bluuebunny/crossref_metadata_2025 huggingface dataset:
Members of the 30- to 32-Kilodalton Mycolyl Transferase Family (Ag85) from Culture Filtrate of
Mycobacterium avium
subsp.
paratuberculosis
Are Immunodominant Th1-Type Antigens Recognized Early upon Infection in Mice and Cattle
Authors: Rosseels Valérie , Marché Sylvie , Roupie Virginie , Govaerts Marc , Godfroid Jacques , Walravens Karl , Huygen Kris
Published: December 2005

The characterization of protective antigens is essential for the development of an effective, subunit-based vaccine against paratuberculosis. Surface-exposed and secreted antigens, present abundantly in mycobacterial culture filtrate (CF), are among the well-known protective antigens of
Mycobacterium tuberculosis
and
Mycobacterium bovis
. Culture filtrate, prepared from
Mycobacterium avium
subsp.
paratuberculosis
ATCC 19698 grown as a surface pellicle on synthetic Sauton medium, was strongly and early recognized in experimentally infected B6
bg/bg
beige mice and cattle, as indicated by elevated spleen cell gamma interferon (IFN-γ) secretion and lymphoproliferative responses of peripheral blood mononuclear cells, respectively. Strong proliferative and ex vivo IFN-γ responses against antigen 85 (Ag85) complex (a major protein component from
M. bovis
BCG culture filtrate) could be detected in cattle as early as 10 weeks after oral
M. avium
subsp.
paratuberculosis
infection. Synthetic peptides from the Ag85A and Ag85B components of this complex were strongly recognized, whereas T-cell responses were weaker against peptides from the Ag85C protein. A promiscuous T-cell epitope spanning amino acids 145 to 162 of Ag85B (identical sequence in
M. bovis
and
M. avium
subsp.
paratuberculosis
) was identified in experimentally infected cattle. Finally, young calves, born from cows with confirmed paratuberculosis, demonstrated proliferative responses to purified, recombinant Ag85A and Ag85B from
M. avium
subsp.
paratuberculosis
. These results indicate that the
M. avium
subsp.
paratuberculosis
Ag85 homologues are immunodominant T-cell antigens that are recognized early in experimental and natural infection of cattle.
Production and Characterization of Guinea Pig Recombinant Gamma Interferon and Its Effect on Macrophage Activation
Authors: Jeevan A. , McFarland C. T. , Yoshimura T. , Skwor T. , Cho H. , Lasco T. , McMurray D. N.
Published: December 2005

Gamma interferon (IFN-γ) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-γ (gpIFN-γ) and reported that BCG vaccination induced a significant increase in the IFN-γ mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain ofMycobacterium tuberculosisstimulated less IFN-γ mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-γ with a histidine tag at the N terminus (His-tagged rgpIFN-γ) inEscherichia coli. rgpIFN-γ was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-γ. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-γ was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-γ did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-γ induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-γ has been successfully produced and characterized in our laboratory. The study of rgpIFN-γ will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.
Comparison of Gamma Interferon-Mediated Antichlamydial Defense Mechanisms in Human and Mouse Cells
Authors: Roshick Christine , Wood Heidi , Caldwell Harlan D. , McClarty Grant
Published: December 2005

Gamma interferon (IFN-γ)-induced effector mechanisms have potent antichlamydial activities that are critical to host defense. The most prominent and well-studied effectors are indoleamine dioxygenase (IDO) and nitric oxide (NO) synthase. The relative contributions of these mechanisms as inhibitors of chlamydial in vitro growth have been extensively studied using different host cells, induction mechanisms, and chlamydial strains with conflicting results. Here, we have undertaken a comparative analysis of cytokine- and lipopolysaccharide (LPS)-induced IDO and NO using an extensive assortment of human and murine host cells infected with human and murine chlamydial strains. Following cytokine (IFN-γ or tumor necrosis factor alpha) and/or LPS treatment, the majority of human cell lines induced IDO but failed to produce NO. Conversely, the majority of mouse cell lines studied produced NO, not IDO. Induction of IDO in human cell lines inhibited growth of L2 and mouse pneumonitis agent, now referred to asChlamydia muridarumMoPn equally in all but two lines, and inhibition was completely reversible by the addition of tryptophan. IFN-γ treatment of mouse cell lines resulted in substantially greater reduction of L2 than MoPn growth. However, despite elevated NO production by murine cells, blockage of NO synthesis with thel-arginine analogueN-monomethyl-l-arginine only partially rescued chlamydial growth, suggesting the presence of another IFN-γ-inducible antichlamydial mechanism unique to murine cells. Moreover, NO generated from the chemical nitric oxide donor sodium nitroprusside showed little direct effect on chlamydial infectivity or growth, indicating a natural resistance to NO. Finally, IFN-γ-inducible IDO expression in human HeLa cells was inhibited following exogenous NO treatment, resulting in a permissive environment for chlamydial growth. In summary, cytokine- and LPS-inducible effectors produced by human and mouse cells differ and, importantly, these host-specific effector responses result in chlamydial strain-specific antimicrobial activities.
Identification of a New Spore Wall Protein from
Encephalitozoon cuniculi
Authors: Xu Yanji , Takvorian Peter , Cali Ann , Wang Fang , Zhang Hong , Orr George , Weiss Louis M.
Published: December 2005

