bluuebunny/crossref_metadata_2025 huggingface dataset:
Pathological and Therapeutic Significance of Cellular Invasion byProteus mirabilisin an Enterocystoplasty Infection Stone Model
Authors: Mathoera Rejiv B. , Kok Dik J. , Verduin Cees M. , Nijman Rien J. M.
Published: November 2002

Proteus mirabilisinfection often leads to stone formation. We evaluated how bacterium-mucin adhesion, invasion, and intracellular crystal formation are related to antibiotic sensitivity and may cause frequent stone formation in enterocystoplasties. Five intestinal (Caco-2, HT29, HT29-18N2, HT29-FU, and HT29-MTX) and one ureter cell line (SV-HUC-1) were incubated in artificial urine with fiveProteus mirabilisstrains. Fluorescence-activated cell sorting (FACS), laser scanning microscopy, and electron microscopy evaluated cellular adhesion and/or invasion, pathologic changes to mitochondria, andP. mirabilis-mucin colocalization (MUC2 and MUC5AC). An MTT (thiazolyl blue tetrazolium bromide) assay and FACS analysis of caspase-3 evaluated the cellular response. Infected cells were incubated with antibiotics at dosages representing the expected urinary concentrations in a 10-year-old, 30-kg child to evaluate bacterial invasion and survival. All cell lines showed colocalization ofP. mirabiliswith human colonic mucin (i.e., MUC2) and human gastric mucin (i.e., MUC5AC). The correlation between membrane mucin expression and invasion was significant and opposite for SV-HUC-1 and HT29-MTX. Microscopically, invasion byP. mirabiliswith intracellular crystal formation and mitochondrial damage was found. Double membranes surrounded bacteria in intestinal cells. Relative resistance to cotrimoxazole and augmentin was found in the presence of epithelial cells. Ciprofloxacin and gentamicin remained effective. Membrane mucin expression was correlated with relative antibiotic resistance. Cell invasion byP. mirabilisand mucin- and cell type-related distribution and response differences indicate bacterial tropism that affects crystal formation and mucosal presence. Bacterial invasion seems to have cell type-dependent mechanisms and prolong bacterial survival in antibiotic therapy, giving a new target for therapeutic optimalization of antibiotic treatment.
Environmental Regulation and Differential Production of Members of the Bdr Protein Family of
Borrelia burgdorferi
Authors: Roberts David M. , Caimano Melissa , McDowell John , Theisen Michael , Holm Arne , Orff Edward , Nelson David , Wikel Stephen , Radolf Justin , Marconi Richard T.
Published: November 2002

Borrelia burgdorferi
B31MI carries 18 plasmid-carried genes that form the
bdr
gene family. The
bdr
genes of
B. burgdorferi
encode proteins that form three distinct subfamilies, the BdrD, BdrE, and BdrF subfamilies.
bdr
orthologs have been demonstrated to be carried by all
Borrelia
species analyzed, and their widespread distribution suggests that they play an important genus-wide functional role. The biological rationale for maintaining 18
bdr
alleles has not been defined. It is our hypothesis that specific paralogs function in different environments and are differentially expressed in response to environmental conditions. As a first step in testing this hypothesis, the production patterns of the Bdr proteins in spirochetes grown under a variety of conditions were assessed through immunoblot analyses. The influence of temperature, serum deprivation, tick feeding, and the mammalian environment on Bdr production was evaluated. These analyses revealed that the synthesis of some Bdr paralogs is environmentally regulated. The production of BdrF
2,
BdrF
1
, BdrE
4
, and BdrE
5
were upregulated in host-adapted bacteria, while the production levels of other Bdr paralogs were influenced by temperature and serum starvation. These observations suggest that different Bdr paralogs function in different biological environments and provide insight into the biological basis for maintaining multiple members of this gene family.
Effect of Trimethoprim-Sulfamethoxazole on Recurrent Bacteriuria and Bacterial Persistence in Mice Infected with Uropathogenic
Escherichia coli
Authors: Schilling Joel D. , Lorenz Robin G. , Hultgren Scott J.
Published: November 2002

One of the more perplexing aspects of urinary tract infections (UTIs) is their high propensity to recur. It has been proposed that recurrent infections are a result of the reintroduction of bacteria from the gastrointestinal tract (GIT) to the urinary tract (UT); however, since a significant subset of recurrent UTIs are caused by an identical bacterial strain, it has been challenging to formally prove this hypothesis for same-strain recurrences by using epidemiologic approaches. We present data here obtained by using a mouse model of UTIs in which it was shown that 36% (5 of 14) of mice infected with uropathogenic
Escherichia coli
(UPEC) will have at least one bacteriuric recurrence, with 21% (3 of 14) having more than one recurrence during a 6-week period after an acute UTI. Intraurethrally infected mice develop UPEC reservoirs in both their feces and their bladders. Ten days of trimethoprim-sulfamethoxazole (SXT) therapy reduces urinary recurrences and eradicates fecal colonization, whereas 3 days of SXT treatment has no effect over a twenty-eight-day observation period despite clearing fecal colonization acutely. Interestingly, SXT is unable to eradicate bacteria from the bladder reservoir even after a 10-day treatment regimen, thus demonstrating that the bladder reservoir can persist even in the face of long-term antibiotic therapy.
Lactoferrin Protects Rabbits fromShigella flexneri-Induced Inflammatory Enteritis
Authors: Gomez Henry F. , Ochoa Theresa J. , Herrera-Insua Irene , Carlin Lily G. , Cleary Thomas G.
Published: November 2002

