bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterial Di-O-Acyl-Trehalose Inhibits Mitogen- and Antigen-Induced Proliferation of Murine T Cells In Vitro
Authors: Saavedra Rafael , Segura Erika , Leyva Rosario , Esparza Luis A. , López-Marı́n Luz M.
Published: July 2002

2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of theMycobacterium tuberculosiscell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains ofM. tuberculosis,orM. fortuitumas a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific forToxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.
Usefulness of Hydatid Cyst Fluid of
Echinococcus granulosus
Developed in Mice with Secondary Infection for Serodiagnosis of Cystic Echinococcosis in Humans
Authors: Mamuti Wulamu , Yamasaki Hiroshi , Sako Yasuhito , Nakaya Kazuhiro , Nakao Minoru , Lightowlers Marshall W. , Ito Akira
Published: July 2002

The aim of this work was to assess the usefulness of hydatid cyst fluid (HCF) of
Echinococcus granulosus
, obtained from mice experimentally infected with hydatid cyst tissue homogenates, for the serodiagnosis of cystic echinococcosis (CE) in humans. The sensitivity and specificity of HCF obtained from mice for the detection of immunoglobulin G (IgG) antibodies in the sera of CE patients were compared with those of HCF from sheep and/or from a human CE patient by using immunoblotting (IB) and an enzyme-linked immunosorbent assay (ELISA). HCFs obtained from three different host species all were highly useful for immunoblotting, and sera from 19 (95%) of 20 CE patients equally recognized the antigen B subunit (approximately 8 kDa). HCF from mice showed a cross-reaction with 9 of 20 alveolar echinococcosis (AE) sera (45%), whereas HCFs from two other host species cross-reacted with 14 of the AE sera (70%). Although 2 (10%) of 20 sera from neurocysticercosis (NCC) patients were false positive with HCF from both sheep and humans, none of these sera showed a positive reaction with HCF from mouse origin. ELISAs with HCFs from both mouse and sheep origins detected all 20 CE and AE sera; however, these ELISAs showed 45% (9 of 20) and 60% (12 of 20) false-positive reactions with 20 NCC sera, respectively. The presence of nonspecific human IgG in HCF obtained from a CE patient prevented us from applying it to the ELISA. HCF of
E. granulosus
, obtained from laboratory mice with a secondary infection with hydatid cyst tissue homogenates, appears to be highly useful for the serodiagnosis of CE in humans and may be useful in domestic animals.
Identification and Characterization of
Yersinia enterocolitica
Genes Induced during Systemic Infection
Authors: Gort Amy Strohmeier , Miller Virginia L.
Published: July 2002

Yersinia enterocolitica
is one of three pathogenic
Yersinia
species that share a tropism for lymphoid tissues. However, infection of an immunocompromised host is likely to result in a systemic infection, which is often fatal. Little is known about the bacterial proteins needed to establish such an infection. The genes that encode these virulence factors are likely to be active only during systemic infection. A library of random
cat
fusions was used to inoculate BALB/c mice. Fusions expressed during a systemic infection were enriched by the administration of chloramphenicol-succinate.
Y. enterocolitica
isolates recovered from the mice were tested for chloramphenicol resistance in vitro. Fusions that were inactive in vitro were analyzed further and found to represent 31 allelic groups. Each was given a
sif
(for systemic infection factor) designation. Based on homology to known proteins, the
sif
genes are likely to encode proteins important for general physiology, transcription regulation, and other functions. During systemic infections, 13 of the
sif-cat
fusions were able to outcompete the wild type in the presence of chloramphenicol-succinate, confirming that the fusions were active. The in vitro expression of several
sif
genes was determined, showing modest changes in response to various growth conditions. A mutation in
sif15
, which encodes a putative outer membrane protein, caused attenuation during systemic infection but not during colonization of the Peyer's patches. Comparisons between the
Y. enterocolitica sif
genes and the previously identified
hre
genes imply that very different groups of genes are active during a systemic infection and during colonization of the Peyer's patches.
Functional Activities and Immunoglobulin Variable Regions of Human and Murine Monoclonal Antibodies Specific for the P1.7 PorA Protein Loop ofNeisseria meningitidis
Authors: Wang Jianfu , Jarvis Gary A. , Achtman Mark , Rosenqvist Einar , Michaelsen Terje E. , Aase Audun , Griffiss J. McLeod
Published: July 2002

