bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the F Antigen-Specific
papA
Alleles of Extraintestinal Pathogenic
Escherichia coli
Using a Novel Multiplex PCR-Based Assay
Authors: Johnson James R. , Stell Adam L. , Scheutz Flemming , O'Bryan Timothy T. , Russo Thomas A. , Carlino Ulrike B. , Fasching Claudine , Kavle Justine , Van Dijk Linda , Gaastra Wim
Published: July 2002

Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic
Escherichia coli
, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of
papA
and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the
papA
alleles among 75
E. coli
isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that
papA
sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of
papA
, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional
pap
fragments. Among the urosepsis isolates, the assay revealed considerable segregation of
papA
alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of
papA
alleles between lineages. Sequencing of
papA
from two strains that were
papA
positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel
papA
variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known
papA
alleles. These findings provide novel insights into the
papA
alleles of extraintestinal pathogenic
E. coli
and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of
papA
alleles available to any laboratory with PCR capability.
Effective Preexposure Tuberculosis Vaccines Fail To Protect When They Are Given in an Immunotherapeutic Mode
Authors: Turner Joanne , Rhoades Elizabeth R. , Keen Marc , Belisle John T. , Frank Anthony A. , Orme Ian M.
Published: July 2002

Two vaccine formulations previously shown to induce protective immunity in mice and prevention of long-term necrosis in guinea pigs were tested as potential immunotherapeutic vaccines in mice earlier infected by aerosol withMycobacterium tuberculosis. Neither vaccine had any effect on the course of the infection in the lungs, but both reduced the bacterial load in the spleen. Similarly, inoculation withMycobacterium bovisBCG had no effect whatsoever and, if given more than once, appeared to induce an increasingly severe pyogranulomatous response in the lungs of these mice.
Antibodies to Tyvelose Exhibit Multiple Modes of Interference with the Epithelial Niche ofTrichinella spiralis
Authors: McVay Catherine S. , Bracken Peter , Gagliardo Lucille F. , Appleton Judith
Published: July 2002

Infection with the parasitic nematodeTrichinella spiralisis initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells againstT. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941–1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment byT. spiralis.
mkp-1
Encoding Mitogen-Activated Protein Kinase Phosphatase 1, a Verotoxin 1 Responsive Gene, Detected by Differential Display Reverse Transcription-PCR in Caco-2 Cells
Authors: Kojima Shihoko , Yanagihara Itaru , Kono Gengo , Sugahara Tomomi , Nasu Hatsumi , Kijima Mika , Hattori Akiko , Kodama Toshio , Nagayama Ken-Ichi , Honda Takeshi
Published: July 2002

The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing
Escherichia coli
is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including
mkp-1
(encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction of
mkp-1
mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced
mkp-1
mRNA, but
mkp-1
mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for
mkp-1
induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.
Intracellular Growth ofLegionella pneumophilainDictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions
Authors: Solomon Jonathan M. , Rupper Adam , Cardelli James A. , Isberg Ralph R.
Published: July 2002

Conditions were established in whichLegionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organismDictyostelium discoideum. By several criteria,L. pneumophilagrew by the same mechanism withinD. discoideumas it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, andL. pneumophila dot/icmmutants, blocked for growth in macrophages and amoebae, also did not grow inD. discoideum. InternalizedL. pneumophilaavoided degradation byD. discoideumand showed evidence of reduced fusion with endocytic compartments. The ability ofL. pneumophilato grow withinD. discoideumdepended on the growth state of the cells.D. discoideumgrown as adherent monolayers was susceptible toL. pneumophilainfection and to contact-dependent cytotoxicity during high-multiplicity infections, whereasD. discoideumgrown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some knownD. discoideummutants were examined for their effect on growth ofL. pneumophila. The coronin mutant and themyoA/Bdouble myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth ofL. pneumophilain a Gβmutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of theL. pneumophila-D. discoideumsystem for genetic analysis of host-pathogen interactions.
Tumor Necrosis Factor Alpha-Mediated Toxic Shock inTrypanosoma cruzi-Infected Interleukin 10-Deficient Mice
Authors: Hölscher Christoph , Mohrs Markus , Dai Wen Juan , Köhler Gabriele , Ryffel Bernhard , Schaub Günter A. , Mossmann Horst , Brombacher Frank
Published: July 2002

