bluuebunny/crossref_metadata_2025 huggingface dataset:
Listeria monocytogenesFlagella Are Used for Motility, Not as Adhesins, To Increase Host Cell Invasion
Authors: O'Neil Heather S. , Marquis Hélène
Published: November 2006

Flagellar structures contribute to the virulence of multiple gastrointestinal pathogens either as the effectors of motility, as adhesins, or as a secretion apparatus for virulence factors.Listeria monocytogenesis a food-borne, gram-positive pathogen that uses flagella to increase the efficiency of epithelial cell invasion (A. Bigot, H. Pagniez, E. Botton, C. Frehel, I. Dubail, C. Jacquet, A. Charbit, and C. Raynaud, Infect. Immun.73:5530-5539, 2005; L. Dons, E. Eriksson, Y. Jin, M. E. Rottenberg, K. Kristensson, C. N. Larsen, J. Bresciani, and J. E. Olsen, Infect. Immun.72:3237-3244, 2004). In this study, we aimed to elucidate the mechanism by which flagella contribute toL. monocytogenesinvasion. To examine the role of flagella as adhesins, invasion and adhesion assays were performed with flagellated motile and nonmotile bacteria and nonflagellated bacteria. We observed that flagellated but nonmotile bacteria do not adhere to or invade human epithelial cells more efficiently than nonflagellated bacteria. These results indicated that flagella do not function as adhesins to enhance the adhesion ofL. monocytogenesto targeted host cells. Instead, it appears that motility is important for tissue culture invasion. Furthermore, we tested whether motility contributes to early colonization of the gastrointestinal tract using a competitive index assay in which mice were infected orally with motile and nonmotile bacteria in a 1:1 ratio. Differential bacterial counts demonstrated that motile bacteria outcompete nonmotile bacteria in the colonization of the intestines at early time points postinfection. This difference is also reflected in invasion of the liver 12 h later, suggesting that flagellum-mediated motility enhancesL. monocytogenesinfectivity soon after bacterial ingestion in vivo.
Antibodies Enhance Interaction of
Vibrio cholerae
with Intestinal M-Like Cells
Authors: Blanco Luz P. , DiRita Victor J.
Published: November 2006

Intestinal M cells bear a receptor for secretory immunoglobulin A (IgA) (sIgA) facing the lumen of the epithelial surfaces. Cells bearing this receptor are also found throughout an experimental monolayer consisting of polarized Caco-2 cells, a colon adenocarcinoma cell line. The presence of antibodies (mainly sIgA) in the lumen of the small intestine led us to explore the participation of the sIgA receptor and antibodies in the interaction of Caco-2-associated M-like cells with the mucosal pathogen
Vibrio cholerae
. Here, we demonstrate that sIgA antibodies isolated from pooled healthy human colostrums, as well as IgG from pooled healthy human serum, can recognize
V. cholerae
. Furthermore, opsonization enhances M-like-cell transcytosis of
V. cholerae
strains. We also show that the cholera toxin (CT) receptor ganglioside GM
1
colocalizes with the sIgA receptor in cells of the epithelial monolayer. Both sIgA and IgG antibodies compete for the attachment of soluble CT subunit B to immobilized GM
1
. Our results indicate that in this in vitro model system of intestinal epithelia, human sIgA and IgG contribute to the uptake of
V. cholerae
by M-like cells, probably through an interaction with GM
1
. Our results support previous findings of others showing that sIgA can act as an endogenous adjuvant and that sIgA is important for the antigen-sampling function of M cells.
Early Neutrophil Recruitment and Aggregation in the Murine Lung Inhibit Germination ofAspergillus fumigatusConidia
Authors: Bonnett Colin R. , Cornish E. Jean , Harmsen Allen G. , Burritt James B.
Published: November 2006

Several types of polymorphonuclear neutrophil (PMN) deficiency are a predisposing condition for fatalAspergillus fumigatusinfection. In order to study the defensive role of PMNs in the lungs, with particular reference to PMN recruitment and antimicrobial oxidant activity, responses to pulmonary instillation ofA. fumigatusconidia were examined. Responses in BALB/c and C57BL/6 mice were compared with those in CXCR2−/−and gp91phox−/−mice, which are known to have delayed recruitment of PMN to the lungs in response to inflammatory stimuli and inactive NADPH oxidase, respectively. In BALB/c mice, PMNs were recruited to the lungs and formed oxidase-active aggregates with conidia, which inhibited germination. In C57BL/6, gp91phox−/−, and CXCR2−/−mice, PMN recruitment was slower and there was increased germination compared to that in BALB/c mice at 6 and 12 h. In gp91phox−/−mice, germination was extensive in PMN aggregates but negligible in alveolar macrophages (AM). Lung sections taken at 6 and 48 h from BALB/c mice showed PMN accumulation at peribronchiolar sites but no germinating conidia. Those from C57BL/6 and CXCR2−/−mice showed germinating conidia at 6 h but not at 48 h and few inflammatory cells. In contrast, those from gp91phox−/−mice showed germination at 6 h with more-extensive hyphal proliferation and tissue invasion at 48 h. These results indicate that when the lungs are exposed to large numbers of conidia, in addition to the phagocytic activity of AM, early PMN recruitment and formation of oxidative-active aggregates are essential in preventing germination ofA. fumigatusconidia.
Caveolin-1-Deficient Mice Show Defects in Innate Immunity and Inflammatory Immune Response during
Salmonella enterica
Serovar Typhimurium Infection
Authors: Medina Freddy A. , de Almeida Cecilia J. , Dew Elliott , Li Jiangwei , Bonuccelli Gloria , Williams Terence M. , Cohen Alex W. , Pestell Richard G. , Frank Philippe G. , Tanowitz Herbert B. , Lisanti Michael P.
Published: November 2006

