bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptospirosis
Authors: Levett Paul N.
Published: July 2002

Leptospirosis is a worldwide zoonotic infection with a much greater incidence in tropical regions and has now been identified as one of the emerging infectious diseases. The epidemiology of leptospirosis has been modified by changes in animal husbandry, climate, and human behavior. Resurgent interest in leptospirosis has resulted from large outbreaks that have received significant publicity. The development of simpler, rapid assays for diagnosis has been based largely on the recognition that early initiation of antibiotic therapy is important in acute disease but also on the need for assays which can be used more widely. In this review, the complex taxonomy of leptospires, previously based on serology and recently modified by a genotypic classification, is discussed, and the clinical and epidemiological value of molecular diagnosis and typing is also evaluated.
Heterologously Expressed
Staphylococcus aureus
Fibronectin-Binding Proteins Are Sufficient for Invasion of Host Cells
Authors: Sinha Bhanu , Francois Patrice , Que Yok-Ai , Hussain Muzaffar , Heilmann Christine , Moreillon Philippe , Lew Daniel , Krause Karl-Heinz , Peters Georg , Herrmann Mathias
Published: July 2002

Staphylococcus aureus
invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between
S. aureus
fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin α
5
β
1
(B. Sinha et al., Cell. Microbiol. 1:101–117, 1999). However, it is unknown whether this mechanism is sufficient for
S. aureus
invasion. To address this question, various
S. aureus
adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in
Staphylococcus carnosus
and
Lactococcus lactis
subsp.
cremoris
. Both noninvasive gram-positive microorganisms are genetically distinct from
S. aureus
, lack any known
S. aureus
surface protein, and do not bind fibronectin. Transformants of
S. carnosus
and
L. lactis
harboring plasmids coding for various
S. aureus
surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to
S. carnosus
and
L. lactis
. Invasion of 293 cells by transformants was comparable to that of strongly invasive
S. aureus
strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus,
S. aureus
FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (
S. aureus
-specific) coreceptors.
Expression of the Nitric Oxide Synthase 2 Gene Is Not Essential for Early Control ofMycobacterium tuberculosisin the Murine Lung
Authors: Cooper Andrea M. , Pearl John E. , Brooks Jason V. , Ehlers Stefan , Orme Ian M.
Published: July 2002

The interleukin-12 and gamma interferon (IFN-γ) pathway of macrophage activation plays a pivotal role in controlling tuberculosis. In the murine model, the generation of supplementary nitric oxide by the induction of the nitric oxide synthase 2 (NOS2) gene product is considered the principal antimicrobial mechanism of IFN-γ-activated macrophages. Using a low-dose aerosol-mediated infection model in the mouse, we have investigated the role of nitric oxide in controllingMycobacterium tuberculosisin the lung. In contrast to the consequences of a systemic infection, a low dose of bacteria introduced directly into the lungs of mice lacking the NOS2 gene is controlled almost as well as in intact animals. This is in contrast to the rapid progression of disease in mice lacking IFN-γ or a key member of the IFN signaling pathway, interferon regulatory factor 1. Thus while IFN-γ is pivotal in early control of bacterial growth in the lung, this control does not completely depend upon the expression of the NOS2 gene. The absence of inducible nitric oxide in the lung does, however, result in increased polymorphonuclear cell involvement and eventual necrosis in the pulmonary granulomas of the infected mice lacking the NOS2 gene.
Optimizing the Germfree Mouse Model for In Vivo Evaluation of Oral
Vibrio cholerae
Vaccine and Vector Strains
Authors: Crean Thomas I. , John Manohar , Calderwood Stephen B. , Ryan Edward T.
Published: July 2002

The germfree mouse model of
Vibrio cholerae
infection can be used to judge immune responses to
V. cholerae
vaccine and vector strains. In the original model, a single oral inoculation was administered on day 0, a booster oral inoculation was administered on day 14, and immune responses were analyzed with samples collected on day 28. Unfortunately, immune responses in this model frequently were low level, and interanimal variability occurred. In order to improve this model, we evaluated various primary and booster
V. cholerae
inoculation schedules. The most prominent systemic and mucosal antibody responses were measured in mice that received a multiple primary inoculation series on days 0, 2, 4, and 6 and booster inoculations on days 28 and 42. These modifications result in improved preliminary evaluation of
V. cholerae
vaccine and vector strains in mice.
Staphylococcal Exfoliative Toxins Cleave α- and β-Melanocyte-Stimulating Hormones
Authors: Rago James V. , Vath Gregory M. , Tripp Timothy J. , Bohach Gregory A. , Ohlendorf Douglas H. , Schlievert Patrick M.
Published: July 2002