The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 VHregion belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 VLregion belonged to the Vλ1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vκ1 family. The Fab fragment of SS269 was cloned and expressed inEscherichia coliand was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.
Pilot Study of
phoP/phoQ
-Deleted
Salmonella enterica
Serovar Typhimurium Expressing
Helicobacter pylori
Urease in Adult Volunteers
Authors: Angelakopoulos Haroula , Hohmann Elizabeth L.
Published: July 2002

Attenuated
Salmonella enterica
serovar Typhi has been studied as an oral vaccine vector. Despite success with attenuated
S. enterica
serovar Typhimurium vectors in animals, early clinical trials of
S. enterica
serovar Typhi expressing heterologous antigens have shown that few subjects have detectable immune responses to vectored antigens. A previous clinical study of
phoP/phoQ
-deleted
S. enterica
serovar Typhi expressing
Helicobacter pylori
urease from a multicopy plasmid showed that none of eight subjects had detectable immune responses to the vectored antigen. In an attempt to further define the variables important for engendering immune responses to vectored antigens in humans, six volunteers were inoculated with 5 × 10
7
to 8 × 10
7
CFU of
phoP/phoQ
-deleted
S. enterica
serovar Typhimurium expressing the same antigen. Two of the six volunteers had fever; none had diarrhea, bacteremia, or other serious side effects. The volunteers were more durably colonized than in previous studies of
phoP/phoQ
-deleted
S. enterica
serovar Typhi. Five of the six volunteers seroconverted to
S. enterica
serovar Typhimurium antigens and had strong evidence of anti-
Salmonella
mucosal immune responses by enzyme-linked immunospot studies. Three of six (three of five who seroconverted to
Salmonella
) had immune responses in the most sensitive assay of urease-specific immunoglobulin production by blood mononuclear cells in vitro. One of these had a fourfold or greater increase in end-point immunoglobulin titer in serum versus urease. Attenuated
S. enterica
serovar Typhimurium appears to be more effective than
S. enterica
serovar Typhi for engendering immune responses to urease. Data suggest that this may be related to a greater stability of antigen-expressing plasmid in
S. enterica
serovar Typhimurium and/or prolonged intestinal colonization. Specific factors unique to nontyphoidal salmonellae may also be important for stimulation of the gastrointestinal immune system.
Potentiality of Interleukin-18 as a Useful Reagent for Treatment and Prevention ofLeishmania majorInfection
Authors: Ohkusu Kazunobu , Yoshimoto Tomohiro , Takeda Kiyoshi , Ogura Takeharu , Kashiwamura Shin-ichiro , Iwakura Yoichiro , Akira Shizuo , Okamura Haruki , Nakanishi Kenji
Published: July 2002

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in natural killer cell activation and the T helper 1 (Th1) cell response, particularly in collaboration with IL-12. Since Th1 cells play a pivotal role in the host defense against infection with intracellular microbes, such asLeishmania major, we investigated whether IL-18 is critically involved in protection againstL. majorinfection by activation of Th1 cells. We administered IL-12 and/or IL-18 daily toL. major-susceptible BALB/c mice. Neither IL-12 (10 ng/mouse) nor IL-18 (1,000 ng/mouse) induced wound healing, while daily injection of IL-12 and IL-18 during the first week after infection strongly protected the mice from footpad swelling by induction and activation of Th1 cells. Furthermore, these mice acquired protective immunity. We also investigated a protective role of endogenous IL-18 by using anti-IL-18 antibody-treated C3H/HeN mice (anL. major-resistant strain) or IL-18 deficient (IL-18−/−) mice with a resistant background (C57BL/6). We found that in the absence of endogenous IL-18, these mice showed prolonged footpad swelling as well as diminished nitric oxide production. However, daily injection of IL-18 into IL-18−/−mice corrected their deficiencies, suggesting that these mice have Th1 cells that produce gamma interferon (IFN-γ) in response to IL-18. Indeed, these mice had normal levels of Th1 cells. Thus, IL-18 is not responsible for inducing Th1 cells but participates in host resistance by its action in stimulating Th1 cells to produce IFN-γ. Our results also indicate the high potentiality of IL-18 as a useful reagent for treatment as well as prevention against reinfection.
Granulocytic Ehrlichiosis in Mice Deficient in Phagocyte Oxidase or Inducible Nitric Oxide Synthase
Authors: Banerjee Rila , Anguita Juan , Fikrig Erol
Published: July 2002