Microsporidia form environmentally resistant spores that are critical for their host-to-host transmission and persistence in the environment. The spore walls of these organisms are composed of two layers, the exospore and the endospore. Two spore wall proteins (SWP1 and SWP2) have been previously identified in members of the Encephalitozoonidae family. These proteins localize to the exospore. The endospore is known to contain chitin, and a putative glycosylphosphatidylinositol (GPI)-anchored chitin deacetylase has been localized to the plasmalemma-endospore interface. Using proteomic techniques, we have identified a new spore wall protein (SWP3) that is located in the endospore. The gene for this protein is located on chromosome 1 and corresponds to the open reading frame ECU01_1270. SWP3 is predicted to have a signal peptide and to be GPI anchored. Consistent with these modifications, two-dimensional electrophoresis demonstrated that SWP3 has an acidic pI and a molecular mass of <20 kDa. By immunoelectron microscopy, this protein was found on the cell surface during sporogony and in the endospore in mature spores. SWP3 has several potential O-glycosylation sites, and it is possible that it is a mannosylated protein like the major polar tube protein (PTP1).
The
lpf
Gene Cluster for Long Polar Fimbriae Is Not Involved in Adherence of Enteropathogenic
Escherichia coli
or Virulence of
Citrobacter rodentium
Authors: Tatsuno Ichiro , Mundy Rosanna , Frankel Gad , Chong Yuwen , Phillips Alan D. , Torres Alfredo G. , Kaper James B.
Published: December 2005

Using the enteropathogenic
Escherichia coli
(EPEC) genome sequence, we found that EPEC E2348/69 has an
lpfABCDE
gene cluster homologous (about 60% identical at the protein level) to the
Salmonella
long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the

lpfABCD
E2

3

genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPEC

ΔlpfABCD
E23

strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the

lpf
E23

gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an
lpf
gene cluster in
Citrobacter rodentium
strain ICC168 (

lpf
cr

). A Δ

lpfA
cr

mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type
C. rodentium
strain and its

lpfA
cr

isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis,

lpf
cr

is not required for
C. rodentium
virulence in either the C3H/HeJ or C57BL/6 mouse model.
Differential Regulation of Cytokine Production by CD1d-Restricted NKT Cells in Response to Superantigen Staphylococcal Enterotoxin B Exposure
Authors: Ragin Melanie J. , Sahu Nisebita , August Avery
Published: December 2005

NKT cells are a heterogeneous population characterized by the ability to rapidly produce cytokines, such as interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-γ) in response to infections by viruses, bacteria, and parasites. The bacterial superantigen staphylococcal enterotoxin B (SEB) interacts with T cells bearing the Vβ3, -7, or -8 T-cell receptors, inducing their expansion and cytokine secretion, leading to death in some cases due to cytokine poisoning. The majority of NKT cells bear the Vβ7 or -8 T-cell receptor, suggesting that they may play a role in regulating this response. Using mice lacking NKT cells (CD1d−/−and Jα18−/−mice), we set out to identify the role of these cells in T-cell expansion, cytokine secretion, and toxicity induced by exposure to SEB. We find that Vβ8+CD4+T-cell populations similarly expand in wild-type (WT) and NKT cell-null mice and that NKT cells did not regulate the secretion of IL-2. By contrast, these cells positively regulated the secretion of IL-4 and IFN-γ production and negatively regulated the secretion of tumor necrosis factor alpha (TNF-α). However, this negative regulation of TNF-α secretion by NKT cells provides only a minor protective effect on SEB-mediated shock in WT mice compared to mice lacking NKT cells. These data suggest that NKT cells may regulate the nature of the cytokine response to exposure to the superantigen SEB and may act as regulatory T cells during exposure to this superantigen.
Identification of a Surface Protein of
Streptococcus suis
and Evaluation of Its Immunogenic and Protective Capacity in Pigs
Authors: Li Yuanyi , Martinez Gabriela , Gottschalk Marcelo , Lacouture Sonia , Willson Philip , Dubreuil J. Daniel , Jacques Mario , Harel Josee
Published: December 2005

A
Streptococcus suis
surface protein reacting with convalescent-phase sera from pigs clinically infected by
S. suis
type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membrane-anchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of
S. suis
Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on
S. suis
. The Sao-specific antibody reacts with cell lysates of 28 of 33
S. suis
serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in
S. suis
species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.
Identification of CsrC and Characterization of Its Role in Epithelial Cell Invasion in
Salmonella enterica
Serovar Typhimurium
Authors: Fortune Doreen R. , Suyemoto Mitsu , Altier Craig
Published: December 2005