The staphylococcal exfoliative toxins (ETs) A and B (ETA and ETB) are 27-kDa exotoxins produced by certain strains ofStaphylococcus aureusand are the causative agents of staphylococcal scalded-skin syndrome. The crystal structures of the ETs strongly indicate that the proteins are members of the serine protease family of enzymes, although protease activity until now has not yet been conclusively demonstrated. Here, we show that the peptide β-melanocyte-stimulating hormone (β-MSH) is cleaved by ETA and that both ETA and ETB are capable of cleaving α-MSH. Both toxins exhibit cleavage at specific glutamic acid residues in MSH peptides. Moreover, biologically inactive mutants of ETA were incapable of cleaving β-MSH.
Role of Activated Protein C inHelicobacter pylori-Associated Gastritis
Authors: Oka Satoko , Gabazza Esteban Cesar , Taguchi Yukiko , Yamaguchi Michihiko , Nakashima Shigehito , Suzuki Koji , Adachi Yukihiko , Imoto Ichiro
Published: July 2002

The protein C (PC) pathway has recently been suggested to play a role in the regulation of the inflammatory response. To further extend the anti-inflammatory effect of activated PC (APC) in vivo, particularly its biological relevance to human disease, the activity of APC in the mucosa of patients withHelicobacter pylori-associated gastritis and the effect of vacuolating cytotoxin (VacA), cytotoxin-associated antigen (CagA), andH. pylorilipopolysaccharide (LPS) on PC activation were evaluated. This study comprised 35 patients with chronic gastritis. There were 20 patients with and 15 withoutH. pyloriinfection. The levels of PC and APC-PC inhibitor (PCI) complex were measured by immunoassays. The level of PC was significantly decreased and the level of APC-PCI complex was significantly increased in biopsy specimens from gastric corpus and antrum in patients withH. pylori-associated gastritis as compared toH. pylori-negative subjects. The concentrations of VacA, CagA, and LPS were significantly correlated with those of the APC-PCI complex in biopsy mucosal specimens from the gastric corpus and antrum.H. pyloriLPS, VacA, and CagA induced a dose-dependent activation of PC on the surface of monocytic cells. APC inhibited the secretion of tumor necrosis factor alpha (TNF-α) induced byH. pyloriLPS. Overall, these results suggest thatH. pyloriinfection is associated with increased APC generation in the gastric mucosa. The inhibitory activity of APC on TNF-α secretion may serve to protectH. pylori-induced gastric mucosal damage.
Heterologous Expression of an Immunogenic Pneumococcal Type 3 Capsular Polysaccharide in
Lactococcus lactis
Authors: Gilbert Christophe , Robinson Karen , Le Page Richard W. F. , Wells Jeremy M.
Published: July 2002

In order to develop a new system for the analysis of capsular biosynthetic pathways we have explored the possibility of expressing type 3 capsular polysaccharide (CPS) from the pathogen
Streptococcus pneumoniae
in
Lactococcus lactis
, an unencapsulated lactic acid bacterium being developed as a vaccine delivery vehicle for mucosal immunization. Only three of the four type 3 CPS biosynthesis genes were found to be necessary for the abundant formation (120 mg liter
−1
) of an extracellular type 3 CPS in
L. lactis
, implying a role for the type 3-specific synthase in the extracellular transport of the CPS or implying the existence of an alternative export system in
L. lactis
. The authenticity of the expressed heterologous polysaccharide was established by chemical and immunological analyses. Proton and carbon nuclear magnetic resonance spectroscopy of CPSs purified from
L. lactis
and
S. pneumoniae
showed that the two CPS structures were identical. When mice were immunized intraperitoneally with 3.5 × 10
6
CFU of live recombinant lactococci expressing a total of approximately 0.5 μg of type 3 CPS, the immune responses elicited appeared identical to those observed in mice inoculated with 0.5 μg of type 3 CPS purified from
S. pneumoniae
. These findings show that
L. lactis
is a useful host in which to study the role and function of genes involved in the production of bacterial capsules. Additionally,
L. lactis
shows potential as a host for the safe production of capsule antigens and as a vaccine delivery vehicle for polysaccharide antigens.
Host Resistance toListeria monocytogenesInfection Is Enhanced but Resistance toStaphylococcus aureusInfection Is Reduced in Acute Graft-versus-Host Disease in Mice
Authors: Miura Tomisato , Mizuki Daisuke , Sasaki Sanae , Hasegawa Suguru , Sashinami Hiroshi , Nakane Akio
Published: July 2002

Acute graft-versus-host disease (GVHD) is characterized by the production of high levels of T helper 1 (Th1)-type cytokines. Bone marrow transplantation from allogeneic C57BL/6 cells to CBF1mice produced acute GVHD. Host resistance to Th1-drivenListeria monocytogeneswas enhanced, whereas host resistance to Th2-drivenStaphylococcus aureuswas reduced during acute GVHD. These results suggest that opposite host responses are observed between Th1-driven and Th2-driven bacterial infections in acute GVHD.
Recombinant Antigen-Enterotoxin A2/B Chimeric Mucosal Immunogens Differentially Enhance Antibody Responses and B7-Dependent Costimulation of CD4+T Cells
Authors: Martin Michael , Hajishengallis George , Metzger Daniel J. , Michalek Suzanne M. , Connell Terry D. , Russell Michael W.
Published: July 2002

