bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Proteins Differentially Expressed in Human Monocytes Exposed toPorphyromonas gingivalisand Its Purified Components by High-Throughput Immunoblotting
Authors: Zhou Qingde , Amar Salomon 
Published: January 2006

ABSTRACTTo characterize the roles ofPorphyromonas gingivalisand its components in disease processes, we investigated the cytokine profiles induced by liveP. gingivalis, its lipopolysaccharide (LPS), and its major fimbrial protein, fimbrillin (FimA). A cytokine antibody array revealed that human monocyte-derived macrophages were induced to produce chemokines (e.g., monocyte chemoattractant protein 1, macrophage inflammatory protein 1β [MIP-1β], and MIP-3α) as early as 1 h after exposure toP. gingivalis, with production declining after 24 h of exposure. As expected, an extensive repertoire of inflammatory mediators increased subsequent to infection, most predominantly tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor. The induction of cytokines byP. gingivaliswas not triggered simply by bacterial cell surface components, since purifiedP. gingivalisLPS and FimA induced similar patterns of cytokines, while the pattern of cytokines induced by liveP. gingivaliswas significantly different, indicating that the host defense system senses live bacteria differently than it does the cell surface components LPS and FimA. To further understand the mechanisms by which liveP. gingivalisand its components exert their effects, we used a high-throughput immunoblot screening approach (Becton-Dickinson PowerBlot) to analyze intracellular proteins involved inP. gingivalisinfection in human macrophages. Exposure of human macrophages to either liveP. gingivalis, its LPS, or its FimA protein led to the up-regulation of 12, 8, and 10 proteins and the down-regulation of 15, 8, and 17 proteins, respectively. The expression of proteins involved in gene transcription (e.g., monocyte enhancer factor 2D [MEF2D], signal transducer and activator of transcription 1 [STAT1], STAT3, STAT6, and IL enhancer binding factors [ILF3]), of protein kinases (e.g., mitogen-activated protein kinase 3 [MAPK3], MAP3K8, double-stranded RNA-activated protein kinase [PRKR], and MAP2K4), and of proteins involved in immune responses (e.g., TNF super family member 6 [TNFSF6] and interferon-induced protein with tetratricopeptide repeat 4 [IFIT4]), apoptosis (e.g., genes associated with retinoid interferon-induced mortality 19 [GRIM19]), and other fundamental cellular processes (e.g., clathrin heavy-chain polypeptide, culreticulin, and Ras-associated protein RAB27A) was found to be modulated differentially byP. gingivalis, LPS, and FimA. These differential changes are interpreted as preferential signal pathway activation in host immune/inflammatory responses toP. gingivalisinfection.
Cytokine Production Associated with Periportal Fibrosis during Chronic Schistosomiasis Mansoni in Humans
Authors: Alves Oliveira L. F. , Moreno E. C. , Gazzinelli G. , Martins-Filho O. A. , Silveira A. M. S. , Gazzinelli A. , Malaquias L. C. C. , LoVerde P. , Leite P. Martins , Correa-Oliveira R. 
Published: January 2006

ABSTRACTVolunteers living in an area where schistosomiasis mansoni is endemic were subjected to ultrasound examination and classified into groups according to the levels of fibrosis diagnosed, namely, absence of indications of fibrosis (group 0), incipient fibrosis (group 1), and moderate/severe fibrosis (group 2). Peripheral blood mononuclear cells (PBMC) collected from the volunteers were stimulated with soluble antigens from adult schistosomes or from schistosome eggs, and the production of the cytokines gamma interferon, tumor necrosis factor alpha, transforming growth factor β (TGF-β), interleukin-4 (IL-4), IL-10, and IL-13 was determined. Potential associations of the level of fibrosis with age, sex, intensity of infection, and cytokine production were investigated between the three groups. Univariate analysis identified associations of age (>50), gender (male), and absence of eggs/g of feces with moderate/severe fibrosis and an association of intensity of infection (>100 eggs) with incipient fibrosis. When cytokine production in PBMC cultures stimulated by soluble egg antigens was categorized as low or high, significant differences in the distribution of IL-13 levels were established between groups 0 and 2. No significant differences were detected between the groups in the cytokines produced by PBMC cultures stimulated with soluble antigens from adult schistosomes. When all variables were tested in multivariate analyses, only IL-13 was strongly associated with fibrosis (odds ratio = 5.8; 95% confidence interval [CI] = 1.1 to 30.5). While high levels of TGF-β appeared to be associated with protection against fibrosis, the strength of the association was low.
Identification of Amino Acid Residues Involved in Heme Binding and Hemoprotein Utilization in the
Porphyromonas gingivalis
Heme Receptor HmuR
Authors: Liu Xinyan , Olczak Teresa , Guo Hwai-Chen , Dixon Dabney W. , Genco Caroline Attardo 
Published: January 2006