Mice deficient in phox (gp91phox−/−) or NOS2 (NOS2−/−) were infected with the agent of human granulocytic ehrlichiosis (HGE) to evaluate the importance of these pathways in the eradication of HGE bacteria. NOS2−/−mice had delayed clearance of the HGE agent in comparison to control or gp91phox−/−mice, suggesting that reactive nitrogen intermediates play a role in the early control of HGE.
Avidity, Potency, and Cross-Reactivity of Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide Serotype 6B
Authors: Sun Yan , Hwang Young-il , Nahm Moon H.
Published: July 2002

Many pneumococcal capsular polysaccharides (PSs) are similar in structure, and a pneumococcal antibody often binds to all of the PSs with a similar structure. Yet, these cross-reactive antibodies may bind to the structurally related pneumococcal capsular PSs with an avidity too low to be effective. If memory B cells producing such weakly cross-reactive antibodies are elicited with pneumococcal conjugate vaccines, the memory cells for low-avidity antibodies could compromise the subsequent immune responses to the cross-reactive PS (original antigenic sin). To investigate these issues, we produced 14 hybridomas secreting monoclonal antibodies (MAbs) to the capsular PS ofStreptococcus pneumoniaeserotype 6B by immunizing BALB/c mice with antigens containing 6B PS and studied their epitope, avidity, in vitro opsonizing capacity, in vivo protective capacity, and “antigen binding titer” by enzyme-linked immunosorbent assay (ELISA) of 6A and 6B capsular PSs. Six MAbs bound to the non-cross-reactive 6B-specific epitope, and seven MAbs bound to the cross-reactive epitope present in both 6A and 6B PSs One MAb (Hyp6BM6) revealed a novel epitope. This epitope was found on 6A PS in solution, but not on 6A PS adsorbed onto the plastic surface of the ELISA plates. The avidity of the MAb for 6A or 6B PS ranged from 7.8 × 106M−1to 4.1 × 1011M−1. No MAbs were weakly cross-reactive, since none of the cross-reactive MAbs showed any tendency toward having less avidity to 6A PS (the cross-reactive PS) than to 6B PS. Avidity influenced the results of several antibody assays. When all of the hybridomas were examined, avidity strongly correlated with the titer of a unit amount of MAb to bind antigen-coated ELISA plates (r= 0.91) or to opsonize pneumococci in vitro (r= −0.85). Because both assay results are avidity dependent, the ELISA and the opsonization assay results were strongly correlated (r= 0.91), regardless of avidity. Avidity also correlated with the potency of a MAb to passively protect mice against pneumococcal infections. When only the immunoglobulin G hybridomas were examined, little increase in opsonizing capacity and in vivo protective potency was observed above 109M−1. Taken together, an ELISA measuring antigen binding titer may be an adequate measure of the protective immunity induced with pneumococcal vaccines, and the absence of a partially cross-reactive MAb suggests that antigenic sin may not be significant in responses to vaccines against theS. pneumoniae6B serotype.
Dynamic Changes in Neutrophil Defensins during Endotoxemia
Authors: Klut M. E. , Whalen B. A. , Hogg J. C.
Published: July 2002

Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis. This study determines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes (PMN) in rabbits treated with LPS. PMN functional activity was assessed by measuring CD18 expression and H2O2production and by examining the lungs. NP-2 and, to a minor extent, NP-5 of circulating PMN increase during endotoxemia. This early increase is concomitant with neutrophilia and elevated CD18 expression and H2O2production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels. A decline in defensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMN and enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage. This study shows that LPS-induced changes in the defensins of circulating PMN correlate with the number and activated condition of these cells and suggests that PMN-derived products implement the inflammatory reaction that leads to lung injury and sepsis.
In Vitro and In Vivo Neutralization of the Relapsing Fever AgentBorrelia hermsiiwith Serotype-Specific Immunoglobulin M Antibodies
Authors: Barbour Alan G. , Bundoc Virgilio
Published: July 2002

The antigenic variation of the relapsing fever agentBorrelia hermsiiis associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.
Down-Regulation of Interleukin-8 Secretion fromMycobacterium tuberculosis-Infected Monocytes by Interleukin-4 and -10 but Not by Interleukin-13
Authors: Ameixa Clara , Friedland Jon S.
Published: July 2002