The ADP-ribosylating enterotoxins, cholera toxin (CT) and theEscherichia coliheat-labile toxin (LT-IIa), have been shown to enhance mucosal and systemic antibody (Ab) responses to coadministered antigens. The purpose of the present study was to compare the ability of the nontoxic A2/B subunits of these toxins, which have distinct targeting properties, to augment the immunogenicity of a genetically coupled protein antigen. Structurally similar chimeric proteins were generated by genetically replacing the toxic A1 subunit of CT or LT-IIa with the saliva-binding region (SBR) from the streptococcal adhesin AgI/II. Intranasal immunization of BALB/c mice with either chimeric protein induced significantly higher plasma and mucosal anti-SBR immunoglobulin A (IgA) and IgG Ab responses than SBR alone. Moreover, compared to SBR–LT-IIaA2/B, SBR-CTA2/B elicited significantly higher levels of plasma IgG1 and salivary IgA anti-SBR Ab responses. Ex vivo and in vitro experiments revealed that SBR-CTA2/B selectively up-regulated B7-2 expression on murine B cells isolated from both the nasal associated lymphoid tissue, cervical lymph nodes, and spleen. In contrast, SBR–LT-IIaA2/B had little effect on B7-1 or B7-2 expression on B220+, CD11b+, or CD11c+cells. Analysis of the functional costimulatory activity of SBR-CTA2/B-treated B cells revealed a significant enhancement in anti-CD3-stimulated CD4+T-cell proliferative responses, and this proliferation was significantly reduced by treatment with anti-B7-2 but not with anti-B7-1 or isotype control Abs. Thus, SBR-CTA2/B and SBR–LT-IIaA2/B exhibit distinct patterns of antibody responses associated with differential effects on B7-2 expression and subsequent costimulatory effects on CD4+T cells.
Development of a Recombinant Antigen Vaccine against Infection with the Filarial WormOnchocerca volvulus
Authors: Abraham David , Leon Ofra , Leon Shalom , Lustigman Sara
Published: July 2002

Efforts to controlOnchocerca volvulus, the etiologic agent of river blindness, have been limited to vector control and drug treatment to eliminate microfilariae, with no means available to prevent infection. The goal of this study was to develop a vaccine against this infection using recombinant antigens that are expressed in the early larval stages of the parasite. Five recombinant antigens, Ov7, Ov64, OvB8, Ov9M, and Ov73k, were identified by screening adult and larval cDNA libraries with antibodies from immune humans, chimpanzees, or rabbits. When mice were immunized with the five individual recombinant antigens, statistically significant reductions in parasite survival were induced in mice immunized with Ov7, OvB8, or Ov64, when administered in alum but not when injected in Freund's complete adjuvant (FCA). Live larvae recovered from control and immunized mice were analyzed to determine their developmental stages. A decrease in the percentage of larvae molting from the third stage to the fourth stage was observed with mice immunized with Ov7, Ov64, or OvB8 in alum but not with mice immunized with Ov9 and Ov73k or with mice immunized with any of the five antigens in FCA. Mice immunized with a cocktail of the three protective antigens developed protective immunity equal to that seen with mice immunized with individual antigens. This study has identified, for the first time, three recombinant antigens capable of inducing protective immunity toO. volvulus. Furthermore, since the antigens functioned with alum as the adjuvant, this vaccine could potentially be used clinically to prevent river blindness in humans.
Fish Monocytes as a Model for Mycobacterial Host-Pathogen Interactions
Authors: El-Etr Sahar H. , Yan Ling , Cirillo Jeffrey D.
Published: July 2002

Mycobacterium marinum, a relatively rapid-growing fish and human pathogen, has become an important model for the investigation of mycobacterial pathogenesis.M. marinumis closely related to theMycobacterium tuberculosiscomplex and causes a disease in fish and amphibians with pathology similar to tuberculosis. We have developed an in vitro model for the study ofM. marinumvirulence mechanisms using the carp monocytic cell line CLC (carp leukocyte culture). We found that fish monocytes can differentiate between pathogenic and nonpathogenic mycobacterial species. Interestingly,M. marinumenters fish monocytes at a 40- to 60-fold-higher rate thanMycobacterium smegmatis. In addition,M. marinumsurvives and replicates in fish monocytes whileM. smegmatisis killed. We also found thatM. marinuminhibits lysosomal fusion in fish monocytes, indicating that these cells may be used to dissect the mechanisms of intracellular trafficking in mycobacteria. We conclude from these observations that monocytic cells from fish, a natural host forM. marinum, provide an extremely valuable model for the identification and characterization of mycobacterial virulence determinants in the laboratory.
Evidence that Mycobacterial PE_PGRS Proteins Are Cell Surface Constituents That Influence Interactions with Other Cells
Authors: Brennan Michael J. , Delogu Giovanni , Chen Yiping , Bardarov Stoyan , Kriakov Jordan , Alavi Mohammad , Jacobs William R.
Published: July 2002