A number of studies have shown an association of pathogens with caveolae. To this date, however, there are no studies showing a role for caveolin-1 in modulating immune responses against pathogens. Interestingly, expression of caveolin-1 has been shown to occur in a regulated manner in immune cells in response to lipopolysaccharide (LPS). Here, we sought to determine the role of caveolin-1 (Cav-1) expression in
Salmonella
pathogenesis. Cav-1
−/−
mice displayed a significant decrease in survival when challenged with
Salmonella enterica
serovar Typhimurium. Spleen and tissue burdens were significantly higher in Cav-1
−/−
mice. However, infection of Cav-1
−/−
macrophages with serovar Typhimurium did not result in differences in bacterial invasion. In addition, Cav-1
−/−
mice displayed increased production of inflammatory cytokines, chemokines, and nitric oxide. Regardless of this, Cav-1
−/−
mice were unable to control the systemic infection of
Salmonella
. The increased chemokine production in Cav-1
−/−
mice resulted in greater infiltration of neutrophils into granulomas but did not alter the number of granulomas present. This was accompanied by increased necrosis in the liver. However, Cav-1
−/−
macrophages displayed increased inflammatory responses and increased nitric oxide production in vitro in response to
Salmonella
LPS. These results show that caveolin-1 plays a key role in regulating anti-inflammatory responses in macrophages. Taken together, these data suggest that the increased production of toxic mediators from macrophages lacking caveolin-1 is likely to be responsible for the marked susceptibility of caveolin-1-deficient mice to
S. enterica
serovar Typhimurium.
Monocyte Chemoattractant Protein 1 Does Not Contribute to Protective Immunity against Pneumococcal Pneumonia
Authors: Dessing Mark C. , de Vos Alex F. , Florquin Sandrine , van der Poll Tom
Published: November 2006

To determine the role of monocyte chemoattractant protein 1 (MCP-1) during pneumococcal pneumonia, MCP-1 knockout and wild-type mice were infected withStreptococcus pneumoniae. Pulmonary MCP-1 levels were strongly correlated to bacterial loads in wild-type mice. However, MCP-1 knockout and wild-type mice were indistinguishable with respect to bacterial growth, inflammatory responses, and lethality.
Dual Roles of
Helicobacter pylori
NapA in Inducing and Combating Oxidative Stress
Authors: Wang Ge , Hong Yang , Olczak Adriana , Maier Susan E. , Maier Robert J.
Published: November 2006

Neutrophil-activating protein (NapA) has been well documented to play roles in human neutrophil recruitment and in stimulating host cell production of reactive oxygen intermediates (ROI). A separate role for NapA in combating oxidative stress within
H. pylori
was implied by studies of various
H. pylori
mutant strains. Here, physiological analysis of a
napA
strain was the approach used to assess the iron-sequestering and stress resistance roles of NapA, its role in preventing oxidative DNA damage, and its importance to mouse colonization. The
napA
strain was more sensitive to oxidative stress reagents and to oxygen, and it contained fourfold more intracellular free iron and more damaged DNA than the parent strain. Pure, iron-loaded NapA bound to DNA, but native NapA did not, presumably linking iron levels sensed by NapA to DNA damage protection. Despite its in vitro phenotype of sensitivity to oxidative stress, the
napA
strain showed normal (like that of the wild type) mouse colonization efficiency in the conventional in vivo assay. By use of a modified mouse inoculation protocol whereby nonviable
H. pylori
is first inoculated into mice, followed by (live) bacterial strain administration, an in vivo role for NapA in colonization efficiency could be demonstrated. NapA is the critical component responsible for inducing host-mediated ROI production, thus inhibiting colonization by the
napA
strain. An animal colonization experiment with a mixed-strain infection protocol further demonstrated that the
napA
strain has significantly decreased ability to survive when competing with the wild type.
H. pylori
NapA has unique and separate roles in gastric pathogenesis.
Identification of Potential Virulence Determinants by
Himar1
Transposition of Infectious
Borrelia burgdorferi
B31
Authors: Botkin Douglas J. , Abbott April N. , Stewart Philip E. , Rosa Patricia A. , Kawabata Hiroki , Watanabe Haruo , Norris Steven J.
Published: November 2006

