bluuebunny/crossref_metadata_2025 huggingface dataset:
Historical Perspective of Foamy Virus Epidemiology and Infection
Authors: Meiering Christopher D. , Maxine L. Linial
Published: July 2002

Foamy viruses (FV) are complex retroviruses which are widespread in many species. Despite being discovered over 40 years ago, FV are among the least well characterized retroviruses. The replication of these viruses is different in many interesting respects from that of all other retroviruses. Infection of natural hosts by FV leads to a lifelong persistent infection, without any evidence of pathology. A large number of studies have looked at the prevalence of primate foamy viruses in the human population. Many of these studies have suggested that FV infections are prevalent in some human populations and are associated with specific diseases. More recent data, using more rigorous criteria for the presence of viruses, have not confirmed these studies. Thus, while FV are ubiquitous in all nonhuman primates, they are only acquired as rare zoonotic infections in humans. In this communication, we briefly discuss the current status of FV research and review the history of FV epidemiology, as well as the lack of pathogenicity in natural, experimental, and zoonotic infections.
Salmonella enterica
Serovar Typhimurium Invasion Is Repressed in the Presence of Bile
Authors: Prouty A. M. , Gunn J. S.
Published: July 2002

As enteric pathogens, the salmonellae have developed systems by which they can sense and adapt appropriately to deleterious intestinal components that include bile. Previously, growth in the presence of bile was shown to repress the transcription of
prgH
, a locus encoding components of the
Salmonella
pathogenicity island I (SPI-1) type III secretion system (TTSS) necessary for eukaryotic cell invasion. This result suggested an existing interaction between salmonellae, bile, and eukaryotic cell invasion. Transcription assays demonstrated that invasion gene regulators (e.g.,
sirC
and
invF
) are repressed by bile. However, bile does not interact with any of the invasion regulators directly but exerts its effect at or upstream of the two-component system at the apex of the invasion cascade, SirA-BarA. As suggested by the repression of invasion gene transcription in the presence of bile, Western blot analysis demonstrated that proteins secreted by the SPI-1 TTSS were markedly reduced in the presence of bile. Furthermore,
Salmonella enterica
serovar Typhimurium grown in the presence of bile was able to invade epithelial cells at only 4% of the level of serovar Typhimurium grown without bile. From these data, we propose a model whereby serovar Typhimurium uses bile as an environmental signal to repress its invasive capacity in the lumen of the intestine, but upon mucous layer penetration and association with intestinal epithelial cells, where the apparent bile concentration would be reduced, the system would become derepressed and invasion would be initiated.
Genetically Determined Disparate Innate and Adaptive Cell-Mediated Immune Responses to PulmonaryMycobacterium bovisBCG Infection in C57BL/6 and BALB/c Mice
Authors: Wakeham Julia , Wang Jun , Xing Zhou
Published: July 2002

The current study was designed to investigate the impact of genetic heterogeneity on host immune responses to pulmonary intracellular infection by using two mouse strains of distinct genetic background, C57BL/6 and BALB/c mice, and a model intracellular pathogen,Mycobacterium bovisBCG. Upon infection, compared to C57BL/6 mice, BALB/c mice developed an earlier response of interleukin 12 (IL-12), gamma interferon (IFN-γ), tumor necrosis factor alpha, and macrophage chemoattractive protein 1, and greater neutrophilic influx to the lung by days 7 and 14. However, the level of these cytokines at days 27, 43, and 71 was much lower in BALB/c mice than in C57BL/6 mice. The magnitude of cellular responses was also much lower in the lung of BALB/c mice around day 27. Histologically, while C57BL/6 mice developed lymphocytic granulomas, BALB/c mice displayed atypical granulomas in the lung. Of importance, the level of type 2 cytokines IL-4 and IL-10 remained low and similar in the lung of both C57BL/6 and BALB/c mice throughout. Furthermore, lymphocytes isolated from systemic and local lymphoid tissues of infected BALB/c mice demonstrated a markedly lower antigen-specific IFN-γ recall response. While the number of mycobacterial bacilli recovered from both the lung and spleen of BALB/c mice was similar to that in C57BL/6 mice at day 14, it was higher than that in C57BL/6 mice at day 43. However, it was eventually leveled off to that in C57BL/6 counterparts later. These results suggest the following: (i) genetic heterogeneity can lead to differential innate and adaptive cell-mediated immune responses to primary pulmonary mycobacterial infection; (ii) it is the level of adaptive, but not innate, immune response that is critical to host resistance; and (iii) a lower type 1 immune response in BALB/c mice is not accompanied by a heightened type 2 response during pulmonary mycobacterial infection.
Construction of Urease-Negative Mutants of
Yersinia enterocolitica
Serotypes O:3 and O:8: Role of Urease in Virulence and Arthritogenicity
Authors: Gripenberg-Lerche Christel , Zhang Lijuan , Ahtonen Päivi , Toivanen Paavo , Skurnik Mikael
Published: July 2002

