bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Tumor Necrosis Factor Alpha Expression and Release from Peritoneal Mouse Macrophages In Vitro in Response to Proliferating Gram-Positive versus Gram-Negative Bacteria
Authors: Cui Wei , Morrison David C. , Silverstein Richard
Published: July 2002

ViableEscherichia coliandStaphylococcus aureusbacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-α) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-α mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-α responses. Further, lipopolysaccharide (fromE. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-α secretion of macrophages in response toS. aureuswas at least equivalent to that observed withE. coli, while appearing at time periods hours later than theE. coli-elicited TNF-α response. Both the magnitude and kinetic profile of the TNF-α responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless,S. aureustreated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-α response, with the appearance of an early TNF-α peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301–2308, 2000). In contrast, imipenem treatment ofE. coli-cocultured macrophages does not significantly alter the observed TNF-α response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-α profiles faithfully reflect circulating levels of TNF-α in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-α) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.
Variability of Outer Membrane Protein P1 and Its Evaluation as a Vaccine Candidate against Experimental Otitis Media due to NontypeableHaemophilus influenzae: an Unambiguous, Multifaceted Approach
Authors: Bolduc Gilles R. , Bouchet Valérie , Jiang Ru-Zhang , Geisselsoder Janet , Truong-Bolduc Que Chi , Rice Peter A. , Pelton Stephen I. , Goldstein Richard
Published: July 2002

Candidate vaccine antigens for preventing otitis media caused by nontypeableHaemophilus influenzae(NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations ofH. influenzae. Twenty-three variants of P1 (≤95% similarity) were identified among 42 strains. When chinchillas were immunized with recombinant P1 (rP1) obtained from one of these isolates (BCH-3), all animals developed antibodies specific for rP1. Immunized animals were protected against disease when challenged with BCH-3, but not with anompP1mutant of BCH-3 or a strain (BCH-2) possessing a heterologous P1 (91% identity). We conclude that (i) while P1 induces protection against NTHI-mediated otitis media, development of a polyvalent vaccine reflecting the variability of P1 would be necessary to construct an efficacious vaccine and (ii) use of a phylogenically characterized collection of representative isolates in concert with gene sequencing, cloning, gene inactivation, and animal testing offers an efficient, rational, and rigorous strategy for evaluating the potential problems associated with variability of vaccine targets and specificity of related immune responses.
Mediation ofCryptosporidium parvumInfection In Vitro by Mucin-Like Glycoproteins Defined by a Neutralizing Monoclonal Antibody
Authors: Cevallos Ana María , Bhat Najma , Verdon Renaud , Hamer Davidson H. , Stein Barry , Tzipori Saul , Pereira Miercio E. A. , Keusch Gerald T. , Ward Honorine D.
Published: July 2002

The protozoan parasiteCryptosporidium parvumis a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells byC. parvumsporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putativeC. parvumadhesion- and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralizedC. parvuminfection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains α-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and α-N-acetylgalactosamine-specific lectins strongly implicate these proteins and their glycotopes as playing a role inC. parvum-host cell interactions.
Impaired Control ofBrucella melitensisInfection inRag1-Deficient Mice
Authors: Izadjoo Mina J. , Polotsky Yury , Mense Mark G. , Bhattacharjee Apurba K. , Paranavitana Chrysanthi M. , Hadfield Ted L. , Hoover David L.
Published: July 2002

After intranasal inoculation,Brucella melitensischronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection inRag1knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion ofRag1(recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration ofB. melitensis16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts ofBrucellaantigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T- and B-cell-independent immunity can controlBrucellainfection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.
New Zealand White Rabbit as a Nonsurgical Experimental Model forSalmonella entericaGastroenteritis
Authors: Hanes D. E. , Robl M. G. , Schneider C. M. , Burr D. H.
Published: July 2002

