bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral Immunization with aSalmonella entericaSerovar Typhi Vaccine Induces Specific Circulating Mucosa-Homing CD4+and CD8+T Cells in Humans
Authors: Lundin B. Samuel , Johansson Camilla , Svennerholm Ann-Mari
Published: September 2002

The kinetics and homing characteristics of T-cell responses in humans after mucosal immunizations have not been well characterized. Therefore, we have investigated the magnitude and duration of such responses as well as the homing receptor expression of antigen-specific peripheral blood T cells by using an oral model vaccine, i.e., the live, attenuatedSalmonella entericaserovar Typhi vaccine (Ty21a). Eight volunteers were each given three doses of the vaccine 2 days apart, and blood samples, from which CD4+and CD8+T cells were selected by the use of magnetic beads, were collected before vaccination and at regular intervals thereafter. To purify the potentially antigen-specific gut-homing T cells, CD45RA−integrin β7+cells were further sorted by flow cytometry. The sorted cells were then stimulated in vitro with the serovar Typhi vaccine strain, and the proliferation of cells and the cytokine production were measured. Following vaccination, there was a large increase in both the proliferation of and the gamma interferon (IFN-γ) production by blood T cells stimulated with the vaccine strain. The responses were seen among both CD4+and CD8+T cells, although the CD8+cells produced the largest amounts of IFN-γ. Peak responses were seen 7 to 14 days after the onset of vaccination. Furthermore, most of the IFN-γ produced by both CD4+and CD8+cells emanated from cells with the potential to home to mucosal tissues, as the integrin β7-expressing memory T cells produced around 10-fold more IFN-γ than the remaining populations. In conclusion, we demonstrate that oral vaccination with a live oral bacterial vaccine induces antigen-specific CD4+and CD8+memory T cells, almost all of which express the gut-homing integrin β7.
VirulentMycobacterium fortuitumRestricts NO Production by a Gamma Interferon-Activated J774 Cell Line and Phagosome-Lysosome Fusion
Authors: da Silva Tânia Regina Marques , de Freitas Juliana Ribeiro , Silva Queilan Chagas , Figueira Cláudio Pereira , Roxo Eliana , Leão Sylvia Cardoso , de Freitas Luiz Antônio Rodrigues , Veras Patrícia Sampaio Tavares
Published: September 2002

The virulence of different isolates ofMycobacteriumhas been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mφ) responses to SmOp and SmTrMycobacterium fortuitumvariants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-γ) were infected with SmOp or SmTrM. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-γ-stimulated Mφ. Indeed, the amount of IFN-γ-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 μM) compared to SmTr infection (3.9 to 4.8 μM) (P= 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-γ-induced NO production by Mφ was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-dl-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-γ-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.
N-Acylhomoserine Lactones Undergo Lactonolysis in a pH-, Temperature-, and Acyl Chain Length-Dependent Manner during Growth ofYersinia pseudotuberculosisandPseudomonas aeruginosa
Authors: Yates Edwin A. , Philipp Bodo , Buckley Catherine , Atkinson Steve , Chhabra Siri Ram , Sockett R. Elizabeth , Goldner Morris , Dessaux Yves , Cámara Miguel , Smith Harry , Williams Paul
Published: September 2002

In gram-negative bacterial pathogens, such asPseudomonas aeruginosaandYersinia pseudotuberculosis, cell-to-cell communication via theN-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures ofY. pseudotuberculosisandP. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the majorP. aeruginosa[N-butanoylhomoserine lactone (C4-HSL) andN-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] andY. pseudotuberculosis[N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL),N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as theN-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, andN-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37°C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require anN-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.
Virulence Effect of
Enterococcus faecalis
Protease Genes and the Quorum-Sensing Locus
fsr
in
Caenorhabditis elegans
and Mice
Authors: Sifri Costi D. , Mylonakis Eleftherios , Singh Kavindra V. , Qin Xiang , Garsin Danielle A. , Murray Barbara E. , Ausubel Frederick M. , Calderwood Stephen B.
Published: September 2002