Lyme disease
Borrelia
organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious
Borrelia burgdorferi
, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in
B. burgdorferi
. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted
Himar1
transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative
fliG-1-
encoded flagellar motor switch protein and the
guaB-
encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious
B. burgdorferi
is feasible and will be of value in studying the pathogenesis of Lyme disease
Borrelia
.
Potential Source of
Francisella tularensis
Live Vaccine Strain Attenuation Determined by Genome Comparison
Authors: Rohmer Laurence , Brittnacher Mitchell , Svensson Kerstin , Buckley Danielle , Haugen Eric , Zhou Yang , Chang Jean , Levy Ruth , Hayden Hillary , Forsman Mats , Olson Maynard , Johansson Anders , Kaul Rajinder , Miller Samuel I.
Published: November 2006

Francisella tularensis
is a bacterial pathogen that causes the zoonotic disease tularemia and is important to biodefense. Currently, the only vaccine known to confer protection against tularemia is a specific live vaccine strain (designated LVS) derived from a virulent isolate of
Francisella tularensis
subsp.
holarctica
. The origin and source of attenuation of this strain are not known. To assist with the design of a defined live vaccine strain, we sought to determine the genetic basis of the attenuation of LVS. This analysis relied primarily on the comparison between the genome of LVS and
Francisella tularensis holarctica
strain FSC200, which differ by only 0.08% of their nucleotide sequences. Under the assumption that the attenuation was due to a loss of function(s), only coding regions were examined in this comparison. To complement this analysis, the coding regions of two slightly more distantly related
Francisella tularensis
strains were also compared against the LVS coding regions. Thirty-five genes show unique sequence variations predicted to alter the protein sequence in LVS compared to the other
Francisella tularensis
strains. Due to these polymorphisms, the functions of 15 of these genes are very likely lost or impaired. Seven of these genes were demonstrated to be under stronger selective constraints, suggesting that they are the most probable to be the source of LVS attenuation and useful for a newly defined vaccine.
Borrelia burgdorferiComplement Regulator-Acquiring Surface Protein 1 of the Lyme Disease Spirochetes Is Expressed in Humans and Induces Antibody Responses Restricted to Nondenatured Structural Determinants
Authors: Rossmann Evelyn , Kitiratschky Veronique , Hofmann Heidelore , Kraiczy Peter , Simon Markus M. , Wallich Reinhard
Published: November 2006

Borrelia burgdorfericomplement regulator-acquiring surface protein 1 (CRASP-1), the dominant factor H and FHL-1-binding protein of the Lyme disease spirocheteB. burgdorferi, is implicated in pathogen persistence and was recently reported to be nonimmunogenic in humans. Here we show that serum samples from Lyme disease patients contain antibodies with exclusive specificity for nondenatured structural determinants of CRASP-1.
Prebiotic Galactooligosaccharides Reduce Adherence of EnteropathogenicEscherichia colito Tissue Culture Cells
Authors: Shoaf Kari , Mulvey George L. , Armstrong Glen D. , Hutkins Robert W.
Published: November 2006

Prebiotic oligosaccharides are thought to provide beneficial effects in the gastrointestinal tract of humans and animals by stimulating growth of selected members of the intestinal microflora. Another means by which prebiotic oligosaccharides may confer health benefits is via their antiadhesive activity. Specifically, these oligosaccharides may directly inhibit infections by enteric pathogens due to their ability to act as structural mimics of the pathogen binding sites that coat the surface of gastrointestinal epithelial cells. In this study, the ability of commercial prebiotics to inhibit attachment of microcolony-forming enteropathogenicEscherichia coli(EPEC) was investigated. The adherence of EPEC strain E2348/69 on HEp-2 and Caco-2 cells, in the presence of fructooligosaccharides, inulin, galactooligosaccharides (GOS), lactulose, and raffinose was determined by cultural enumeration and microscopy. Purified GOS exhibited the greatest adherence inhibition on both HEp-2 and Caco-2 cells, reducing the adherence of EPEC by 65 and 70%, respectively. In addition, the average number of bacteria per microcolony was significantly reduced from 14 to 4 when GOS was present. Adherence inhibition by GOS was dose dependent, reaching a maximum at 16 mg/ml. When GOS was added to adhered EPEC cells, no displacement was observed. The expression of BfpA, a bundle-forming-pilus protein involved in localized adherence, was not affected by GOS, indicating that adherence inhibition was not due to the absence of this adherence factor. In addition, GOS did not affect autoaggregation. These observations suggest that some prebiotic oligosaccharides may have antiadhesive activity and directly inhibit the adherence of pathogens to the host epithelial cell surface.
Induction of the Antimicrobial Peptide CRAMP in the Blood-Brain Barrier and Meninges after Meningococcal Infection
Authors: Bergman Peter , Johansson Linda , Wan Hong , Jones Allison , Gallo Richard L. , Gudmundsson Gudmundur H. , Hökfelt Tomas , Jonsson Ann-Beth , Agerberth Birgitta
Published: November 2006