Interleukin-8 (IL-8), a CXC chemokine, has a central role in leukocyte recruitment to areas of granuloma formation in tuberculosis. In the present studies, we investigated the effect of the TH2-derived cytokines IL-4, IL-10, and IL-13 onMycobacterium tuberculosis-induced IL-8 secretion from purified human monocytes. Our results demonstrate that IL-4 and IL-10 have a down-regulatory effect on IL-8 secretion and that this effect is dose dependent. IL-10 has a greater effect than IL-4 on secretion, and autologous IL-10 secreted fromM. tuberculosis-infected monocytes also down-regulates IL-8 secretion. The down-regulatory effect is partly a result of reduced IL-8 mRNA accumulation analyzed by reverse transcription-PCR. When combined, 1 μM IL-4 and IL-10 had an additive effect in decreasing IL-8 secretion and transcription; there was no synergy of action. IL-13 did not have any significant effect on IL-8 gene expression or secretion. The inhibitory effect of IL-10 but not of IL-4 is associated with decreased nuclear binding of the key activating transcription factor NF-κB. We show for the first time thatM. tuberculosiscauses up-regulation of nuclear binding of Oct-1 detected by electromobility gel shift assay. However, neither AP-1 nor Oct-1 nuclear binding was altered by IL-4 or IL-10. In summary, this study demonstrates that type 2 responses have an important role in the regulation ofM. tuberculosis-induced IL-8 expression but that the mechanisms by which the different cytokines act are distinct.
Gut Colonization of Mice withactA-Negative Mutant ofListeria monocytogenesCan Stimulate a Humoral Mucosal Immune Response
Authors: Manohar Muniraj , Baumann Donald O. , Bos Nicolaas A. , Cebra John J.
Published: July 2002

We usedListeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants,actA-negative (ΔactA)L. monocytogenes, to restrict infection largely to the gut. The ΔactAmutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyer's patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates ofL. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in theListeriasonicates.
Identification of
Listeria monocytogenes
In Vivo-Induced Genes by Fluorescence-Activated Cell Sorting
Authors: Wilson Rebecca L. , Tvinnereim A. R. , Jones Bradley D. , Harty John T.
Published: July 2002

Listeria monocytogenes
is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species. Genetic analysis of
Listeria
pathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of
Listeria
into the host cell. To identify additional genes that are induced in vivo and may be required for
L. monocytogenes
pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragments of the
L. monocytogenes
chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the
L. monocytogenes actA
mutant DP-L1942. Fluorescence-activated cell sorting (FACS) was used to isolate
L. monocytogenes
clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular. Using this strategy, several genes were identified, including
actA
, that exhibited such an expression profile. In-frame deletions of two of these genes, one encoding the putative
L. monocytogenes
uracil DNA glycosylase (
ung
) and one encoding a protein with homology to the
Bacillus subtilis
YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type
L. monocytogenes
10403s. The
L. monocytogenes
10403s
ung
deletion mutant was not attenuated for virulence in mice, while the
yhdP
mutant exhibited a three- to sevenfold reduction in virulence.
In Vivo Activation of Dendritic Cells and T Cells duringSalmonella entericaSerovar Typhimurium Infection
Authors: Yrlid Ulf , Svensson Mattias , Håkansson Anders , Chambers Benedict J. , Ljunggren Hans-Gustaf , Wick Mary Jo
Published: July 2002

The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) andSalmonella entericaserovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice givenS. entericaserovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+MHC-II+splenocytes. Furthermore, splenic DC and T cells were activated followingSalmonellainfection. This was shown by increased surface expression of CD86 and CD40 on CD11c+MHC-II+cells and increased CD44 and CD69 expression on CD4+and CD8+T cells.Salmonella-specific gamma interferon (IFN-γ)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency ofSalmonella-specific CD4+T cells producing IFN-γ was greater than that of specific CD8+T cells producing IFN-γ in the same infected animal. This supports the argument that the predominant source of IFN-γ production by cells of the specific immune response is CD4+T cells. Finally, DC that phagocytosed live or heat-killedSalmonellain vitro primed bacterium-specific IFN-γ-producing CD4+and CD8+T cells as well as cytolytic effector cells following administration into naı̈ve mice. Together these data suggest that DC are involved in priming naı̈ve T cells toSalmonellain vivo.
Discrete Protein Determinant Directs the Species-Specific Adherence of
Porphyromonas gingivalis
to Oral Streptococci
Authors: Demuth Donald R. , Irvine Douglas C. , Costerton J. W. , Cook Guy S. , Lamont Richard J.
Published: July 2002