The csr regulatory system of
Salmonella
regulates the expression of the genes of
Salmonella
pathogenicity island 1 (SPI1) required for the invasion of epithelial cells. This system consists of the posttranscriptional regulator CsrA and an untranslated regulatory RNA, CsrB, that opposes the action of CsrA. Here we identify and characterize the role of a second regulatory RNA, CsrC, whose ortholog was discovered previously in
Escherichia coli
. We show that a mutant of
csrC
has only mild defects in invasion and the expression of SPI1 genes, as does a mutant of
csrB
, but that a double
csrB csrC
mutant is markedly deficient in these properties, suggesting that the two regulatory RNAs play redundant roles in the control of invasion. We further show that CsrC, like CsrB, is controlled by the BarA/SirA two-component regulator but that a
csrB csrC
mutant exhibits a loss of invasion equivalent to that of a
barA
or
sirA
mutant, indicating that much of the effect of BarA/SirA on invasion functions through its control of CsrB and CsrC. In addition to their control by BarA/SirA, each regulatory RNA is also controlled by other components of the csr system. The loss of
csrB
was found to increase the level of CsrC by sevenfold, while the loss of
csrC
increased CsrB by nearly twofold. Similarly, the overexpression of
csrA
increased CsrC by nearly 11-fold and CsrB by 3-fold and also significantly increased the stability of both RNAs.
T-Helper-2 Cytokine Responses to Sj97 Predict Resistance to Reinfection withSchistosoma japonicum
Authors: Leenstra Tjalling , Acosta Luz P. , Wu Hai-Wei , Langdon Gretchen C. , Solomon Julie S. , Manalo Daria L. , Su Li , Jiz Mario , Jarilla Blanca , Pablo Archie O. , McGarvey Stephen T. , Olveda Remigio M. , Friedman Jennifer F. , Kurtis Jonathan D.
Published: December 2005

Although schistosomiasis is effectively treated with Praziquantel, rapid reinfection with rebound morbidity precludes effective control based on chemotherapy alone and justifies current efforts to develop vaccines for these parasites. Using a longitudinal treatment-reinfection study design with 616 participants 7 to 30 years of age, we evaluated the relationship between cytokine responses toSchistosoma japonicumsoluble adult worm extract (SWAP), Sj97, Sj22.6, and Sj67, measured 4 weeks after treatment with Praziquantel, and resistance to reinfection in a population from Leyte, The Philippines, whereS. japonicumis endemic.S. japonicumtransmission was high: 54.8% and 91.1% were reinfected within 6 and 18 months, respectively. A Th2 bias in the following cytokine ratios, interleukin-4 (IL-4)/IL-12, IL-5/IL-12, IL-13/IL-12, IL-4/gamma-IFN (IFN-γ), IL-5/IFN-γ, and IL-13/IFN-γ, in response to SWAP predicted a 1.4- to 2.9-month longer time to reinfection (P< 0.05) and a 27 to 55% lower intensity of reinfection (P< 0.05). Similarly, a Th2 bias in response to Sj97 predicted a 1.6- to 2.2-month longer time to reinfection (P< 0.05) and a 30 to 41% lower intensity of reinfection (P< 0.05). Only a high IL-5/IL-10 ratio in response to Sj22.6 predicted a 3.0-month-longer time to reinfection (P= 0.03). Cytokine responses to Sj67 were not associated with protection. In a large population-based treatment-reinfection study we found that Th2 responses to SWAP and Sj97 consistently predicted resistance to reinfection. These findings underscore Th2-type immune responses as central in human resistance toS. japonicumand support Sj97 as a leading vaccine candidate for this parasite.
Complement and Fc Function Are Required for Optimal Antibody Prophylaxis againstPneumocystis cariniiPneumonia
Authors: Wells Jesse , Haidaris Constantine G. , Wright Terry W. , Gigliotti Francis
Published: December 2005

Pneumocystis cariniiis an opportunistic fungal pathogen that causesP. cariniipneumonia (PCP) in the immunocompromised host. We investigated the role of antibody Fc-mediated function in passive prophylaxis against the development of PCP in SCID mice. By comparison of anti-mouseP. cariniiimmunoglobulin G1 monoclonal antibody (MAb) 4F11(G1) and its F(ab′)2 derivative in an intranasal immunoprophylaxis model, we determined that Fc-mediated function is required for maximum effect of this antibody. Comparison of efficacy of antibody prophylaxis in SCID mice depleted of complement to that in nondepleted mice demonstrated that complement fixation by MAb 4F11(G1) is also necessary for optimal effect of passively administered antibody, although residual protection was observed in complement-depleted SCID mice. The necessity of complement for optimal PCP prophylaxis by MAb 4F11(G1) suggests that complement may play a role in antibody-mediated protection against development of PCP.
Effect of Inactivation of the Global Oxidative Stress Regulator
oxyR
on the Colonization Ability of
Escherichia coli
O1:K1:H7 in a Mouse Model of Ascending Urinary Tract Infection
Authors: Johnson James R. , Clabots Connie , Rosen Henry
Published: December 2005

