bluuebunny/crossref_metadata_2025 huggingface dataset:
Reservoir Competency of Goats for the Ap-Variant 1 Strain of
Anaplasma phagocytophilum
Authors: Massung Robert F. , Mather Thomas N. , Levin Michael L.
Published: January 2006

Field-collected ticks were used to infect goats with either Ap-ha, a strain associated with human disease, or a variant strain, Ap-Variant 1, of
Anaplasma phagocytophilum
. Goats were shown to be competent as a reservoir for Ap-Variant 1, and challenge and immunosuppression studies were used to further examine infection in the goat model.
Pathogen-Accelerated Atherosclerosis Occurs Early after Exposure and Can Be Prevented via Immunization
Authors: Miyamoto Takanari , Yumoto Hiromichi , Takahashi Yusuke , Davey Michael , Gibson Frank C. , Genco Caroline Attardo
Published: January 2006

Here we report on early inflammatory events associated with
Porphyromonas gingivalis
-accelerated atherosclerosis in apolipoprotein E knockout (ApoE
−/−
) mice. Animals challenged with
P. gingivalis
presented with increased macrophage infiltration, innate immune marker expression, and atheroma without elevated systemic inflammatory mediators. This early local inflammatory response was prevented in mice immunized with
P. gingivalis
. We conclude that localized up-regulation of innate immune markers early after infection, rather than systemic inflammation, contributes to pathogen-accelerated atherosclerosis.
Calcium-Regulated Type III Secretion of Yop Proteins by an
Escherichia coli hha
Mutant Carrying a
Yersinia pestis
pCD1 Virulence Plasmid
Authors: Bartra Sara Schesser , Jackson Michael W. , Ross Julia A. , Plano Gregory V.
Published: January 2006

A series of four large deletions that removed a total of ca. 36 kb of DNA from the ca. 70-kb
Yersinia pestis
pCD1 virulence plasmid were constructed using lambda Red-mediated recombination.
Escherichia coli hha
deletion mutants carrying the virulence plasmid with the deletions expressed a functional calcium-regulated type III secretion system. The
E. coli hha
/pCD1 system should facilitate molecular studies of the type III secretion process.
Role of Phase Variation of Type 1 Fimbriae in a Uropathogenic
Escherichia coli
Cystitis Isolate during Urinary Tract Infection
Authors: Snyder Jennifer A. , Lloyd Amanda L. , Lockatell C. Virginia , Johnson David E. , Mobley Harry L. T.
Published: January 2006

Type 1 fimbrial phase-locked mutants of uropathogenic
Escherichia coli
cystitis isolate F11 were used to assess the role of the invertible element during urinary tract infection. Compared to the wild type, the phase-locked off mutant was attenuated, and constitutive production of type 1 fimbriae by the phase-locked on mutant did not provide a competitive advantage.
A DltA Mutant of
Haemophilus ducreyi
Is Partially Attenuated in Its Ability To Cause Pustules in Human Volunteers
Authors: Janowicz Diane , Leduc Isabelle , Fortney Kate R. , Katz Barry P. , Elkins Christopher , Spinola Stanley M.
Published: January 2006

Haemophilus ducreyi
produces two outer membrane proteins, called DltA (
H. ducreyi
lectin A) and DsrA (
H. ducreyi
serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (
P
= 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.
Salmonella enterica
Serovar Typhimurium
ompS1
and
ompS2
Mutants Are Attenuated for Virulence in Mice
Authors: Rodríguez-Morales Olivia , Fernández-Mora Marcos , Hernández-Lucas Ismael , Vázquez Alejandra , Puente José Luis , Calva Edmundo
Published: January 2006

Salmonella enterica
serovar Typhimurium mutants with mutations in the
ompS1
and
ompS2
genes, which code for quiescent porins, were nevertheless highly attenuated for virulence in a mouse model, indicating a role in pathogenesis. Similarly, a strain with a mutation in the gene coding for LeuO, a positive regulator of
ompS2
, was also attenuated.
Killed
Candida albicans
Yeasts and Hyphae Inhibit Gamma Interferon Release by Murine Natural Killer Cells
Authors: Murciano Celia , Villamón Eva , O'Connor José-Enrique , Gozalbo Daniel , Gil M. Luisa
Published: January 2006