Rabbits orally challenged withSalmonella entericadeveloped a dose-dependent diarrheal disease comparable to human salmonellosis. ViableSalmonellaorganisms recovered from the intestine and deep tissues indicate local and systemic infections. Therefore, results show that the rabbit can be used as a model for diarrheal disease and sequelae associated with salmonellosis.
Mouse Cytokine Profiles Associated withBrucella abortusRB51 Vaccination orB. abortus2308 Infection
Authors: Pasquali Paolo , Adone Rosanna , Gasbarre Louis C. , Pistoia Claudia , Ciuchini Franco
Published: July 2002

This study indicated that mice immunized withBrucella abortusRB51 bacteria and subsequently challenged withB. abortus2308 were protected from reinfection. After vaccination, both Th1 and Th2 cytokine patterns were observed. Of those, the early production of gamma interferon seems to have the prominent role in inducing an immunologically based protection.
toxB
Gene on pO157 of Enterohemorrhagic
Escherichia
coli
O157:H7 Is Required for Full Epithelial Cell Adherence Phenotype
Authors: Tatsuno Ichiro , Horie Masanori , Abe Hiroyuki , Miki Takeyoshi , Makino Kozo , Shinagawa Hideo , Taguchi Haruhiko , Kamiya Shigeru , Hayashi Tetsuya , Sasakawa Chihiro
Published: July 2002

Adherence of enterohemorrhagic
Escherichia
coli
(EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection. An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors. In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the
toxB
gene to be involved in the full adherence phenotype. A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir. Introduction of a mini-pO157 plasmid (pIC37) composed of the
toxB
and
ori
regions restored full adherence capacity to O157Cu, including production and secretion of the proteins. In contrast, introduction of a pO157 mutant possessing
toxB
::Km into O157Cu could not restore the full adherence phenotype. Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu. These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.
Enhanced Antimycobacterial Response to RecombinantMycobacterium bovisBCG Expressing Latency-Associated Peptide
Authors: Marshall Ben G. , Wangoo Arun , O'Gaora Peadar , Cook H. Terry , Shaw Rory J. , Young Douglas B.
Published: July 2002

With a view to exploring the role of transforming growth factor β (TGF-β) during mycobacterial infection, recombinant clones of bacillus Calmette-Guérin (BCG) were engineered to express the natural antagonist of TGF-β, latency-activated peptide (LAP). Induction of TGF-β activity was reduced when macrophages were infected with BCG expressing the LAP construct (LAP-BCG). There was a significant reduction in the growth of LAP-BCG in comparison to that of control BCG following intravenous infection in a mouse model. The enhanced control of mycobacterial replication was associated with an increase in the production of gamma interferon by splenocytes challenged during the acute stage of infection but with a diminished recall response assessed after 13 weeks. Organ weight and hydroxyproline content, representing tissue pathology, were also lower in mice infected with LAP-BCG. The results are consistent with the hypothesis that TGF-β has a detrimental effect on mycobacterial immunity. While a reduction in TGF-β activity augments the initial response to BCG vaccination, early bacterial clearance may adversely affect the induction of a long-term memory response by LAP-BCG.
Staphylococcus aureus
Cap5O Has UDP-ManNAc Dehydrogenase Activity and Is Essential for Capsule Expression
Authors: Portolés Marta , Kiser Kevin B. , Bhasin Navneet , Chan Kenneth H. N. , Lee Jean C.
Published: July 2002

The
Staphylococcus aureus
serotype 5 capsular polysaccharide (CP5) has a repeating unit composed of (→4)-3-
O
-acetyl-β-
d
-ManNAcA-(1→4)-α-
l
-FucNAc (1→3)-β-
d
-FucNAc-(1→)