Antimicrobial peptides are present in most living species and constitute important effector molecules of innate immunity. Recently, we and others have detected antimicrobial peptides in the brain. This is an organ that is rarely infected, which has mainly been ascribed to the protective functions of the blood-brain barrier (BBB) and meninges. Since the bactericidal properties of the BBB and meninges are not known, we hypothesized that antimicrobial peptides could play a role in these barriers. We addressed this hypothesis by infecting mice with the neuropathogenic bacterium
Neisseria meningitidis
. Brains were analyzed for expression of the antimicrobial peptide CRAMP by immunohistochemistry in combination with confocal microscopy. After infection, we observed induction of CRAMP in endothelial cells of the BBB and in cells of the meninges. To explore the functional role of CRAMP in meningococcal disease, we infected mice deficient of the CRAMP gene. Even though CRAMP did not appear to protect the brain from invasion of meningococci, CRAMP knockout mice were more susceptible to meningococcal infection than wild-type mice and exhibited increased meningococcal growth in blood, liver, and spleen. Moreover, we could demonstrate that carbonate, a compound that accumulates in the circulation during metabolic acidosis, makes meningococci more susceptible to CRAMP.
CXCL16 Regulates Cell-Mediated Immunity toSalmonella entericaSerovar Enteritidis via Promotion of Gamma Interferon Production
Authors: Fahy Olivier L. , Townley Scott L. , McColl Shaun R.
Published: November 2006

CXCL16 is a recently discovered multifaceted chemokine that has been shown not only to recruit activated T lymphocytes but also to play a direct role in the binding and phagocytosis of bacteria by professional antigen-presenting cells. In this study, we investigated the role of CXCL16 in vivo in the regulation of the immune response using a murine model ofSalmonella entericaserovar Enteritidis infection. The expression of CXCL16 was strongly upregulated in the spleens and livers of animals developing an immune response to a primary acute infection but not in the Peyer's patches. Animals developing a secondary response after reexposure to the bacteria displayed a similar pattern of expression. During the primary response, prior treatment with neutralizing antibodies to CXCL16 induced a significant increase in bacterial burden in the spleen and liver. The production of gamma interferon (IFN-γ) by the lymphocytes in the spleen was decreased by anti-CXCL16 treatment. In comparison, during the secondary response, anti-CXCL16 treatment also significantly increased bacterial burden in both the spleen and liver but had no effect on IFN-γ production. No role was found for CXCL16 in the production of antibody against SefA, a major surface antigen ofS. enteritidis. Together, these results demonstrate a role for CXCL16 in the control of bacterial colonization of target organs and, more specifically, in the regulation of the cell-mediated arm of the primary response toS. enteritidis.
CXCL10/Gamma Interferon-Inducible Protein 10-Mediated Protection againstLeishmania amazonensisInfection in Mice
Authors: Vasquez Rene E. , Soong Lynn
Published: November 2006

Leishmania amazonensiscan cause progressive disease in most inbred strains of mice. We have previously shown thatL. amazonensis-infected C57BL/6 mice have profound impairments in expression of proinflammatory cytokines and chemokines and in activation of antigen-specific CD4+T cells. These impairments are independent of interleukin-4 (IL-4) but partially due to IL-10 production. The precise mechanism of pathogenesis associated withL. amazonensisinfection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and for the eventual control of the infection. In this study, we examined the roles of CXCL10/gamma interferon-inducible protein 10 (IP-10) and CCL2/monocyte chemoattractant protein 1 (MCP-1) in the activation of the macrophage effector function in vitro and their efficacy in ameliorating infection in vivo. Bone marrow-derived macrophages of both BALB/c and C57BL/6 mice were treated with increasing concentrations of recombinant chemokines prior to infection with either stationary-phase promastigotes or tissue-derived amastigotes. We found that treatment with IP-10 or MCP-1 significantly reduced parasite burdens, in a dose-dependent manner, and triggered nitric oxide production. When susceptible C57BL/6 mice were injected locally with IP-10 followingL. amazonensisinfection, there was a significant delay in lesion development and a reduction in parasite burdens, accompanied by 7- and 3.5-fold increases in gamma interferon and IL-12 secretion, respectively, in restimulated lymph node cells. This study confirms that IP-10 plays a protective role in promoting the reduction of intracellular parasites and thereby opens new avenues for therapeutic control of nonhealing cutaneous leishmaniasis in the New World.
Helicobacter pylori
VacA Toxin Promotes Bacterial Intracellular Survival in Gastric Epithelial Cells
Authors: Terebiznik M. R. , Vazquez C. L. , Torbicki K. , Banks D. , Wang T. , Hong W. , Blanke S. R. , Colombo M. I. , Jones N. L.
Published: November 2006