To survive within the host urinary tract,
Escherichia coli
strains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of
Escherichia coli
as a possible virulence factor in UTI by determining the impact of
oxyR
inactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47
phox−/−
) mice. The
oxyR
and
oxyS
genes of wild-type
E. coli
strain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an
oxyRS
mutant. During in vitro growth in broth or human urine, the
oxyRS
mutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and
oxyRS
mutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H
2
O
2
, which, like the altered growth kinetics observed with
oxyRS
inactivation, were reversed by restoration of
oxyR
on a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its
oxyRS
mutant (by >1 log
10
) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an
oxyR
-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47
phox−/−
C57BL mice were similarly susceptible, and the
oxyR
mutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47
phox
genotype, as in CBA/J mice. Within the
E. coli
Reference collection, 94% of strains were positive for
oxyR
. These findings fulfill the second and third of Koch's molecular postulates for
oxyR
as a candidate virulence-facilitating factor in
E. coli
and indicate that
oxyR
is a broadly prevalent potential target for future preventive interventions against UTI due to
E. coli
. They also suggest that neutrophil phagocyte oxidase is not critical for defense against
E. coli
UTI and that the major oxidative stresses against which OxyR protects
E. coli
within the host milieu are not phagocyte derived.
Roles of Macrophages and Neutrophils in the Early Host Response toBacillus anthracisSpores in a Mouse Model of Infection
Authors: Cote Christopher K. , Van Rooijen Nico , Welkos Susan L.
Published: December 2005

The development of new approaches to combat anthrax requires that the pathogenesis and host response toBacillus anthracisspores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection byB. anthracisin a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminatedB. anthracisstrain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the saline-pretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection byB. anthracisspores.
Two Domains within theMycoplasma hyopneumoniaeCilium Adhesin Bind Heparin
Authors: Jenkins Cheryl , Wilton Jody L. , Minion F. Chris , Falconer Linda , Walker Mark J. , Djordjevic Steven P.
Published: December 2005

Mycoplasma hyopneumoniaeis the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide.M. hyopneumoniaeadheres to and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (Mhp183, P97) is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognized step in pathogenesis, knowledge of glycosaminoglycan-binding proteins inM. hyopneumoniaeis lacking. However, heparin and other sulfated polysaccharides are known to block the binding ofM. hyopneumoniaeto purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for the ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N terminus and 301 amino acids of the C terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27 ± 0.02 μM and 1.89 ± 0.33 μM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multifunctional role in the adherence ofM. hyopneumoniaeto host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.
CpG-Oligodeoxynucleotide Is a Potent Adjuvant with anEntamoeba histolyticaGal-Inhibitable Lectin Vaccine against Amoebic Liver Abscess in Gerbils
Authors: Ivory Catherine P. A. , Keller Kathy , Chadee Kris
Published: December 2005

The protozoan parasiteEntamoeba histolyticacauses invasive amoebiasis characterized by amoebic dysentery and liver abscesses (ALA). TheE. histolyticagalactose/N-acetyl-d-galactosamine-inhibitable lectin (Gal-lectin), an immunogenic surface molecule involved in colonization and invasion, is a promising vaccine candidate against amoebiasis. Gal-lectin is known to induce Th1 cytokines in macrophages and spleen cells in vitro, and a Th1 response is thought to be protective against ALA. In this study, we report the use of cytosine guanine oligodeoxynucleotide (CpG-ODN) as adjuvant to augment Th1 responses against Gal-lectin in the gerbil model of ALA. Gerbils were vaccinated intramuscularly with the native Gal-lectin plus CpG-ODN or a paired non-CpG control GpC-ODN, and control gerbils received CpG-ODN alone. One week after the last boost gerbils were challenged intrahepatically with 106amoebae. Gerbils receiving CpG-ODN as adjuvant with Gal-lectin were completely protected against the development of ALA, whereas 50% of gerbils receiving GpC-ODN and Gal-lectin developed ALA and 85% of controls developed ALA. Stronger lymphoproliferation in response to the Gal-lectin and higher prechallenge titers of serum Gal-lectin-specific antibodies, capable of blocking amoebic adherence, were observed when CpG-ODN was used as adjuvant. Gerbils vaccinated with CpG-ODN and Gal-lectin also had significantly higher levels of gamma interferon, interleukin-12 (IL-12), and IL-2 mRNA than controls. These data indicate that CpG-ODN can enhance the Th1 responses, which improve the protective effects of Gal-lectin. This is the first report of the use of CpG as a potent Th1 adjuvant with Gal-lectin to increase protection against ALA formation.
Plasmid Interleukin-23 (IL-23), but Not Plasmid IL-27, Enhances the Protective Efficacy of a DNA Vaccine againstMycobacterium tuberculosisInfection
Authors: Wozniak Teresa M. , Ryan Anthony A. , Triccas James A. , Britton Warwick J.
Published: December 2005