The expression of two
Enterococcus faecalis
extracellular virulence-related proteins, gelatinase (GelE) and serine protease (SprE), has been shown to be positively regulated by the
fsr
quorum-sensing system. We recently developed a novel system for studying
E. faecalis
pathogenicity that involves killing of the nematode worm
Caenorhabditis elegans
and showed that an
E. faecalis fsrB
mutant (strain TX5266) exhibited attenuated killing. We explore here the role of the
fsr
/
gelE
-
sprE
locus in pathogenicity by comparing results obtained in the nematode system with a mouse peritonitis model of
E. faecalis
infection. Insertion mutants of
fsrA
(TX5240) and
fsrC
(TX5242), like
fsrB
(TX5266), were attenuated in their ability to kill
C. elegans
. A deletion mutant of
gelE
(TX5264) and an insertion mutant of
sprE
(TX5243) were also attenuated in
C. elegans
killing, although to a lesser extent than the
fsr
mutants. Complementation of
fsrB
(TX5266) with a 6-kb fragment containing the entire
fsr
locus restored virulence in both the nematode and the mouse peritonitis models. The
fsr
mutants were not impaired in their ability to colonize the nematode intestine. These data show that extracellular proteases and the quorum-sensing
fsr
system are important for
E. faecalis
virulence in two highly divergent hosts: nematodes and mice.
Interleukin-4 Receptor α Chain and STAT6 Signaling Inhibit Gamma Interferon but Not Th2 Cytokine Expression within Schistosome Granulomas
Authors: Metwali Ahmed , Blum Arthur , Elliott David E. , Weinstock Joel V.
Published: September 2002

Compared to wild-type (WT) mice, schistosome granulomas in Stat6 knockout (KO) mice lacked eosinophils and had Th1 features. Interleukin-4 (IL-4) acts through Stat6 in assisting Th2 cell development. The importance of Stat6 for Th2-cell development within schistosome granulomas had not been explored. Therefore we studied gamma interferon (IFN-γ), IL-4, and IL-5 production in granulomas from Stat6 KO and WT mice. Dispersed granuloma cells from Stat6 KO and WT mice made similar amounts of IL-4 and IL-5. Only Stat6 KO granuloma cells released IFN-γ. Granuloma T cells contained most of the IL-4, IL-5, and IFN-γ mRNA and secreted these cytokines. In Stat6 KO mice, 16.6% of the granuloma cells were CD4+. Of these, 10.7% stained for IFN-γ and/or IL-4 by intracytoplasmic flow analysis. Few CD4−T cells stained positively. The IL-4-producing T cells did not stain for DX5 or with labeled α-GalCer CD1d tetramer, suggesting an absence of NK T cells. Thus, conventional Th cells in Stat6 KO granulomas produce IFN-γ and Th2 cytokines. Stat6 limits IFN-γ production but is unnecessary for Th2-cell development or localization within the granuloma.
Legionella pneumophila feoABPromotes Ferrous Iron Uptake and Intracellular Infection
Authors: Robey Marianne , Cianciotto Nicholas P.
Published: September 2002

In order to determine the role of ferrous iron transport inLegionellapathogenesis, we identified and mutated thefeoBgene in virulentLegionella pneumophilastrain 130b. As it is inEscherichia coli, theL. pneumophila feoBgene was contained within a putativefeoABoperon.L. pneumophila feoBinsertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss ofL. pneumophilaFeoB. However, theL. pneumophila feoBmutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition offeoBintrans. In unsupplemented buffered yeast extract broth, thefeoBmutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2′-dipyridyl. ThefeoBmutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of thefeoBmutant was noted within iron-depletedHartmannella vermiformisamoebae and human U937 cell macrophages. The reduced intracellular infectivity of thefeoBmutant was complemented by the introduction of a plasmid containingfeoAB. TheL. pneumophila feoBgene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate thatL. pneumophilaFeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.
Genetic Analysis of a
Cryptosporidium parvum
Human Genotype 1 Isolate Passaged through Different Host Species
Authors: Akiyoshi D. E. , Feng X. , Buckholt M. A. , Widmer G. , Tzipori S.
Published: September 2002

Cryptosporidium parvum
TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the
Cryptosporidium
oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and β-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of
C. parvum
genotype 1 and is currently used in several research projects including human volunteer studies.
Secreted Metalloprotease Gene Family of
Microsporum canis
Authors: Brouta Frédéric , Descamps Frédéric , Monod Michel , Vermout Sandy , Losson Bertrand , Mignon Bernard
Published: September 2002