n

. Sixteen chromosomal genes (
cap5A
through
cap5P
) are involved in the synthesis of CP5. We recently demonstrated that Cap5P, a 2-epimerase, catalyzes the conversion of UDP–
N
-acetyl glucosamine (UDP-GlcNAc) to UDP–
N
-acetylmannosamine (UDP-ManNAc). In this study, we show that UDP-ManNAc is oxidized to UDP–
N
-acetylmannosaminuronic acid (UDP-ManNAcA) by a UDP-ManNAc dehydrogenase encoded by
S. aureus cap5O
. We expressed Cap5O in
Escherichia coli
and purified the recombinant protein. The UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incubating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, NAD
+
, and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH formation. The product of the reaction was confirmed as UDP-ManNAcA by gas chromatography-mass spectroscopy. A
cap5O
mutation, created by deletion of 727 bp in the 5′ end of the gene, was introduced by allelic replacement into
S. aureus
Reynolds, rendering it CP5 negative. Mice inoculated intravenously or subcutaneously with the wild-type strain Reynolds had greater numbers of
S. aureus
recovered from their kidneys (
P
= 0.019) or their subcutaneous abscesses (
P
= 0.0018), respectively, than did animals inoculated with the
cap5O
mutant. The results of this study indicate that
S. aureus cap5O
is essential for capsule production and that capsule promotes staphylococcal virulence in mouse models of abscess formation.
Molecular Characterization ofStreptococcus pneumoniaeType 4, 6B, 8, and 18C Capsular Polysaccharide Gene Clusters
Authors: Jiang Sheng-Mei , Wang Lei , Reeves Peter R.
Published: July 2002

Capsular polysaccharide (CPS) is a major virulence factor inStreptococcus pneumoniae. CPS gene clusters ofS. pneumoniaetypes 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5′ end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3′ end of the gene cluster. Comparison of the four dTDP-l-rhamnose pathway genes (rmlgenes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation ofrmlgenes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed forrmlgenes of O-antigen gene clusters ofSalmonella enterica. Our data indicate that the conserved first four genes at the 5′ ends and the relatively conservedrmlgenes at the 3′ ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identifiedwzxandwzygenes for all sequenced CPS gene clusters by use of motifs.
Canine Feces as a Reservoir of Extraintestinal PathogenicEscherichia coli
Authors: Johnson James R. , Stell Adam L. , Delavari Parissa
Published: July 2002

To test the canine reservoir hypothesis of extraintestinal pathogenicEscherichia coli(ExPEC), 63 environmental canine fecal deposits were evaluated for the presence of ExPEC by a combination of selective culturing, extended virulence genotyping, hemagglutination testing, O serotyping, and PCR-based phylotyping. Overall, 30% of canine fecal samples (56% of those that yielded viableE. coli) containedpapG-positiveE. coli, usually as the predominantE. colistrain and always possessingpapGallele III (which encodes variant III of the P-fimbrial adhesin molecule PapG). Multiple other virulence-associated genes typical of human ExPEC were prevalent among the canine fecal isolates. According to serotyping, virulence genotyping, and random amplified polymorphic DNA analysis, over 50% ofpapG-positive fecalE. colicould be directly correlated with specific human clinical isolates from patients with cystitis, pyelonephritis, bacteremia, or meningitis, including archetypal human ExPEC strains 536, CP9, and RS218. Five canine fecal isolates and (clonally related) archetypal human pyelonephritis isolate 536 were found to share a novel allele ofpapA(which encodes the P-fimbrial structural subunit PapA). These data confirm that ExPEC representing known virulent clones are highly prevalent in canine feces, which consequently may provide a reservoir of ExPEC for acquisition by humans.
Requirement of Non-T Cells That Produce Gamma Interferon for Prevention of Reactivation ofToxoplasma gondiiInfection in the Brain
Authors: Kang Hoil , Suzuki Yasuhiro
Published: July 2002