Yersinia enterocolitica
serotype O:3 and O:8 urease-negative mutants unable to express the 19-kDa β subunit of urease were constructed and tested for virulence and arthritogenicity. Our results indicate that urease is needed for full virulence in oral infections and that it is not an arthritogenic factor in the rat model.
Malaria Infection Induces Rapid Elevation of the Soluble Fas Ligand Level in Serum and Subsequent T Lymphocytopenia: Possible Factors Responsible for the Differences in Susceptibility of Two Species ofMacacaMonkeys toPlasmodium coatneyiInfection
Authors: Matsumoto Jun , Kawai Satoru , Terao Keiji , Kirinoki Masashi , Yasutomi Yasuhiro , Aikawa Masamichi , Matsuda Hajime
Published: July 2002

The intraerythrocytic stage of the simian malaria parasitePlasmodium coatneyi(CDC strain) was intravenously inoculated into two species of macaques with different susceptibilities to infection with this parasite, including four Japanese macaques (Macaca fuscata) and three cynomolgus macaques (M. fascicularis). The Japanese macaques infected withP. coatneyideveloped severe clinical manifestations similar to those of severe human malaria and eventually became moribund, while the infected cynomolgus macaques, natural hosts of the parasite, exhibited no severe manifestation of disease except anemia and finally recovered from the infection. In the infected Japanese macaques, peripheral CD4+and CD8+T-cell populations were markedly decreased and fragmentation of chromosomal DNA in peripheral blood mononuclear cells was detected during the terminal period of infection, suggesting that apoptotic cell death was responsible at least in part for the T lymphocytopenia. Furthermore, soluble Fas ligand levels in sera of the infected Japanese macaques increased gradually to a markedly high level of 28.83 ± 10.56 pg/ml (n= 4) when the animals became moribund. On the other hand, none of the infected cynomolgus monkeys exhibited either T lymphocytopenia or elevated soluble Fas ligand level. These findings suggest that differences in immune response between the two species of macaque tested accounted for the contrasting outcomes after infection with the same isolate of malarial parasite, and in particular that a profound T lymphocytopenia due to Fas-derived apoptosis played a role in the fatal course of malaria in the infected Japanese macaques.
Characterization of In Vitro DNA Binding Sites of the EUO Protein of
Chlamydia psittaci
Authors: Zhang Li , Howe Martha M. , Hatch Thomas P.
Published: July 2002

The EUO gene of chlamydia is highly expressed early in the developmental cycle, relative to other genes, but continues to be expressed throughout the active growth phases. The precise function of EUO protein is not known, but it binds to DNA in vitro. In this study, we developed a selection and amplification scheme for identifying chlamydial genomic fragments to which EUO preferentially binds in vitro. The scheme involved mixing recombinant EUO with a
Chlamydia psittaci
genomic library in a pBluescript plasmid vector in vitro, trapping EUO-bound plasmid clones on filters, and amplifying the clones in
Escherichia coli
. After nine rounds of enrichment, the EUO binding sites of the three most highly enriched clones were identified by DNase I footprint analysis. All three clones had multiple binding sites of various sizes with no clear distinguishing feature other than they were AT-rich and were usually not located in putative promoter regions. We used limited site-specific mutagenesis to characterize the strongest binding site of the most-highly-enriched clone, which represented about 50% of the population after nine rounds. This mutagenesis identified a core binding site of 15 nucleotides (nt) whose sequence was used to find related sequences within each of the strong binding sites in the other two clones. Using the frequency of bases at specific positions within this group of sequences as a guide, we carried out trial-and-error searching with many related sequences, eliminating those which identified nonfootprinted sites. This process led us to the consensus 15-nt sequence AHGAAAWVTYTWDAY, which, when allowing two mismatches, picked out all of the strong binding sites and no nonfootprinting sites within the three enriched clones. This sequence may be useful for predicting additional possible EUO binding sites in the chlamydial genome.
Gamma Interferon Dominates the Murine Cytokine Response to the Agent of Human Granulocytic Ehrlichiosis and Helps To Control the Degree of Early Rickettsemia
Authors: Akkoyunlu Mustafa , Fikrig Erol
Published: July 2002