ABSTRACT

We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in
Porphyromonas gingivalis.
To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of
P. gingivalis
A7436
hmuR
mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both
P. gingivalis
and
Escherichia coli
. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of
P. gingivalis
when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the
P. gingivalis
HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.
Polynucleotide Phosphorylase Negatively ControlsspvVirulence Gene Expression inSalmonella enterica
Authors: Ygberg Sofia Eriksson , Clements Mark O. , Rytkönen Anne , Thompson Arthur , Holden David W. , Hinton Jay C. D. , Rhen Mikael 
Published: January 2006

ABSTRACTMutational inactivation of the cold-shock-associated exoribonuclease polynucleotide phosphorylase (PNPase; encoded by thepnpgene) inSalmonella entericaserovar Typhimurium was previously shown to enable the bacteria to cause chronic infection and to affect the bacterial replication in BALB/c mice (M. O. Clements et al., Proc. Natl. Acad. Sci. USA99:8784-8789, 2002). Here, we report that PNPase deficiency results in increased expression ofSalmonellaplasmid virulence (spv) genes under in vitro growth conditions that allow induction ofspvexpression. Furthermore, whole-genome microarray-based transcriptome analyses of bacteria growing inside murine macrophage-like J774.A.1 cells revealed six genes as being significantly up-regulated in the PNPase-deficient background, which includedspvABC,rtcB,entC, and STM2236. Mutational inactivation of thespvRregulator diminished the increased expression ofspvobserved in thepnpmutant background, implying that PNPase acts upstream of or at the level of SpvR. Finally, competition experiments revealed that the growth advantage of thepnpmutant in BALB/c mice was dependent onspvRas well. Combined, our results support the idea that inS. entericaPNPase, apart from being a regulator of the cold shock response, also functions in tuning the expression of virulence genes and bacterial fitness during infection.
Anthrax Lethal Toxin Induces Ketotifen-Sensitive Intradermal Vascular Leakage in Certain Inbred Mice
Authors: Gozes Yehoshua , Moayeri Mahtab , Wiggins Jason F. , Leppla Stephen H. 
Published: January 2006

ABSTRACT

Bacillus anthracis
lethal toxin (LT) is a bipartite toxin composed of protective antigen (PA) and lethal factor (LF). Injection of LT produces clinical signs characteristic of anthrax infection, including pleural edema and vascular collapse in various animal models. We utilized the classic Miles leakage assay to quantify vascular leakage in mice. LT injected intradermally induced leakage as early as 15 to 25 min in some inbred mouse strains, but not in others, whereas PA or LF individually did not induce leakage. A third component of anthrax toxin, edema factor, did not induce leakage alone or with PA. Leakage was quantified in eight mouse strains, and no correlation was found between sensitivity to intradermal leakage and sensitivity to the lethality of systemically administered LT. The leakage could be inhibited by ketotifen, an inhibitor of mast cell degranulation, but not by azelastine, a histamine receptor 1 antagonist, or by ketanserin, a serotonin 5-HT2A receptor antagonist. LT was cytotoxic to MC/9 mast cells (in vitro) by 7 h after toxin treatment but did not induce histamine release from these cells. Mast cell-deficient mice exhibited the leakage event and had no increased resistance to systemic LT. Human umbilical vein endothelial cells were resistant to LT over 12 h, with only 20% of cells succumbing by 24 h, suggesting that endothelial cell killing is not the cause of the rapid LT-mediated leakage event. We describe here a ketotifen-sensitive vascular leakage event induced by LT which is the most rapid in vivo or in vitro LT-mediated effect reported to date.
Intranasal Exposure to Bacterial Superantigens Induces Airway Inflammation in HLA Class II Transgenic Mice
Authors: Rajagopalan Govindarajan , Iijima Koji , Singh Manisha , Kita Hirohito , Patel Robin , David Chella S. 
Published: January 2006