The elucidation of the genomic sequence ofMycobacterium tuberculosisrevealed the presence of a novel multigene family designated PE/PE_PGRS that encodes numerous, highly related proteins of unknown function. In this study, we demonstrate that a transposon insertion in a PE_PGRS gene (1818PE_PGRS) found inMycobacterium bovisBCG Pasteur, which is the BCG homologue of theM.tuberculosisH37Rv geneRv1818c, introduces new phenotypic properties to this BCG strain. These properties include dispersed growth in liquid medium and reduced infection of macrophages. Complementation of the 1818PE_PGRS::Tn5367mutant with the wild-type gene restores both aggregative growth (clumping) in liquid medium and reestablishes infectivity of macrophages to levels equivalent to those for the parent BCG strain. Western blot analysis using antisera raised against the 1818PE_PGRSprotein shows that PE_PGRS proteins are found in cell lysates of BCG andM.tuberculosisH37Ra and in the cell wall fraction ofM.tuberculosisH37Rv. Moreover, immunofluorescent labeling of mycobacteria indicates that certain PE_PGRS proteins are localized at the cell surface of BCG andM.tuberculosis. Together these results suggest that certain PE_PGRS proteins may be found at the surface of mycobacteria and influence both cell surface interactions among mycobacteria as well as the interactions of mycobacteria with macrophages.
Nippocystatin, a Cysteine Protease Inhibitor fromNippostrongylus brasiliensis, Inhibits Antigen Processing and Modulates Antigen-Specific Immune Response
Authors: Dainichi Teruki , Maekawa Yoichi , Ishii Kazunari , Zhang Tianqian , Nashed Baher Fawzy , Sakai Tohru , Takashima Miwa , Himeno Kunisuke
Published: July 2002

During infection, parasites evade the host immune system by modulating or exploiting the immune system; e.g., they suppress expression of major histocompatibility complex class II molecules or secrete cytokine-like molecules. However, it is not clear whether helminths disturb the immune responses of their hosts by controlling the antigen-processing pathways of the hosts. In this study, we identified a new cysteine protease inhibitor, nippocystatin, derived from excretory-secretory (ES) products of an intestinal nematode,Nippostrongylus brasiliensis. Nippocystatin, which belongs to cystatin family 2, consists of 144 amino acids and is secreted as a 14-kDa mature form. In vivo treatment of ovalbumin (OVA)-immunized mice with recombinant nippocystatin (rNbCys) profoundly suppressed OVA-specific proliferation of splenocytes but not non-antigen-specific proliferation of splenocytes. OVA-specific cytokine production was also greatly suppressed in rNbCys-treated mice. Although the serum levels of both OVA-specific immunoglobulin G1 (IgG1) and IgG2a were not affected by rNbCys treatment, OVA-specific IgE was preferentially downregulated in rNbCys-treated mice. In vitro rNbCys inhibited processing of OVA by lysosomal cysteine proteases from the spleens of mice. Mice with anti-nippocystatin antibodies became partially resistant to infection withN. brasiliensis. Based on these findings,N. brasiliensisappears to skillfully evade host immune systems by secreting nippocystatin, which modulates antigen processing in antigen-presenting cells of hosts.
BrkA Protein ofBordetella pertussisInhibits the Classical Pathway of Complement after C1 Deposition
Authors: Barnes Michael G. , Weiss Alison A.
Published: July 2002

Bordetella pertussisproduces a 73-kDa protein,BrkA (Bordetellaresistance to killing), which inhibits the bactericidal activity of complement. In this study we characterized the step in the complement cascade where BrkA acts, using three strains: a wild-type strain, a strain containing an insertional disruption ofbrkA, and a strain containing two copies of thebrkAlocus. Following incubation with 10% human serum, killing was greatest for the BrkA mutant, followed by that for the wild-type strain, while the strain with two copies ofbrkAwas the most resistant. Complement activation was monitored by enzyme-linked immunosorbent assay (ELISA) or Western blotting. ELISAs for SC5b-9, the soluble membrane attack complex, showed that production of SC5b-9 was greatest with thebrkAmutant, less with the wild type, and least with the strain containing two copies ofbrkA. Deposition of complement proteins on the bacteria was monitored by Western blotting. A decrease in deposition on the bacteria of C4, C3, and C9 corresponded with decreased complement sensitivity. Deposition of C1, however, was not affected by the presence of BrkA. These studies show that BrkA inhibits the classical pathway of complement activation and prevents accumulation of deposited C4.
Meningococcal Outer Membrane Vesicle Vaccine Given Intranasally Can Induce Immunological Memory and Booster Responses without Evidence of Tolerance
Authors: Bakke Hilde , Lie Kristian , Haugen Inger Lise , Korsvold Gro Ellen , Høiby E. Arne , Næss Lisbeth Meyer , Holst Johan , Aaberge Ingeborg S. , Oftung Fredrik , Haneberg Bjørn
Published: July 2002