The cytokine response to the agent of human granulocytic ehrlichiosis (HGE) was assessed in a murine infection model and the role of gamma interferon (IFN-γ), a cytokine that is crucial for host defenses against intracellular pathogens, was investigated by using IFN-γ-deficient mice. The agent of HGE (aoHGE) is an obligate intracellular bacterium that survives within neutrophils: morulae (vacuoles containing HGE organisms) are evident in polymorphonuclear leukocytes of experimentally infected immunocompetent mice for 1 to 2 weeks. We now show that IFN-γ levels increase during early infection of C3H/HeN or C57BL/6 mice with HGE bacteria. Moreover, in response to aoHGE extracts or concanavalin A, splenocytes from ehrlichia-infected mice produced more IFN-γ and less interleukin-4 than controls, suggesting that aoHGE partially skewed the immune response towards a Th1 phenotype. Absolute concentration of morulae containing neutrophils in blood was 122 ± 22 cells/μl on day 8. The bacterial DNA burden was also highest on day 8 and then declined after IFN-γ levels peaked. In contrast, IFN-γ-deficient mice had a markedly elevated HGE bacteria burden with morulae concentration of 282 ± 48 cells/μl on day 5 (P= 0.004) and 242 ± 63 cells/μl on day 8 (P= 0.005). Rickettsemia resolved in immunocompetent and IFN-γ deficient mice after 2 weeks, while both the immunocompetent and the IFN-γ-deficient mice had increased serum antibodies against aoHGE antigens at this time point. These data demonstrate that the HGE agent elicits a prominent IFN-γ response in mice and that IFN-γ is important in controlling the degree of rickettsemia during the early phase of infection, while IFN-γ independent mechanisms play a role at later time points.
Antibodies against the
Plasmodium falciparum
Receptor Binding Domain of EBA-175 Block Invasion Pathways That Do Not Involve Sialic Acids
Authors: Narum David L. , Haynes J. David , Fuhrmann Steven , Moch Kathy , Liang Hong , Hoffman Stephen L. , Sim B. Kim Lee
Published: July 2002

The 175-kDa
Plasmodium falciparum
erythrocyte binding protein (EBA-175) binds to its receptor, sialic acids on glycophorin A. The binding region within EBA-175 is a cysteine-rich region identified as region II. Antibodies against region II block the binding of native EBA-175 to erythrocytes. We identified a
P. falciparum
strain, FVO, that could not invade erythrocytes devoid of sialic acids due to prior neuraminidase treatment, and in addition, we used a strain, 3D7, that could invade such sialic acid-depleted erythrocytes. We used these two strains to study the capacity of anti-region II antibodies to inhibit FVO and 3D7 parasite development in vitro. Analysis of growth-inhibitory effects of purified FVO anti-region II immunoglobulin G (IgG) with the FVO and 3D7 strains resulted in similar levels of growth inhibition. FVO and 3D7 strains were inhibited between 28 and 56% compared to control IgG. There appeared to be no intracellular growth retardation or killing of either isolate, suggesting that invasion was indeed inhibited. Incubation of recombinant region II with anti-region II IgG reversed the growth inhibition. These results suggest that antibodies against region II can also interfere with merozoite invasion pathways that do not involve sialic acids. The fact that EBA-175 has such a universal and yet susceptible role in erythrocyte invasion clearly supports its inclusion in a multivalent malaria vaccine.
rpoS
Gene Function Is a Disadvantage for
Escherichia coli
BJ4 during Competitive Colonization of the Mouse Large Intestine
Authors: Krogfelt Karen A. , Hjulgaard Marianne , Sørensen Kristine , Cohen Paul S. , Givskov Michael
Published: July 2002

The ability of
Escherichia coli
to survive stress during growth in different environments is, in large part, dependent on
rpoS
and the genes that comprise the
rpoS
regulon.
E. coli
BJ4 and an isogenic BJ4
rpoS
mutant were used to examine the influence of the
rpoS
gene on
E. coli
colonization of the streptomycin-treated mouse large intestine. Colonization experiments in which the wild-type
E. coli
BJ4 and its
rpoS
mutant were fed individually as well as simultaneously to mice suggested that
E. coli
BJ4 does not face prolonged periods of nutrient starvation in the mouse large intestine and that the
rpoS
regulon is not expressed during long-term colonization after adaptation of the bacteria to the gut environment.
The Lipopolysaccharide Structures ofSalmonella entericaSerovar Typhimurium andNeisseria gonorrhoeaeDetermine the Attachment of Human Mannose-Binding Lectin to Intact Organisms
Authors: Devyatyarova-Johnson Marina , Rees Ian H. , Robertson Brian D. , Turner Malcolm W. , Klein Nigel J. , Jack Dominic L.
Published: July 2002

Mannose-binding lectin (MBL) is an important component of the innate immune system. It binds to the arrays of sugars commonly presented by microorganisms and activates the complement system independently of antibody. Despite detailed knowledge of the stereochemical basis of MBL binding, relatively little is known about how bacterial surface structures influence binding of the lectin. Using flow cytometry, we have measured the binding of MBL to a range of mutants ofSalmonella entericaserovar Typhimurium andNeisseria gonorrhoeaewhich differ in the structure of expressed lipopolysaccharide (LPS). For both organisms, the possession of core LPS structures led to avid binding of MBL, which was abrogated by the addition of O antigen (Salmonellaserovar Typhimurium) or sialic acid (N. gonorrhoeae). Truncation of the LPS within the core led to lower levels of MBL binding. It was not possible to predict the magnitude of MBL binding from the identity of the LPS terminal sugar alone, indicating that the three-dimensional disposition of LPS molecules is probably also of importance in determining MBL attachment. These results further support the hypothesis that LPS structure is a major determinant of MBL binding.
The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains
Authors: Frisardi Marta , Ghosh Sudip K. , Field Jessica , Van Dellen Katrina , Rogers Rick , Robbins Phillips , Samuelson John
Published: July 2002