Killed yeasts and hyphae of
Candida albicans
inhibit gamma interferon secretion by highly purified murine NK cells in response to the Toll-like receptor ligands lipopolysaccharide and zymosan. This effect, which is also observed in the presence of NK-activating cytokines (interleukin-2 [IL-2], IL-12, and IL-15), may represent a novel mechanism of immune evasion that contributes to the virulence of
C. albicans
.
Human Tumor Necrosis Factor is a Chemoattractant for the ParasiteEntamoeba histolytica
Authors: Blazquez Samantha , Zimmer Christophe , Guigon Ghislaine , Olivo-Marin Jean-Christophe , Guillén Nancy , Labruyère Elisabeth
Published: January 2006

In an analysis of the molecular factors triggering amebiasis, we investigated the chemotaxis ofEntamoeba histolyticatoward tumor necrosis factor (TNF) in vitro, using quantitative imaging techniques. Our findings enabled us to propose a hitherto unknown role for TNF as a chemokinetic and chemoattractant agent for this parasite.
Induction of In Vivo Antipolysaccharide Immunoglobulin Responses to IntactStreptococcus pneumoniaeIs More Heavily Dependent on Btk-Mediated B-Cell Receptor Signaling than Antiprotein Responses
Authors: Khan Abdul Q. , Sen Goutam , Guo Shuling , Witte Owen N. , Snapper Clifford M.
Published: January 2006

The relative role of Btk-dependent B-cell receptor (BCR) signaling in the induction of antipolysaccharide (anti-PS) and antiprotein immunoglobulin (Ig) responses to an intact extracellular bacterium in vivo is unknown. Btklowmice exhibit reduced BCR signaling but largely restore B-cell development. Btklowmice immunized with intactStreptococcus pneumoniaeelicit reduced anti-PS but normal antiprotein Ig responses. Immunization of Btklowmice with PS-protein conjugate in saline results in an even more profound defect in the anti-PS but not antiprotein response, which is largely restored by use of a CpG-containing oligodeoxynucleotide as an adjuvant. These data demonstrate a greater dependence on Btk-mediated BCR signaling for physiologic anti-PS relative to antiprotein responses, as well as the existence of a compensatory Toll-like-receptor-mediated signaling pathway naturally triggered in response to intact bacterial pathogens.
Macrophages Isolated from Chickens Genetically Resistant or Susceptible to Systemic Salmonellosis Show Magnitudinal and Temporal Differential Expression of Cytokines and Chemokines following
Salmonella enterica
Challenge
Authors: Wigley Paul , Hulme Scott , Rothwell Lisa , Bumstead Nat , Kaiser Pete , Barrow Paul
Published: January 2006

Macrophages from inbred chickens that are resistant to salmonellosis show greater and more rapid expression of proinflammatory chemokines and cytokines, including the key Th1-inducing cytokine interleukin-18, upon
Salmonella
challenge than those from susceptible birds. This suggests the possibility that salmonellosis resistant-line macrophages signal more effectively and rapidly and are more able to induce protective Th1 adaptive responses.
Upregulation of Immunoregulatory Src Homology 2 Molecule Containing Inositol Phosphatase and Mononuclear Cell Hyporesponsiveness in Oral Mucosa during Chronic Periodontitis
Authors: Muthukuru Manoj , Cutler Christopher W.
Published: January 2006

