
Authors: Spencer John S. , Marques Maria Angela M. , Lima Monica C. B. S. , Junqueira-Kipnis Ana Paula , Gregory Bruce C. , Truman Richard W. , Brennan Patrick J.
Published: July 2002

The sequence of theMycobacterium lepraehomologue of ESAT-6 shows only 36% amino acid correspondence to that fromMycobacterium tuberculosis. Anti-M. lepraeESAT-6 polyclonal and monoclonal antibodies and T-cell hybridomas reacted only with the homologous protein and allowed identification of the B- and T-cell epitopes. The protein is expressed inM. lepraeand appears in the cell wall fraction. Thus,M. lepraeESAT-6 shows promise as a specific diagnostic agent for leprosy.
Role of Glutathione Metabolism of
Treponema denticola
in Bacterial Growth and Virulence Expression
Authors: Chu Lianrui , Dong Zheng , Xu Xiaoping , Cochran David L. , Ebersole Jefferey L.
Published: July 2002

Hydrogen sulfide (H
2
S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis.
Treponema denticola
, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H
2
S. The metabolic pathway leading to H
2
S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for
T. denticola
to produce H
2
S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H
2
S production by this microorganism. In addition to H
2
S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H
2
S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in
T. denticola
responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56°C) and freezing (−20°C), by sonication, and by exposure to
N
α-
p
-tosyl-
l
-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete
T. denticola
; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.
Peripheral Blood Mononuclear Cell Activation Induced byLeptospira interrogansGlycolipoprotein
Authors: Diament Decio , Brunialti Milena Karina Colo , Romero Eliete Calo , Kallas Esper Georges , Salomao Reinaldo
Published: July 2002

Leptospira interrogansglycolipoprotein (GLP) has been implicated in pathological and functional derangement seen in leptospirosis. The goal of this study was to evaluate GLP's ability to induce cellular activation, as assessed by cytokine production and expression of surface activation markers. GLP extracted from either pathogenicL. interrogansserovar Copenhageni or nonpathogenicLeptospira biflexaserovar Patoc (GLPp) was used to stimulate peripheral blood mononuclear cell cultures from healthy donors. Supernatant cytokine levels were measured by enzyme-linked immunosorbent assay. Expression of CD69 and HLA-DR on lymphocytes and monocytes, as well as lipopolysaccharide (LPS) binding, were measured by flow cytometry. At 6 h of incubation, GLP induced a significant rise in tumor necrosis factor alpha levels, which dropped progressively until 72 h of incubation. Interleukin-10 peak levels were obtained at between 24 and 48 h, with sustained levels until 72 h of incubation. The response magnitude was proportional to the GLP dose. CD69 expression on T lymphocytes and monocytes increased significantly, as did HLA-DR expression on monocytes. GLPp induced no CD69 or HLA-DR expression. GLP did not block biotinylated LPS binding to monocytes, suggesting that different pathways are used to induce cell activation. In conclusion, GLP induces cellular activation and may play a major role in the pathogenesis of leptospirosis.
Coexpression of Interleukin-12 Chains by a Self-Splicing Vector Increases the Protective Cellular Immune Response of DNA andMycobacterium bovisBCG Vaccines againstMycobacterium tuberculosis
Authors: Palendira Umaimainthan , Kamath Arun T. , Feng Carl G. , Martin Ela , Chaplin Paul J. , Triccas James A. , Britton Warwick J.
Published: July 2002