Keratinolytic proteases secreted by dermatophytes are likely to be virulence-related factors.
Microsporum canis
, the main agent of dermatophytosis in dogs and cats, causes a zoonosis that is frequently reported. Using
Aspergillus fumigatus
metalloprotease genomic sequence (
MEP
) as a probe, three genes (
MEP1
,
MEP2
, and
MEP3
) were isolated from an
M. canis
genomic library. They presented a quite-high percentage of identity with both
A. fumigatus MEP
and
Aspergillus oryzae
neutral protease I genes. At the amino acid level, they all contained an HEXXH consensus sequence, confirming that these
M. canis
genes (
MEP
genes) encode a zinc-containing metalloprotease gene family. Furthermore,
MEP3
was found to be the gene encoding a previously isolated
M. canis
43.5-kDa keratinolytic metalloprotease, and was successfully expressed as an active recombinant enzyme in
Pichia pastoris
. Reverse transcriptase nested PCR performed on total RNA extracted from the hair of
M. canis
-infected guinea pigs showed that at least MEP2 and MEP3 are produced during the infection process. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.
Relationship of the Glyoxylate Pathway to the Pathogenesis of
Cryptococcus neoformans
Authors: Rude Thomas H. , Toffaletti Dena L. , Cox Gary M. , Perfect John R.
Published: September 2002

Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of
Cryptococcus neoformans
cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed
icl1
mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a
C. neoformans
infection. Compounds that specifically target only ICL1 are unlikely to cripple
C. neoformans
growth in vivo.
Modulation of an Interleukin-12 and Gamma Interferon Synergistic Feedback Regulatory Cycle of T-Cell and Monocyte Cocultures byPorphyromonas gingivalisLipopolysaccharide in the Absence or Presence of Cysteine Proteinases
Authors: Yun Peter L. W. , DeCarlo Arthur A. , Collyer Charles , Hunter Neil
Published: September 2002

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-γ) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-γ have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogenPorphyromonas gingivalisstimulated monocytes primed with IFN-γ to release IL-12, thereby enhancing IFN-γ accumulation in T-cell populations. P. gingivalisLPS was shown to enhance IL-12 induction of IFN-γ in T cells in a manner independent from TNF-α contribution. The levels of T-cell IL-12 receptors were not affected byP. gingivalisLPS and played only a minor role in the magnitude of the IFN-γ response. These data suggest that LPS fromP. gingivalisestablishes an activation loop with IL-12 and IFN-γ with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-γ accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-γ levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) ofP. gingivaliscould lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.
The Divergently Transcribed
Streptococcus parasanguis
Virulence-Associated
fimA
Operon Encoding an Mn
2+
-Responsive Metal Transporter and
pepO
Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated
Authors: Oetjen Joyce , Fives-Taylor Paula , Froeliger Eunice H.
Published: September 2002

The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The
Streptococcus parasanguis fimA
operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the
Streptococcus gordonii
ScaR binding domain. Mapping of the promoter and regulatory regions of
fimA
and the divergently transcribed
pepO
gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap.
fimA
had one transcriptional start site, whereas
pepO
had three. Analysis of truncated versions of the
pepO
promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the
fimA
operon promoter and the
pepO
promoter are not coordinately regulated. The
fimA
operon is responsive to changes in Mn
2+
concentration, but the
pepO
promoter is not. A
S. parasanguis fimA
mutant showed a growth deficiency under conditions of limiting Mn
2+
. This deficiency was not alleviated by compensation with either Mg
2+
or Fe
3+
. Wild-type
S. parasanguis
could take up Mn
2+
and Fe
3+
, while the
fimA
mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn
2+
and Fe
3+
. FimA expression itself was shown to be responsive to Mn
2+
concentration, but not to availability of Fe
3+
or Mg
2+
.
Combined Treatment with Interleukin-12 and Indomethacin Promotes Increased Resistance in BALB/c Mice with EstablishedLeishmania majorInfections
Authors: Li Jian , Padigel Udaikumar M. , Scott Phillip , Farrell Jay P.
Published: September 2002

Following infection of susceptible BALB/c mice withLeishmania major, early production of interleukin-4 (IL-4) is associated with the development of a nonprotective Th2 response and the development of progressive disease. Treatment of mice with IL-12 at the time of infection can promote the activation of a protective Th1 response; however, IL-12 treatment of mice with established infections has little effect on the progress of lesion development. This may be due to a down-regulation of the IL-12 receptor β2 chain (IL-12Rβ2) that accompanies the expansion of IL-4-producing Th2 cells. We have examined whether prostaglandins function to regulate in vivo responsiveness to IL-12. Mice treated with indomethacin are responsive to treatment with exogenous IL-12 through at least the first 2 weeks of infection and, unlike control mice treated with IL-12, develop an enhanced Th1-type response associated with increased enhanced resistance to infection. Cells from indomethacin-treated mice also exhibit enhanced production of gamma interferon (IFN-γ) following in vitro stimulation with IL-12. Although in vivo indomethacin treatment did not appear to influence IL-12 production in infected mice, cells from indomethacin-treated mice did express higher levels of IL-12Rβ2, suggesting that prostaglandins may play a role in the loss of IL-12 responsiveness observed during nonhealingL. majorinfections.
Characterization of a Murine Model ofUreaplasma urealyticumPneumonia
Authors: Viscardi Rose M. , Kaplan Jennifer , Lovchik Judith C. , He Ju Ren , Hester Lisa , Rao Srinivas , Hasday Jeffrey D.
Published: September 2002