Shigellaspecies cause bacillary dysentery in humans by invasion, intracellular multiplication, spread to adjacent cells, and induction of brisk inflammatory responses in the intestinal epithelium. In vitro data suggest that lactoferrin, a glycoprotein present in human mucosal secretions, has a role in protection from bacterial enteric infections. We sought to determine the activity of lactoferrin in vivo, using the concentration present in human colostrum, to investigate its effect on the development of clinical and pathological evidence of inflammation in a rabbit model of enteritis. Lactoferrin protected rabbits infected withShigella flexnerifrom developing inflammatory intestinal disease. Typical histological changes in ill animals included villous blunting with sloughing of epithelial cells, submucosal edema, infiltration of leukocytes, venous congestion, and hemorrhage. Lactoferrin at a concentration normally found in human colostrum blocks development ofS. flexneri-induced inflammatory enteritis.
The Bacterial Redox Protein Azurin Induces Apoptosis in J774 Macrophages through Complex Formation and Stabilization of the Tumor Suppressor Protein p53
Authors: Yamada Tohru , Goto Masatoshi , Punj Vasu , Zaborina Olga , Kimbara Kazuhide , Das Gupta T. K. , Chakrabarty A. M.
Published: November 2002

Two redox proteins, azurin and cytochrome
c
551
elaborated by
Pseudomonas aeruginosa
, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.
Comparative Genomics Identifies the Genetic Islands That Distinguish
Neisseria meningitidis
, the Agent of Cerebrospinal Meningitis, from Other
Neisseria
Species
Authors: Perrin Agnès , Bonacorsi Stéphane , Carbonnelle Etienne , Talibi Driss , Dessen Philippe , Nassif Xavier , Tinsley Colin
Published: November 2002

Neisseria meningitidis
colonizes the nasopharynx and, unlike commensal
Neisseria
species, is capable of entering the bloodstream, crossing the blood-brain barrier, and invading the meninges. The other pathogenic
Neisseria
species,
Neisseria gonorrhoeae
, generally causes an infection which is localized to the genitourinary tract. In order to investigate the genetic basis of this difference in disease profiles, we used a strategy of genomic comparison. We used DNA arrays to compare the genome of
N. meningitidis
with those of
N. gonorrhoeae
and
Neisseria lactamica
, a commensal of the nasopharynx. We thus identified sequences conserved among a representative set of virulent strains which are either specific to
N. meningitidis
or shared with
N. gonorrhoeae
but absent from
N. lactamica
. Though these bacteria express dramatically different pathogenicities, these meningococcal sequences were limited and, in contrast to what has been found in other pathogenic bacterial species, they are not organized in large chromosomal islands.
Interleukin-18 and Gamma Interferon Production by Oral Epithelial Cells in Response to Exposure toCandida albicansor Lipopolysaccharide Stimulation
Authors: Rouabhia Mahmoud , Ross Geneviève , Pagé Nathalie , Chakir Jamila
Published: November 2002

Oral candidiasis is a collective name for a group of disorders caused by the dimorphic fungusCandida albicans. Host defenses againstC. albicansessentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral epithelial cells secrete a variety of cytokines and chemokines in response to oral microorganisms and sinceC. albicansis closely associated with oral epithelial cells as a commensal organism, we wanted to determine whether interleukin-18 (IL-18) and gamma interferon (IFN-γ) were produced by oral epithelial cells in response toC. albicansinfection and lipopolysaccharide (LPS) stimulation. Our results showed that IL-18 mRNA and protein were constitutively expressed by oral epithelial cells and were down-regulated byCandidainfections but increased following LPS stimulation. BothC. albicansand LPS significantly decreased pro-IL-18 (24 kDa) levels and increased active IL-18 (18 kDa) levels. This effect was IL-1β-converting-enzyme dependent. The increase in active IL-18 protein levels promoted the production of IFN-γ by infected cells. No effect was obtained with LPS. Although produced only at an early stage, secreted IFN-γ seemed to be a preferential response by oral epithelial cells toC. albicansgrowth. These results provide additional evidence for the contribution of oral epithelial cells to local (direct contact) and systemic (IL-18 and IFN-γ production) defense against exogenous stimulation such asC. albicansinfection or LPS stimulation.
Vaginal and Oral Epithelial Cell Anti-CandidaActivity
Authors: Nomanbhoy Fatema , Steele Chad , Yano Junko , Fidel, Paul L.
Published: November 2002