More effective vaccines againstMycobacterium tuberculosismay contribute to the control of this major human pathogen. DNA vaccines encoding single mycobacterial proteins stimulate antimycobacterial T-cell responses and induce partial protection againstM. tuberculosisin animal models. The protective efficacy of these vaccines encoding a single antigen, however, has been less than that afforded by the current vaccine,Mycobacterium bovisbacillus Calmette-Guérin (BCG). The heterodimeric cytokine interleukin-12 (IL-12) potentiates the induction and maintenance of the type 1 helper T-cell response. We have developed a novel self-splicing vector based on the 2A protein of foot-and-mouth disease virus that permits the coordinate expression of both chains of IL-12 (p2AIL12). Coimmunization with this vector and DNA expressingM. tuberculosisantigen 85B or MPT64 enhanced the specific lymphocyte proliferative response and increased the frequency of specific gamma interferon-secreting T cells against the whole protein and a defined CD8+T-cell epitope on MPT64. Further, coimmunizing with p2AIL12 significantly increased the protective efficacy of DNA-85 in the lung against an aerosol challenge withM. tuberculosisto the level achieved with BCG. Therefore, codelivery of an IL-12-secreting plasmid may be a potent strategy for enhancing the protective efficacy of vaccines againstM. tuberculosis.
RecombinantOchrobactrum anthropiExpressingBrucella abortusCu,Zn Superoxide Dismutase Protects Mice againstB. abortusInfection Only after Switching of Immune Responses to Th1 Type
Authors: He Yongqun , Vemulapalli Ramesh , Schurig Gerhardt G.
Published: July 2002

The members of the genusBrucellaare gram-negative, facultatively intracellular bacterial pathogens that cause brucellosis in many animal species and humans. Although live, attenuated vaccines are available to protect several animal species from the disease, there is no safe and effective vaccine for human use. Here we report that a bacterium that is closely related toBrucellaspecies,Ochrobactrum anthropi, can be used as a vaccine vector for the delivery ofBrucellaantigens to mice, leading to the elicitation of protective immunity against brucellosis.Brucella abortusCu,Zn superoxide dismutase (SOD), a protectiveBrucellaantigen, was expressed in large amounts inO. anthropistrain 49237 by use of the broad-host-range plasmid pBBR1MCS. NeitherO. anthropistrain 49237 nor the recombinantO. anthropistrain 49237SOD, expressingB. abortusCu,Zn SOD, provided protection against virulentBrucellainfection in mice. Analysis of immune responses indicated that strains 49237 and 49237SOD stimulated a mix of Th1 and Th2 type responses in the mice. After the immune response was switched to a Th1-biased response by addition of oligonucleotides containing unmethylated CpG motifs, bothO. anthropistrain 49237 and the recombinantO. anthropistrain 49237SOD induced protection in mice. However, the protection conferred by strain 49237SOD was significantly better than that induced by the parental strain, 49237.
Modulation of
Borrelia burgdorferi
Stringent Response and Gene Expression during Extracellular Growth with Tick Cells
Authors: Bugrysheva Julia , Dobrikova Elena Y. , Godfrey Henry P. , Sartakova Marina L. , Cabello Felipe C.
Published: July 2002

Borrelia burgdorferi
N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support
B. burgdorferi
N40 growth. Growth of
B. burgdorferi
N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in
relA
/
spoT
mRNA, an eightfold net decrease in
bmpD
mRNA, and a fourfold increase in
rpsL-bmpD
mRNA compared to growth of
B. burgdorferi
in BSK-H medium. As a result, the polycistronic
rpsL-bmpD
mRNA level increased from 3 to 100% of the total
bmpD
message. These observations demonstrate that there are reciprocal interactions between
B. burgdorferi
and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in
B. burgdorferi
growing in BSK-H medium is ameliorated in
B. burgdorferi
growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of
B. burgdorferi
with tick cells in vitro when it is coupled with genetic methods to isolate and complement
B. burgdorferi
mutants.
Chlamydia trachomatis
Induces Remodeling of the Actin Cytoskeleton during Attachment and Entry into HeLa Cells
Authors: Carabeo Reynaldo A. , Grieshaber Scott S. , Fischer Elizabeth , Hackstadt Ted
Published: July 2002