Ureaplasma urealyticumrespiratory tract colonization in preterm infants has been associated with a high incidence of pneumonia and the development of bronchopulmonary dysplasia. However, study of this human pathogen has been hampered by the absence of animal models. We have developed the first juvenile mouse model ofUreaplasmapneumonia and characterized the histopathology during the month following inoculation. C3H/HeN mice were inoculated intratracheally with a mouse-adapted clinicalUreaplasmaisolate (biovar 2) or sham inoculated with 10B broth. Culture of lung homogenates and PCR of DNA from bronchoalveolar lavage fluid (BAL) confirmed the presence ofUreaplasmain 100% of inoculated animals at 1 day, 60% at 2 days, 50% at 3 days, and 25% at 7 and 14 days.Ureaplasmawas undetectable 28 days postinoculation. There were marked changes in BAL and interstitial-cell composition with increased number of polymorphonuclear leukocytes 1 to 2 days and 14 days postinoculation and macrophages at 2 and 14 days postinoculation. TheUreaplasmainfection caused a persistent focal loss of airway ciliated epithelium and a mild increase in interstitial cellularity. There were no differences in BAL protein concentration during the first 28 days, suggesting that pulmonary vascular endothelial barrier integrity remained intact. Comparison of BAL cytokine and chemokine concentrations revealed low levels of tumor necrosis factor alpha (TNF-α) at 3 days and monocyte chemoattractant protein 1 at 7 days inUreaplasma-infected mice but a trend toward increased TNF-α at 14 days and increased granulocyte-macrophage colony-stimulating factor and interleukin-10 at 28 days. These data suggest thatUreaplasmaalone may cause limited inflammation and minimal tissue injury in the early phase of infection but may promote a mild chronic inflammatory response in the later phase of infection (days 14 to 28), similar to the process that occurs in human newborns.
Identification of a Streptolysin S-Associated Gene Cluster and Its Role in the Pathogenesis ofStreptococcus iniaeDisease
Authors: Fuller Jeffrey D. , Camus Alvin C. , Duncan Carla L. , Nizet Victor , Bast Darrin J. , Thune Ronald L. , Low Donald E. , de Azavedo Joyce C. S.
Published: September 2002

Streptococcus iniaecauses meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported thatS. iniaeis responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001).S. iniaeproduces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible forS. iniaecytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associatedS. iniaestrains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS)sagoperon for streptolysin S (SLS) biosynthesis. Confirmation that theS. iniaecytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negativesagBmutants were compared to their wild-typeS. iniaeparent strains in the murine model and in human whole-blood killing assays. These studies demonstrated thatS. iniaeSLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.
Nuclear Targeting ofPorphyromonas gingivalisW50 Protease in Epithelial Cells
Authors: Scragg Margaret A. , Alsam Asil , Rangarajan Minnie , Slaney Jennifer M. , Shepherd Philip , Williams David M. , Curtis Michael A.
Published: September 2002

Porphyromonas gingivalisis an important pathogen associated with destructive periodontal disease and is able to invade the epithelial cell barrier. Its cysteine proteases are recognized as major virulence factors, and in this study, we examined the interaction of the arginine-specific protease with epithelial cells in culture. Three cell lines (KB, HeLa, and SCC4) were incubated with strain W50 culture supernatant; stained with monoclonal antibody 1A1, which recognizes an epitope on the adhesin (β) component of the cysteine protease-adhesin (α/β) heterodimer; and viewed using immunofluorescence microscopy. Within 1 h, the protease traversed the plasma membrane and was localized around the nucleus before becoming concentrated in the cytoplasm after 24 to 48 h. In contrast, the purified arginine-specific heterodimeric protease (HRgpA) rapidly entered the nucleus within 15 to 30 min. This nuclear targeting (i) was seen with active andNα-p-tosyl-l-lysine chloromethyl ketone (TLCK)-inactivated HRgpA, indicating it was independent of the proteolytic activity; (ii) occurred at both 4 and 37°C; and (iii) failed to occur with the monomeric protease (RgpAcat), indicating the importance of the adhesin chain of the HRgpA protease to this process. Rapid cell entry was also observed with recombinant catalytic (α) and adhesin (β) chains, with the latter again targeting the nuclear area. After 48 h of incubation with HRgpA, significant dose-dependent stimulation of metabolic activity was observed (measured by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and a doubling of mitotic activity combined with the presence of apoptotic cells indicated that HRgpA may interfere with cell cycle control mechanisms. These effects were seen with both active and TLCK-inactivated protease, confirming that they were not dependent on proteolytic activity, and thus provide new insights into the functioning of thisP. gingivalisprotease.
Independent Translocation of Two Micronemal Proteins in Developing
Plasmodium falciparum
Merozoites
Authors: Healer Julie , Crawford Simon , Ralph Stuart , McFadden Geoff , Cowman Alan F.
Published: September 2002