We examined the mechanism of resistance against reactivation of infection withToxoplasma gondiiin the brain. BALB/c-background gamma interferon (IFN-γ)-knockout (IFN-γ−/−) and control mice were infected and treated with sulfadiazine beginning 4 days after infection for 3 weeks. After discontinuation of treatment, IFN-γ−/−mice succumbed to toxoplasmic encephalitis (TE) and died, whereas control animals did not develop TE and survived. Adoptive transfer of immune spleen cells from infected control mice did not prevent development of TE or mortality in the IFN-γ−/−mice. To examine whether the failure of the cell transfer to protect against TE is unique to IFN-γ−/−mice, athymic nude and SCID mice that lack T cells were infected and injected with the immune spleen or T cells in the same manner as IFN-γ−/−mice. Whereas control nude and SCID mice that had not received the immune cells developed severe TE and died after discontinuation of sulfadiazine, those that had received the cells did not develop TE and survived. Before cell transfer, IFN-γ mRNA was detected in brains of infected nude and SCID but not in brains of IFN-γ−/−mice. IFN-γ mRNA was also detected in brains of infected SCID mice depleted of NK cells by treatment with anti-asialo GM1 antibody, and such animals did not develop TE after receiving immune T cells. Thus, IFN-γ production by non-T cells, in addition to T cells, is required for prevention of reactivation ofT. gondiiinfection in the brain. The IFN-γ-producing non-T cells do not appear to be NK cells.
Pathogenic YeastsCryptococcus neoformansandCandida albicansProduce Immunomodulatory Prostaglandins
Authors: Noverr Mairi C. , Phare Susan M. , Toews Galen B. , Coffey Michael J. , Huffnagle Gary B.
Published: July 2002

Enhanced prostaglandin production during fungal infection could be an important factor in promoting fungal colonization and chronic infection. Host cells are one source of prostaglandins; however, another potential source of prostaglandins is the fungal pathogen itself. Our objective was to determine if the pathogenic yeastsCryptococcus neoformansandCandida albicansproduce prostaglandins and, if so, to begin to define the role of these bioactive lipids in yeast biology and disease pathogenesis.C. neoformansandC. albicansboth secreted prostaglandins de novo or via conversion of exogenous arachidonic acid. Treatment with cyclooxygenase inhibitors dramatically reduced the viability of the yeast and the production of prostaglandins, suggesting that an essential cyclooxygenase like enzyme may be responsible for fungal prostaglandin production. A PGE series lipid was purified from bothC. albicansandC. neoformansand was biologically active on both fungal and mammalian cells. Fungal PGExand synthetic PGE2enhanced the yeast-to-hypha transition inC. albicans. Furthermore, in mammalian cells, fungal PGExdown-modulated chemokine production, tumor necrosis factor alpha production, and splenocyte proliferation while up-regulating interleukin 10 production. These are all activities previously documented for mammalian PGE2. Thus, eicosanoids are produced by pathogenic fungi, are critical for growth of the fungi, and can modulate host immune functions. The discovery that pathogenic fungi produce and respond to immunomodulatory eicosanoids reveals a virulence mechanism that has potentially great implications for understanding the mechanisms of chronic fungal infection, immune deviation, and fungi as disease cofactors.
Improved Immunogenicity and Protective Efficacy of a Tuberculosis DNA Vaccine Encoding Ag85 by Protein Boosting
Authors: Tanghe Audrey , D'Souza Sushila , Rosseels Valérie , Denis Olivier , Ottenhoff Thomas H. M. , Dalemans Wilfried , Wheeler Carl , Huygen Kris
Published: July 2002

C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 fromMycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 μg of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-γ) response in spleen two- to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-γ-producing CD4+T cells in DNA-primed–protein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenousM. tuberculosisH37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4+helper T cells in mediating protection.
ExtensiveMycobacterium bovisBCG Infection of Liver Parenchymal Cells in Immunocompromised Mice
Authors: Mills John W. , Ryan Lynn , LaCourse Ronald , North Robert J.
Published: July 2002