Helicobacter pylori
colonizes the gastric epithelium of at least 50% of the world's human population, playing a causative role in the development of chronic gastritis, peptic ulcers, and gastric adenocarcinoma. Current evidence indicates that
H. pylori
can invade epithelial cells in the gastric mucosa. However, relatively little is known about the biology of
H. pylori
invasion and survival in host cells. Here, we analyze both the nature of and the mechanisms responsible for the formation of
H. pylori
's intracellular niche. We show that in AGS cells infected with
H. pylori
, bacterium-containing vacuoles originate through the fusion of late endocytic organelles. This process is mediated by the VacA-dependent retention of the small GTPase Rab7. In addition, functional interactions between Rab7 and its downstream effector, Rab-interacting lysosomal protein (RILP), are necessary for the formation of the bacterial compartment since expression of mutant forms of RILP or Rab7 that fail to bind each other impaired the formation of this unique bacterial niche. Moreover, the VacA-mediated sequestration of active Rab7 disrupts the full maturation of vacuoles as assessed by the lack of both colocalization with cathepsin D and degradation of internalized cargo in the
H. pylori
-containing vacuole. Based on these findings, we propose that the VacA-dependent isolation of the
H. pylori
-containing vacuole from bactericidal components of the lysosomal pathway promotes bacterial survival and contributes to the persistence of infection.
Effect of Different Forms of Adenylate Cyclase Toxin ofBordetella pertussison Protection Afforded by an Acellular Pertussis Vaccine in a Murine Model
Authors: Cheung Gordon Y. C. , Xing Dorothy , Prior Sandra , Corbel Michael J. , Parton Roger , Coote John G.
Published: November 2006

Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) ofBordetella pertussiswere compared for the ability to enhance protection againstB. pertussisin mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P> 0.05) protection againstB. pertussisin a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P< 0.05) reductions in bacterial numbers in the lungs after intranasal challenge compared with those for control mice. When administered with ACV, both CyaA and CyaA* further reduced bacterial numbers in the lungs of mice after intranasal challenge compared with those for ACV-immunized mice, but the enhanced protection was only significant (P< 0.05) with CyaA*. Coadministration of CyaA* with ACV caused a significant (P< 0.05) increase in immunoglobulin G2a antibody levels against pertactin compared with those in mice immunized with ACV alone. Spleen cells from mice immunized with ACV plus CyaA* secreted larger amounts of interleukin-5 (IL-5), IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did cells from mice immunized with ACV plus CyaA or ACV alone after stimulation in vitro with a mixture ofB. pertussisantigens. Spleen cells from mice immunized with ACV plus CyaA* also secreted larger amounts of IFN-γ and GM-CSF than did cells from mice immunized with CyaA* alone after stimulation in vitro with CyaA*. Macrophages from mice immunized with ACV plus CyaA* produced significantly (P< 0.05) higher levels of nitric oxide than did macrophages from mice immunized with CyaA* alone, ACV alone, or ACV plus CyaA after stimulation in vitro with a mixture ofB. pertussisantigens or heat-killedB. pertussiscells. These data suggest that the enhancement of protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses toB. pertussisantigens.
Mycobacterium avium
subsp.
paratuberculosis
PtpA Is an Endogenous Tyrosine Phosphatase Secreted during Infection
Authors: Bach Horacio , Sun Jim , Hmama Zakaria , Av-Gay Yossef
Published: November 2006

Adaptive gene expression in prokaryotes is mediated by protein kinases and phosphatases. These regulatory proteins mediate phosphorylation of histidine or aspartate in two-component systems and serine/threonine or tyrosine in eukaryotic and eukaryote-like protein kinase systems. The genome sequence of
Mycobacterium avium
subsp.
paratuberculosis
, the causative agent of Johne's disease, does not possess a defined tyrosine kinase. Nevertheless, it encodes for protein tyrosine phosphatases. Here, we report that Map1985, is a functional low-molecular tyrosine phosphatase that is secreted intracellularly upon macrophage infection. This finding suggests that Map1985 might contribute to the pathogenesis of
Mycobacterium avium
subsp.
paratuberculosis
by dephosphorylating essential macrophage signaling and/or adaptor molecules.
Mutation of the Maturase Lipoprotein Attenuates the Virulence ofStreptococcus equito a Greater Extent than Does Loss of General Lipoprotein Lipidation
Authors: Hamilton Andrea , Robinson Carl , Sutcliffe Iain C. , Slater Josh , Maskell Duncan J. , Davis-Poynter Nick , Smith Ken , Waller Andrew , Harrington Dean J.
Published: November 2006

Streptococcus equiis the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis ofS. equivirulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities ofS. equito colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (ΔprtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-typeS. equistrain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (Δlgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and Δlgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the Δlgt190-685mutant did still exhibit signs of disease. In contrast, only the ΔprtM138-213mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the Δlgt190-685mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.
Chlamydial Protease-Like Activity Factor Induces Protective Immunity against Genital Chlamydial Infection in Transgenic Mice That Express the Human HLA-DR4 Allele
Authors: Murthy Ashlesh K. , Cong Yu , Murphey Cathi , Guentzel M. Neal , Forsthuber Thomas G. , Zhong Guangming , Arulanandam Bernard P.
Published: November 2006