Using interleukin-10 (IL-10)-deficient (IL-10−/−) mice, previous studies revealed a pathological immune response after infection withTrypanosoma cruzithat is associated with CD4+T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals.T. cruzi-infected IL-10−/−mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-γ), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-α). Despite this early resistance, IL-10−/−mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-α overproduction. Indeed, high production of systemic TNF-α significantly correlated with mortality, and moribund mice died with critically high TNF-α concentrations in the blood. Consequent treatment with anti-TNF-α antiserum attenuated pathological changes inT. cruzi-infected IL-10−/−mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-α concentrations and the time of death. Since elevated serum IL-12 and IFN-γ concentrations were not affected by the administration of antiserum, these studies suggest that TNF-α is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-α-mediated toxic shock.
Expression and Immunological Analysis of the Plasmid-BornemlpGenes ofBorrelia burgdorferiStrain B31
Authors: Porcella Stephen F. , Fitzpatrick Cecily A. , Bono James L.
Published: July 2002

A lipoprotein gene family first identified inBorrelia burgdorferistrain 297, designated 2.9LPand recently renamedmlp, was found on circular and linear plasmids in the genome sequence ofB. burgdorferistrain B31-M1. Sequence analyses of the B31mlpgenes and physically linked variant gene families indicated thatmlpgene heterogeneity is unique and unrelated to location or linkage to divergent sequences. Evidence of recombination between B31mlpalleles was also detected. Northern blot analysis of cultured strain B31 indicated that themlpgenes were not expressed at a temperature (23°C) characteristic of that of ticks in the environment. In striking contrast, expression of manymlpgenes increased substantially when strain B31 was shifted to 35°C, a temperature change mimicking that occurring in the natural transmission cycle of the spirochete from tick to mammal. Primer extension analysis of themlpmRNA transcripts suggested that sigma 70-like promoters are involved inmlpexpression during temperature shift conditions. Antibodies were made against strain B31 Mlp proteins within the first 4 weeks after experimental mouse infection. Importantly, Lyme disease patients also had serum antibodies reactive with purified recombinant Mlp proteins from strain B31, a result indicating that humans are exposed to Mlp proteins during infection. Taken together, the data indicate that strain B31mlpgenes encode a diverse array of lipoproteins which may participate in early infection processes in the mammalian host.
Suppression of Macrophage Activation with CNI-1493 Increases Survival in Infant Rats with Systemic
Haemophilus influenzae
Infection
Authors: Granert Carl , Abdalla Hana , Lindquist Lars , Diab Asim , Bakhiet Moiz , Tracey Kevin J. , Andersson Jan
Published: July 2002

CNI-1493, a potent macrophage deactivator, was used to treat infant rats systemically infected with
Haemophilus influenzae
type b (Hib). CNI-1493 was injected 1 h prior to bacterial inoculation and 24 h later and resulted in a 75 percent increased rate of survival compared to that for untreated controls. The effect of CNI-1493 on the inflammatory response was studied by immunohistochemical detection of individual tumor necrosis factor alpha (TNF-α)-, interleukin 1 beta (IL-1β)-, and gamma interferon (IFN-γ)-producing cells in the spleen. A significant reduction of the incidence of TNF-α- and IL-1β-expressing cells was found for CNI-1493-treated animals. IFN-γ expression was not suppressed by CNI-1493, indicating that cytokine inhibition was specific in macrophages. CNI-1493 significantly reduced the number of infiltrating granulocytes in the brain from that for controls. This study provides evidence that CNI-1493 protects against lethal Hib infection by deactivating the inflammatory cascade in infant rats.
Clonal Associations amongStaphylococcus aureusIsolates from Various Sites of Infection
Authors: Booth Mary C. , Pence Lisa M. , Mahasreshti Param , Callegan Michelle C. , Gilmore Michael S.
Published: July 2002