A histologic study was performed on the livers of wild-type (WT), severe combined immunodeficient (SCID), hydrocortisone acetate (HC)-treated WT, and HC-treated SCID mice infected intravenously with 105CFU ofMycobacterium bovisBCG. It was found that infection progressed faster in SCID mice than in WT mice and that HC treatment caused exacerbation of infection in both types of mice. In all cases infection in the liver was confined to granulomas that were populated predominantly by macrophages. Higher levels of infection in HC-treated SCID mice, but not HC-treated WT mice, were associated with extensive infection and destruction of parenchymal cells at the margins of granulomas. The results indicate that in the absence of T-cell-mediated immunity and of HC-sensitive T-cell-independent defense mechanisms, macrophages are incapable of restricting BCG growth and of confining infection to their cytoplasm. Consequently, BCG bacilli are released into the extracellular environment, where they are ingested by neighboring parenchymal cells.
Key Role of Teichoic Acid Net Charge in
Staphylococcus aureus
Colonization of Artificial Surfaces
Authors: Gross Matthias , Cramton Sarah E. , Götz Friedrich , Peschel Andreas
Published: July 2002

Staphylococcus aureus
is responsible for a large percentage of infections associated with implanted biomedical devices. The molecular basis of primary adhesion to artificial surfaces is not yet understood. Here, we demonstrate that teichoic acids, highly charged cell wall polymers, play a key role in the first step of biofilm formation. An
S. aureus
mutant bearing a stronger negative surface charge due to the lack of
d
-alanine esters in its teichoic acids can no longer colonize polystyrene or glass. The mutation abrogates primary adhesion to plastic while production of the glucosamine-based polymer involved in later steps of biofilm formation is not affected. Our data suggest that repulsive electrostatic forces can lead to reduced staphylococcal biofilm formation, which could have considerable impact on the design of novel implanted materials.
Immunization with Recombinant Opc Outer Membrane Protein from
Neisseria meningitidis
: Influence of Sequence Variation and Levels of Expression on the Bactericidal Immune Response against Meningococci
Authors: Jolley Keith A. , Appleby Lynn , Wright J. Claire , Christodoulides Myron , Heckels John E.
Published: July 2002

The
opc
gene from
Neisseria meningitidis
was cloned into the pRSETA vector, and recombinant protein was expressed at high levels in
Escherichia coli
. The protein was readily purified by affinity chromatography and used for immunization with conventional Al(OH)
3
adjuvant or after incorporation into liposomes and Zwittergent micelles. The resulting sera were analyzed for their ability to recognize purified recombinant protein and “native” protein in an enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Immunization with Al(OH)
3
induced high levels of antibodies which reacted with the purified protein but did not recognize whole cells. In contrast, liposomes and micelles induced antibodies which reacted with the native protein in whole cells. The addition of monophosphoryl lipid A (MPLA) to either liposomes or micelle preparations increased the magnitude of the immune response and induced a wider range of immunoglobulin subclasses. This was associated with the ability of the sera to induce complement-mediated killing of the homologous strain. The most effective bactericidal activity was observed with Opc protein incorporated into liposomes containing MPLA. The magnitude of the bactericidal effect was strongly influenced by the level of expression of the Opc protein and was abolished by limited variation in the sequence of the protein expressed by heterologous strains.
Deletion ofwboAEnhances Activation of the Lectin Pathway of Complement inBrucella abortusandBrucella melitensis
Authors: Fernandez-Prada Carmen M. , Nikolich Mikeljon , Vemulapalli Ramesh , Sriranganathan Nammalwar , Boyle Stephen M. , Schurig Gerhardt G. , Hadfield Ted L. , Hoover David L.
Published: July 2002

Brucellaspp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. ThewboAgene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain inBrucella. In this study, the sensitivity to serum of smooth, virulentBrucella melitensis16M andB. abortus2308, roughwboAmutants VTRM1, RA1, and WRR51 derived from these twoBrucellaspecies, and theB. abortusvaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. RoughB. abortusmutants were more sensitive to the bactericidal action of NHS than were roughB. melitensismutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived fromB. melitensis16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived fromB. abortus2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing ofBrucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.
Role of Gingipains in Growth of
Porphyromonas gingivalis
in the Presence of Human Serum Albumin
Authors: Grenier Daniel , Imbeault Sandra , Plamondon Pascale , Grenier Gilbert , Nakayama Koji , Mayrand Denis
Published: July 2002