We have studied the ability of outer membrane vesicle (OMV) vaccines fromNeisseria meningitidisserogroup B to induce vaccine-specific antibody and spleen cell proliferative responses in mice after being administered intranasally (i.n.) and/or subcutaneously (s.c.). A series of four weekly i.n. doses (25 μg) without adjuvant or a single s.c. dose (2.5 μg) with aluminum hydroxide was followed 2 months later by secondary i.n. or s.c. immunizations. After i.n. priming, both immunoglobulin G (IgG) antibody responses in serum, measured by enzyme-linked immunosorbent assay, and IgA antibodies in saliva and extracts of feces were significantly boosted by later i.n. immunizations. The IgG antibody responses in serum were also significantly augmented by secondary s.c. immunization after i.n. as well as s.c. priming. Sera from mice immunized i.n. reached the same level of bactericidal activity as after s.c. immunizations. The s.c. immunizations alone, however, had no effect on mucosal IgA antibody responses, but could prime for booster antibody responses in secretions to later i.n. immunizations. The i.n. immunizations also led to marked OMV-specific spleen cell proliferation in vitro. Both serum antibody responses and spleen cell proliferation were higher after i.n. priming and later s.c. immunizations than after s.c. immunizations alone. There was thus no evidence that i.n. priming had induced immunological tolerance within the B- or T-cell system. Our results indicate that a nonproliferating meningococcal OMV vaccine given i.n. can induce immunological memory and that it may be favorably combined with similar vaccines for injections.
Recognition of Lewis x Derivatives Present on Mucins by Flagellar Components of
Pseudomonas aeruginosa
Authors: Scharfman Andree , Arora Shiwani K. , Delmotte Philippe , Van Brussel Edwige , Mazurier Joel , Ramphal Reuben , Roussel Philippe
Published: July 2002

Pseudomonas aeruginosa
binds to human respiratory mucins by mechanisms involving flagellar component-receptor interactions. The adhesion of
P. aeruginosa
strain PAK is mediated by the flagellar cap protein, FliD, without the involvement of flagellin. Two distinct types of FliD proteins have been identified in
P. aeruginosa
: A type, found in strain PAK, and B type, found in strain PAO1. In the present work, studies performed with the
P. aeruginosa
B-type strain PAO1 indicate that both the FliD protein and the flagellin of this strain are involved in the binding to respiratory mucins. Using polyacrylamide-based fluorescent glycoconjugates in a flow cytometry assay, it was previously demonstrated that
P. aeruginosa
recognizes Le
x
(or Lewis x) derivatives found at the periphery of human respiratory mucins. The aim of the present work was therefore to determine whether these carbohydrate epitopes (or glycotopes) are receptors for FliD proteins and flagellin. The results obtained by both flow cytometry and a microplate adhesion assay indicate that the FliD protein of strain PAO1 is involved in the binding of glycoconjugates bearing Le
x
or sialyl-Le
x
determinants, while the binding of flagellin is restricted to the glycoconjugate bearing Le
x
glycotope. In contrast, the type A cap protein of
P. aeruginosa
strain PAK is not involved in the binding to glycoconjugates bearing Le
x
, sialyl-Le
x
, or sulfosialyl-Le
x
glycotopes. This study demonstrates a clear association between a specific
Pseudomonas
adhesin and a specific mucin glycotope and demonstrates that fine specificities exist in mucin recognition by
P. aeruginosa
.
Accumulation of Human Heat Shock Protein 60-Reactive T Cells in the Gingival Tissues of Periodontitis Patients
Authors: Yamazaki Kazuhisa , Ohsawa Yutaka , Tabeta Koichi , Ito Harue , Ueki Kaoru , Oda Taro , Yoshie Hiromasa , Seymour Gregory J.
Published: July 2002

Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 andPorphyromonas gingivalisGroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) β-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not toP. gingivalisGroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class II antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-γ) in periodontitis patients, whereasP. gingivalisGroEL did not induce any skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-γ or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type 1 cytokine profile in their peripheral blood T-cell pools.
Protection of Mice againstCoccidioides immitisIntranasal Infection by Vaccination with Recombinant Antigen 2/PRA
Authors: Shubitz Lisa , Peng Tao , Perrill Robert , Simons Julie , Orsborn Kris , Galgiani John N
Published: July 2002