A molecular epidemiological analysis was undertaken to identify lineages ofStaphylococcus aureusthat may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405S. aureusclinical isolates collected from various infection types and geographic locations was performed. Five distinctS. aureuslineages (SALs 1, 2, 4, 5, and 6) were identified, which accounted for 19.01, 9.14, 22.72, 10.12, and 4.69% of isolates, respectively. In addition, 85 lineages which occurred with frequencies of <2.5% were identified and were termed “sporadic.” The most prevalent lineage was methicillin-resistantS. aureus(SAL 4). The second most prevalent lineage, SAL 1, was also isolated at a high frequency from the anterior nares of healthy volunteers, suggesting that its prevalence among clinical isolates may be a consequence of high carriage rates in humans. Gene-specific PCR was carried out to detect genes for a number of staphylococcal virulence traits.tstandcnawere found to be significantly associated with prevalent lineages compared to sporadic lineages. When specific infection sites were examined, SAL 4 was significantly associated with respiratory tract infection, while SAL 2 was enriched among blood isolates. SAL 1 and SAL 5 were clonally related to SALs shown by others to be widespread in the clinical isolate population. We conclude from this study that at least five phylogenetic lineages ofS. aureusare highly prevalent and widely distributed among clinical isolates. The traits that confer on these lineages a propensity to infect may suggest novel approaches to antistaphylococcal therapy.
Environmental Signals Controlling Production of Hemagglutinin/Protease in
Vibrio cholerae
Authors: Benitez Jorge A. , Silva Anisia J. , Finkelstein Richard A.
Published: July 2002

Vibrio cholerae
hemagglutinin/protease (Hap) was induced upon nutrient limitation and strongly repressed by glucose. Hap was not produced in a mutant defective in the cyclic AMP (cAMP) receptor protein, suggesting that intracellular cAMP levels mediate Hap expression. No difference was found in Hap production between an
rpoS
deletion mutant and its isogenic wild-type precursor, indicating that the alternate ς
s
factor is not essential for Hap expression. Based on these and previous results, we discuss the role of Hap in the pathogenesis of cholera.
Characterization of a
Brucella
Species 25-Kilobase DNA Fragment Deleted from
Brucella abortus
Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide
Authors: Vizcaı́no Nieves , Cloeckaert Axel , Zygmunt Michel S. , Fernández-Lago Luis
Published: July 2002

In the present study we completed the nucleotide sequence of a
Brucella melitensis
16M DNA fragment deleted from
B. abortus
that accounts for 25,064 bp and show that the other
Brucella
spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the
B. melitensis
16M DNA flanking both sides of the fragment deleted from
B. abortus
, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to
omp31
, coding for an immunogenic protein located in the
Brucella
outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysacharides from other bacteria, suggesting that they might be involved in the synthesis of a
Brucella
polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in
B. abortus
and would be a polysaccharide not identified until present in the genus
Brucella
, since all of the known polysaccharides are synthesized in all smooth
Brucella
species. Discovery of a novel polysaccharide not synthesized in
B. abortus
might be interesting for a better understanding of the pathogenicity and host preference differences observed between the
Brucella
species. However, the possibility that the genes detected in the DNA fragment deleted in
B. abortus
no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class
Proteobacteria
, with some of its members synthesizing extracellular polysaccharides and, as
Brucella
spp., living in association with eukaryotic cells.
Enzyme Degradation and Proinflammatory Activity in Arthritogenic and NonarthritogenicEubacterium aerofaciensCell Walls
Authors: Zhang Xiang , Rimpiläinen Marja , Šimelyte Egle , Toivanen Paavo
Published: July 2002

Two almost-identical strains ofEubacterium aerofaciensisolated from the normal human gut flora were used. The cell wall (CW) of one strain with a peptidoglycan (PG) type A4α induces chronic arthritis in the rat after a single intraperitoneal injection, whereas CW of the other with PG type A4β induces only a transient acute arthritis. The CW of the arthritogenicE. aerofacienswas a twofold-more-potent stimulator of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein 1 (MCP-1) than the nonarthritogenic CW. After degradation with mutanolysin, the capacity of the arthritogenic PG to stimulate production of TNF-α and MCP-1 was significantly increased, whereas that of the nonarthritogenic PG was significantly decreased. In other words, after enzyme degradation the arthritogenic PG had a four- to fivefold-stronger stimulatory capacity than that of the enzyme-treated nonarthritogenic PG. These findings indicate that the arthritogenicity of CW or a PG is not dependent on the enzyme resistance alone but also on how the PG fragments released by enzyme degradation stimulate the production of proinflammatory cytokines.
Influence ofMycobacterium bovisBacillus Calmette Guérin on In Vitro Induction of CD1 Molecules in Human Adherent Mononuclear Cells
Authors: Giuliani Anna , Prete Salvatore P. , Graziani Grazia , Aquino Angelo , Balduzzi Alessandra , Sugita Masahiko , Brenner Michael B. , Iona Elena , Fattorini Lanfranco , Orefici Graziella , Porcelli Steven A. , Bonmassar Enzo
Published: July 2002