Porphyromonas gingivalis
, a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction. The aim of the present study was to investigate the contribution of Arg- and Lys-gingipains produced by
P. gingivalis
to its growth. Although all of the proteins studied were degraded by
P. gingivalis
, only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM). Growth studies with site-directed gingipain-deficient mutants of
P. gingivalis
revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg- or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain. Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin. Incubation of resting
P. gingivalis
ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells. Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants. Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not. The critical role of proteinases in the growth of
P. gingivalis
was further investigated using specific Arg- and Lys-gingipain inhibitors. Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of
P. gingivalis
in a defined medium containing a human protein as the sole carbon and nitrogen source.
Comparative Sequence Analysis of the Plasmid-Encoded Regulator of Enteropathogenic
Escherichia coli
Strains
Authors: Okeke Iruka N. , Borneman Jade A. , Shin Sooan , Mellies Jay L. , Quinn Laura E. , Kaper James B.
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (
per
). Considerable variation in gene content of EAF plasmids from different strains was seen. However,
bfpA
, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and
per
genes were found in 96.8% of strains. Sequence analysis of the
per
operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the
perA
gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in
perA
leading to premature truncation and consequent inactivation of the gene were identified. Cloned
perA
, -
B
, and -
C
genes from these strains, unlike those from strains with a functional operon, failed to activate the
LEE1
operon and
bfpA
transcriptional fusions or to complement a
per
mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive
per
operons were deficient in virulence protein expression. The context in which the
perABC
operon occurs on the EAF plasmid varies. The sequence upstream of the
per
promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS
1294
-homologous sequence.
RS1 Element of
Vibrio cholerae
Can Propagate Horizontally as a Filamentous Phage Exploiting the Morphogenesis Genes of CTXΦ
Authors: Faruque Shah M. , Asadulghani  , Kamruzzaman M. , Nandi Ranjan K. , Ghosh A. N. , Nair G. Balakrish , Mekalanos John J. , Sack David A.
Published: July 2002

In toxigenic
Vibrio cholerae
, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying
ctxAB
genes and genes required for CTXΦ morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXΦ is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated
rstC
. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXΦ genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic
V. cholerae
strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km
r
) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between
ig1
and
rstC
. Introduction of pRS1-Km into a
V. cholerae
O1 classical biotype strain, O395, led to the production of extracellular Km
r
transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmΦ). Analysis of
V. cholerae
strains for susceptibility to RS1-KmΦ showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX
−
) O1 and O139 strains which carried genes encoding the CTXΦ receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXΦ, the RS1Φ genome also integrated into the host chromosomes by using the
att
RS sequence. However, only transductants of RS1-KmΦ which also harbored the CTXΦ genome produced a detectable level of extracellular RS1-KmΦ. This suggested that the core genes of CTXΦ are also required for the morphogenesis of RS1Φ. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in
V. cholerae
, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXΦ.
Bacteriophage Therapy Rescues Mice Bacteremic from a Clinical Isolate of Vancomycin-Resistant
Enterococcus faecium
Authors: Biswas Biswajit , Adhya Sankar , Washart Paul , Paul Brian , Trostel Andrei N. , Powell Bradford , Carlton Richard , Merril Carl R.
Published: July 2002

Colonization of the gastrointestinal tract with vancomycin-resistant
Enterococcus faecium
(VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 10
9
CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 × 10
8
PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.
In Vivo Rho GTPase-Activating Protein Activity of
Pseudomonas aeruginosa
Cytotoxin ExoS
Authors: Krall Rebecca , Sun Jianjun , Pederson Kristin J. , Barbieri Joseph T.
Published: July 2002