The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from
Entamoeba invadens
. Jacob, which was acidic and had an apparent molecular mass of ∼100 kDa, contained sugars that bound to concanavalin A and ricin. The
jacob
gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the
E. histolytica
plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose,
E. invadens
formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the
E. invadens
cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.
Adherence ofStaphylococcus aureusto Endothelial Cells: Influence of Capsular Polysaccharide, Global Regulatoragr, and Bacterial Growth Phase
Authors: Pöhlmann-Dietze Petra , Ulrich Martina , Kiser Kevin B. , Döring Gerd , Lee Jean C. , Fournier Jean-Michel , Botzenhart Konrad , Wolz Christiane
Published: July 2002

The adherence ofStaphylococcus aureusto human endothelial cells (EC) is probably an important step in the pathogenesis of systemic staphylococcal infections. We examined the influence of type 5 capsular polysaccharide (CP5) production, the global regulatoragr, and the bacterial growth phase onS. aureusadherence to EC. WhereasS. aureusNewman showed maximal adherence to EC in the logarithmic phase of growth, an isogenicagrmutant showed maximal adherence in the stationary growth phase.S. aureusadherence to EC and CP5 expression were negatively correlated: a mutation in theagrlocus diminished CP5 production and led to increased adherence. Likewise, induction of CP5 expression by addition of NaCl to the growth medium resulted in reduced staphylococcal adherence to EC.S. aureusNewman cells that adhered to EC did not express CP5. A Newmancap5Omutant was acapsular and showed significantly greater adherence to EC than the parental strain did (P< 0.005). Complementation of thecap5Omutation intransrestored CP5 expression and reduced EC adherence to a level similar to that of the parental strain. The enhanced adherence shown by thecap5Omutant was similar in magnitude to that of theagrmutant or thecap5O agrdouble mutant. Cells of thecap5Omutant andcap5O agrdouble mutant harvested from stationary-phase cultures adhered significantly better than did cells harvested in the exponential growth phase. These data are consistent with the postexponential andagr-independent expression byS. aureusof at least one putative EC adhesin, whose binding domain may be masked by CP5.
T-Cell-Independent Responses toBorrelia burgdorferiAre Critical for Protective Immunity and Resolution of Lyme Disease
Authors: McKisic Maureen D. , Barthold Stephen W.
Published: July 2002

The humoral immune response toBorrelia burgdorferiduring persistent infection is critical to both protective and disease-resolving immunity. This study examined the role of B cells in the absence of T cells during these events, using mice with selected immune dysfunctions. At 6 weeks postinfection, an interval at which arthritis resolves in immunocompetent mice, arthritis severity was equivalent among immunocompetent mice, αβ+-T-cell-deficient mice, and mice lacking both αβ+and γδ+T cells. Arthritis severity was worse in SCID mice, which lack T and B lymphocytes. Carditis regressed in immunocompetent mice and those lacking both αβ+and γδ+T cells but remained active in mice lacking only αβ+T cells and in SCID mice. Mice lacking only αβ+T cells and those lacking both αβ+and γδ+T cells generated immunoglobulin M (IgM) and IgG3B. burgdorferi-reactive antibodies. Sera from infected immunocompetent mice, mice lacking only αβ+T cells, and mice lacking both αβ+and γδ+T cells passively protected naive mice against challenge inoculation withB. burgdorferi. However, only sera from infected immunocompetent mice, but not sera from infected T-cell-deficient mice, were able to resolve arthritis when passively transferred to actively infected SCID mice. These data demonstrate that B-cell activation during a T-cell-independent response may be critical for resolution of arthritis and carditis and that protective antibodies are generated during this response.
Identification of a New Iron-Regulated Virulence Gene,
ireA
, in an Extraintestinal Pathogenic Isolate of
Escherichia coli
Authors: Russo Thomas A. , Carlino Ulrike B. , Johnson James R.
Published: July 2002