Candida albicansis the causative agent of acute and recurrent vulvovaginal candidiasis (VVC), a common mucosal infection affecting significant numbers of women in their reproductive years. While any murine host protective role for cell-mediated immunity (CMI), humoral immunity, and innate resistance by neutrophils against the vaginal infection appear negligible, significant in vitro growth inhibition ofCandidaspecies by vaginal and oral epithelial cell-enriched cells has been observed. Both oral and vaginal epithelial cell anti-Candidaactivity has a strict requirement for cell contact toC. albicanswith no role for soluble factors, and oral epithelial cells inhibitC. albicansthrough a cell surface carbohydrate moiety. The present study further evaluated the inhibitory mechanisms by murine vaginal epithelial cells and the fate ofC. albicansby oral and vaginal epithelial cells. Similar to human oral cells, anti-Candidaactivity produced by murine vaginal epithelial cells is unaffected by enzymatic cleavage of cell surface proteins and lipids but sensitive to periodic acid cleavage of surface carbohydrates. Analysis of specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose and mannose-containing carbohydrates, also similar to oral cells. Staining for live and deadCandidain the coculture with fluorescein diacetate (FDA) and propidium iodide (PI), respectively, showed a clear predominance of live organisms, suggesting a static rather than cidal action. Together, the results suggest that oral and vaginal epithelial cells retard or arrest the growth rather than killC. albicansthrough an as-yet-unidentified carbohydrate moiety in a noninflammatory manner.
Effect ofToxoplasma gondiiInfection Kinetics on Trophoblast Cell Population inCalomys callosus, a Model of Congenital Toxoplasmosis
Authors: Ferro E. A. V. , Silva D. A. O. , Bevilacqua E. , Mineo J. R.
Published: November 2002

This work evaluated the kinetics of events that occur in the placenta ofCalomys callosusafterToxoplasma gondiiinfection. Animals on the first day of pregnancy (dop) and virgin nonpregnant females were perorally infected with 20 cysts ofT. gondiistrain ME49. After 100 days of infection, the virgin animals were mated and received an additional 20 cysts on the first dop. The placentas and the embryos from both acutely and chronically infected animals were analyzed up to day 20 of pregnancy by morphological and immunocytochemical assays. Noninfected and infected animals exhibited placenta with normal morphology. From the seventh dop and infection onwards, liver and spleen cells of the infected animals contained several parasitophorous vacuoles. On the 13th day, the maternal blood present at the placental blood spaces containedT. gondii-infected leukocytes. Infected placental cells were only seen on the 15th dop, being the trophoblast giant cells, the first cell type to contain signs of the parasite internalization, followed by labyrinth zone cells 24 h later and spongiotrophoblast cells only after the 19th dop. Fetal liver and brain were infected byT. gondiiconcomitantly to the labyrinth cell infection. No signals of infection were observed on placentas and embryos from chronically infected animals. Therefore, considering the sequence of events leading to the infection of the various organs, it could be hypothesized that the placenta is infected later on during pregnancy, which may be related to the defense roles played by this structure. However, trophoblast giant cells are unable to completely stop the progression ofT. gondiiinfection towards the fetal tissues.C. callosuswas demonstrated to be a suitable experimental model to study the dynamics of congenital toxoplasmosis.
Characterization of Two Novel Pyrogenic Toxin Superantigens Made by an Acute Rheumatic Fever Clone ofStreptococcus pyogenesAssociated with Multiple Disease Outbreaks
Authors: Smoot Laura M. , McCormick John K. , Smoot James C. , Hoe Nancy P. , Strickland Ian , Cole Robert L. , Barbian Kent D. , Earhart Cathleen A. , Ohlendorf Douglas H. , Veasy L. George , Hill Harry R. , Leung Donald Y. M. , Schlievert Patrick M. , Musser James M.
Published: November 2002

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designatedspeLandspeM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS).speLandspeMwere located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS.speLandspeMwere transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vβ1, Vβ5.1, and Vβ23, and SpeM had specificity for Vβ1 and Vβ23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.
Functional Comparison of Serine Protease Autotransporters of
Enterobacteriaceae
Authors: Dutta Pinaki R. , Cappello Renato , Navarro-García Fernando , Nataro James P.
Published: November 2002