For pathogens to survive in the human oral cavity, they must identify a suitable niche in the complex multispecies biofilm that exists on oral tissues. The periodontal pathogen
Porphyromonas gingivalis
adheres to
Streptococcus gordonii
by interacting with a specific region of the streptococcal SspB polypeptide, designated BAR. However, it does not adhere to
Streptococcus mutans,
which expresses SpaP, a highly conserved homolog of SspB. Comparison of the predicted secondary structure of BAR with the corresponding region of SpaP suggested that the substitution of Asn for Gly
1182
and Val for Pro
1185
in SspB may confer a unique local structure that is not conserved in SpaP. A synthetic peptide of 26 amino acids that encompassed residues 1167 to 1193 of SspB promoted avid adherence of
P. gingivalis
, whereas a peptide derived from the region corresponding to BAR in SpaP was inactive. Substitution of Gly
1182
and Pro
1185
for Asn
1182
and Val
1185
in SspB by site-specific mutation generated proteins that were predicted to assume an SpaP-like secondary structure, and the purified proteins did not promote
P. gingivalis
adherence. Furthermore,
Enterococcus faecalis
strains expressing the site-specific mutants did not support adherence of
P. gingivalis
cells. In contrast,
P. gingivalis
adhered efficiently to
E. faecalis
strains expressing intact SspB or SspB-SpaP chimeric proteins containing BAR. These results suggest that a region of SspB consisting of 26 amino acids is sufficient to mediate the adherence of
P. gingivalis
to
S. gordonii
and that the species specificity of adherence arises from its interaction with a discrete structural determinant of SspB that is not conserved in SpaP.
dksA
Is Required for Intercellular Spread of
Shigella flexneri
via an RpoS-Independent Mechanism
Authors: Mogull Scott A. , Runyen-Janecky Laura J. , Hong Mei , Payne Shelley M.
Published: July 2002

Pathogenesis of
Shigella flexneri
is dependent on the ability of the bacterium to invade and spread within epithelial cells. In this study, we identified
dksA
as a gene necessary for intercellular spread in, but not invasion of, cultured cells. The
S. flexneri dksA
mutant exhibited sensitivity to acid and oxidative stress, in part due to an effect of DksA on production of RpoS. However, an
S. flexneri rpoS
mutant formed plaques on tissue culture monolayers, thus excluding DksA regulation of RpoS as the mechanism responsible for the inability of the
dksA
mutant to spread intercellularly. Intracellular analysis of the
dksA
mutant indicates that it survived and divided within the Henle cell cytoplasm, but the
dksA
mutant cells were elongated, and some exhibited filamentation in the intracellular environment. Some of the
S. flexneri dksA
mutant cells showed aberrant localization of virulence protein IcsA, which may inhibit spread between epithelial cells.
Effects ofMycobacterium bovisBCG Infection on Regulation ofl-Arginine Uptake and Synthesis of Reactive Nitrogen Intermediates in J774.1 Murine Macrophages
Authors: Peteroy-Kelly Marcy , Venketaraman Vishwanath , Connell Nancy D.
Published: July 2002

The generation of nitric oxide (NO) by activated macrophages is believed to control mycobacterial infection in the murine system. In this study we examined the effect ofMycobacterium bovisBCG infection on thel-arginine-dependent NO pathway in J774.1 murine macrophages. We have confirmed previous results by demonstrating that stimulation of J774.1 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) results in an increase in the uptake of3H-labeledl-arginine and a concomitant increase in the production of NO. We have also shown that BCG can mimic LPS treatment, leading to enhancedl-[3H]arginine uptake by IFN-γ-stimulated macrophages. Lipoarabinomannan, a component of the BCG cell wall that is structurally similar to LPS, is not responsible for the uptake stimulation in IFN-γ stimulated macrophages. Although we demonstrated that there was a 2.5-fold increase in NO production by macrophages 4 h after LPS–IFN-γ stimulation, BCG infection (with or without IFN-γ stimulation) did not lead to the production of NO by the macrophages by 4 h postinfection. At 24 h postinfection, the infected macrophages that were stimulated with IFN-γ produced amounts of NO similar to those of macrophages stimulated with LPS–IFN-γ. This suggests that there are multiple regulatory pathways involved in the production of NO. Finally, our data suggest that increased expression of the arginine permease, MCAT2B, after 4 h of LPS–IFN-γ treatment or BCG infection–IFN-γ treatment is not sufficient to account for the increases inl-[3H]arginine uptake detected. This suggests that the activity of thel-arginine transporter(s) is also altered in response to macrophage activation.
Analysis of Factors Affecting Surface Expression and Immunogenicity of Recombinant Proteins Expressed by Gram-Positive Commensal Vectors
Authors: Bolken Tové C. , Franke Christine A. , Jones Kevin F. , Bell Richard H. , Swanson Ryan M. , King David S. , Fischetti Vincent A. , Hruby Dennis E.
Published: July 2002