Our laboratory is studying an extraintestinal pathogenic isolate of
Escherichia coli
(CP9) as a model pathogen. We have been using human urine, ascites, and blood ex vivo to identify genes with increased expression in these media relative to expression in Luria-Bertani (LB) broth. Such genes may represent new or unrecognized virulence traits. In this study, we report the identification of a new gene,
ireA
(iron-responsive element). This gene has an open reading frame of 2,049 nucleotides, and its peptide has a molecular mass of 75.3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its expression is increased a mean of 3.6-fold in human urine, 16.2-fold in human ascites, and 6.6-fold in human blood relative to expression in LB medium, and it is Fe repressible. IreA also exhibits peptide similarities (48 to 56%) to previously identified proteins that function as siderophore receptors, suggesting that IreA is involved in iron acquisition. PCR-based analysis of
ireA
's phylogenetic distribution detected
ireA
in none (0%) of 14 fecal isolates that represented probable commensal strains, but in 13 (26%) of 50 random urine and blood clinical isolates (
P
= 0.05) and in 5 (100%) of 5 representatives of the J96-like, clonal group of which CP9 is a member (
P
< 0.001). In a mouse urinary tract infection model, the presence of
ireA
contributed significantly to CP9's ability to colonize the bladder (
P
< 0.02), evidence that IreA is a urovirulence factor. Taken together, these findings demonstrate that
ireA
encodes a new virulence factor, which is likely involved in Fe acquisition.
Neospora caninum
Microneme Protein NcMIC3: Secretion, Subcellular Localization, and Functional Involvement in Host Cell Interaction
Authors: Naguleswaran Arunasalam , Cannas Angela , Keller Nadine , Vonlaufen Nathalie , Schares Gereon , Conraths Franz J. , Björkman Camilla , Hemphill Andrew
Published: July 2002

In apicomplexan parasites, host cell adhesion and subsequent invasion involve the sequential release of molecules originating from secretory organelles named micronemes, rhoptries, and dense granules. Microneme proteins have been shown to be released at the onset of the initial contact between the parasite and the host cell and thus mediate and establish the physical interaction between the parasite and the host cell surface. This interaction most likely involves adhesive domains found within the polypeptide sequences of most microneme proteins identified to date. NcMIC3 is a microneme-associated protein found in
Neospora caninum
tachyzoites and bradyzoites, and a large portion of this protein is comprised of a stretch of four consecutive epidermal growth factor (EGF)-like domains. We determined the subcellular localization of NcMIC3 prior to and following host cell invasion and found that NcMIC3 was secreted onto the tachyzoite surface immediately following host cell lysis in a temperature-dependent manner. Surface-exposed NcMIC3 could be detected up to 2 to 3 h following host cell invasion, and at later time points the distribution of the protein was again restricted to the micronemes. In vitro secretion assays using purified tachyzoites showed that following secretion onto the surface, NcMIC3 was largely translocated towards the posterior end of the parasite, employing a mechanism which requires a functional actin microfilament system. Following this, the protein remained bound to the parasite surface, since it could not be detected in a soluble form in respective culture supernatants. Secretion of NcMIC3 onto the surface resulted in an outward exposure of the EGF-like domains and coincided with an increased capacity of
N. caninum
tachyzoites to adhere to Vero cell monolayers in vitro, a capacity which could be inhibited by addition of antibodies directed against the EGF-like domains. NcMIC3 is a prominent component of Triton X-100 lysates of tachyzoites, and cosedimentation assays employing prefixed Vero cells showed that the protein binds to the Vero cell surface. In addition, the EGF-like domains, expressed as recombinant proteins in
Escherichia coli
, also interacted with the Vero cell surface, while binding of NcSRS2 and NcSAG1, the major immunodominant surface antigens, was not as efficient. Our data are indicative of a functional role of NcMIC3 in host cell infection.
Infection withMycobacterium aviumDifferentially Regulates the Expression of Iron Transport Protein mRNA in Murine Peritoneal Macrophages
Authors: Zhong Wangjian , Lafuse William P. , Zwilling Bruce S.
Published: July 2002

Iron is an important element for the growth of microorganisms as well as in the defense of the host by serving as a catalyst for the generation of free radicals via the Fenton/Haber-Weiss reactions. The iron transporter natural resistance-associated macrophage protein 1 (Nramp1) confers resistance to the growth of a variety of intracellular pathogens includingMycobacterium avium. Recently several other proteins that are involved in iron transport, including the highly homologous iron transporter Nramp2 and the transferrin receptor-associated protein HFE (hereditary hemochromatosis protein), have been described. The relationship of these proteins to host defense and to the growth of intracellular pathogens is not known. Here, we report that infection withM. aviumdifferentially regulates mRNA expression of the proteins associated with iron transport in murine peritoneal macrophages. Both Nramp1 and Nramp2 mRNA levels increase following infection, while the expression of transferrin receptor mRNA decreases. The level of expression of HFE mRNA remains unchanged. The difference in the expression of the mRNA of these proteins following infection or cytokine stimulation suggests that they may play an important role in host defense by maintaining a delicate balance between iron availability for host defense and at the same time limiting iron availability for microbial growth.
Identification and Characterization of the Hemophore-Dependent Heme Acquisition System of
Yersinia pestis
Authors: Rossi Maria-Silvia , Fetherston Jacqueline D. , Létoffé Sylvie , Carniel Elisabeth , Perry Robert D. , Ghigo Jean-Marc
Published: July 2002