Protection against intracellular pathogens such asMycobacterium tuberculosisrequires the development of Th1-like T-cell responses. This in turn is dependent on the pattern of cytokine produced from dendritic cells (DCs) after infection. Three heterodimeric cytokines, interleukin-12 (IL-12), IL-23, and IL-27, as well as IL-18, contribute to the differentiation and expansion of naive CD4+T cells. In this study we compared the effects of plasmids expressing both chains of IL-12, IL-23, or IL-27 as adjuvants for DNA immunization againstM. tuberculosisinfection. The genes encoding p19 and p40 chains of IL-23 or EBI3 and p28 chains of IL-27 were cloned on either side of a self-cleaving peptide from the FMDV2A protein. The secretion of functional cytokines from transfected cells was detected with bioassays. Supernatant from p2AIL-23-transfected cells induced the release of IL-17 from activated lymphocytes, confirming the presence of bioactive IL-23. Further, supernatant from p2AIL-27-transfected cells stimulated a significant increase in the proliferation of peptide-stimulated transgenic CD4+T cells. In initial experiments,M. tuberculosisinfection of DCs was more potent at inducing IL-12 and IL-23 secretion than infection with the vaccine strainMycobacterium bovisbacille Calmette-Guérin (BCG), and no significant upregulation of IL-27 was observed. Coimmunization of C57BL/6 mice with DNA expressingM. tuberculosisantigen 85B (Ag85B; DNA85B) and plasmids expressing IL-23 or IL-12 stimulated stronger Ag85B-specific T-cell proliferative and IFN-γ responses than DNA85B alone, whereas the addition of p2AIL-27 had no effect. Interestingly, DNA85B codelivered with p2AIL-12, but not p2AIL-23, reduced the immunoglobulin G antibody response. Both p2AIL-23 and p2AIL-12, but not p2AIL-27, enhanced the protective efficacy of DNA85B against aerosolM. tuberculosischallenge. Therefore, both p2AIL-23 and p2AIL-12 are valuable as cytokine adjuvants for increasing the protective antituberculosis immunity induced by DNA vaccines.
Dihydrolipoamide Acyltransferase Is Critical forMycobacterium tuberculosisPathogenesis
Authors: Shi Shuangping , Ehrt Sabine
Published: December 2005

Mycobacterium tuberculosishas evolved to persist in host macrophages, where it faces a nutrient-poor environment and is exposed to oxidative and nitrosative stress. To defend itself against oxidative/nitrosative stress,M. tuberculosisexpresses an NADH-dependent peroxidase and peroxynitrite reductase that is encoded byahpC,ahpD,lpd, anddlaT. In addition to its central role in the peroxynitrite reductase complex,dlaT(Rv2215) also encodes the E2 component of pyruvate dehydrogenase. Here we demonstrate that inactivation ofdlaTin the chromosome of H37Rv resulted in a mutant (H37RvΔdlaT) that displayed phenotypes associated with DlaT's role in metabolism and in defense against nitrosative stress. The H37RvΔdlaTstrain showed retarded growth in vitro and was highly susceptible to killing by acidified sodium nitrite. Mouse macrophages readily killed intracellular H37RvΔdlaTorganisms, and in micedlaTwas required for full virulence.
Identification of the Insulin-Like Growth Factor II Receptor as a Novel Receptor for Binding and Invasion by
Listeria monocytogenes
Authors: Gasanov Uta , Koina Craig , Beagley Kenneth W. , Aitken R. John , Hansbro Philip M.
Published: December 2005

The gram-positive bacterium
Listeria monocytogenes
causes a life-threatening disease known as listeriosis. The mechanism by which
L. monocytogenes
invades mammalian cells is not fully understood, but the processes involved may provide targets to prevent and treat listeriosis. Here, for the first time, we have identified the insulin-like growth factor II receptor (IGFIIR; also known as the cation-independent mannose 6-phosphate receptor
CI
M6PR or CD222) as a novel receptor for binding and invasion of
Listeria
species. Random peptide phage display was employed to select a peptide sequence by panning with immobilized
L. monocytogenes
cells; this peptide sequence corresponds to a sequence within the mannose 6-phosphate binding site of the IGFIIR. All
Listeria
spp. specifically bound the labeled peptide but not a control peptide, which was demonstrated using fluorescence spectrophotometry and fluorescence-activated cell sorting. Further evidence for binding of the receptor by
L. monocytogenes
and
L. innocua
was provided by affinity purification of the bovine IGFIIR from fetal calf serum by use of magnetic beads coated with cell preparations of
Listeria
spp. as affinity matrices. Adherence to and invasion of mammalian cells by
L. monocytogenes
was significantly inhibited by both the synthetic peptide and mannose 6-phosphate but not by appropriate controls. These observations indicate a role for the IGFIIR in the adherence and invasion of
L. monocytogenes
of mammalian cells, perhaps in combination with known mechanisms. Ligation of IGFIIR by
L. monocytogenes
may be a novel mechanism that contributes to the regulation of infectivity, possibly in combination with other mechanisms.
TheompAGene inChlamydia trachomatisDiffers in Phylogeny and Rate of Evolution from Other Regions of the Genome
Authors: Brunelle Brian W. , Sensabaugh George F.
Published: December 2005