ExoS is a bifunctional type III cytotoxin secreted by
Pseudomonas aeruginosa,
which comprises a C-terminal ADP ribosyltransferase domain and an N-terminal Rho GTPase-activating protein (GAP) domain. In vitro, ExoS is a Rho GAP for Rho, Rac, and Cdc42; however, the in vivo modulation of Rho GTPases has not been addressed. Using a transient transfection system and delivery by
P. aeruginosa
, interactions were examined between the Rho GAP domain of ExoS and Rho GTPases in CHO cells. Rho GTPases were expressed as green fluorescent protein (GFP) fusion proteins to facilitate quantitation. GFP fusions of wild-type and dominant active Rho, Rac, and Cdc42 localized to discrete regions of CHO cells and appeared functional based upon their modulation of the actin cytoskeleton. Coexpression of the Rho GAP domain of ExoS changed the intracellular distribution of GFP-Rac and GFP-Cdc42 from a predominately membrane location to a cytosolic location. Coexpression of the Rho GAP domain of ExoS did not change the distribution of GFP-Rho, which was primarily in the cytosol. Coexpression of dominant active Rac (DARac) and DACdc42 inhibited actin reorganization by the Rho GAP domain but did not maintain the formation of actin stress fibers, which indicated that Rho had been inactivated. Similar results were observed when ExoS was delivered into CHO cells by
P. aeruginosa
. These data indicate that in vivo the Rho GAP activity of ExoS stimulates the reorganization of the actin cytoskeleton by inhibition of Rac and Cdc42 and stimulates actin stress fiber formation by inhibition of Rho.
Yersinia enterocoliticaas a Vehicle for a Naked DNA Vaccine EncodingBrucella abortusBacterioferritin or P39 Antigen
Authors: Al-Mariri Ayman , Tibor Anne , Lestrate Pascal , Mertens Pascal , De Bolle Xavier , Letesson Jean-Jacques
Published: July 2002

Brucellais a facultative intracellular parasite that causes brucellosis in animals and humans. The protective immune response againstBrucellainvolves both humoral and cell-mediated immunity. In previous studies, we demonstrated that the T-dominantBrucellaantigens bacterioferritin (BFR) and P39 administered either as CpG adjuvant recombinant proteins or as naked-DNA plasmids induced a specific Th1-biased immune response in mice. In order to improve the protection conferred by the BFR and P39 vaccines and to evaluate the additive role of antilipopolysaccharide (anti-LPS) antibodies, we used live attenuatedYersinia enterocoliticaserotypes O:3 and O:9 as delivery vectors for naked-DNA plasmids encoding these BFR and P39 antigens. Following two intragastric immunizations in BALB/c mice, theYersiniavectors harboring a DNA vaccine encoding BFR or P39 induced antigen-specific serum immunoglobulin and Th1-type responses (both lymphocyte proliferation and gamma interferon production) among splenocytes. Moreover, as expected, antibodies recognizingBrucella abortus544 lipopolysaccharide were detected in O:9-immunized mice but not in O:3-treated animals. Animals immunized with O:9 organisms carrying pCI or with O:9 organisms alone were found to be significantly resistant to infection byB. abortus544. Our data demonstrated that pCI plasmids encoding BFR or P39 and delivered with live attenuated strains ofYersiniaO:3 or O:9 can trigger Th1-type responses. The fact than only O:9 vectors induced a highly significant protective immunity againstB. abortus544 infection pointed out the crucial role of anti-LPS antibodies in protection. The best protection was conferred by a serotype O:9 strain carrying pCIP39, confirming the importance of the P39 T-cell antigen in this mechanism.
Protection against Bacterial Superantigen Staphylococcal Enterotoxin B by Passive Vaccination
Authors: LeClaire Ross D. , Hunt Robert E. , Bavari Sina
Published: July 2002