Nonpeptide antigens (including glycolipids of microbial origin) can be presented to T cells by CD1 molecules expressed on monocyte-derived dendritic cells. These HLA unrestricted responses appear to play a role in host immunity againstMycobacterium tuberculosisand other pathogenic bacteria. It is known that vaccination withMycobacterium bovisbacillus Calmette-Guérin (BCG) has limited efficacy in many clinical settings, although the reasons for its inadequacy remain unclear. Here we have investigated the influence of BCG on the induction of CD1b on human monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF), which is believed to be the principal inducer of this antigen-presenting molecule. Although BCG alone led to a slight induction of CD1b expression, this agent reduced markedly the ability of GM-CSF to induce high levels of CD1b that were typically observed in uninfected cells. Inhibition of CD1b expression in BCG-infected monocytes was apparent at both the mRNA transcript and CD1b protein levels. Down-regulation of CD1b expression by BCG was mediated, at least in part,by one or more soluble factors and could not be reversed with high concentrations of GM-CSF or a variety of other cytokines. The present results suggest that BCG could diminish the efficiency of CD1-restricted T-cell responses against nonpeptide mycobacterial antigens by reducing CD1 expression on antigen-presenting cells. These findings have potential implications for understanding the nature of the immune response elicited by BCG in humans and suggest potential strategies that could be important for the development of better vaccines for the prevention of tuberculosis.
Cryptococcus neoformansInduces Macrophage Inflammatory Protein 1α (MIP-1α) and MIP-1β in Human Microglia: Role of Specific Antibody and Soluble Capsular Polysaccharide
Authors: Goldman David , Song Xianyuan , Kitai Ryuhei , Casadevall Arturo , Zhao Meng-Liang , Lee Sunhee C.
Published: July 2002

We characterized the expression of the β-chemokines macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and RANTES by primary human microglia after exposure toCryptococcus neoformans.In the absence of specific antibody,C. neoformansfailed to elicit a chemokine response, while in the presence of specific antibody, microglia produced MIP-1α and MIP-1β in amounts comparable to those induced by lipopolysaccharide. RANTES was also induced but at much lower levels. In addition to MIP-1α and MIP-1β mRNA, we observed a robust induction of monocyte chemoattractant protein 1 and interleukin-8 mRNA following incubation of microglia with opsonizedC. neoformans. In contrast, cryptococcal polysaccharide did not induce a chemokine response even when specific antibody was present and inhibited the MIP-1α induction associated with antibody-mediated phagocytosis ofC. neoformans. The role of the Fc receptor in the observed chemokine induction was explored in several experiments. Treatment of microglia with cytochalasin D inhibited internalization ofC. neoformansbut did not affect MIP-1α induction. In contrast, treatment with herbimycin A, a tyrosine kinase inhibitor, inhibited MIP-1α induction. Microglia stimulated with immobilized murine immunoglobulin also produced MIP-1α and RANTES (MIP-1α > RANTES). Our results show that microglia produce several chemokines when stimulated byC. neoformansin the presence of specific antibody and that this process is likely to be mediated by Fc receptor activation. This response can be down-regulated by cryptococcal capsular polysaccharide. These findings suggest a mechanism by whichC. neoformansinfections fail to induce strong inflammatory responses in patients with cryptococcal meningoencephalitis and have important implications for antibody therapy.
Flagellum of
Legionella pneumophila
Positively Affects the Early Phase of Infection of Eukaryotic Host Cells
Authors: Dietrich Claudia , Heuner Klaus , Brand Bettina C. , Hacker Jörg , Steinert Michael
Published: July 2002

Legionella pneumophila
, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology of
Legionella
, the
flaA
gene of
L. pneumophila
Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant
L. pneumophila
Corby KH3 was nonflagellated. The redelivery of the intact
flaA
gene into the chromosome (
L. pneumophila
Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the
flaA
mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of
L. pneumophila
positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.
Genetic Regulation of Acquired Immune Responses to Antigens ofMycobacterium tuberculosis:a Study of Twins in West Africa
Authors: Jepson Annette , Fowler Amanda , Banya Winston , Singh Mahavir , Bennett Steve , Whittle Hilton , Hill Adrian V. S.
Published: July 2002