There is no licensed vaccine available against
Chlamydia trachomatis
, the leading cause of bacterial sexually transmitted disease. We have found that intranasal immunization with recombinant chlamydial protease-like activity factor (CPAF) induces CD4
+
T-cell- and gamma interferon (IFN-γ)-dependent protective immunity against murine genital chlamydial infection, thus making CPAF a viable vaccine candidate for further characterization. HLA-DR4 is the predominant allele involved in chlamydial antigen presentation to CD4
+
T cells in humans. We used engineered mice that lack endogenous major histocompatibility complex class II (MHC-II) alleles but express a human HLA allele (HLA-DR4 transgenic [tg] mice) to examine primary immune and CPAF-mediated responses against genital
Chlamydia muridarum
challenge. Upon primary bacterial exposure, HLA-DR4 tg mice developed
Chlamydia
-specific IFN-γ and antibody production and resolved the infection within 30 days, similar to challenged conventional C57BL/6 animals. Moreover,
C. muridarum
-challenged HLA-DR4 tg mice exhibited CPAF-specific antibody and IFN-γ production. Upon CPAF-plus-interleukin-12 (IL-12) vaccination, HLA-DR4 tg animals exhibited robust CPAF-specific IFN-γ production and elevated titers of anti-CPAF total antibody and immunoglobulin G2a (IgG2a) and lower titers of IgG2b and IgG1 antibodies. HLA-DR4 tg and C57BL/6 mice vaccinated with CPAF plus IL-12 resolved the primary genital chlamydial infection significantly earlier than mock-immunized animals, whereas similarly vaccinated MHC class II-deficient mice displayed minimal antigen-specific immune responses and failed to resolve the infection even at 30 days postchallenge. Together, these results demonstrate the importance of human HLA-DR4 molecules in the recognition and presentation of CPAF epitopes, leading to the generation of protective antichlamydial immunity and making these mice a valuable model for mapping HLA-DR4-restricted chlamydial epitopes.
A Linear Peptide Containing Minimal T- and B-Cell Epitopes ofPlasmodium falciparumCircumsporozoite Protein Elicits Protection against Transgenic Sporozoite Challenge
Authors: Calvo-Calle J. Mauricio , Oliveira Giane A. , Watta Carol Othoro , Soverow Jonathan , Parra-Lopez Carlos , Nardin Elizabeth H.
Published: November 2006

An effective malaria vaccine is needed to address the public health tragedy resulting from the high levels of morbidity and mortality caused byPlasmodiumparasites. The first protective immune mechanism identified in the irradiated sporozoite vaccine, the “gold standard” for malaria preerythrocytic vaccines, was sporozoite-neutralizing antibody specific for the repeat region of the surface circumsporozoite (CS) protein. Previous phase I studies demonstrated that a branched peptide containing minimal T- and B-cell epitopes ofPlasmodium falciparumCS protein elicited antirepeat antibody and CD4+-T-cell responses comparable to those observed in volunteers immunized with irradiatedP. falciparumsporozoites. The current study compares the immunogenicity of linear versus tetrabranched peptides containing the same minimal T- and B-cell epitopes, T1BT*, comprised of a CS-derived universal Th epitope (T*) synthesized in tandem with the T1 and B repeats ofP. falciparumCS protein. A simple 48-mer linear synthetic peptide was found to elicit antisporozoite antibody and gamma interferon-secreting T-cell responses comparable to the more complex tetrabranched peptides in inbred strains of mice. The linear peptide was also immunogenic in outbred nonhuman primates (Aotus nancymaae), eliciting antibody titers equivalent to those induced by tetrabranched peptides. Importantly, the 48-mer linear peptide administered in adjuvants suitable for human use elicited antibody-mediated protection against challenge with rodent malaria transgenic sporozoites expressingP. falciparumCS repeats. These findings support further evaluation of linear peptides as economical, safe, and readily produced malaria vaccines for the one-third of the world's population at risk of malaria infection.
Limited Cross-Reactivity among Domains of the
Plasmodium falciparum
Clone 3D7 Erythrocyte Membrane Protein 1 Family
Authors: Joergensen Louise , Turner Louise , Magistrado Pamela , Dahlbäck Madeleine A. , Vestergaard Lasse S. , Lusingu John P. , Lemnge Martha , Salanti Ali , Theander Thor G. , Jensen Anja T. R.
Published: November 2006

The
var
gene-encoded
Plasmodium falciparum
erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of
P. falciparum
clone 3D7 into groups A and B/A (category A) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains sharing cross-reactive antibody epitopes. Identification of groups of serological cross-reacting molecules is pivotal for the development of vaccines based on PfEMP1.
Helicobacter pylori
HP1512 Is a Nickel-Responsive NikR-Regulated Outer Membrane Protein
Authors: Davis Gregg S. , Flannery Erika L. , Mobley Harry L. T.
Published: November 2006