Several key protein structural attributes were altered in an effort to optimize expression and immunogenicity of a foreign protein (M protein from
Streptococcus pyogenes
) exposed on the surface of
Streptococcus gordonii
commensal bacterial vectors: (i) a shorter N-terminal region, (ii) the addition of a 94-amino-acid spacer, and (iii) the addition of extra C-repeat regions (CRR) from the M6 protein. A decrease in the amount of cell surface M6 was observed upon deletion of 10 or more amino acid residues at the N terminus. On the other hand, reactivity of monoclonal antibody to surface M6 increased with the addition of the spacer adjacent to the proline- and glycine-rich region, and an increase in epitope dosage was obtained by adding another CRR immediately downstream of the original CRR. The results obtained should facilitate the design of improved vaccine candidates using this antigen delivery technology.
Demonstration of a Novel Glycolytic Pathway in the Hyperthermophilic Archaeon
Thermococcus zilligii
by
13
C-Labeling Experiments and Nuclear Magnetic Resonance Analysis
Authors: Xavier Karina B. , da Costa Milton S. , Santos Helena
Published: July 2002

The operation of a novel glycolytic pathway was demonstrated in nongrowing cells of
Thermococcus zilligii
by analysis of the isotopic enrichment in the end products derived from fermentation of
13
C-labeled glucose. The new pathway involved the formation of formate, derived from C-1 in glucose, via cleavage of a six-carbon carboxylic acid.
Flavonoid-Induced Expression of a Symbiosis-Related Gene in the Cyanobacterium
Nostoc punctiforme
Authors: Cohen Michael F. , Yamasaki Hideo
Published: July 2002

The flavonoid naringin was found to induce the expression of
hrmA
, a gene with a symbiotic phenotype in the cyanobacterium
Nostoc punctiforme
. A comparative analysis of several flavonoids revealed the 7-
O
-neohesperidoside, 4′-OH, and C-2-C-3 double bond in naringin as structural determinants of its
hrmA
-inducing activity.
Cell-Associated Pheromone Peptide (cCF10) Production and Pheromone Inhibition in
Enterococcus faecalis
Authors: Buttaro B. A. (Leonard) , Antiporta M. H. , Dunny G. M.
Published: July 2002

In
Enterococcus faecalis
, the peptide cCF10 acts as a pheromone, inducing transfer of the conjugative plasmid pCF10 from plasmid-containing donor cells to plasmid-free recipient cells. In these studies, it was found that a substantial amount of cCF10 associates with the envelope of the producing cell. Pheromone activity was detected in both wall and membrane fractions, with the highest activity associated with the wall. Experiments examining the effects of protease inhibitor treatments either prior to or following cell fractionation suggested the presence of a cell envelope-associated pro-cCF10 that can be processed to mature cCF10 by a maturase or protease. A pCF10-encoded membrane protein, PrgY, was shown to prevent self-induction of donor cells by reducing the level of pheromone activity in the cell wall fraction.
Bacillus subtilis comZ
(
yjzA
) Negatively Affects Expression of
comG
but Not
comK
Authors: Ogura Mitsuo , Tanaka Teruo
Published: July 2002

The
yjzA
open reading frame, along with
med
, constitutes an operon. Disruption of
yjzA
caused a five-fold enhancement of
comG
expression, thereby leading to a three-fold-higher transformation efficiency. The expression of
comK
and the other three late competence operons was not affected significantly in the
yjzA
-deficient mutant.
A Hydrogen Peroxide-Forming NADH Oxidase That Functions as an Alkyl Hydroperoxide Reductase in
Amphibacillus xylanus
Authors: Niimura Youichi , Nishiyama Yoshitaka , Saito Daisuke , Tsuji Hirokazu , Hidaka Makoto , Miyaji Tatsurou , Watanabe Toshiro , Massey Vincent
Published: July 2002

The
Amphibacillus xylanus
NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with β-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing
Salmonella enterica
AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812–5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 × 10
6
M
−1
s
−1
. Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH
2
reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH
2
is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The
A. xylanus ahpC
gene was cloned and overexpressed in
Escherichia coli
. When the NADH oxidase was coupled with
A. xylanus
AhpC, the peroxidase activity was not inhibited by oxygen. The
V
max
values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s
−1
. The

K
m

values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.
Proteolysis of the McpA Chemoreceptor Does Not Require the
Caulobacter
Major Chemotaxis Operon
Authors: Tsai Jeng-Wen , Alley M. R. K.
Published: July 2002