The role of genetic factors in clinical tuberculosis is increasingly recognized; how such factors regulate the immune response toMycobacterium tuberculosisin healthy individuals is unclear. In this study of 255 adult twin pairs residing in The Gambia, West Africa, it is apparent that memory T-cell responses to secreted mycobacterial antigens (85-kDa antigen complex, “short-term culture filtrate,” and peptides from the ESAT-6 protein), as well as to the 65-kDa heat shock protein, are subject to effective genetic regulation. The delayed hypersensitivity response to intradermal tuberculin also demonstrates significant genetic variance, while quantitative T-cell and antibody responses to the 38-kDa cell membrane protein appear to be determined largely by environmental factors. Such findings have implications for vaccine development.
Impact of Genotypic Variation ofBorrelia burgdorferiSensu Stricto on Kinetics of Dissemination and Severity of Disease in C3H/HeJ Mice
Authors: Wang Guiqing , Ojaimi Caroline , Iyer Radha , Saksenberg Victoria , McClain Steve A. , Wormser Gary P. , Schwartz Ira
Published: July 2002

Various genotypes ofBorrelia burgdorferisensu stricto have been previously identified among a large collection of isolates cultured from patients with Lyme disease in the United States. Furthermore, association of specific genotypes with hematogenous dissemination early in the disease course has been observed. The present study assessed kinetics of spirochete dissemination and disease severity in C3H/HeJ mice infected with two different genotypes ofB. burgdorferi. Spirochete load in plasma and ear and other tissue samples of infected mice was measured by quantitative PCR, and these data were compared to those obtained by culture and histopathologic analysis. In mice infected with isolate BL206 (a type 1 strain), the peak number of spirochetes was observed in plasma between day 4 and 7, in heart and ear tissue on day 14, and in joints on day 28 postinoculation. There was a correlation between the peak number of spirochetes in plasma on day 4 or 7 and that in ear biopsy and joint specimens on day 14. By contrast, spirochete burdens in plasma of mice infected with isolate B356 (a type 3 strain) were 16- and 5-fold lower than those of BL206-infected mice on days 7 and 14 of infection, respectively. Similarly, approximately 6- and 13-fold fewer spirochetes were detected in the heart tissues of B356-infected mice compared to BL206-infected mice. Histopathologically, severe arthritis and aortitis were noted only in mice infected with isolate BL206. Spirochete dissemination and disease severity vary significantly in mice infected with distinct genotypes ofB. burgdorferi, suggesting that genotypic differences in the infecting spirochetes play a key role in the pathogenesis and development of clinical disease.
Evidence of Recombination in
Porphyromonas gingivalis
and Random Distribution of Putative Virulence Markers
Authors: Frandsen Ellen V. G. , Poulsen Knud , Curtis Michael A. , Kilian Mogens
Published: July 2002

The association of
Porphyromonas gingivalis
to periodontal disease is not clearly understood. Similar proportions of
P. gingivalis
may be cultivated from both inactive and actively degrading periodontal pockets. Differences in virulence among strains of
P. gingivalis
exist, but the molecular reason for this remains unknown. We examined the population structure of
P. gingivalis
to obtain a framework in which to study pathogenicity in relation to evolution. Phylogenetic trees derived from the sequencing of fragments of four housekeeping genes,
ahp, thy, rmlB
, and
infB
, in 57 strains were completely different with no correlation between clustering of strains in the four dendrograms. Combining the various alleles of the four gene fragments sequenced resulted in 41 different sequence types. The index of association, I
A
, based on a single representative of each sequence type was 0.143 ± 0.202, indicating a population at linkage equilibrium. Inclusion of all isolates for the calculation of I
A
resulted in a value of 0.206 ± 0.171. This suggests an epidemic population structure supported by the finding of genetically identical strains in different parts of the world. We observed a random distribution of two virulence-associated mobile genetic elements, the
ragB
locus and the insertion sequence IS
1598
, among 132 strains tested. In conclusion,
P. gingivalis
has a nonclonal population structure characterized by frequent recombination. Our study suggests that particular genotypes, possibly with increased pathogenic potential, may spread successfully in the human population.
Variation in the Infectivity of
Listeria monocytogenes
Isolates following Intragastric Inoculation of Mice
Authors: Barbour Angela H. , Rampling Anita , Hormaeche Carlos E.
Published: July 2002