The plasmid-encoded toxin (Pet) of enteroaggregative
Escherichia coli
(EAEC) belongs to a family of high-molecular-weight serine protease autotransporters of
Enterobacteriaceae
(SPATEs) which also includes Pic from EAEC and
Shigella flexneri
, EspC from enteropathogenic
E. coli
, EspP from enterohemorrhagic
E. coli
, Sat from uropathogenic
E. coli
, Tsh from avian pathogenic
E. coli
, and SepA from
S. flexneri
. Phylogenetic analysis shows the SPATE proteins to represent a distinct subfamily of autotransporters with amino acid identities ranging from 35 to 55%, providing a powerful resource to direct structure-function studies. In this study, we show that these related proteins are proteases with divergent substrate specificities, suggesting different functions. The cleavage profile of oligopeptides was found to be unique for each SPATE protein. The SPATEs showed proteolytic activity for several substrates, namely mucin, pepsin, human coagulation factor V, and erythroid spectrin. The cleavage of spectrin has been hypothesized as the mechanism through which Pet induces cytopathic effects. However, whereas Pet, Sat, and EspC cleaved spectrin, only Pet and Sat elicited cytopathic effects; the remaining SPATEs did not cause any morphological changes to HEp-2 cell monolayers. EspC and Pet exhibited acid-dissociable binding to HEp-2 cells. However, Pet was more efficient at entering HEp-2 cells, suggesting a basis for the different abilities of these two proteases to damage cells. Our data suggest that, despite the homologies observed among these proteins, the SPATEs have different pathogenetic functions only partly dependent on their substrate specificities.
Concomitant Cytosolic Delivery of Two Immunodominant Listerial Antigens bySalmonella entericaSerovar Typhimurium Confers Superior Protection against Murine Listeriosis
Authors: Igwe Emeka I. , Geginat Gernot , Rüssmann Holger
Published: November 2002

During its interaction with host cells,Salmonella entericaserovar Typhimurium employs a type III secretion system for cytosolic targeting of virulence factors. This protein translocation mechanism is a useful tool for heterologous antigen delivery by attenuatedSalmonellavaccine carrier strains. In the present study, we used theYersiniaouter protein E (YopE) as a carrier molecule forSalmonellatype III-dependent cytosolic delivery of the immunodominant CD8 T-cell antigens listeriolysin O (LLO) and p60 ofListeria monocytogenes. It is shown that concomitant translocation of hybrid YopE/LLO and YopE/p60 proteins bySalmonellaled to antigen presentation and CD8 T-cell priming efficacies comparable to those of translocation of single listerial antigens. However, simultaneous translocation of LLO and p60 significantly surpassed single cytosolic antigen delivery in the ability to protect againstListeria. For the first time, this study demonstrates that concomitant expression of two independent antigens via the same recombinant plasmid leads to superior protection against a challenge with an intracellular bacterial pathogen. In conclusion, these findings emphasize the versatility ofSalmonellatype III-mediated heterologous antigen delivery for the induction of cytotoxic T-lymphocyte-mediated immunity.
Construction of anActinobacillus pleuropneumoniaeSerotype 2 Prototype Live Negative-Marker Vaccine
Authors: Tonpitak Walaiporn , Baltes Nina , Hennig-Pauka Isabel , Gerlach Gerald F.
Published: November 2002

Deletions were introduced into theureCandapxIIAgenes of anActinobacillus pleuropneumoniaeserotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.
Genetic Requirements for
Salmonella
-Induced Cytopathology in Human Monocyte-Derived Macrophages
Authors: Browne Sara H. , Lesnick Marc L. , Guiney Donald G.
Published: November 2002

Infection of human macrophages with
Salmonella enterica
serovar Typhimurium or
Salmonella enterica
serovar Dublin produces delayed cytotoxicity characterized by cell detachment and associated apoptosis. Using a site-specific mutant in the SpvB active site, we verify that the ADP-ribosylation activity of SpvB is required for delayed cytotoxicity in human macrophages infected with
Salmonella
. SipB and the type III protein secretion system (TTSS) encoded by
Salmonella
pathogenicity island 1 (SPI1) are not involved, whereas the SPI2 TTSS is absolutely required for SpvB-dependent cytotoxicity. Furthermore, we show that infection of macrophage cultures with wild-type or
sipB
mutant bacteria led to a complete loss of polymerized actin in over half of the cells after 24 h. In contrast, macrophages infected with the
spvB
or SPI2 (
ssaV
or
ssaJ
) mutant strain retained normal F-actin filaments, despite similar numbers of intracellular bacteria. We conclude that SpvB and a functional SPI2 TTSS are essential for
Salmonella
-induced delayed cytotoxicity of human macrophages.
The Furin Inhibitor Hexa-d-Arginine Blocks the Activation ofPseudomonas aeruginosaExotoxin A In Vivo
Authors: Sarac Miroslav S. , Cameron Angus , Lindberg Iris
Published: November 2002

ThePseudomonas aeruginosaexotoxin A (PEA) protein requires furin-mediated cleavage for manifestation of toxicity. We show here that the small stable furin inhibitor hexa-d-arginine amide effectively blocks PEA-induced cell lysis and is itself noncytotoxic. Administration of hexa-d-arginine to PEA-treated mice significantly improves their survival rate and also decreases circulating levels of tumor necrosis factor alpha.
Gene Discovery in the Freshwater Fish Parasite
Trypanosoma carassii
: Identification of
trans
-Sialidase-Like and Mucin-Like Genes
Authors: Agüero Fernán , Campo Vanina , Cremona Laura , Jäger Adriana , Di Noia Javier M. , Overath Peter , Sánchez Daniel O. , Frasch Alberto Carlos
Published: November 2002