Apical membrane antigen 1 of
Plasmodium falciparum
(PfAMA1) contains an N-terminal propeptide that is removed prior to the translocation of the mature protein onto the merozoite surface. We localized unprocessed PfAMA1 to the microneme organelles of the intraerythrocytic schizont. The results have suggested that the processed form of PfAMA1 translocates from the microneme compartment independently of another microneme protein, EBA175, which is also involved in the invasion of human erythrocytes.
Characterization of a Pore-Forming Cytotoxin Expressed by
Salmonella enterica
Serovars Typhi and Paratyphi A
Authors: Oscarsson Jan , Westermark Marie , Löfdahl Sven , Olsen Björn , Palmgren Helena , Mizunoe Yoshimitsu , Wai Sun Nyunt , Uhlin Bernt Eric
Published: September 2002

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the
clyA
gene that has been characterized so far only in
Escherichia coli
. Using DNA sequence analysis and PCR, we established that
clyA
is conserved in the human-specific typhoid
Salmonella enterica
serovars Typhi and Paratyphi A and that the entire
clyA
gene locus is absent in many other
S. enterica
serovars, including Typhimurium. The gene products, designated ClyA
STy
and ClyA
SPa
, show ≥90% amino acid identity to
E. coli
cytolysin A, ClyA
EC
, and they are immunogenically related. The
Salmonella
proteins showed a pore-forming activity and are hence functional homologues to ClyA
EC
. The chromosomal
clyA
STy
gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of
clyA
STy
reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the
clyA
STy
chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned
clyA
STy
gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic
Salmonella
strain in which the genetic determinant has been identified.
Binding of
Clostridium difficile
Surface Layer Proteins to Gastrointestinal Tissues
Authors: Calabi Emanuela , Calabi Franco , Phillips Alan D. , Fairweather Neil F.
Published: September 2002

Clostridium difficile
is the etiological agent of antibiotic-associated diarrhea, a potentially serious condition frequently affecting elderly hospitalized patients. While tissue damage is primarily induced by two toxins, the mechanism of gut colonization, and particularly the role of bacterial adherence to the mucosa, remains to be clarified. Previous studies have shown binding of
C. difficile
whole cells to cultured cell lines and suggested the existence of multiple adhesins, only one of which has been molecularly characterized. In this paper, we have investigated tissue binding of
C. difficile
surface layer proteins (SLPs), which are the predominant outer surface components and are encoded by the
slpA
gene. The adherence of
C. difficile
to HEp-2 cells was studied by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis, which showed that antibodies to the high-molecular-weight (MW) SLP inhibited adherence. Immunohistochemical analysis of human gastrointestinal tissue sections revealed strong binding both to the surface epithelium lining the digestive cavities and to the subjacent lamina propria, while glands were negative. A similar pattern was observed in the mouse. By using purified recombinant SLPs, we show that binding is largely mediated by the high-MW SLP. By Western blotting analysis, we have identified two potential ligands of the
C. difficile
SLPs, one of which may be specific to the gut. By using purified extracellular matrix components immobilized on nitrocellulose, we also show SLP binding to collagen I, thrombospondin, and vitronectin, but not to collagen IV, fibronectin, or laminin. These results raise the possibility that the SLPs play a role both in the initial colonization of the gut by
C. difficile
and in the subsequent inflammatory reaction.
Variation in the Expression of a
Plasmodium falciparum
Protein Family Implicated in Erythrocyte Invasion
Authors: Taylor Helen M. , Grainger Munira , Holder Anthony A.
Published: September 2002