Yersinia pestis
possesses a heme-protein acquisition system (Hmu) that allows it to utilize heme and heme-protein complexes as the sole sources of iron. Analysis of the
Y. pestis
CO92 genomic sequence revealed a second heme-protein acquisition gene cluster that shares homology with the hemophore-dependent heme acquisition system (Has system) of
Serratia marcescens
. This locus consisted of the
hasR

yp

receptor gene, the
hasA

yp

hemophore gene, and genes encoding components of the HasA
yp
dedicated ABC transporter factor (
hasDE

yp

), as well as a
tonB
homologue (
hasB
yp
). By using a reconstituted secretion system in
Escherichia coli
, we showed that HasA
yp
is a secreted heme-binding protein and that expression of HasA
yp
is iron regulated in
E. coli
. The use of a transcriptional reporter fusion showed that the
hasRADEB
promoter is Fur regulated and has increased activity at 37°C. Hemoglobin utilization via the Has
yp
system was studied with both
E. coli
and
Y. pestis
, for which
has
and
has hmu
mutant strains were used. No contribution of the Has system to heme utilization was observed in either
E. coli
or
Y. pestis
under the conditions we tested. Previously it was shown that a deletion of the Hmu system had no effect on the virulence of
Y. pestis
in a mouse model of bubonic plague. An Hmu
−
Has
−
double mutant also retained full virulence in this model of infection. This report constitutes the first attempt to investigate the contribution of the hemophore-dependent heme acquisition system in bacterial pathogenicity.
Analysis ofBorrelia burgdorferivlsEGene Expression and Recombination in the Tick Vector
Authors: Indest Karl J. , Howell Jerrilyn K. , Jacobs Mary B. , Scholl-Meeker Dorothy , Norris Steven J. , Philipp Mario T.
Published: July 2002

Expression and recombination of the antigenic variationvlsEgene of the Lyme disease spirocheteBorrelia burgdorferiwere analyzed in the tick vector. To assessvlsEexpression,Ixodes scapularisnymphs infected with theB. burgdorferistrain B31 were fed on mice for 48 or 96 h or to repletion and then crushed and acetone fixed either immediately thereafter (ticks collected at the two earlier time points) or 4 days after repletion. Unfed nymphs also were examined. At all of the time points investigated, spirochetes were able to bind a rabbit antibody raised against the conserved invariable region 6 of VlsE, as assessed by indirect immunofluorescence, but not preimmune serum from the same rabbit. This same antibody also bound to B31 spirochetes cultivated in vitro. Intensity of fluorescence appeared highest in cultured spirochetes, followed by spirochetes present in unfed ticks. Only a dim fluorescent signal was observed on spirochetes at the 48 and 96 h time points and at day 4 postrepletion. Expression ofvlsEin vitro was affected by a rise in pH from 7.0 to 8.0 at 34°C. Hence,vlsEexpression appears to be sensitive to environmental cues of the type found in theB. burgdorferinatural history. To assessvlsErecombination, nymphs were capillary fed theB. burgdorferiB31 clonal isolate 5A3. Ticks thus infected were either left to rest for 4 weeks (Group I) or fed to repletion on a mouse (Group II). The contents of each tick from both groups were cultured and 10B. burgdorfericlones from the spirochetal isolate of each tick were obtained. ThevlsEcassettes from several of these clones were amplified by PCR and sequenced. Regardless of whether the isolate was derived from Group I or Group II ticks, no changes were observed in thevlsEsequence. In contrast,vlsEcassettes amplified fromB. burgdorfericlones derived from a mouse that was infected with B31-5A3 capillary-fed nymphs showed considerable recombination. It follows thatvlsErecombination does not occur in the tick vector.
Human Macrophage Gamma Interferon Decreases Gene Expression but Not Replication ofMycobacterium tuberculosis: Analysis of the Host-Pathogen Reciprocal Influence on Transcription in a Comparison of Strains H37Rv and CMT97
Authors: Cappelli Giulia , Volpe Pietro , Sanduzzi Alessandro , Sacchi Alessandra , Colizzi Vittorio , Mariani Francesca
Published: July 2002

Mycobacterium tuberculosisis an intracellular pathogen that readily survives and replicates in human macrophages (MΦ). Host cells have developed different mycobactericidal mechanisms, including the production of inflammatory cytokines. The aim of this study was to compare the MΦ response, in terms of cytokine gene expression, to infection with theM. tuberculosislaboratory strain H37Rv and the clinicalM. tuberculosisisolate CMT97. Both strains induce the production of interleukin-12 (IL-12) and IL-16 at comparable levels. However, the clinical isolate induces a significantly higher and more prolonged MΦ activation, as shown by reverse transcription-PCR analysis of IL-1β, IL-6, IL-10, transforming growth factor beta, tumor necrosis factor alpha, and gamma interferon (IFN-γ) transcripts. Interestingly, when IFN-γ transcription is high, the number ofM. tuberculosisgenes expressed decreases and vice versa, whereas no mycobactericidal effect was observed in terms of bacterial growth. Expression of 11 genes was also studied in the twoM. tuberculosisstrains by infecting resting or activated MΦ and compared to bacterial intracellular survival. In both cases, a peculiar inverse correlation between expression of these genes and multiplication was observed. The number and type of genes expressed by the two strains differed significantly.
Alveolar Macrophage Deactivation in Murine Septic Peritonitis: Role of Interleukin 10
Authors: Reddy Raju C. , Chen Gina H. , Newstead Michael W. , Moore Tom , Zeng Xianying , Tateda Kazuhiro , Standiford Theodore J.
Published: July 2002

Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.
Mycobacterium avium
Invades the Intestinal Mucosa Primarily by Interacting with Enterocytes
Authors: Sangari Felix J. , Goodman Joseph , Petrofsky Mary , Kolonoski Peter , Bermudez Luiz E.
Published: July 2002

Previous studies have demonstrated that
Mycobacterium avium
can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally,
M. avium
preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of
M. avium
binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of
M. avium
into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of
M. avium
to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches,
M. avium
was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells.
Recombinant
Neisseria meningitidis
Transferrin Binding Protein A Protects against Experimental Meningococcal Infection
Authors: West David , Reddin Karen , Matheson Mary , Heath Robert , Funnell Simon , Hudson Michael , Robinson Andrew , Gorringe Andrew
Published: July 2002

To better characterize the vaccine potential of
Neisseria meningitidis
transferrin binding proteins (Tbps), we have overexpressed TbpA and TbpB from
Neisseria meningitidis
isolate K454 in
Escherichia coli
. The ability to bind human transferrin was retained by both recombinant proteins, enabling purification by affinity chromotography. The recombinant Tbps were evaluated individually and in combination in a mouse intraperitoneal-infection model to determine their ability to protect against meningococcal infection and to induce cross-reactive and bactericidal antibodies. For the first time, TbpA was found to afford protection against meningococcal challenge when administered as the sole immunogen. In contrast to the protection conferred by TbpB, this protection extended to a serogroup C isolate and strain B16B6, a serogroup B isolate with a lower-molecular-weight TbpB than that from strain K454. However, serum from a TbpB-immunized rabbit was found to be significantly more bactericidal than that from a TbpA-immunized animal. Our evidence demonstrates that TbpA used as a vaccine antigen may provide protection against a wider range of meningococcal strains than does TbpB alone. This protection appears not to be due to complement-mediated lysis and indicates that serum bactericidal activity may not always be the most appropriate predictor of efficacy for protein-based meningococcal vaccines.
Haemophilus ducreyi
Associates with Phagocytes, Collagen, and Fibrin and Remains Extracellular throughout Infection of Human Volunteers
Authors: Bauer Margaret E. , Goheen Michael P. , Townsend Carisa A. , Spinola Stanley M.
Published: July 2002

In a previous study,
Haemophilus ducreyi
was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis.
H. ducreyi
was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts.
H. ducreyi
colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular
H. ducreyi
. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that
H. ducreyi
remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.
Identification of the Exported Proteins of the Oral Opportunistic Pathogen
Actinobacillus actinomycetemcomitans
by Using Alkaline Phosphatase Fusions
Authors: Ward John , Fletcher Julie , Nair Sean P. , Wilson Michael , Williams Rachel J. , Poole Stephen , Henderson Brian
Published: July 2002

A
phoA
fusion library of
Actinobacillus actinomycetemcomitans
genomic DNA has been screened to identify genes encoding exported and secreted proteins. A total of 8,000 colonies were screened, and 80 positive colonies were detected. From these, 48 genes were identified with (i) more than half having homology to known or hypothetical
Haemophilus influenzae
genes, (ii) 14 having no ascribed function, and (iii) 4 having very limited or no homology to known genes. The proteins encoded by these genes may, by virtue of their presence on the cell surface, be novel virulence determinants.
Complement Evasion byBorrelia burgdorferi: Serum-Resistant Strains Promote C3b Inactivation
Authors: Alitalo Antti , Meri Taru , Rämö Lasse , Jokiranta T. Sakari , Heikkilä Tero , Seppälä Ilkka J. T. , Oksi Jarmo , Viljanen Matti , Meri Seppo
Published: July 2002

The most characteristic features of the Lyme disease pathogens, theBorrelia burgdorferisensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whetherB. burgdorferiis able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii,B. burgdorferisensu stricto, andBorrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation.B. burgdorferisensu stricto andB. afzeliistrains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistantB. afzelii(n= 3) andB. burgdorferisensu stricto (n= 1) strains but not in the presence of C-sensitiveB. garinii(n= 7) strains or control bacteria (Escherichia coli,Staphylococcus aureus,andEnterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistantB. burgdorferis.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.
Cloning, Overexpression, Purification, and Immunobiology of an 85-Kilodalton Outer Membrane Protein fromHaemophilus ducreyi
Authors: Thomas Kevin L. , Leduc Isabelle , Olsen Bonnie , Thomas Christopher E. , Cameron D. William , Elkins Christopher
Published: July 2002