Strains ofChlamydia trachomatisare classified into serovars based on nucleotide sequence differences inompA, the gene that encodes the major outer membrane protein. Phylogenetic characterization of strains based onompA, however, results in serovar groupings that are inconsistent with the distinguishing features ofC. trachomatispathobiology, e.g., tissue tropisms and disease presentation. We have compared nucleotide sequences at multiple sites distributed around the chlamydial genome from 18 strains representing 16 serovars; sampled regions included genes encoding housekeeping enzymes (totaling 2,073 bp), intergenic noncoding segments (1,612 bp), and a gene encoding a second outer membrane protein (porB; 1,023 bp), with theompAsequence (1,194 bp) used for reference. These comparative analyses revealed substantial variation in nucleotide substitution patterns among the sampled regions, with average pairwise sequence differences ranging from 0.15% for the housekeeping genes to 12.1% forompA. Phylogenetic characterization of the sampled genomic sequences yielded a strongly supported tree that divides the strains into groupings consistent withC. trachomatisbiology and which has a topology quite distinct from theompAtree. This phylogenetic incongruity can be accounted for by recombination of theompAgene between different genomic backgrounds. We found, however, no evidence of recombination within or between any of the sampled regions around theC. trachomatisgenome apart fromompA. Parallel analysis of published sequence data on four members of thepmpgene family are consistent with the phylogenetic analyses reported here.
Construction and Immunological Characterization of a Novel Nontoxic Protective Pneumolysin Mutant for Use in Future Pneumococcal Vaccines
Authors: Kirkham Lea-Ann S. , Kerr Alison R. , Douce Gill R. , Paterson Gavin K. , Dilts Deborah A. , Liu Dai-Fang , Mitchell Tim J.
Published: December 2005

Pneumolysin, the pore-forming toxin produced by
Streptococcus pneumoniae
, may have an application as an immunogenic carrier protein in future pneumococcal conjugate vaccines. Most of the 90
S. pneumoniae
serotypes identified produce pneumolysin; therefore, this protein may confer non-serotype-specific protection against pneumococcal infections such as pneumonia, meningitis, and otitis media. However, as pneumolysin is highly toxic, a nontoxic form of pneumolysin would be a more desirable starting point in terms of vaccine production. Previous pneumolysin mutants have reduced activity but retain residual toxicity. We have found a single amino acid deletion that blocks pore formation, resulting in a form of pneumolysin that is unable to form large oligomeric ring structures. This mutant is nontoxic at concentrations greater than 1,000 times that of the native toxin. We have demonstrated that this mutant is as immunogenic as native pneumolysin without the associated effects such as production of the inflammatory mediators interleukin-6 and cytokine-induced neutrophil chemoattractant KC, damage to lung integrity, and hypothermia in mice. Vaccination with this mutant protects mice from challenge with
S. pneumoniae
. Incorporation of this mutant pneumolysin into current pneumococcal vaccines may increase their efficacy.
Identification and Characterization of a Novel Bacterial Virulence Factor That Shares Homology with Mammalian Toll/Interleukin-1 Receptor Family Proteins
Authors: Newman Ruchi M. , Salunkhe Prabhakar , Godzik Adam , Reed John C.
Published: December 2005

Many important bacterial virulence factors act as mimics of mammalian proteins to subvert normal host cell processes. To identify bacterial protein mimics of components of the innate immune signaling pathway, we searched the bacterial genome database for proteins with homology to the Toll/interleukin-1 receptor (TIR) domain of the mammalian Toll-like receptors (TLRs) and their adaptor proteins. A previously uncharacterized gene, which we have namedtlpA(for TIR-like protein A), was identified in theSalmonella entericaserovar Enteritidis genome that is predicted to encode a protein resembling mammalian TIR domains, We show that overexpression of TlpA in mammalian cells suppresses the ability of mammalian TIR-containing proteins TLR4, IL-1 receptor, and MyD88 to induce the transactivation and DNA-binding activities of NF-κB, a downstream target of the TIR signaling pathway. In addition, TlpA mimics the previously characterizedSalmonellavirulence factor SipB in its ability to induce activation of caspase-1 in a mammalian cell transfection model. Disruption of the chromosomaltlpAgene rendered a virulent serovar Enteritidis strain defective in intracellular survival and IL-1β secretion in a cell culture infection model using human THP1 macrophages. Bacteria with disruptedtlpAalso displayed reduced lethality in mice, further confirming an important role for this factor in pathogenesis. Taken together, our findings demonstrate that the bacterial TIR-like protein TlpA is a novel prokaryotic modulator of NF-κB activity and IL-1β secretion that contributes to serovar Enteritidis virulence.
The Asymptomatic BacteriuriaEscherichia coliStrain 83972 Outcompetes UropathogenicE. coliStrains in Human Urine
Authors: Roos Viktoria , Ulett Glen C. , Schembri Mark A. , Klemm Per
Published: December 2005