Helicobacter pylori
is dependent upon the production of the highly abundant and active metalloenzyme urease for colonization of the human stomach. Thus,
H. pylori
has an absolute requirement for the transition metal nickel, a required cofactor for urease. To investigate the contribution of genes that are factors in this process, microarray analysis comparing the transcriptome of wild-type
H. pylori
26695 cultured in brucella broth containing fetal calf serum (BBF) alone or supplemented with 100 μM NiCl
2
suggested that HP1512 is repressed in the presence of 100 μM supplemental nickel. When measured by comparative real-time quantitative PCR (qPCR), HP1512 transcription was reduced 43-fold relative to the value for the wild type when cultured in BBF supplemented with 10 μM NiCl
2
. When grown in unsupplemented BBF, urease activity of an HP1512::
cat
mutant was significantly reduced compared to the wild type, 4.9 ± 0.5 μmol/min/mg of protein (
n
= 7) and 17.1 ± 4.9 μmol/min/mg of protein (
n
= 13), respectively (
P
< 0.0001). In silico analysis of the HP1511-HP1512 (HP1511-1512) intergenic region identified a putative NikR operator upstream of HP1512. Gel shift analysis with purified recombinant NikR verified nickel-dependent binding of
H. pylori
NikR to the HP1511-1512 intergenic region. Furthermore, comparative real-time qPCR of four nickel-related genes suggests that mutation of HP1512 results in reduced intracellular nickel concentration relative to wild-type
H. pylori
26695. Taken together, these data suggest that HP1512 encodes a NikR-nickel-regulated outer membrane protein.
NikR Mediates Nickel-Responsive Transcriptional Repression of the
Helicobacter pylori
Outer Membrane Proteins FecA3 (HP1400) and FrpB4 (HP1512)
Authors: Ernst Florian D. , Stoof Jeroen , Horrevoets Wannie M. , Kuipers Ernst J. , Kusters Johannes G. , van Vliet Arnoud H. M.
Published: November 2006

The transition metal nickel plays an important role in gastric colonization and persistence of the important human pathogen
Helicobacter pylori
, as it is the cofactor of the abundantly produced acid resistance factor urease. Nickel uptake through the inner membrane is mediated by the NixA protein, and the expression of NixA is controlled by the NikR regulatory protein. Here we report that NikR also controls the nickel-responsive expression of the FecA3 (HP1400) and FrpB4 (HP1512) outer membrane proteins (OMPs), as well as the nickel-responsive expression of an ExbB-ExbD-TonB system, which may function in energization of outer membrane transport. Transcription and expression of the
frpB4
and
fecA3
genes were repressed by nickel in wild-type
H. pylori
26695, but they were independent of nickel and derepressed in an isogenic
nikR
mutant. Both the
frpB4
and
fecA3
genes were transcribed from a promoter directly upstream of their start codon. Regulation by NikR was mediated via nickel-dependent binding to specific operators overlapping either the +1 or −10 sequence in the
frpB4
and
fecA3
promoters, respectively, and these operators contained sequences resembling the proposed
H. pylori
NikR recognition sequence (TATWATT-N
11
-AATWATA). Transcription of the HP1339-1340-1341 operon encoding the ExbB2-ExbD2-TonB2 complex was also regulated by nickel and NikR, but not by Fur and iron. In conclusion,
H. pylori
NikR controls nickel-responsive expression of the HP1400 (FecA3) and HP1512 (FrpB4) OMPs. We hypothesize that these two NikR-regulated OMPs may participate in the uptake of complexed nickel ions and that this process is energized by the NikR-regulated ExbB2-ExbD2-TonB2 system, another example of the specific adaptation of
H. pylori
to the gastric lifestyle.
The 13C4 Monoclonal Antibody That Neutralizes Shiga Toxin Type 1 (Stx1) Recognizes Three Regions on the Stx1 B Subunit and Prevents Stx1 from Binding to Its Eukaryotic Receptor Globotriaosylceramide
Authors: Smith Michael J. , Carvalho Humberto M. , Melton-Celsa Angela R. , O'Brien Alison D.
Published: November 2006

The 13C4 monoclonal antibody (MAb) recognizes the B subunit of Stx1 (StxB1) and neutralizes the cytotoxic and lethal activities of Stx1. However, this MAb does not bind to the B polypeptide of Stx2, despite the 73% amino acid sequence similarity between StxB1 and StxB2. When we compared the amino acid sequences of StxB1 and StxB2, we noted three regions of dissimilarity (amino acids 1 to 6, 25 to 32, and 54 to 61) located near each other on the crystal structure of StxB1. To identify the 13C4 epitope, we generated seven Stx1/Stx2 B chimeric polypeptides that contained one, two, or three of the dissimilar StxB1 regions. The 13C4 MAb reacted strongly with StxB1 and the triple-chimeric B subunit but not with the other chimeras. Mice immunized with the triple-chimeric B subunit survived a lethal challenge with Stx1 but not Stx2, substantiating the identified regions as the 13C4 MAb epitope and suggesting that the incorporation of this epitope into StxB2 altered sites necessary for anti-Stx2-neutralizing Ab production. Next, single amino acid substitutions were made in StxB1 to mimic Stx1d, a variant not recognized by the 13C4 MAb. The 13C4 MAb reacted strongly to StxB1 with the T1A or G25A mutations but not with the N55T change. Finally, we found that the 13C4 MAb blocked the binding of Stx1 to its receptor, globotriaosyl ceramide. Taken together, these results indicate that the 13C4 MAb prevents the interaction of Stx1 with its receptor by binding three nonlinear regions of the molecule that span receptor recognition sites on StxB1, one of which includes the essential residue 55N.
Identification of MglA-Regulated Genes Reveals Novel Virulence Factors in
Francisella tularensis
Authors: Brotcke Anna , Weiss David S. , Kim Charles C. , Chain Patrick , Malfatti Stephanie , Garcia Emilio , Monack Denise M.
Published: November 2006