The PfRH protein family of
Plasmodium falciparum
is implicated in erythrocyte invasion. Here we report variations in the sequence, transcription, and protein expression of four different members of this family in three parasite lines, 3D7, T996, and FCB1. There are sequence polymorphisms in
PfRH1
,
PfRH2a
,
PfRH2b
, and
PfRH3
, ranging from variations across repeat regions to a 585-bp deletion in the 3′ end of
PfRH2b
in T996. Not all the genes are transcribed: although all members of the family are transcribed in 3D7 and T996,
PfRH2a
and
PfRH2b
are not transcribed in FCB1. The PfRH1, PfRH2a, and PfRH2b proteins are expressed in late schizonts and merozoites and are located in apical organelles and on the apical surface. However, the PfRH1 protein does not appear to be correctly targeted to the apex in 3D7 and T996. In contrast, the PfRH1 protein is present at the apical end of FCB1 merozoites, but the PfRH2a and PfRH2b proteins are undetectable. The apparent redundancy in the PfRH family of proteins at the level of gene number and sequence and the variations in transcription and protein expression may allow the parasite to use alternative invasion pathways.
Candida-Specific Antibodies during Experimental Vaginal Candidiasis in Mice
Authors: Wozniak Karen L. , Wormley Floyd L. , Fidel Paul L.
Published: September 2002

Protective host defense mechanisms against vaginalCandida albicansinfections are poorly understood. Although cell-mediated immunity (CMI) is the predominant host defense mechanism against most mucosalCandidainfections, the role of CMI against vaginal candidiasis is uncertain, both in humans and in an experimental mouse model. The role of humoral immunity is equally unclear. While clinical observations suggest a minimal role for antibodies against vaginal candidiasis, an experimental rat model has provided evidence for a protective role forCandida-specific immunoglobulin A (IgA) antibodies. Additionally,Candidavaccination-induced IgM and IgG3 antibodies are protective in a mouse model of vaginitis. In the present study, the role of infection-induced humoral immunity in protection against experimental vaginal candidiasis was evaluated through the quantification ofCandida-specific IgA, IgG, and IgM antibodies in serum and vaginal lavage fluids of mice with primary and secondary (partially protected) infection. In naïve mice, total, but notCandida-specific, antibodies were detected in serum and lavage fluids, consistent with lack of yeast colonization in mice. In infected mice,Candida-specific IgA and IgG antibodies were induced in serum with anamnestic responses to secondary infection. In lavage fluid, whileCandida-specific antibodies were detectable, concentrations were extremely low with no anamnestic responses in mice with secondary infection. The incorporation of alternative protocols—including infections in a different strain of mice, prolongation of primary infection prior to secondary challenge, use of different enzyme-linked immunosorbent assay capture antigens, and concentration of lavage fluid—did not enhance localCandida-specific antibody production or detection. Additionally, antibodies were not removed from lavage fluids by being bound toCandidaduring infection. Together, these data suggest that antibodies are not readily present in vaginal secretions of infected mice and thus have a limited natural protective role against infection.
TheMycobacterium tuberculosisPhagosome in Human Macrophages Is Isolated from the Host Cell Cytoplasm
Authors: Clemens Daniel L. , Lee Bai-Yu , Horwitz Marcus A.
Published: September 2002

Knowledge of whetherMycobacterium tuberculosisresides within a relatively impermeable membrane-bound vacuole or is free within the cytoplasm within its host cell is central to an understanding of the immunobiology of this intracellular parasite but is a matter of controversy. To explore this issue, we assessed the accessibility of medium-size protein molecules (Fab fragments of 50,000 Da) toM. tuberculosiswithin human macrophages. We infected the macrophages with wild-type or green fluorescent protein-expressingM. tuberculosis, microinjected Fab fragments directed against a major surface antigen ofM. tuberculosisinto the host cell, and assayed the accessibility of the bacteria to the Fab fragments by both immunofluorescence microscopy and immunogold electron microscopy. Whereas microinjected intact immunoglobulin G molecules against cytoplasmic early endosomal antigen 1 readily stained this antigen, microinjected Fab fragments againstM. tuberculosisdid not stain the bacterium within its phagosome. In contrast, microinjected Fab fragments againstListeria monocytogenes, an intracellular bacterium known to permeabilize its phagosomal membrane, strongly stained this bacterium. Our study shows thatM. tuberculosisresides in an isolated phagosome that is relatively impermeable to cytoplasmic constituents.
Immortalization of Human Urethral Epithelial Cells: a Model for the Study of the Pathogenesis of and the Inflammatory Cytokine Response to
Neisseria gonorrhoeae
Infection
Authors: Harvey Hillery A. , Post Deborah M. B. , Apicella Michael A.
Published: September 2002