To elucidate the host cell machinery utilized by
Chlamydia trachomatis
to invade epithelial cells, we examined the role of the actin cytoskeleton in the internalization of chlamydial elementary bodies (EBs). Treatment of HeLa cells with cytochalasin D markedly inhibited the internalization of
C. trachomatis
serovar L2 and D EBs. Association of EBs with HeLa cells induced localized actin polymerization at the site of attachment, as visualized by either phalloidin staining of fixed cells or the active recruitment of GFP-actin in viable infected cells. The recruitment of actin to the specific site of attachment was accompanied by dramatic changes in the morphology of cell surface microvilli. Ultrastructural studies revealed a transient microvillar hypertrophy that was dependent upon
C. trachomatis
attachment, mediated by structural components on the EBs, and cytochalasin D sensitive. In addition, a mutant CHO cell line that does not support entry of
C. trachomatis
serovar L2 did not display such microvillar hypertrophy following exposure to L2 EBs, which is in contrast to infection with serovar D, to which it is susceptible. We propose that
C. trachomatis
entry is facilitated by an active actin remodeling process that is induced by the attachment of this pathogen, resulting in distinct microvillar reorganization throughout the cell surface and the formation of a pedestal-like structure at the immediate site of attachment and entry.
Isolation and Characterization of Nonchemotactic CheZ Mutants of
Escherichia coli
Authors: Boesch Kristin C. , Silversmith Ruth E. , Bourret Robert B.
Published: July 2002

The
Escherichia coli
CheZ protein stimulates dephosphorylation of CheY, a response regulator in the chemotaxis signal transduction pathway, by an unknown mechanism. Genetic analysis of CheZ has lagged behind biochemical and biophysical characterization. To identify putative regions of functional importance in CheZ, we subjected
cheZ
to random mutagenesis and isolated 107 nonchemotactic CheZ mutants. Missense mutations clustered in six regions of
cheZ
, whereas nonsense and frameshift mutations were scattered reasonably uniformly across the gene. Intragenic complementation experiments showed restoration of swarming activity when compatible plasmids containing genes for the truncated CheZ
1–189
peptide and either CheZA65V, CheZL90S, or CheZD143G were both present, implying the existence of at least two independent functional domains in each chain of the CheZ dimer. Six mutant CheZ proteins, one from each cluster of loss-of-function missense mutations, were purified and characterized biochemically. All of the tested mutant proteins were defective in their ability to dephosphorylate CheY-P, with activities ranging from 0.45 to 16% of that of wild-type CheZ. There was good correlation between the phosphatase activity of CheZ and the ability to form large chemically cross-linked complexes with CheY in the presence of the CheY phosphodonor acetyl phosphate. In consideration of both the genetic and biochemical data, the most severe functional impairments in this set of CheZ mutants seemed to be concentrated in regions which are located in a proposed large N-terminal domain of the CheZ protein.
The Flagellar Hook Protein, FlgE, of
Salmonella enterica
Serovar Typhimurium Is Posttranscriptionally Regulated in Response to the Stage of Flagellar Assembly
Authors: Bonifield Heather R. , Yamaguchi Shigeru , Hughes Kelly T.
Published: July 2002

We investigated the posttranscriptional regulation of
flgE
, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the
flgE
gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook–basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly.
Recombination Is Essential for Viability of an
Escherichia coli dam
(DNA Adenine Methyltransferase) Mutant
Authors: Marinus M. G.
Published: July 2002

Double mutants of
Escherichia coli dam
(DNA adenine methyltransferase) strains with
ruvA
,
ruvB
, or
ruvC
could not be constructed, whereas
dam
derivatives with
recD
,
recF
,
recJ
, and
recR
were viable. The
ruv
gene products are required for Holliday junction translocation and resolution of recombination intermediates. A
dam recG
(Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a
dam lexA
(Ind
−
) host was abrogated by the simultaneous presence of plasmids encoding both
recA
and
ruvAB
. This result indicates that of more than 20 SOS genes, only
recA
and
ruvAB
need to be derepressed to allow for
dam
mutant survival. The presence of
mutS
or
mutL
mutations allowed the construction of
dam lexA
(Ind
−
) derivatives. The requirement for
recA
,
recB
,
recC
,
ruvA
,
ruvB
,
ruvC
, and possibly
recG
gene expression indicates that recombination is essential for viability of
dam
bacteria probably to repair DNA double-strand breaks. The effect of
mutS
and
mutL
mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of
dam
cells to certain DNA-damaging agents.
Cell Cycle Arrest in Archaea by the Hypusination Inhibitor
N
1
-Guanyl-1,7-Diaminoheptane
Authors: Jansson B. P. Mattias , Malandrin Laurence , Johansson Hans E.
Published: July 2002