A total of 1,921 expressed sequence tags (ESTs) were obtained from bloodstream trypomastigotes of
Trypanosoma carassii
, a parasite of economic importance due to its high prevalence in fish farms. Analysis of the data set allowed us to identify a
trans
-sialidase (TS)-like gene and three ESTs coding for putative mucin-like genes. TS activity was detected in cell extracts of bloodstream trypomastigotes. We have also used the sequence information obtained to identify genes that have not been previously described in trypanosomatids. (Additional information on these ESTs can be found at
http://genoma.unsam.edu.ar/projects/tca
.)
Generalized Immunological Decline during Long-Term Experimental Infection withMycobacterium avium
Authors: Gilbertson Brad , Cheers Christina
Published: November 2002

Terminal loss of immune responsiveness in C57BL/10 mice intranasally infected withMycobacterium aviumwas observed in both spleen and lung. It was nonspecific and related to the duration of infection, not the age of the mice. While there was loss of total T cells, the remaining cells were less efficient at gamma interferon production.
Nasal Immunization with Homogenate and Peptide Antigens Induces Protective Immunity against
Trichinella spiralis
Authors: McGuire Carolann , Chan Weng C. , Wakelin Derek
Published: November 2002

Mice were successfully immunized against the intestinal nematode
Trichinella spiralis
by intranasal administration of a 30-mer peptide antigen with cholera toxin B. Immunized mice developed antigen-specific serum immunoglobulin G1, intestinal immunoglobulin A, and a type 2-biased cytokine response. Intranasal immunization therefore generates the Th2-mediated responses required for immunity against intestinal parasites.
Cloning and Characterization of Genes Encoding Homologues of the B Subunit of Cholera Toxin and the
Escherichia coli
Heat-Labile Enterotoxin from Clinical Isolates of
Citrobacter freundii
and
E. coli
Authors: Karasawa Tadahiro , Ito Hideaki , Tsukamoto Teizo , Yamasaki Shinji , Kurazono Hisao , Faruque Shah M. , Nair G. Balakrish , Nishibuchi Mitsuaki , Takeda Yoshifumi
Published: November 2002

We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of
Escherichia coli
from a clinical isolate of
Citrobacter freundii
that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA). The gene encoding the ELISA-positive factor,
cfxB
, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame,
cfxA
, with a 56-nucleotide intergenic space. The
cfxB
gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively. However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT. DNA probes developed from the sequences of
cfxA
and
cfxB
were used to screen 67
C. freundii
isolates and 152
E. coli
isolates from diarrheal patients by colony blot hybridization. Two strains,
C. freundii
48 and
E. coli
176, reacted with both DNA probes under conditions of high stringency. We cloned homologues of the
cfxA
and
cfxB
genes from
E. coli
176 and designated them
ecxA
and
ecxB
, respectively. The
ecxA
gene and the
ecxB
gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively. The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.
IroN Functions as a Siderophore Receptor and Is a Urovirulence Factor in an Extraintestinal Pathogenic Isolate of
Escherichia coli
Authors: Russo Thomas A. , McFadden Catherine D. , Carlino-MacDonald Ulrike B. , Beanan Janet M. , Barnard Travis J. , Johnson James R.
Published: November 2002

IroN was recently identified in the extracellular pathogenic
Escherichia coli
strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of
iroN
contributed significantly to CP9's ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence.
Neuraminidase Expressed by
Streptococcus pneumoniae
Desialylates the Lipopolysaccharide of
Neisseria meningitidis
and
Haemophilus influenzae
: a Paradigm for Interbacterial Competition among Pathogens of the Human Respiratory Tract
Authors: Shakhnovich Elizabeth A. , King Samantha J. , Weiser Jeffrey N.
Published: November 2002

Both
Neisseria meningitidis
and
Haemophilus influenzae
are capable of mimicking host structures by decorating their lipopolysaccharides with sialic acid. We show that a neuraminidase expressed by
Streptococcus pneumoniae
(NanA) is able to desialylate the cell surfaces of both these species, which reside in and possibly compete for the same host niche.
Rough Lipopolysaccharide from
Brucella abortus
and
Escherichia coli
Differentially Activates the Same Mitogen-Activated Protein Kinase Signaling Pathways for Tumor Necrosis Factor Alpha in RAW 264.7 Macrophage-Like Cells
Authors: Jarvis Bruce W. , Harris Tajie H. , Qureshi Nilofer , Splitter Gary A.
Published: November 2002

The intracellular, gram-negative pathogen
Brucella abortus
establishes chronic infections in host macrophages while downregulating cytokines such as tumor necrosis factor alpha (TNF-α). When producing TNF-α,
Brucella abortus
rough lipopolysaccharide (LPS) activates the same mitogen-activated protein kinase signaling pathways (ERK and JNK) as
Escherichia coli
LPS, but
Brucella
LPS is a much less potent agonist.
Inhibition of
Neisseria gonorrhoeae
by
Lactobacillus
Species That Are Commonly Isolated from the Female Genital Tract
Authors: St. Amant Diane C. , Valentin-Bon Iris E. , Jerse Ann E.
Published: November 2002