The degradation of the McpA chemoreceptor in
Caulobacter crescentus
accompanies the swarmer cell to the stalked-cell differentiation event. To further analyze the requirements for its degradation, we have constructed a series of strains that have deletions in the
mcpA
gene and in the
mcpA
chemotaxis operon. Internal deletions of the
mcpA
gene demonstrate that the highly conserved domain (signalling unit) and the methylation domains are not required for cell cycle-regulated proteolysis. The deletion of the chemotaxis operon, which is absolutely required for chemotaxis and McpA chemoreceptor methylation, has no effect on McpA proteolysis.
Comparison of the UDP-
N
-Acetylmuramate:
l
-Alanine Ligase Enzymes from
Mycobacterium tuberculosis
and
Mycobacterium leprae
Authors: Mahapatra Sebabrata , Crick Dean C. , Brennan Patrick J.
Published: July 2002

In the peptidoglycan of
Mycobacterium leprae
,
l
-alanine of the side chain is replaced by glycine. When expressed in
Escherichia coli
, MurC (UDP-
N
-acetyl-muramate:
l
-alanine ligase) of
M. leprae
showed

K
m

and
V
max
for
l
-alanine and glycine similar to those of
Mycobacterium tuberculosis
MurC, suggesting that another explanation should be sought for the presence of glycine.
Catabolite Repression Control by Crc in 2xYT Medium Is Mediated by Posttranscriptional Regulation of
bkdR
Expression in
Pseudomonas putida
Authors: Hester Kathryn L. , Madhusudhan K. T. , Sokatch John R.
Published: July 2002

The effect of growth in 2xYT medium on catabolite repression control in
Pseudomonas putida
has been investigated using the
bkd
operon, encoding branched-chain keto acid dehydrogenase. Crc (catabolite repression control protein) was shown to be responsible for repression of
bkd
operon transcription in 2xYT. BkdR levels were elevated in a
P. putida crc
mutant, but
bkdR
transcript levels were the same in both wild type and
crc
mutant. This suggests that the mechanism of catabolite repression control in rich media by Crc involves posttranscriptional regulation of the bkdR message.
Regulation of Cold Shock-Induced RNA Helicase Gene Expression in the Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Chamot Danuta , Owttrim George W.
Published: July 2002

Expression of the
Anabaena
sp. strain PCC 7120 RNA helicase gene
crhC
is induced by cold shock.
crhC
transcripts are not detectable at 30°C but accumulate at 20°C, and levels remain elevated for the duration of the cold stress. Light-derived metabolic capability, and not light per se, is required for
crhC
transcript accumulation. Enhanced
crhC
mRNA stability contributes significantly to the accumulation of
crhC
transcripts, with the
crhC
half-life increasing sixfold at 20°C. The accumulation is reversible, with the cells responding more rapidly to temperature downshifts than to upshifts, as a result of the lack of active mRNA destabilization and the continuation of
crhC
transcription, at least transiently, after a temperature upshift. Translational inhibitors do not induce
crhC
expression to cold shock levels, indicating that inhibition of translation is only one of the signals required to activate the cold shock response in
Anabaena
. Limited amounts of protein synthesis are required for the cold shock-induced accumulation of
crhC
transcripts, as normal levels of accumulation occur in the presence of tetracycline but are abolished by chloramphenicol. Regulation of
crhC
expression may also extend to the translational level, as CrhC protein levels do not correlate completely with the pattern of mRNA transcript accumulation. Our experiments indicate that the regulation of
crhC
transcript accumulation is tightly controlled by both temperature and metabolic activity at the levels of transcription, mRNA stabilization, and translation.
Novel Missense Mutations That Affect the Transport Function of MalK, the ATP-Binding-Cassette Subunit of the
Salmonella enterica
Serovar Typhimurium Maltose Transport System
Authors: Hunke Sabine , Landmesser Heidi , Schneider Erwin
Published: July 2002

We report on novel mutations in the
malK
gene of
Salmonella enterica
serovar Typhimurium, encoding the ATPase subunit of the maltose transporter (MalFGK
2
). Biochemical analysis suggests that (i) L86 might be involved in a signaling step during substrate translocation and (ii) E306 may be critical for the structural integrity of the protein.
ssrA
(tmRNA) Plays a Role in
Salmonella enterica
Serovar Typhimurium Pathogenesis
Authors: Julio Steven M. , Heithoff Douglas M. , Mahan Michael J.
Published: July 2002