ABSTRACTStaphylococcus aureusis widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections.S. aureuscan elaborate a variety of superantigen exotoxins in “carrier” or “pathogenic” states.Streptococcus pyogenescan also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.
Cathepsin B Homologue at the Interface between a Parasitic Nematode and Its Intermediate Host
Authors: Duffy Michael S. , Cevasco Deanne K. , Zarlenga Dante S. , Sukhumavasi Woraporn , Appleton Judith A. 
Published: January 2006

ABSTRACTParelaphostrongylus tenuisis a parasitic nematode that causes a debilitating neurologic disease in many North American cervids and domestic livestock species. We produced a PCR-based cDNA library from infective larvae (L3) in order to identify molecules that mediate parasitism. A dominant 1,250-bp amplicon encoded a homologue of cathepsin B cysteine proteases. The sequence incorporated a C29G substitution in the putative active site. Antibodies generated against a recombinant form detected the native protein (PtCPR-1) in Western blot assays of L3, but not adult worm, extracts. Immunohistochemical methods revealed that PtCPR-1 synthesis was restricted to larval stages within the snail intermediate host (Triodopsissp.), beginning as early as 2 days postinfection (dpi) of snails. The protein was present in the intestine and luminal contents and was lost from larvae over time. Concurrent studies showed that larvae induced an immune response in snails beginning at 1 dpi. Layers of hemocytes encapsulated larvae immediately after infection, and granuloma-like structures formed around parasites in chronic infections. Loss of PtCPR-1 from L3 and its accumulation in host tissues coincided with degeneration of granuloma architecture 90 to 105 dpi. Fully developed L3 emerged from the snail at this time. Our data implicate PtCPR-1 in larval development and possibly in the emergence ofP. tenuisfrom the intermediate host. Emerged L3 survived desiccation and cold stress, suggesting that they could remain infectious in the environment. Molecules promoting emergence would facilitate dispersal of L3 and increase the likelihood of transmission to definitive hosts.
Biogenesis of
Leishmania major
-Harboring Vacuoles in Murine Dendritic Cells
Authors: Körner Ulrich , Fuss Veronika , Steigerwald Jutta , Moll Heidrun 
Published: January 2006

ABSTRACT

In mammalian hosts,
Leishmania
sp. parasites are obligatory intracellular organisms that invade macrophages and dendritic cells (DC), where they reside in endocytic organelles termed parasitophorous vacuoles (PV). Most of the present knowledge of the characteristics of PV harboring
Leishmania
sp. is derived from studies with infected macrophages. Since DC play a key role in host resistance to leishmaniasis, there is a need to understand the properties and biogenesis of PV in
Leishmania
sp.-infected DC. Therefore, we determined the acquisition of endosomal and lysosomal molecules by
Leishmania major
-containing compartments in DC at different maturation stages, using fluorescence labeling and confocal microscopy. The results show that newly formed phagosomes in DC rapidly develop into late endosomal compartments. However, the small GTPase Rab7, which regulates late fusion processes, was found only in PV of mature bone marrow-derived DC (BMDC); it was absent in immature BMDC, suggesting an arrest of their PV biogenesis at the stage of late endosomes. Indeed, fusion assays with endocytic tracers demonstrated that the fusion activity of
L. major
-harboring PV toward lysosomes is higher in mature BMDC than in immature BMDC. The inhibition of PV-lysosome fusion in DC is dependent upon the viability and life cycle stage of the parasite, because live promastigotes blocked the fusion almost completely, whereas killed organisms and amastigotes induced a considerable level of fusion activity. The differences in the fusion competences of immature and mature DC may be relevant for their distinct functional activities in the uptake, transport, and presentation of parasite antigens.
Analysis of Antibodies Directed against Merozoite Surface Protein 1 of the Human Malaria ParasitePlasmodium falciparum
Authors: Woehlbier Ute , Epp Christian , Kauth Christian W. , Lutz Rolf , Long Carole A. , Coulibaly Boubacar , Kouyaté Bocar , Arevalo-Herrera Myriam , Herrera Sócrates , Bujard Hermann 
Published: January 2006