We investigated the ability of two overlapping fragments of staphylococcal enterotoxin B (SEB), which encompass the whole toxin, to induce protection and also examined if passive transfer of chicken anti-SEB antibodies raised against the holotoxin could protect rhesus monkeys against aerosolized SEB. Although both fragments of SEB were highly immunogenic, the fragments failed to protect mice whether they were injected separately or injected together. Passive transfer of antibody generated in chickens (immunoglobulin Y [IgY]) against the whole toxin suppressed cytokine responses and was protective in mice. All rhesus monkeys treated with the IgY specific for SEB up to 4 h after challenge survived lethal SEB aerosol exposure. These findings suggest that large fragments of SEB may not be ideal for productive vaccination, but passive transfer of SEB-specific antibodies protects nonhuman primates against lethal aerosol challenge. Thus, antibodies raised in chickens against the holotoxin may have potential therapeutic value within a therapeutic window of opportunity after SEB encounter.
Mycoplasma hyopneumoniaeIncreases Intracellular Calcium Release in Porcine Ciliated Tracheal Cells
Authors: Park Seung-Chun , Yibchok-Anun Sirintorn , Cheng Henrique , Young Theresa F. , Thacker Eileen L. , Minion F. Chris , Ross Richard F. , Hsu Walter H.
Published: July 2002

We investigated the effects of intact pathogenicMycoplasma hyopneumoniae, nonpathogenicM. hyopneumoniae, andMycoplasma flocculareon intracellular free Ca2+concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]iof 103 ± 3 nM (n= 217 cells). The [Ca2+]iincreased by 250 ± 19 nM (n= 47 cells) from the basal level within 100 s of the addition of pathogenicM. hyopneumoniaestrain 91-3 (300 μg/ml), and this increase lasted ∼60 s. In contrast, nonpathogenicM. hyopneumoniaeandM. flocculareat concentrations of 300 μg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenicM. hyopneumoniaestill increased [Ca2+]iin tracheal cells. Pretreatment with thapsigargin (1 μM for 30 min), which depleted the Ca2+store in the endoplasmic reticulum, abolished the effect ofM. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 μM for 100 s), an inhibitor of phospholipase C, also abolished the effect ofM. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins Giand Go, increased [Ca2+]iin ciliated tracheal cells. These results suggest that pathogenicM. hyopneumoniaeactivates receptors that are coupled to Gior Go, which in turn activates a phospholipase C pathway, thereby releasing Ca2+from the endoplasmic reticulum. Thus, an increase in Ca2+may serve as a signal for the pathogenesis ofM. hyopneumoniae.
Differential Regulation of the Mitogen-Activated Protein Kinases by Pathogenic and Nonpathogenic Mycobacteria
Authors: Roach Shannon K. , Schorey Jeffrey S.
Published: July 2002

Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-α) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenicMycobacterium aviumstrains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected withM. aviumproduced lower levels of TNF-α, interleukin 1β, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for theMycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains ofM. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated withM. aviuminfections suggests a novel point of immune intervention by this mycobacterial species.
Mice Lacking Monocyte Chemoattractant Protein 1 Have Enhanced Susceptibility to an Interstitial Polymicrobial Infection Due to Impaired Monocyte Recruitment
Authors: Chae P. , Im M. , Gibson F. , Jiang Y. , Graves D. T.
Published: July 2002

Monocyte chemoattractant protein 1 (MCP-1) is an important chemokine that induces monocyte recruitment in a number of different pathologies, including infection. To investigate the role of MCP-1 in protecting a host from a chronic interstitial polymicrobial infection, dental pulps of MCP-1−/−mice and controls were inoculated with six different oral pathogens. In this model the recruitment of leukocytes and the impact of a genetic deletion on the susceptibility to infection can be accurately assessed by measuring the progression of soft tissue necrosis and osteolytic lesion formation. The absence of MCP-1 significantly impaired the recruitment of monocytes, which at later time points was threefold higher in the wild-type mice than in MCP-1−/−mice (P< 0.05). The consequence was significantly enhanced rates of soft tissue necrosis and bone resorption (P< 0.05). We also determined that the MCP-1−/−mice were able to recruit polymorphonuclear leukocytes (PMNs) to a similar or greater extent as controls and to produce equivalent levels ofPorphyromonas gingivalis-specific total immunoglobulin G (IgG) and IgG1. These results point to the importance of MCP-1 expression and monocyte recruitment in antibacterial defense and demonstrate that antibacterial defense is not due to an indirect effect on PMN recruitment or modulation of the adaptive immune response.