Subcutaneous vaccination with recombinant antigen 2/PRA (rAg2/PRA) protected BALB/c mice against intranasal infection withCoccidioides immitis. Subcutaneously vaccinated C57BL/6 mice and intranasally vaccinated BALB/c mice were protected against larger numbers of infecting spores. Weight loss correlated with lethality, but histologic appearance did not. These studies support rAg2/PRA vaccination to prevent coccidioidomycosis.
Control of Initiation of Sporulation by Replication Initiation Genes in
Bacillus subtilis
Authors: Lemon Katherine P. , Kurtser Iren , Wu Judy , Grossman Alan D.
Published: July 2002

Initiation of spore formation in
Bacillus subtilis
appears to depend on initiation of DNA replication. This regulation was first identified using a temperature-sensitive mutation in
dnaB
. We found that mutations in the replication initiation genes
dnaA
and
dnaD
also inhibit sporulation, indicating that inhibition of sporulation is triggered by general defects in the function of replication initiation proteins.
Analysis of Guanine Nucleotide Binding and Exchange Kinetics of the
Escherichia coli
GTPase Era
Authors: Sullivan S. M. , Mishra R. , Neubig R. R. , Maddock J. R.
Published: July 2002

Era is an essential
Escherichia coli
guanine nucleotide binding protein that appears to play a number of cellular roles. Although the kinetics of Era guanine nucleotide binding and hydrolysis have been described, guanine nucleotide exchange rates have never been reported. Here we describe a kinetic analysis of guanine nucleotide binding, exchange, and hydrolysis by Era using the fluorescent mant (
N
-methyl-3′-
O
-anthraniloyl) guanine nucleotide analogs. The equilibrium binding constants (

K
D

) for mGDP and mGTP (0.61 ± 0.12 μM and 3.6 ± 0.80 μM, respectively) are similar to those of the unmodified nucleotides. The single turnover rates for mGTP hydrolysis by Era were 3.1 ± 0.2 mmol of mGTP hydrolyzed/min/mol in the presence of 5 mM MgCl
2
and 5.6 ± 0.3 mmol of mGTP hydrolyzed/min/mol in the presence of 0.2 mM MgCl
2
. Moreover, Era associates with and exchanges guanine nucleotide rapidly (on the order of seconds) in both the presence and absence of Mg
2+
. We suggest that models of Era function should reflect the rapid exchange of nucleotides in addition to the GTPase activity inherent to Era.
Effect of
CIS
on Activity in
trans
of the Replication Initiator Protein of an IncB Plasmid
Authors: Praszkier J. , Murthy S. , Pittard A. J.
Published: July 2002

RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in
cis
. The
cis
activity of RepA is thought to be mediated by
CIS
, a 166-bp region of DNA separating the coding region of
repA
from the origin of replication (
ori
) of pMU720. To investigate the
trans
activity of RepA, the
repA
gene, without its cognate
ori
, was cloned on a multicopy plasmid, pSU39. The
ori
on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl-β-
d
-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned
ori
by RepA produced in
trans
from the pSU39 derivatives. The effect of
CIS
, when present either on the RepA-producing or the
ori
plasmid or both, on the efficiency of replication of the
ori
plasmid in vivo, was determined. The presence of
CIS
, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the
ori
plasmid. This inhibitory activity of
CIS
was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast,
CIS
had no effect when present on the
ori
plasmid. Initiation of replication from the
ori
in
trans
was independent of transcription into
CIS
.
Genetic Analysis of Sliding Motility in
Mycobacterium smegmatis
Authors: Recht Judith , Martínez Asunción , Torello Sandra , Kolter Roberto
Published: July 2002

A screen for nonsliding mutants of
Mycobacterium smegmatis
yielded 20 mutants with transposon insertions in the
mps
gene, which is involved in glycopeptidolipid biosynthesis. One mutant had an insertion in a gene predicted to encode a membrane transport protein. All mutants lacked glycopeptidolipids and were unable to form biofilms on polyvinyl chloride.
Characterization of
Escherichia coli
DNA Lesions Generated within J774 Macrophages
Authors: Schlosser-Silverman Eliana , Elgrably-Weiss Maya , Rosenshine Ilan , Kohen Ron , Altuvia Shoshy
Published: July 2002

Macrophages are armed with multiple oxygen-dependent and -independent bactericidal properties. However, the respiratory burst, generating reactive oxygen species, is believed to be a major cause of bacterial killing. We exploited the susceptibility of
Escherichia coli
in macrophages to characterize the effects of the respiratory burst on intracellular bacteria. We show that
E. coli
strains recovered from J774 macrophages exhibit high rates of mutations. We report that the DNA damage generated inside macrophages includes DNA strand breaks and the modification 8-oxo-2′-deoxyguanosine, which are typical oxidative lesions. Interestingly, we found that under these conditions, early in the infection the majority of
E. coli
cells are viable but gene expression is inhibited. Our findings demonstrate that macrophages can cause severe DNA damage to intracellular bacteria. Our results also suggest that protection against the macrophage-induced DNA damage is an important component of the bacterial defense mechanism within macrophages.
Varying the Abundance of O Antigen in
Rhizobium etli
and Its Effect on Symbiosis with
Phaseolus vulgaris
Authors: Noel K. Dale , Forsberg Lennart S. , Carlson Russell W.
Published: July 2002