Escherichia coliis the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenicE. coli(UPEC), which causes symptomatic urinary tract infections (UTI), very little is known about the mechanisms by which these strains colonize the human urinary tract. The prototype ABUE. colistrain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. Deliberate colonization of UTI-susceptible individuals withE. coli83972 has been used successfully as an alternative approach for the treatment of patients who are refractory to conventional therapy. Colonization with strain 83972 appears to prevent infection with UPEC strains in such patients despite the fact that this strain is unable to express the primary adhesins involved in UTI, viz. P and type 1 fimbriae. Here we investigated the growth characteristics ofE. coli83972 in human urine and show that it can outcompete a representative spectrum of UPEC strains for growth in urine. The unique ability of ABUE. coli83972 to outcompete UPEC in urine was also demonstrated in a murine model of human UTI, confirming the selective advantage over UPEC in vivo. Comparison of global gene expression profiles ofE. coli83972 grown in lab medium and human urine revealed significant differences in expression levels in the two media; significant down-regulation of genes encoding virulence factors such as hemolysin, lipid A, and capsular polysaccharides was observed in cells grown in urine. Clearly, divergent abilities of ABUE. coliand UPEC to exploit human urine as a niche for persistence and survival suggest that these key differences may be exploited for preventative and/or therapeutic approaches.
Microbial Pattern Recognition Receptors Mediate M-Cell Uptake of a Gram-Negative Bacterium
Authors: Tyrer Peter , Foxwell A. Ruth , Cripps Allan W. , Apicella Michael A. , Kyd Jennelle M.
Published: December 2005

The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and α5β1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and α5β1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed α5β1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.
Fusobacterium nucleatum
Transports Noninvasive
Streptococcus cristatus
into Human Epithelial Cells
Authors: Edwards Andrew M. , Grossman Tracy J. , Rudney Joel D.
Published: December 2005

Analysis of human buccal epithelial cells frequently reveals an intracellular polymicrobial consortium of bacteria. Although several oral bacteria have been demonstrated to invade cultured epithelial cells, several others appear unable to internalize. We hypothesized that normally noninvasive bacteria may gain entry into epithelial cells via adhesion to invasive bacteria.
Fusobacterium nucleatum
is capable of binding to and invading oral epithelial cells. By contrast,
Streptococcus cristatus
binds weakly to host cells and is not internalized.
F. nucleatum
and
S. cristatus
coaggregate strongly via an arginine-sensitive interaction. Coincubation of KB or TERT-2 epithelial cells with equal numbers of
F. nucleatum
and
S. cristatus
bacteria led to significantly increased numbers of adherent and internalized streptococci.
F. nucleatum
also promoted invasion of KB cells by other oral streptococci and
Actinomyces naeslundii
. Dissection of fusobacterial or streptococcal adhesive interactions by using sugars, amino acids, or antibodies demonstrated that this phenomenon is due to direct attachment of
S. cristatus
to adherent and invading
F. nucleatum
. Inhibition of
F. nucleatum
host cell attachment and invasion with galactose, or fusobacterial-streptococcal coaggregation by the arginine homologue
l
-canavanine, abrogated the increased
S. cristatus
adhesion to, and invasion of, host cells. In addition, polyclonal antibodies to
F. nucleatum
, which inhibited fusobacterial attachment to both KB cells and
S. cristatus
, significantly decreased invasion by both species. Similar decreases were obtained when epithelial cells were pretreated with cytochalasin D, staurosporine, or cycloheximide. These studies indicate that
F. nucleatum
may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly.
CD4+-T-Cell Effector Functions and Costimulatory Requirements Essential for Surviving Mucosal Infection withCitrobacter rodentium
Authors: Bry Lynn , Brigl Manfred , Brenner Michael B.
Published: December 2005