Epidemiological studies suggest H
2
O
2
-producing lactobacilli protect women against gonorrhea. Here we demonstrate that
Lactobacillus crispatus
and
Lactobacillus jensenii
, the most common lactobacilli in the female genital tract, inhibit gonococci in both acidic and neutral pH conditions. Inhibition was neutralized by bovine catalase, suggesting that H
2
O
2
is the primary mediator of inhibition.
Leptospires Are Killed In Vitro by Both Oxygen-Dependent and -Independent Reactions
Authors: Murgia Rossella , Garcia Rodolfo , Cinco Marina
Published: November 2002

This study reports for the first time that leptospires are killed by H
2
O
2
and by low-molecular-weight primary granule components, which are agents normally released by neutrophils upon stimulation. Although both pathogenic and nonpathogenic strains were sensitive to H
2
O
2
-mediated killing, nonpathogenic organisms were found to be more susceptible. In addition, the killing of leptospires by H
2
O
2
was found to be independent of the presence of the neutrophil primary granule component myeloperoxidase and therefore not a consequence of halogenation reactions. We have also determined that leptospires are significantly sensitive only to primary granule components and, among those, to proteins and/or peptides of less than 30 kDa.
PeptidoglycanN-Acetylglucosamine Deacetylase, a Putative Virulence Factor inStreptococcus pneumoniae
Authors: Vollmer Waldemar , Tomasz Alexander
Published: November 2002

Many glucosamine residues of the pneumococcal peptidoglycan (PG) are not acetylated, which makes the PG resistant to lysozyme. A capsular type III mutant with an inactivatedpgdAgene (encoding the peptidoglycanN-acetylglucosamine deacetylase A) became hypersensitive to exogenous lysozyme and showed reduced virulence in the intraperitoneal mouse model.
Expression of NADPH Oxidase-Dependent Resistance to Listeriosis in Mice Occurs during the First 6 to 12 Hours of Liver Infection
Authors: LaCourse Ronald , Ryan Lynn , North Robert J.
Published: November 2002

Wild-type mice inoculated withListeria monocytogenesintravenously were capable of reducing the bacterial load in their livers by 90% within 6 h. In contrast, mice with deletions of the gene for NADPH oxidase were incapable of expressing this early oxygen-dependent anti-Listeriadefense and consequently showed higher levels of liver infection at later times.
Plasmodium falciparum
Serine Repeat Protein, a New Target of Monocyte-Dependent Antibody-Mediated Parasite Killing
Authors: Soe Soe , Singh Subhash , Camus Daniel , Horii Toshihiro , Druilhe Pierre
Published: November 2002

Using monoclonal antibodies and human affinity-purified antibodies specific to the
Plasmodium falciparum
126-kDa serine-rich protein, SERP, we found that these antibodies have no direct effect upon merozoite invasion at the concentrations tested but can cooperate with blood monocytes to strongly inhibit
P. falciparum
in vitro growth.
Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation
Authors: Ohuchi Masanobu , Ohuchi Reiko , Sakai Tatsuya , Matsumoto Akira
Published: November 2002

Deletion of oligosaccharide side chains near the receptor binding site of influenza virus A/USSR/90/77 (H1N1) hemagglutinin (HA) enhanced the binding of HA to erythrocyte receptors, as was also observed with A/FPV/Rostock/34 (H7N1). Correlated with the enhancement of binding activity, the cell fusion activity of HA was reduced. A mutant HA in which three oligosaccharide side chains were deleted showed the highest level of binding and the lowest level of fusion among the HAs tested. The cell fusion activity of the oligosaccharide deletion mutant of HA, however, was drastically elevated when the binding activity was reduced by deletion of four amino acids adjacent to the receptor binding site. Thus, a reciprocal relationship was observed between the receptor binding and the cell fusion activities of H1/USSR HA. No difference was observed, however, in lipid mixing activity, so-called hemifusion, between wild-type (WT) and oligosaccharide deletion mutant HAs. Soluble dye transfer testing showed that even the HA with the lowest cell fusion activity was able to form fusion pores through which a small molecule such as calcein could pass. However, electron microscopic studies revealed that a large molecule such as hemoglobin hardly passed through the fusion pores formed by the mutant HA, whereas hemoglobin did efficiently pass through those formed by the WT HA. These results suggested that interference in the process of dilation of fusion pores occurs when the binding of HA to the receptor is too tight. Since the viral nucleocapsid is far larger than hemoglobin, appropriate receptor binding affinity is important for virus entry.
Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+T Cells in Infected Children Correlate Positively with Plasma Viral Load
Authors: Buseyne Florence , Scott-Algara Daniel , Porrot Françoise , Corre Béatrice , Bellal Nassima , Burgard Marianne , Rouzioux Christine , Blanche Stéphane , Rivière Yves
Published: November 2002