The infectivities of 66
Listeria monocytogenes
isolates were assessed by intragastric inoculation of mice. Eight were poorly infective. Serovars 4b and 1/2 were more infective than serovars 3 and 4nonb. A noninfective isolate was cleared more rapidly from the cecum than were infective isolates, suggesting that survival in the gut may relate to infectivity.
Leptospiral Proteins Recognized during the Humoral Immune Response to Leptospirosis in Humans
Authors: Guerreiro Hygia , Croda Júlio , Flannery Brendan , Mazel Mary , Matsunaga James , Reis Mitermayer Galvão , Levett Paul N. , Ko Albert I. , Haake David A.
Published: July 2002

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus
Leptospira
. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.
Construction and Characterization of Genetically Defined
aro omp
Mutants of Enterotoxigenic
Escherichia coli
and Preliminary Studies of Safety and Immunogenicity in Humans
Authors: Turner Arthur K. , Terry Tamsin D. , Sack David A. , Londoño-Arcila Patricia , Darsley Michael J.
Published: July 2002

Enterotoxigenic
Escherichia coli
(ETEC) is a leading cause of diarrhea in travelers to countries where the disease is endemic and causes a major disease burden in the indigenous population, particularly children. We describe here the generation and preclinical characterization of candidate strains of ETEC which are intended to provide the basis of a live attenuated oral vaccine to prevent this disease. It has been shown previously that a spontaneously arising toxin-negative variant ETEC strain, E1392/75-2A, could confer 75% protection against challenge when administered to volunteers. Unfortunately this strain induced mild diarrhea in 15% of recipients. To eliminate the unacceptable reactogenicity of strain E1392/75-2A, it was further attenuated by introducing three different combinations of defined deletion mutations into the chromosome. A mouse intranasal model of immunization was developed and used to show that all of the strains were immunogenic. Immune responses against colonization factor antigens (CFAs) were particularly strong when the bacterial inocula were grown on “CFA agar,” which induces strong expression of these antigens. Two of the strains were selected for a phase I dose escalation safety study with healthy adult volunteers. Freshly grown organisms were harvested from CFA agar plates and administered to volunteers as a suspension containing from 5 × 10
7
to 5 × 10
9
CFU. The vaccine was well tolerated at all doses and induced significant immune responses in all recipients at the highest dose of either strain. The results provide the basis for further clinical evaluation of these vaccine candidates.
Transcutaneous Immunization Using Colonization Factor and Heat-Labile Enterotoxin Induces Correlates of Protective Immunity for EnterotoxigenicEscherichia coli
Authors: Yu Jianmei , Cassels Frederick , Scharton-Kersten Tanya , Hammond Scott A. , Hartman Antoinette , Angov Evelina , Corthésy Blaise , Alving Carl , Glenn Gregory
Published: July 2002

EnterotoxigenicEscherichia coli(ETEC) diarrheal disease is a worldwide problem that may be addressed by transcutaneous delivery of a vaccine. In several human settings, protective immunity has been associated with immune responses toE. colicolonization factors and to the heat-labile toxin that induces the diarrhea. In this set of animal studies, transcutaneous immunization (TCI) using recombinant colonization factor CS6 and cholera toxin (CT) or heat-labile enterotoxin (LT) as the adjuvant induced immunoglobulin G (IgG) and IgA anti-CS6 responses in sera and stools and antibody responses that recognized CS6 antigen in its native configuration. The antitoxin immunity induced by TCI was also shown to protect against enteric toxin challenge. Although immunization with LT via the skin induced mucosal secretory IgA responses to LT, protection could also be achieved by intravenous injection of the immune sera. Finally, a malaria vaccine antigen, merzoite surface protein 142administered with CT as the adjuvant, induced both merzoite surface protein antibodies and T-cell responses while conferring protective antitoxin immunity, suggesting that both antiparasitic activity and antidiarrheal activity can be obtained with a single vaccine formulation. Overall, our results demonstrate that relevant colonization factor and antitoxin immunity can be induced by TCI and suggest that an ETEC traveler's diarrhea vaccine could be delivered by using a patch.
Human Gingival Fibroblasts Rescue Butyric Acid-Induced T-Cell Apoptosis
Authors: Kurita-Ochiai Tomoko , Ochiai Kuniyasu , Suzuki Naoto , Otsuka Kichibee , Fukushima Kazuo
Published: July 2002