The facultative intracellular bacterium
Francisella tularensis
causes the zoonotic disease tularemia.
F. tularensis
resides within host macrophages in vivo, and this ability is essential for pathogenesis. The transcription factor MglA is required for the expression of several
Francisella
genes that are necessary for replication in macrophages and for virulence in mice. We hypothesized that the identification of MglA-regulated genes in the
Francisella
genome by transcriptional profiling of wild-type and
mglA
mutant bacteria would lead to the discovery of new virulence factors utilized by
F. tularensis
. A total of 102 MglA-regulated genes were identified, the majority of which were positively regulated, including all of the
Francisella
pathogenicity island (FPI) genes. We mutated novel MglA-regulated genes and tested the mutants for their ability to replicate and induce cytotoxicity in macrophages and to grow in mice. Mutations in MglA-regulated genes within the FPI (
pdpB
and
cds2
) as well as outside the FPI (FTT0989,
oppB
, and FTT1209c) were either attenuated or hypervirulent in macrophages compared to the wild-type strain. All of these mutants exhibited decreased fitness in vivo in competition experiments with wild-type bacteria. We have identified five new
Francisella
virulence genes, and our results suggest that characterizations of additional MglA-regulated genes will yield further insights into the pathogenesis of this bacterium.
Surfactant Protein D Increases Fusion ofMycobacterium tuberculosis- Containing Phagosomes with Lysosomes in Human Macrophages
Authors: Ferguson J. Scott , Martin Jennifer L. , Azad Abul K. , McCarthy Travis R. , Kang Peter B. , Voelker Dennis R. , Crouch Erika C. , Schlesinger Larry S.
Published: November 2006

Lung surfactant protein D (SP-D) binds toMycobacterium tuberculosissurface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.
Alleles 308A and 238A in the Tumor Necrosis Factor Alpha Gene Promoter Do Not Increase the Risk of Severe Malaria in Children with
Plasmodium falciparum
Infection in Mali
Authors: Cabantous Sandrine , Doumbo Ogobara , Ranque Stéphane , Poudiougou Belco , Traore Abdoulaye , Hou Xunya , Keita Marouf M. , Cisse Mahamadou B. , Dessein Alain J. , Marquet Sandrine
Published: November 2006

The hypothesis that tumor necrosis factor (TNF) aggravates malaria in children is supported by observations that TNF polymorphisms and high TNF levels have been associated with cerebral malaria. Nevertheless, severe malaria was not associated with polymorphisms located at positions −308A and −238A in the TNF alpha gene promoter or with a high TNF level in plasma in children from Bamako, Mali.
Leishmania chagasiT-Cell Antigens Identified through a Double Library Screen
Authors: Martins Daniella R. A. , Jeronimo Selma M. B. , Donelson John E. , Wilson Mary E.
Published: November 2006

Control of human visceral leishmaniasis in regions where it is endemic is hampered in part by limited accessibility to medical care and emerging drug resistance. There is no available protective vaccine.Leishmaniaspp. protozoa express multiple antigens recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form ofLeishmania chagasi, the cause of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T-cell antigens and T-dependent B-cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide by screening with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second-step screen for their ability to cause proliferation and gamma interferon responses in T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The corresponding antigens were derived from glutamine synthetase, a transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected withL. chagasimounted both cellular and antibody responses to these proteins. Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines.
Population structure effect on reproduction of a rare plant: beyond population size effect
Authors: Luzuriaga Arantzazu L. , Escudero Adrián , Albert María José , Giménez-Benavides Luis
Published: November 2006

Several studies have evaluated the effect of population size on plant reproductive output, but there are few studies concerning the effect of other population structure variables on plant reproduction. In this study, we went beyond population size effect and we tested the effect of some population spatial variables such as plant density, plant aggregation, population structure, and population identity on plant reproductive success and fitness in large populations (>1000 individuals) of Centaurea hyssopifolia Vahl., an endemic species of central Spain. We analysed the reproductive output (capitula, flowers, and seeds) and another two components of plant fitness (seed mass and germinability) in 350 individuals from seven populations in 2002 and 2003. All populations were similar in size but differed in other population spatial characteristics (density, plant aggregation pattern, population plant size structure). We used Morisita’s aggregation index to characterize the level of plant aggregation within populations. Population plant size structure variable determined the proportion of different sized plants in a population. We used generalized linear mixed models to model the contribution of these population spatial variables to several reproductive parameters. Our results showed that whereas the aggregation index exerted a positive control on plant seed set, plant density had a negative effect, and plant population size structure did not significantly influence any response variable. Density only exerted a negative effect on seed set. On the contrary, plant aggregation had a positive effect. Seed mass was also larger in more aggregated populations, although no differences in seed germinability were observed. We detected the effect of population attributes on reproduction in terms of seed set, but we did not detect any effect on other overall reproductive variables measured at plant level. To sum up, our results showed that if population fragments were large enough, spatial population attributes became crucial factors for plant reproductive output and fitness. These easily measurable population variables may improve the conservation management of rare plant species.