HIV-specific CD8+T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-γ)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+T cells from HIV-infected children. We studied 20 HLA-A∗0201-positive HIV-infected children. The IFN-γ production in response to stimulation with two HLA-A∗0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/106peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+T cells were able to produce IFN-γ. Most importantly, we found that the frequencies of IFN-γ-producing CD8+T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+T cells. In conclusion, our results show that the ex vivo-activated IFN-γ-producing HIV-specific CD8+-T-cell subset is dependent upon continuous antigenic stimulation.
Virus-Specific CD8+Lymphocytes Share the Same Effector-Memory Phenotype but Exhibit Functional Differences in Acute Hepatitis B and C
Authors: Urbani Simona , Boni Carolina , Missale Gabriele , Elia Gianfranco , Cavallo Cristina , Massari Marco , Raimondo Giovanni , Ferrari Carlo
Published: November 2002

Hepatitis B and hepatitis C viruses (HBV and HCV) are both noncytopathic and can cause acute and chronic infections of the liver. Although they share tropism for the same organ, development of chronic hepatitis is much more frequent following HCV infection, suggesting different mechanisms of viral persistence. In this study, we show that circulating HBV- and HCV-specific tetramer-positive CD8 cells during the acute phase of hepatitis B and C belong almost entirely to an effector-memory subset (CCR7−CD45RA−). Despite this phenotypic similarity, HBV- and HCV-specific CD8 cells show striking functional differences. HBV-specific tetramer-positive CD8 cells express high perforin content ex vivo, expand vigorously, and display efficient cytotoxic activity and gamma interferon (IFN-γ) production upon peptide stimulation. A comparable degree of functional efficiency is maintained after the resolution of hepatitis B. In contrast, HCV-specific CD8 cells in the acute phase of hepatitis C express significantly lower levels of perforin molecules ex vivo and show depressed CD8 function in terms of proliferation, lytic activity, and IFN-γ production, irrespective of the final outcome of the disease. This defect is transient, because HCV-specific CD8 cells can progressively improve their function in patients with self-limited hepatitis C, while the CD8 function remains persistently depressed in subjects with a chronic evolution.
Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated Viruses
Authors: Qiu Jianming , Nayak Ramnath , Tullis Gregory E. , Pintel David J.
Published: November 2002

We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5
rep/cap
plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7- and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.
Elevated Levels of Circulating Interleukin-18 in Human Immunodeficiency Virus-Infected Individuals: Role of Peripheral Blood Mononuclear Cells and Implications for AIDS Pathogenesis
Authors: Ahmad Rasheed , Sindhu Sardar T. A. , Toma Emil , Morisset Richard , Ahmad Ali
Published: November 2002

Originally identified as the gamma interferon-inducing factor, interleukin-18 (IL-18) was rediscovered as a proinflammatory cytokine related to the IL-1 family of cytokines that plays an important role in both innate and adaptive immune responses against viruses and intracellular pathogens. Despite its importance in inducing and regulating immune responses, relatively little is known about its production in HIV infection. We report here significantly (P< 0.05) elevated levels of this cytokine in the sera of human immunodeficiency virus (HIV)-infected/AIDS patients compared to those of HIV-seronegative healthy persons. Surprisingly, the peripheral blood mononuclear cells (PBMC) from HIV-infected/AIDS patients were compromised in the ability to upregulate IL-18 gene expression and produce this cytokine with and without lipopolysaccharide (LPS) stimulation. A significant positive correlation (P< 0.05) existed between the concentration of IL-18 in serum and its production from PBMC of HIV-seronegative healthy individuals but not those of HIV-infected/AIDS patients. Furthermore, the patients' PBMC expressed relatively reduced levels of activated caspase-1 constitutively as well as in response to LPS stimulation. Our data suggest the involvement of transforming growth factor beta (TGF-β) in suppressing IL-18 production from the patients' PBMC for the following reasons. (i) In in vitro studies it suppressed the production of IL-18 from PBMC. (ii) Its levels were significantly higher in the plasma of patients compared to that of control subjects. (iii) A significant negative correlation existed between the concentrations of TGF-β in plasma and of IL-18 in serum of the patients. The elevated levels of IL-18 in the serum of HIV-infected individuals may contribute to AIDS pathogenesis, whereas its compromised production from their PBMC in response to stimuli may reduce their innate defense to opportunistic intracellular pathogens.
Cytoplasmic Expression of mRNAs Containing the Internal Ribosome Entry Site and 3′ Noncoding Region of Hepatitis C Virus: Effects of the 3′ Leader on mRNA Translation and mRNA Stability
Authors: Kong Li Kuo , Sarnow Peter
Published: November 2002