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of
Rhizobium etli
mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn
5
insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the
R. etli
CE3
lps
genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host
Phaseolus vulgaris
, whereas the mutant suppressed by DNA from
lps
region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other
R. etli
mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on
P. vulgaris
. The latter strains were mutated in
lps
region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamine.
Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation
Authors: Hugo N. , Meyer C. , Armengaud J. , Gaillard J. , Timmis K. N. , Jouanneau Y.
Published: July 2002

The
xylT
gene product, a component of the xylene catabolic pathway of
Pseudomonas putida
mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in
Escherichia coli
, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were −286 mV and −323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an
M
r
of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing
xylT
or its analogues were introduced into an XylT-null mutant of
P. putida
which is unable to grow on
p
-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.
The
nrfA
and
nirB
Nitrite Reductase Operons in
Escherichia coli
Are Expressed Differently in Response to Nitrate than to Nitrite
Authors: Wang Henian , Gunsalus Robert P.
Published: July 2002

Escherichia coli
possesses two distinct nitrite reductase enzymes encoded by the
nrfA
and
nirB
operons. The expression of each operon is induced during anaerobic cell growth conditions and is further modulated by the presence of either nitrite or nitrate in the cells' environment. To examine how each operon is expressed at low, intermediate, and high levels of either nitrate or nitrite, anaerobic chemostat culture techniques were employed using
nrfA-lacZ
and
nirB-lacZ
reporter fusions. Steady-state gene expression studies revealed a differential pattern of nitrite reductase gene expression where optimal
nrfA-lacZ
expression occurred only at low to intermediate levels of nitrate and where
nirB-lacZ
expression was induced only by high nitrate conditions. Under these conditions, the presence of high levels of nitrate suppressed
nrfA
gene expression. While either NarL or NarP was able to induce
nrfA-lacZ
expression in response to low levels of nitrate, only NarL could repress at high nitrate levels. The different expression profile for the alternative nitrite reductase operon encoded by
nirBDC
under high-nitrate conditions was due to transcriptional activation by either NarL or NarP. Neither response regulator could repress
nirB
expression. Nitrite was also an inducer of
nirB
and
nrfA
gene expression, but nitrate was always the more potent inducer by >100-fold. Lastly, since
nrfA
operon expression is only induced under low-nitrate concentrations, the NrfA enzyme is predicted to have a physiological role only where nitrate (or nitrite) is limiting in the cell environment. In contrast, the
nirB
nitrite reductase is optimally synthesized only when nitrate or nitrite is in excess of the cell's capacity to consume it. Revised regulatory schemes are presented for NarL and NarP in control of the two operons.
Polyamine Transport and Role of
potE
in Response to Osmotic Stress in
Escherichia coli
Authors: Schiller Dirk , Kruse Daniela , Kneifel Helmut , Krämer Reinhard , Burkovski Andreas
Published: July 2002

When transport of polyamines in
Escherichia coli
was examined, putrescine excretion was observed under two different physiological conditions: (i) strictly correlated to growth and (ii) following a hyperosmotic shock. Spermidine was not excreted. Characterization of a deletion mutant showed that PotE is not involved in these transport processes.
Characterization of SotA and SotB, Two
Erwinia chrysanthemi
Proteins Which Modify Isopropyl-β-
d
-Thiogalactopyranoside and Lactose Induction of the
Escherichia coli lac
Promoter
Authors: Condemine Guy
Published: July 2002

The expression, in
Escherichia coli
, of variants of the
Erwinia chrysanthemi
secretion genes
outB
and
outS
under the P
tac
promoter is toxic to the cells. During attempts to clone
E. chrysanthemi
genes able to suppress this toxicity, I identified two genes,
sotA
and
sotB
, whose products are able to reduce the isopropyl-β-
d
-thiogalactopyranoside (IPTG) induction of the
E. coli lac
promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression of
sotA
and
sotB
is not regulated by their substrates, but
sotA
is activated by the cyclic AMP receptor protein (CRP), while
sotB
is repressed by CRP. Lactose is weakly toxic for
E. chrysanthemi
. This toxicity is increased in a
sotB
mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.
The TetA(K) Tetracycline/H
+
Antiporter from
Staphylococcus aureus
: Mutagenesis and Functional Analysis of Motif C
Authors: Ginn Samantha L. , Brown Melissa H. , Skurray Ronald A.
Published: July 2002

Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from
Staphylococcus aureus
; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H
+
activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.
Purification and Characterization of the DeoR Repressor of
Bacillus subtilis
Authors: Zeng Xianmin , Saxild Hans H. , Switzer Robert L.
Published: July 2002

Transcription of the
Bacillus subtilis dra-nupC-pdp
operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in
Escherichia coli
and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the
dra-nupC-pdp
operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.
Characterization of the Gene Cluster Involved in Isoprene Metabolism in
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Leemhuis Hans , Spelberg Jeffrey H. Lutje , Janssen Dick B.
Published: July 2002

The genes involved in isoprene (2-methyl-1,3-butadiene) utilization in
Rhodococcus
sp. strain AD45 were cloned and characterized. Sequence analysis of an 8.5-kb DNA fragment showed the presence of 10 genes of which 2 encoded enzymes which were previously found to be involved in isoprene degradation: a glutathione
S
-transferase with activity towards 1,2-epoxy-2-methyl-3-butene (
isoI
) and a 1-hydroxy-2-glutathionyl-2-methyl-3-butene dehydrogenase (
isoH
). Furthermore, a gene encoding a second glutathione
S
-transferase was identified (
isoJ
). The
isoJ
gene was overexpressed in
Escherichia coli
and was found to have activity with 1-chloro-2,4-dinitrobenzene and 3,4-dichloro-1-nitrobenzene but not with 1,2-epoxy-2-methyl-3-butene. Downstream of
isoJ
, six genes (
isoABCDEF
) were found; these genes encoded a putative alkene monooxygenase that showed high similarity to components of the alkene monooxygenase from
Xanthobacter
sp. strain Py2 and other multicomponent monooxygenases. The deduced amino acid sequence encoded by an additional gene (
isoG
) showed significant similarity with that of α-methylacyl-coenzyme A racemase. The results are in agreement with a catabolic route for isoprene involving epoxidation by a monooxygenase, conjugation to glutathione, and oxidation of the hydroxyl group to a carboxylate. Metabolism may proceed by fatty acid oxidation after removal of glutathione by a still-unknown mechanism.
areCBA
Is an Operon in
Acinetobacter
sp. Strain ADP1 and Is Controlled by AreR, a ς
54
-Dependent Regulator
Authors: Jones Rheinallt M. , Williams Peter A.
Published: July 2002

The
areCBA
genes in
Acinetobacter
sp. strain ADP1, determining growth on benzyl alkanoates, are shown to be transcribed as a single operon and regulated by
areR
, which encodes a regulatory protein of the NtrC/XylR family. Assays of the Are enzymes and of two insertions of
lacZ
as a reporter gene have shown that the operon is induced by benzyl acetate, benzyl alcohol, and benzaldehyde, as well as 2- and 4-hydroxybenzyl acetates and benzyl propionate and butyrate. Two adjacent sites of transcriptional initiation were 97 and 96 bp upstream of the start codon for
areC
, near a ς
54
-dependent −12, −24 promoter. Inactivation of
areR
and
rpoN
(for RNA polymerase ς
54
) drastically reduced growth rates on the Are substrates and induction of the operon.
Reverse Genetics of
Escherichia coli
Glycerol Kinase Allosteric Regulation and Glucose Control of Glycerol Utilization In Vivo
Authors: Holtman C. Kay , Pawlyk Aaron C. , Meadow Norman D. , Pettigrew Donald W.
Published: July 2002

Reverse genetics is used to evaluate the roles in vivo of allosteric regulation of
Escherichia coli
glycerol kinase by the glucose-specific phosphocarrier of the phosphoenolpyruvate:glycose phosphotransferase system, IIA
Glc
(formerly known as III
glc
), and by fructose 1,6-bisphosphate. Roles have been postulated for these allosteric effectors in glucose control of both glycerol utilization and expression of the
glpK
gene. Genetics methods based on homologous recombination are used to place
glpK
alleles with known specific mutations into the chromosomal context of the
glpK
gene in three different genetic backgrounds. The alleles encode glycerol kinases with normal catalytic properties and specific alterations of allosteric regulatory properties, as determined by in vitro characterization of the purified enzymes. The
E. coli
strains with these alleles display the glycerol kinase regulatory phenotypes that are expected on the basis of the in vitro characterizations. Strains with different
glpR
alleles are used to assess the relationships between allosteric regulation of glycerol kinase and specific repression in glucose control of the expression of the
glpK
gene. Results of these studies show that glucose control of glycerol utilization and glycerol kinase expression is not affected by the loss of IIA
Glc
inhibition of glycerol kinase. In contrast, fructose 1,6-bisphosphate inhibition of glycerol kinase is the dominant allosteric control mechanism, and glucose is unable to control glycerol utilization in its absence. Specific repression is not required for glucose control of glycerol utilization, and the relative roles of various mechanisms for glucose control (catabolite repression, specific repression, and inducer exclusion) are different for glycerol utilization than for lactose utilization.