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor
N
1
-guanyl-1,7-diaminoheptane (GC
7
) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC
7
, whereas the bacterium
Escherichia coli
is not, (ii) GC
7
causes rapid and reversible arrest of growth of the archaeon
Sulfolobus acidocaldarius
, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.
Streptococcus gordonii
Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes
Authors: Loo C. Y. , Corliss D. A. , Ganeshkumar N.
Published: July 2002

Viridans streptococci, which include
Streptococcus gordonii
, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of
S. gordonii
Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn
916
transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of
S. gordonii
Challis were identified based on Southern hybridization with a Tn
916
-based probe and DNA sequences of the Tn
916
-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.
Characterization of a 12-Kilodalton Rhodanese Encoded by
glpE
of
Escherichia coli
and Its Interaction with Thioredoxin
Authors: Ray W. Keith , Zeng Gang , Potters M. Benjamin , Mansuri Aqil M. , Larson Timothy J.
Published: July 2002

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from
Escherichia coli
. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The
glpE
gene, a member of the
sn
-glycerol 3-phosphate (
glp
) regulon of
E. coli
, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The

K
m

s for SSO
3
2−
and CN
−
were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a
k
cat
of 230 s
−1
. Thioredoxin 1 from
E. coli
, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent

K
m

of 34 μM when thiosulfate was near its

K
m

, suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (∼17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (
http://www.ncbi.nlm.nih.gov/COG/
) for which sulfurtransferase activity has been confirmed.
Identification of SopE2, a
Salmonella
Secreted Protein Which Is Highly Homologous to SopE and Involved in Bacterial Invasion of Epithelial Cells
Authors: Bakshi C. S. , Singh V. P. , Wood M. W. , Jones P. W. , Wallis T. S. , Galyov E. E.
Published: July 2002

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying
Salmonella
pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the
sopE2
gene which is present and conserved in pathogenic strains of
Salmonella
. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.
Divergence of Eukaryotic Secretory Components: the
Candida albicans
Homolog of the
Saccharomyces cerevisiae
Sec20 Protein Is N Terminally Truncated, and Its Levels Determine Antifungal Drug Resistance and Growth
Authors: Weber Yvonne , Santore Uwe J. , Ernst Joachim F. , Swoboda Rolf K.
Published: July 2002

Sec20p is a component of the yeast
Saccharomyces cerevisiae
secretory pathway that does not have a close homolog in higher eukaryotic cells. To verify the function of Sec20p in other fungal species, we characterized the gene encoding a Sec20p homolog in the human fungal pathogen
Candida albicans
. The deduced protein has 27% identity with, but is missing about 100 N-terminal residues compared to
S. cerevisiae
Sec20p, which is part of the cytoplasmic tail interacting with the cytoplasmic protein Tip20p. Because a strain lacking both
C. albicans SEC20
alleles could not be constructed, we placed
SEC20
under transcriptional control of two regulatable promoters,
MET3p
and
PCK1p
. Repression of
SEC20
expression in these strains prevented (
MET3p-SEC20
allele) or retarded (
PCK1p-SEC20
allele) growth and led to the appearance of extensive intracellular membranes, which frequently formed stacks. Reduced
SEC20
expression in the
PCK1p-SEC20
strain did not affect morphogenesis but led to a series of hypersensitivity phenotypes including supersensitivity to aminoglycoside antibiotics, to nystatin, to sodium dodecyl sulfate, and to cell wall inhibitors. These results demonstrate the occurrence and function of Sec20p in a fungal species other than
S. cerevisiae
, but the lack of the N-terminal domain and the apparent absence of a close
TIP20
homolog in the
C. albicans
genome also indicate a considerable diversity in mechanisms of retrograde vesicle traffic in eukaryotes.
Roles of LcrG and LcrV during Type III Targeting of Effector Yops by
Yersinia enterocolitica
Authors: DeBord Kristin L. , Lee Vincent T. , Schneewind Olaf
Published: July 2002

Yersinia enterocolitica
target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of
lcrG
caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations in
lcrV
reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused Δ
lcrGV
yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in
lcrG
alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.