Translation initiation in many eukaryotic mRNAs is modulated by an interaction between the cap binding protein complex, bound to the 5′ end of the mRNA, and the polyadenosine binding protein, bound to the 3′-terminal polyadenosine sequences. A few cellular and viral mRNAs, such as the hepatitis C virus (HCV) mRNA genome, lack 3′-terminal polyadenosine sequences. For such mRNAs, the question of whether their 3′-end sequences also regulate the initiation phase of protein synthesis via an interaction with their 5′ ends has received intense scrutiny. For HCV mRNA, various experimental designs have led to conflicting interpretations, that the 3′ end of the RNA can modulate translation initiation either in a positive or in a negative fashion. To examine the possibility of end-to-end communication in HCV in detail, mRNAs containing the HCV internal ribosome entry site linked to a luciferase coding region, followed by different 3′ noncoding regions, were expressed in the cytoplasm of cultured cells by T7 RNA polymerase. The intracellular translation efficiencies, steady-state levels, stabilities, and 3′-end sequences of these chimeric RNAs were examined. It was found that the HCV 3′ noncoding region modulates neither the translation nor the stability of the mRNAs. Thus, there is no detectable end-to-end communication in cytoplasmically expressed chimeric mRNAs containing the HCV noncoding regions. However, it remains an open question whether end-to-end communication occurs in full-length HCV mRNAs in the infected liver.
Covalent Modifications of the Ebola Virus Glycoprotein
Authors: Jeffers Scott A. , Sanders David Avram , Sanchez Anthony
Published: November 2002

The role of covalent modifications of the Ebola virus glycoprotein (GP) and the significance of the sequence identity between filovirus and avian retrovirus GPs were investigated through biochemical and functional analyses of mutant GPs. The expression and processing of mutant GPs with altered N-linked glycosylation, substitutions for conserved cysteine residues, or a deletion in the region of O-linked glycosylation were analyzed, and virus entry capacities were assayed through the use of pseudotyped retroviruses. Cys-53 was the only GP
1
(∼130 kDa) cysteine residue whose replacement resulted in the efficient secretion of GP
1
, and it is therefore proposed that it participates in the formation of the only disulfide bond linking GP
1
to GP
2
(∼24 kDa). We propose a complete cystine bridge map for the filovirus GPs based upon our analysis of mutant Ebola virus GPs. The effect of replacement of the conserved cysteines in the membrane-spanning region of GP
2
was found to depend on the nature of the substitution. Mutations in conserved N-linked glycosylation sites proved generally, with a few exceptions, innocuous. Deletion of the O-linked glycosylation region increased GP processing, incorporation into retrovirus particles, and viral transduction. Our data support a common evolutionary origin for the GPs of Ebola virus and avian retroviruses and have implications for gene transfer mediated by Ebola virus GP-pseudotyped retroviruses.
African Swine Fever Virus Polyproteins pp220 and pp62 Assemble into the Core Shell
Authors: Andrés Germán , Alejo Alí , Salas José , Salas María L.
Published: November 2002

African swine fever virus (ASFV), a complex enveloped DNA virus, expresses two polyprotein precursors, pp220 and pp62, which after proteolytic processing give rise to several major components of the virus particle. We have analyzed the structural role of polyprotein pp62, the precursor form of mature products p35 and p15, in virus morphogenesis. Densitometric analysis of one- and two-dimensional gels of purified virions showed that proteins p35 and p15, as well as the pp220-derived products, are present in equimolecular amounts in the virus particle. Immunoelectron microscopy revealed that the pp62-derived products localize at the core shell, a matrix-like domain placed between the DNA-containing nucleoid and the inner envelope, where the pp220-derived products are also localized. Pulse-chase experiments indicated that the processing of both polyprotein precursors is concomitant with virus assembly. Furthermore, using inducible ASFV recombinants, we show that pp62 processing requires the expression of the pp220 core precursor, whereas the processing of both precursors pp220 and pp62 is dependent on expression of the major capsid protein p72. Interestingly, when p72 expression is blocked, unprocessed pp220 and pp62 polyproteins assemble into aberrant zipper-like elements consisting of an elongated membrane-bound protein structure reminiscent of the core shell. Moreover, the two polyproteins, when coexpressed in COS cells, interact with each other to form zipper-like structures. Together, these findings indicate that the mature products derived from both polyproteins, which collectively account for about 30% of the virion protein mass, are the basic components of the core shell and that polyprotein processing represents a maturational process related to ASFV morphogenesis.
Evidence that Replication of the Antitumor Adenovirus ONYX-015 Is Not Controlled by the p53 and p14
ARF
Tumor Suppressor Genes
Authors: Edwards Sara J. , Dix Brett R. , Myers Colleen J. , Dobson-Le Deirdre , Huschtscha Lily , Hibma Merilyn , Royds Janice , Braithwaite Antony W.
Published: November 2002

The adenovirus mutant ONYX-015 is in phase III clinical trials as a novel antitumor therapy. Its apparent efficacy is thought to be due to its ability to replicate selectively in tumor cells defective in the signaling pathway for p53. Recent data have shown that p14
ARF
, a positive regulator of p53, inhibits ONYX-015 replication in cells with a wild-type p53, a phenotype that characterizes normal cells. We, however, found that ONYX-015 activates p53 in tumor cells and in normal cells and that this can occur without p14
ARF
induction. We also show that ONYX-015 is not attenuated in cells with functional p53, whether or not p14
ARF
is expressed, and that where attenuation does occur, it is cell type specific.