ABSTRACTThe 190-kDa merozoite surface protein 1 (MSP-1) ofPlasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera ofAotusmonkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria.
Intracellular Gene Expression Profile of
Listeria monocytogenes
Authors: Chatterjee Som Subhra , Hossain Hamid , Otten Sonja , Kuenne Carsten , Kuchmina Katja , Machata Silke , Domann Eugen , Chakraborty Trinad , Hain Torsten 
Published: January 2006

ABSTRACT

Listeria monocytogenes
is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality.
L. monocytogenes
is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with
L. monocytogenes
strain EGD-e and examined the gene expression profile of
L. monocytogenes
inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic
Listeria innocua
CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced
L. monocytogenes
F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of
L. monocytogenes
in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by
L. monocytogenes
to escape the host cell responses.
Expression Microarray and Mouse Virulence Analysis of Four Conserved Two-Component Gene Regulatory Systems in Group A
Streptococcus
Authors: Sitkiewicz Izabela , Musser James M. 
Published: January 2006

ABSTRACT

Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the ΔM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including
scpA
, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.
Role of
AFR1
, an ABC Transporter-Encoding Gene, in the In Vivo Response to Fluconazole and Virulence of
Cryptococcus neoformans
Authors: Sanguinetti Maurizio , Posteraro Brunella , La Sorda Marilena , Torelli Riccardo , Fiori Barbara , Santangelo Rosaria , Delogu Giovanni , Fadda Giovanni 
Published: January 2006

ABSTRACT

We have recently demonstrated that upregulation of the ATP binding cassette (ABC) transporter-encoding gene
AFR1
in
Cryptococcus neoformans
is involved in the in vitro resistance to fluconazole of this yeast. In the present study, we investigated the role of
AFR1
in the in vivo response to fluconazole in a mouse model of systemic cryptococcosis. Mice were infected with a wild-type fluconazole-susceptible strain of
C. neoformans
, strain BPY22; an
afr1
mutant, BPY444, which displayed hypersusceptibility to fluconazole in vitro; or an
AFR1-
overexpressing strain, BPY445, which exhibited in vitro resistance to the drug. In each of the three groups, infected animals were randomly assigned to fluconazole treatment or untreated-control subgroups. As expected, fluconazole prolonged survival and reduced fungal tissue burdens (compared with no treatment) in BPY22- and BPY444-infected mice, whereas it had no significant effects in mice infected with BPY445. When the pathogenicities of these strains in mice were investigated, strain BPY445 was significantly more virulent than BPY22 following inhalational or intravenous inoculation, but mice infected with BPY444 survived significantly longer than BPY22-infected animals only when infection was acquired via the respiratory tract. In in vitro macrophage infection studies, strain BPY445 also displayed enhanced intracellular survival compared with strains BPY22 and BPY444, suggesting that its increased virulence may be due to its reduced vulnerability to the antimicrobial factors produced by phagocytic cells. These findings indicate that the upregulation of the
AFR1
gene is an important factor in either determining the in vivo resistance to fluconazole or influencing the virulence of
C. neoformans
.