We have identified an 85-kDa outer membrane protein that is expressed by all tested strains ofHaemophilus ducreyi. Studies of related proteins from other pathogenic bacteria, includingHaemophilus influenzae, Pasteurella multocida, Neisseria gonorrhoeae, Neisseria meningitidis, andShigella dysenteriae, suggested a role for these proteins in pathogenesis and immunity. In keeping with the first such described protein fromHaemophilus influenzaetype B, we termed theH. ducreyiprotein D15. The gene encoding theH. ducreyiD15 protein was cloned and sequenced, and the deduced amino acid sequence was found to be most similar to sequences of the D15-related proteins from otherPasteurellaspp. The arrangement of the flanking genes was similar to that ofH. influenzaeRd and suggested that D15 was part of a multigene operon. Attempts to make a null mutation of the D15 gene were unsuccessful, paralleling results in other D15 gene studies. Overexpression ofH. ducreyiD15 inEscherichia coliresulted in a source of recombinant D15 (rD15) from which it was readily purified. rD15 was immunogenic, and it was found that immunization of rabbits with an rD15 vaccine preparation conferred partial protection against a virulent challenge infection. Antisera to an N-terminal peptide recognized all tested strains ofH. ducreyi.
Dr Operon-Associated Invasiveness of
Escherichia coli
from Pregnant Patients with Pyelonephritis
Authors: Goluszko Pawel , Niesel David , Nowicki Bogdan , Selvarangan Rangaraj , Nowicki Stella , Hart Audrey , Pawelczyk Edyta , Das Margaret , Urvil Petri , Hasan Rafia
Published: July 2002

We used a gentamicin protection assay to assess the ability of gestational pyelonephritis isolates of
Escherichia coli
to invade HeLa cells. The ability to enter HeLa cells was strongly associated with the presence of Dr operons coding for Dr adhesins. In contrast, the nonivasive isolates predominantly expressed
papG
, coding for P fimbriae.
Escherichia coliEnterotoxin B Subunit Triggers Apoptosis of CD8+T Cells by Activating Transcription Factor c-Myc
Authors: Soriani Marco , Williams Neil A. , Hirst Timothy R.
Published: July 2002

Heat-labile enterotoxin from enterotoxinogenicEscherichia coliis not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8+T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8+T cells but not of CD4+T cells triggers the specific up-regulation of the transcription factorc-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-mycantisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IκBα turnover, suggesting that elevated Myc expression may be dependent on NF-κB. When CD8+T cells were pretreated with inhibitors of IκBα turnover and NF-κB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.
Coupled Phase-Variable Expression and Epitope Masking of Selective Surface Lipoproteins Increase Surface Phenotypic Diversity in
Mycoplasma hominis
Authors: Zhang Qijing , Wise Kim S.
Published: July 2002

A new mechanism expanding mycoplasmal surface diversity is described. Exposure of surface epitopes on a constitutively expressed membrane protein (P56) of
Mycoplasma hominis
was subject to high-frequency phase variation due to phase-variable expression of the P120 antigen and its selective masking of P56 epitopes. Phase-variable masking may confer previously unrealized adaptive capabilities on mycoplasmas.
Borrelia burgdorferi
RevA Antigen Is a Surface-Exposed Outer Membrane Protein Whose Expression Is Regulated in Response to Environmental Temperature and pH
Authors: Carroll James A. , El-Hage Nazira , Miller Jennifer C. , Babb Kelly , Stevenson Brian
Published: July 2002

Borrelia burgdorferi
, the causative agent of Lyme disease, produces RevA protein during the early stages of mammalian infection.
B. burgdorferi
apparently uses temperature as a cue to its location, producing proteins required for infection of warm-blooded animals at temperatures corresponding to host body temperature, but does not produce such virulence factors at cooler, ambient temperatures. We have observed that
B. burgdorferi
regulates expression of RevA in response to temperature, with the protein being synthesized by bacteria cultivated at 34°C but not by those grown at 23°C. Tissues encountered by
B. burgdorferi
during its infectious cycle vary in their pH values, and the level of RevA expression was also found to be dependent upon pH of the culture medium. The cellular localization of RevA was also analyzed. Borrelial inner and outer membranes were purified by isopycnic centrifugation, and membrane fractions were conclusively identified by immunoblot analysis using antibodies raised against the integral inner membrane protein MotB and outer membrane-associated Erp lipoproteins. Immunoblot analyses indicated that RevA is located in the
B. burgdorferi
outer membrane. These analyses also demonstrated that an earlier report (H. A. Bledsoe et al., Infect. Immun. 176:7447–7455, 1994) had misidentified such
B. burgdorferi
membrane fractions. RevA was further demonstrated to be exposed to the external environment, where it could facilitate interactions with host tissues.