bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTFollowing 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

ABSTRACTUreaplasma 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

ABSTRACTStreptococcus 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

ABSTRACTPorphyromonas 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTProtective 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

ABSTRACTKnowledge 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

ABSTRACT

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

ABSTRACT

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.