We previously demonstrated that butyric acid, an extracellular metabolite from periodontopathic bacteria, induces cytotoxicity and apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we used a cell-to-cell interaction system to examine the contribution of gingival fibroblasts to the regulation of T-cell death induced by butyric acid. Butyric acid slightly suppressed fibroblast viability in a concentration-dependent fashion. However, DNA fragmentation assays indicated that butyric acid did not induce apoptosis for up to 21 h in human gingival fibroblasts (Gin 1, F41-G, and H. pulp cells). The culture supernatants were assayed for interleukin 1α (IL-1α), IL-1β, IL-6, IL-8, IL-11, tumor necrosis factor alpha, and transforming growth factor β, but only the IL-6, IL-8, and IL-11 levels were significantly increased by addition of butyric acid. Butyric acid- or Fas-induced Jurkat-cell apoptosis was attenuated when Jurkat cells were cocultured with either F41-G or Gin 1 cells that had been preincubated for 6 h with butyric acid. IL-8 slightly stimulated butyric acid- or Fas-induced Jurkat-cell apoptosis in a dose-dependent manner, although a low dose of IL-8 had a mildly inhibitory effect on apoptosis. In contrast, IL-6 and IL-11 significantly suppressed butyric acid- or Fas-induced apoptosis in a dose-dependent fashion. Furthermore, the addition of monoclonal antibodies against human IL-6 and IL-11 to cocultures of gingival fibroblasts and Jurkat cells partially eliminated T-cell recovery. These results suggest that the proinflammatory cytokines such as IL-6 and IL-11, produced in fibroblasts stimulated with butyric acid, are involved in the attenuation of T-cell apoptosis by gingival fibroblasts.
Autonomous Expression of the
slo
Gene of the Bicistronic
nga
-
slo
Operon of
Streptococcus pyogenes
Authors: Savic Dragutin J. , McShan William M. , Ferretti Joseph J.
Published: July 2002

A recent model for cytolysin-mediated translocation in
Streptococcus pyogenes
proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (
nga
) and streptolysin O (
slo
) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (
nga
). We expand this model by showing that
slo
possesses its own autonomous promoter, which is located 155 bp upstream of the
slo
gene. Under experimental conditions in which
S. pyogenes
is grown in THY medium, the strength of the
slo
promoter, as measured by the activity of a
lacZ
reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that
sloR
, a
S. pyogenes
gene that closely resembles the
Clostridium perfringens pfoR
gene, exerts a negative effect on the expression of the
slo
gene.
Purification and Characterization of the R64 Shufflon-Specific Recombinase
Authors: Gyohda Atsuko , Komano Teruya
Published: July 2002

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene,
rci
, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.
Altered Stationary-Phase Response in a
Borrelia burgdorferi rpoS
Mutant
Authors: Elias Abdallah F. , Bono James L. , Carroll James A. , Stewart Philip , Tilly Kit , Rosa Patricia
Published: July 2002

The homolog of the chromosomally encoded stationary-phase sigma factor
RpoS
in
Borrelia burgdorferi
was inactivated using

gyrB
r

as a selectable marker. Two-dimensional nonequilibrium pH gradient electrophoresis of stationary-phase cell lysates identified at least 11 differences between the protein profiles of the
rpoS
mutant and wild-type organisms. Wild-type
B. burgdorferi
had a growth phase-dependent resistance to 1 N NaCl, similar to the stationary-phase response reported for other bacteria. The
B. burgdorferi rpoS
mutant strain was less resistant to osmotic stress in stationary phase than the isogenic
rpoS
wild-type organism. The results indicate that the
B. burgdorferi rpoS
homolog influences protein composition and participates in stationary-phase-dependent osmotic resistance. This
rpoS
mutant will be useful for studying regulation of gene expression in response to changing environmental conditions.
Interaction of the FimB Integrase with the
fimS
Invertible DNA Element in
Escherichia coli
In Vivo and In Vitro
Authors: Burns Lesley S. , Smith Stephen G. J. , Dorman Charles J.
Published: July 2002