Citrobacter rodentiumcauses an attaching and effacing infection of the mouse colon. Surprisingly, protective adaptive immunity against this mucosal pathogen requires a systemic T-cell-dependent antibody response. To define CD4+T-cell effector functions promoting this systemic defense of infected epithelial surfaces, studies were undertaken in weaning-age mice lacking costimulatory molecules CD28 or CD40L or cytokines gamma interferon (IFN-γ) or interleukin-4 (IL-4). Adoptive transfer of CD4+T cells from wild-type, CD28−/−, CD40L−/−, or IFN-γ−/−donors to CD4−/−recipients delineated functions of these CD4+T-cell-expressed molecules on the outcome of infection. Wild-type and IL-4−/−mice successfully resolved infection, while 70% of IFN-γ−/−mice survived. In contrast, all CD28−/−mice succumbed during acute infection. While fewer than half of CD40L−/−mice succumbed acutely, surviving mice failed to clear infection, resulting in progressive mucosal destruction, polymicrobial sepsis, and death 1 to 2 weeks later than in CD28−/−mice. Downstream of CD28-mediated effects, CD4+T-cell-expressed CD40L proved essential for generating acute pathogen-specific immunoglobulin M (IgM) and early IgG, which reduced pathogen burdens. However, deficiency of CD4+T-cell-expressed IFN-γ did not adversely impact survival or development of protective antibody in adoptively transferred CD4−/−recipients, though it impacted Th1 antibody responses. These findings demonstrate that CD4+T-cell-expressed CD40L promotes the rapid production of protective systemic antibody during acute infection, while deficiencies of IL-4 or of CD4+T-cell-expressed IFN-γ can be overcome. These findings have important implications for understanding the role of T-helper-cell responses during infections involving mucosal surfaces.
Identification of the
Vibrio vulnificus wbpP
Gene and Evaluation of Its Role in Virulence
Authors: Park Na Young , Lee Jeong Hyun , Kim Myung Won , Jeong Hee Gon , Lee Byung Cheol , Kim Tae Sung , Choi Sang Ho
Published: December 2005

A
wbpP
gene encoding a putative UDP-
N
-acetyl-
d
-glucosamine C
4
epimerase was identified and cloned from
Vibrio vulnificus
. The functions of the
wbpP
gene, assessed by the construction of an isogenic mutant and by evaluating its phenotype changes, demonstrated that WbpP is essential in both the pathogenesis and the capsular polysaccharide biosynthesis of
V. vulnificus
.
Association between Epithelial Cell Death and Invasion by Microspheres Conjugated to
Porphyromonas gingivalis
Vesicles with Different Types of Fimbriae
Authors: Inaba Hiroaki , Kawai Shinji , Kato Takahiro , Nakagawa Ichiro , Amano Atsuo
Published: December 2005

Microspheres
(MS) conjugated to
Porphyromonas gingivalis
vesicles with type
II fimbriae were the most efficient human epithelial cell invaders
among the six types. Cell death was induced by MS, though becoming less
frequent over time, with invasion efficiency partially related to cell
death and gingipains likely the major
cause.
Novel Three-Dimensional Organoid Model for Evaluation of the Interaction of Uropathogenic
Escherichia coli
with Terminally Differentiated Human Urothelial Cells
Authors: Smith Yarery C. , Grande Kerian K. , Rasmussen Susan B. , O'Brien Alison D.
Published: December 2005

Human bladder 5637 cells cultivated under microgravity conditions formed organoids that displayed characteristics of in vivo tissue-specific differentiation. Uropathogenic
Escherichia coli
(UPEC) strain CP9 colonized and penetrated the organoids and induced α-hemolysin-mediated exfoliation of uroepithelial cells. We propose these uro-organoids as models that simulate the interactions between UPEC and terminally differentiated human urothelium.
Immunization with Persistent Attenuated Δ
lpg2 Leishmania major
Parasites Requires Adjuvant To Provide Protective Immunity in C57BL/6 Mice
Authors: Kébaïer Chahnaz , Uzonna Jude E. , Beverley Stephen M. , Scott Phillip
Published: December 2005

Leishmania major
parasites lacking the GDP-mannose transporter, termed Δ
lpg2
parasites, fail to induce disease in mice but persist long-term. We previously found that Δ
lpg2
organisms protect BALB/c mice from virulent
L. major
challenge. In contrast, we report here that Δ
lpg2
parasites induce protective immunity in C57BL/6 mice only when administered with CpG-containing oligodeoxynucleotides, indicating that parasite persistence alone is not sufficient to maintain protective immunity to
L. major
.
Molecular Characterization of the
Escherichia coli
Asymptomatic Bacteriuria Strain 83972: the Taming of a Pathogen
Authors: Klemm Per , Roos Viktoria , Ulett Glen C. , Svanborg Catharina , Schembri Mark A.
Published: December 2005

Escherichia coli
83972 is a clinical asymptomatia bacteriuric isolate that is able to colonize the human urinary bladder without inducing an immune response. Here we demonstrate that one of the mechanisms by which this strain has become attenuated is through the mutation of its genes encoding type 1 and P fimbriae.
ON AN ELEMENTARY APPROACH TO THE LEBESGUE–NAGELL EQUATION
Authors: MOLLIN R. A.
Published: December 2005

We discuss the feasibility of an elementary solution to the Diophantine equation of the form x2 + D = yn, where D > 1, n ≥ 3 and x > 0, called the Lebesgue–Nagell equation, which has recently been solved for 1 ≤ D ≤ 100 in [1].