The primary human urethral epithelial cells developed by our laboratory have been immortalized by transduction with a retroviral vector expressing the human papillomavirus E6E7 oncogenes. Analysis of telomerase expression and comparison to that in primary cells revealed detectable levels in the transduced human urethral epithelial cells. Immortalized urethral cells could be passaged over 20 times. Immunofluorescence microscopy studies showed that the immortalized cells were phenotypically similar and responded to gonococcal infection similarly to primary cells. Specifically, positive cytokeratin staining showed that the immortalized cells are keratinocytes; cell surface levels of human asialoglycoprotein receptor increase following gonococcal infection, and, like the primary cells, the immortalized urethral epithelial cells are CD14 negative. Using enzyme-linked immunosorbent assay, we found that interleukin-6 (IL-6) and IL-8 levels in primary urethral epithelial cell supernatants increase after challenge with
N. gonorrhoeae
. Likewise, the immortalized urethral epithelial cells produced higher levels of IL-6 and IL-8 cytokines in response to gonococcal infection. Cells challenged with a gonococcal lipid A
msbB
mutant produced reduced IL-6 and IL-8 levels when compared to the parent strain. Additionally, these data suggest that the 1291
msbB
lipooligosaccharide may suppress cytokine induction.
Coxiella burnetii
Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics
Authors: Berón Walter , Gutierrez Maximiliano G. , Rabinovitch Michel , Colombo Maria I.
Published: September 2002

The obligate intracellular bacterium
Coxiella burnetii
, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with
C. burnetii
phase II; 48 h later, bacteria
-
containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked
Coxiella
vacuole formation. These autophagosomal features suggest that
Coxiella
vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of
C. burnetii
phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy.
Coxiella
-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.
Cellular Localization of
Babesia bovis
Merozoite Rhoptry-Associated Protein 1 and Its Erythrocyte-Binding Activity
Authors: Yokoyama Naoaki , Suthisak Boonchit , Hirata Haruyuki , Matsuo Tomohide , Inoue Noboru , Sugimoto Chihiro , Igarashi Ikuo
Published: September 2002

The cellular localization of
Babesia bovis
rhoptry-associated protein 1 (RAP-1) and its erythrocyte-binding affinity were examined with anti-RAP-1 antibodies. In an indirect immunofluorescent antibody test, RAP-1 was detectable in all developmental stages of merozoites and in extracellular merozoites. In the early stage of merozoite development, RAP-1 appears as a dense accumulation, which later thins out and blankets the host cell cytoplasm, but retains a denser mass around newly formed parasite nuclei. The preferential accumulations of RAP-1 on the inner surface of a host cell membrane and bordering the parasite's outer surface were demonstrable by immunoelectron microscopy. An erythrocyte-binding assay with the lysate of merozoites demonstrated RAP-1 binding to both bovine and equine erythrocytes. Anti-RAP-1 monoclonal antibody 1C1 prevented the interaction of RAP-1 with bovine erythrocytes and significantly inhibited parasite proliferation in vitro. With the recombinant RAP-1, the addition of increasing concentrations of Ca
2+
accentuated its binding affinity with bovine erythrocytes. The present findings lend support to an earlier proposition of an erythrocytic binding role for RAP-1 expressed in
B. bovis
merozoites and, possibly, its involvement in the escape of newly formed merozoites from host cells.
Isolation of
Chlamydia pneumoniae
Clonal Variants by a Focus-Forming Assay
Authors: Gieffers Jens , Belland Robert J. , Whitmire William , Ouellette Scot , Crane Deborah , Maass Matthias , Byrne Gerald I. , Caldwell Harlan D.
Published: September 2002

Chlamydia pneumoniae
is an obligate intracellular prokaryotic human pathogen that causes community-acquired respiratory infection and has been associated with atherosclerosis and cardiovascular disease. Unexpected results from genomic sequencing indicate that significant intrastrain polymorphism exists for some
C. pneumoniae
isolates. These polymorphisms could reflect genotypes with differing disease-causing characteristics. A definitive means to test this hypothesis is to obtain genetically homogeneous clonal populations of the pathogen and test them in models of infection and disease. To date, methods for cloning
C. pneumoniae
have not been reported. In this study, we describe the isolation of clonal variants with genetic differences in the
tyrP
locus from a polymorphic respiratory isolate, using a novel focus-forming assay. These results now allow investigations on the biology and pathogenesis of
C. pneumoniae
clonal genovars that could lead to new insights into the pathogenesis of this important human pathogen.
Identification of theStaphylococcus aureus etdPathogenicity Island Which Encodes a Novel Exfoliative Toxin, ETD, and EDIN-B
Authors: Yamaguchi Takayuki , Nishifuji Koji , Sasaki Megumi , Fudaba Yasuyuki , Aepfelbacher Martin , Takata Takashi , Ohara Masaru , Komatsuzawa Hitoshi , Amagai Masayuki , Sugai Motoyuki
Published: September 2002