Escherichia coli ssrA
encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that
ssrA
plays a role in
Salmonella enterica
serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site,
attL
, encoded within
ssrA
, serves as the site of integration of a region of
Salmonella
-specific sequence; adjacent to the 5′ end of
ssrA
is another region of
Salmonella
-specific sequence with extensive homology to predicted proteins encoded within the unlinked
Salmonella
pathogenicity island SPI4.
S. enterica
serovar Typhimurium
ssrA
mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that
ssrA
plays a role in the pathogenesis of
Salmonella
, serves as an attachment site for
Salmonella
-specific sequences, and is required for the growth of phage P22.
A New IS
4
Family Insertion Sequence, IS
4Bsu
1, Responsible for Genetic Instability of Poly-γ-Glutamic Acid Production in
Bacillus subtilis
Authors: Nagai Toshiro , Phan Tran Lam-Son , Inatsu Yasuhiro , Itoh Yoshifumi
Published: July 2002

Certain
Bacillus subtilis
strains, such as
B. subtilis
(
natto
) starter strains for the manufacture of natto (fermented soybeans), produce capsular poly-γ-glutamate (γPGA). In
B. subtilis
(
natto
), γPGA synthesis is controlled by the ComP-ComA two-component regulatory system and thereby induced at the beginning of the stationary growth phase. We have found a new insertion sequence (IS), designated IS
4Bsu1
, in the
comP
gene of a spontaneous γPGA-negative mutant of
B. subtilis
(
natto
) NAF4. IS
4Bsu1
(1,406 bp), the first IS discovered in
B. subtilis
, encodes a putative transposase (Tpase) with a predicted
M
r
of 34,895 (374 residues) which displays similarity to the Tpases of IS
4
family members. Southern blot analyses have identified 6 to 11 copies of IS
4Bsu1
, among which 6 copies were at the same loci, in the chromosomes of
B. subtilis
(
natto
) strains, including NAF4, three commercial starters, and another three γPGA-producing
B. subtilis
(
natto
) strains. All of the eight spontaneous γPGA
−
mutants, which were derived from five independent NAF4 cultures, had a new additional IS
4Bsu1
copy in
comP
at six different positions within 600 bp of the 5′-terminal region. The target sites of IS
4Bsu1
were determined to be AT-rich 9-bp sequences by sequencing the flanking regions of IS
4Bsu1
in mutant
comP
genes. These results indicate that IS
4Bsu1
transposes by the replicative mechanism, in contrast to other IS
4
members that use the conservative mechanism, and that most, if not all, of spontaneous γPGA
−
mutants appear to have resulted from the insertion of IS
4Bsu1
exclusively into
comP
. The presence of insertion hot spots in
comP
, which is essential for γPGA synthesis, as well as high transposition activity, would account for the high frequency of spontaneous γPGA
−
mutation by IS
4Bsu1
in
B. subtilis
(
natto
).
Renaturation of
Bacillus thermoglucosidasius
HrcA Repressor by DNA and Thermostability of the HrcA-DNA Complex In Vitro
Authors: Watanabe Kunihiko , Yamamoto Takeshi , Suzuki Yuzuru
Published: July 2002

HrcA, a negative control repressor for chaperone expression from the obligate thermophile
Bacillus thermoglucosidasius
KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified
B. thermoglucosidasius
HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize
B. thermoglucosidasius
HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii)
B. thermoglucosidasius
HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii)
B. thermoglucosidasius
HrcA binding to the CIRCE element was stable at below 50°C whereas the complex was rapidly denatured at 70°C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.
Identification and Distribution of New Insertion Sequences in the Genome of Alkaliphilic
Bacillus halodurans
C-125
Authors: Takami Hideto , Han Chang-Gyun , Takaki Yoshihiro , Ohtsubo Eiichi
Published: July 2002

Fifteen kinds of new insertion sequences (ISs), IS
641
to IS
643
, IS
650
to IS
658
, IS
660
, IS
662
, and IS
663
, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic
Bacillus halodurans
C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS
650
, IS
653
, IS
660
, and IS
663
generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS
650
, IS
653
, and IS
657
carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS
642
, IS
643
, IS
654
, IS
655
, IS
657
, and IS
658
) belong to a separate IS family (IS
630
, IS
21
, IS
256
, IS
3
, IS
200
/IS
605
, and IS
30
, respectively) as a new member. Also, IS
651
and IS
652
were characterized as new members of the IS
L3
family. Significant similarity was found between the transposase (Tpase) sequences between IS
650
and IS
653
(78.2%), IS
651
and IS
652
(56.3%), IS
656
and IS
662
(71.0%), and IS
660
and IS
663
(44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS
651
in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and
l
-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.