The FimB protein is a site-specific recombinase that inverts the
fimS
genetic switch in
Escherichia coli
. Based on amino acid sequence analysis alone, FimB has been assigned to the integrase family of tyrosine recombinases. We show that amino acid substitutions at positions R47, H141, R144, and Y176, corresponding to highly conserved members of the catalytic motif of integrase proteins, render FimB incapable of inverting the
fimS
element in vivo. The arginine substitutions reduced the ability of FimB to bind to
fimS
in vivo or in vitro, while the substitution R144Q resulted in a protein unable to bind independently to the half sites located at the left end of
fimS
in phase-on bacteria. These data confirm that FimB is an integrase and suggest that residue R144 has a role in binding to a specific component of the
fim
switch.
Interacting Regulatory Circuits Involved in Orderly Control of Photosynthesis Gene Expression in
Rhodobacter sphaeroides
2.4.1
Authors: Oh Jeong-Il , Eraso Jesus M. , Kaplan Samuel
Published: July 2002

FnrL, the homolog of the global anaerobic regulator Fnr, is required for the induction of the photosynthetic apparatus in
Rhodobacter sphaeroides
2.4.1. Thus, the precise role of FnrL in photosynthesis (PS) gene expression and its interaction(s) with other regulators of PS gene expression are of considerable importance to our understanding of the regulatory circuitry governing spectral complex formation. Using a CcoP and FnrL double mutant strain, we obtained results which suggested that FnrL is not involved in the transduction of the inhibitory signal, by which PS gene expression is “silenced,” emanating from the
cbb
3
oxidase encoded by the
ccoNOQP
operon under aerobic conditions. The dominant effect of the
ccoP
mutation in the FnrL mutant strain with respect to spectral complex formation under aerobic conditions and restoration of a PS-positive phenotype suggested that inactivation of the
cbb
3
oxidase to some extent bypasses the requirement for FnrL in the formation of spectral complexes. Additional analyses revealed that anaerobic induction of the
bchE
,
hemN
, and
hemZ
genes, which are involved in the tetrapyrrole biosynthetic pathways, requires FnrL. Thus, FnrL appears to be involved at multiple loci involved in the regulation of PS gene expression. Additionally,
bchE
was also shown to be regulated by the PrrBA two-component system, in conjunction with
hemN
and
hemZ
. These and other results to be discussed permit us to more accurately describe the role of FnrL as well as the interactions between the FnrL, PrrBA, and other regulatory circuits in the regulation of PS gene expression.
Phosphorelay as the Sole Physiological Route of Signal Transmission by the Arc Two-Component System of
Escherichia coli
Authors: Kwon Ohsuk , Georgellis Dimitris , Lin E. C. C.
Published: July 2002

The Arc two-component system, comprising a tripartite sensor kinase (ArcB) and a response regulator (ArcA), modulates the expression of numerous genes involved in respiratory functions. In this study, the steps of phosphoryl group transfer from phosphorylated ArcB to ArcA were examined in vivo by using single copies of wild-type and mutant
arcB
alleles. The results indicate that the signal transmission occurs solely by His-Asp-His-Asp phosphorelay.
All Major Regions of FtsK Are Required for Resolution of Chromosome Dimers
Authors: Boyle David S. , Grant Dawn , Draper G. Craig , Donachie William D.
Published: July 2002

Resolution of chromosome dimers, by site-specific recombination between
dif
sites, is carried out in
Escherichia coli
by XerCD recombinase in association with the FtsK protein. We show here that a variety of altered FtsK polypeptides, consisting of the N-terminal (cell division) domain alone or with deletions in the proline-glutamine-rich part of the protein, or polypeptides consisting of the C-terminal domain alone are all unable to carry out
dif
recombination. Alteration of the putative nucleotide-binding site also abolishes the ability of FtsK to carry out recombination between
dif
sites.
Cross-Linked Complex between Oligomeric Periplasmic Lipoprotein AcrA and the Inner-Membrane-Associated Multidrug Efflux Pump AcrB from
Escherichia coli
Authors: Zgurskaya Helen I. , Nikaido Hiroshi
Published: July 2002

In
Escherichia coli
, the intrinsic levels of resistance to multiple antimicrobial agents are produced through expression of the three-component multidrug efflux system AcrAB-TolC. AcrB is a proton-motive-force-dependent transporter located in the inner membrane, and AcrA and TolC are accessory proteins located in the periplasm and the outer membrane, respectively. In this study, these three proteins were expressed separately, and the interactions between them were analyzed by chemical cross-linking in intact cells. We show that AcrA protein forms oligomers, most probably trimers. In this oligomeric form, AcrA interacts specifically with AcrB transporter independently of substrate and TolC.