We identified a novel pathogenicity island inStaphylococcus aureuswhich contains open reading frames (ORFs) similar to the exfoliative toxin (ET) gene, glutamyl endopeptidase gene, andedin-Bgene in tandem and the phage resistance gene, flanked byhsdM,hsdS(restriction and modification system), and IS256. The protein encoded by the ET-like gene showed 40, 59, and 68% amino acid sequence identities with exfoliative toxin A (ETA), exfoliative toxin B (ETB), andStaphylococcus hyicusETB (ShETB), respectively. When injected into neonatal mice, the recombinant protein derived from the ET-like gene induced exfoliation of the skin with loss of cell-to-cell adhesion in the upper part of the epidermis as observed in histological examinations, just as was found in neonatal mice injected with ETA or ETB. Western blot analysis indicated that the recombinant protein is serologically distinct from ETA and ETB. Therefore, the product encoded by this new ORF is a new ET member produced byS. aureusand is termed ETD. ETD did not induce blisters in 1-day-old chickens. In the skins of mice injected with ETD, cell surface staining of desmoglein 1 (Dsg1), a cadherin type cell-to-cell adhesion molecule in desmosomes, was abolished without affecting that of desmoglein 3 (Dsg3). Furthermore, in vitro incubation of the recombinant extracellular domains of Dsg1 and Dsg3 with the recombinant protein demonstrated that both mouse and human Dsg1, but not Dsg3, were directly cleaved in a dose-dependent manner. These results demonstrate that ETD and ETA induce blister formation by identical pathophysiological mechanisms. Clinical strains positive foredin-Bwere suggested to be clonally associated, and alledin-B-positive strains tested were positive foretd. Among 18etd-positive strains, 12 produced ETD extracellularly. Interestingly, these strains are mainly isolated from other sources of infections and not from patients with bullous impetigo or staphylococcal scalded-skin syndrome. This strongly suggests that ETD might play a pathogenic role in a broader spectrum of bacterial infections than previously considered.
Discrete Proteolysis of Focal Contact and Adherens Junction Components in
Porphyromonas gingivalis-
Infected Oral Keratinocytes: a Strategy for Cell Adhesion and Migration Disabling
Authors: Hintermann Edith , Haake Susan Kinder , Christen Urs , Sharabi Andrew , Quaranta Vito
Published: September 2002

Adhesive interactions of cells are critical to tissue integrity. We show that infection with
Porphyromonas gingivalis
, a major pathogen in the periodontal disease periodontitis, interferes with both cell-matrix and cell-cell adhesion in the oral keratinocyte cell line HOK-16. Thus, infected cells showed reduced adhesion to extracellular matrix, changes in morphology from spread to rounded, and impaired motility on purified matrices in Transwell migration assays and scratch assays. Western blot analysis of
P. gingivalis
-challenged HOK-16 cells revealed proteolysis of focal contact components (e.g., focal adhesion kinase), adherens junction proteins (e.g., catenins), and adhesion signaling molecules (e.g., the tyrosine kinase SRC). Proteolysis was selective, since important components of adherens junctions (E-cadherin) or signaling molecules (extracellular signal-regulated kinases ERK1/2) were not degraded. The virulence factors gingipains, cysteine proteinases expressed by
P. gingivalis
, are likely responsible for this proteolytic attack, since they directly digested specific proteins in pull-down experiments, and their proteolytic activity was blocked by the cysteine proteinase inhibitor
N-
α-
p
-tosyl-
l
-lysine chloromethyl ketone and also by a caspase inhibitor. Proteolysis was strain dependent, such that ATCC 33277 and 381 had high proteolytic potential, whereas W50 showed almost no proteolytic activity. These findings may help explain the formation of gingival pockets between cementum and periodontal epithelium, a hallmark of periodontitis. Furthermore, they illustrate a new pathogenetic paradigm of infection whereby bacteria may disrupt the integrity of epithelia.
Requirement for Tumor Necrosis Factor Receptor 2 Expression on Vascular Cells To Induce Experimental Cerebral Malaria
Authors: Stoelcker Benjamin , Hehlgans Thomas , Weigl Karin , Bluethmann Horst , Grau Georges E. , Männel Daniela N.
Published: September 2002

Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.