Our group and others have shown in vitro that repeated exposure of human mononuclear cells (MNC) to lipopolysaccharide can induce endotoxin tolerance, evidenced by downregulation of TLR2 and TLR4 mRNA and surface protein; moreover, the ability of the MNC to secrete inflammatory cytokines is reduced. In situ studies performed on diseased and healthy gingiva suggest that a similar pattern of endotoxin tolerance occurs in human oral mucosa with chronic periodontitis (CP). We hypothesized that this represents a fundamental immunoregulatory mechanism to restore immune homeostasis and protect the host from further tissue damage. In the current study, we extend these published studies by providing evidence that Src homology 2 containing inositol phosphatase, an inhibitor of NF-κB activation and a negative regulator of the immune response, is upregulated in the oral mucosa during CP compared to its level during gingival health. We have also isolated MNC from patients with CP and those with healthy gingiva and show that MNC from CP subjects have a reduced capacity to upregulate TLR2, TLR4, and interleukin-1β in response to endotoxin. Thus, we provide more definitive evidence for a basic mechanism of immunoregulation in the oral mucosa.
Development of a Guinea Pig Immune Response-Related Microarray and Its Use To Define the Host Response followingMycobacterium bovisBCG Vaccination
Authors: Tree Julia A. , Elmore Michael J. , Javed Sajid , Williams Ann , Marsh Philip D.
Published: January 2006

Immune responses in the guinea pig model are understudied because of a lack of commercial reagents. We have developed a custom-made guinea pig oligonucleotide microarray (81 spots) and have examined the gene expression profile of splenocytes restimulated in vitro fromMycobacterium bovisBCG-vaccinated and naive animals. Eleven genes were significantly (P< 0.05) up-regulated following vaccination, indicating a Th1-type response. These results show that microarrays can be used to more fully define immune profiles of guinea pigs.
Analysis of the Function of EnteropathogenicEscherichia coliEspB by Random Mutagenesis
Authors: Luo Wensheng , Donnenberg Michael S.
Published: January 2006

EnteropathogenicEscherichia coli(EPEC) is an important cause of infantile diarrhea, especially in developing countries. EspB, a key virulence factor of EPEC, is required for the attaching and effacing effect characteristic of EPEC and enterohemorrhagicE. coliand has been posited to play several functions in the process of infection. Attaching and effacing activity is associated with the accumulation of filamentous actin beneath adherent bacteria as measured in the fluorescence actin staining (FAS) test. To determine whether different domains of EspB are responsible for different functions, 42 plasmids carrying mutatedespBwere introduced into anespBdeletion mutant. Two major groups ofespBmutants were identified. One group of 17 mutants exhibited positive FAS results and normal levels of hemolytic activity. Another group of 22 mutants exhibited negative FAS results and low levels of hemolytic activity. Three mutants were exceptional. One mutant was FAS positive but had significantly reduced hemolytic activity. Conversely, a second mutant was FAS negative but had full hemolytic activity. A third mutant had a significantly reduced FAS level compared to the wild type but full hemolytic activity. The results of EspF and Tir translocation assays confirmed that FAS-negative insertions disrupt effector translocation and mutants with FAS-positive insertions retain protein translocation activity. These results suggest that EspB has distinct domain functions involved in effector translocation that can be distinguished from its role as a component of the translocation pore.
Multifunctional Role of Choline Binding Protein G in Pneumococcal Pathogenesis
Authors: Mann B. , Orihuela C. , Antikainen J. , Gao G. , Sublett J. , Korhonen T. K. , Tuomanen E.
Published: January 2006

Members of the choline binding protein (Cbp) family are noncovalently bound to phosphorylcholine residues on the surface of
Streptococcus pneumoniae
. It has been suggested that CbpG plays a role in adherence and increase virulence both at the mucosal surface and in the bloodstream, but the function of this protein has been unclear. A new sequence analysis indicated that CbpG is a possible member of the S1 family of multifunctional surface-associated serine proteases. Clinical isolates contained two alleles of
cbpG
, and one-third of the strains expressed a truncated protein lacking the C-terminal, cell wall-anchoring choline binding domain. CbpG on the surface of pneumococci (full length) or released into the supernatant (truncated) showed proteolytic activity for fibronectin and casein, as did CbpG expressed on lactobacilli or as a purified full-length or truncated recombinant protein. Recombinant CbpG (rCbpG)-coated beads adhered to eukaryotic cells, and TIGR4 mutants lacking CbpG or having a truncated CbpG protein showed decreased adherence in vitro and attenuation of disease in mouse challenge models of colonization, pneumonia, and bacteremia. Immunization with rCbpG was protective in an animal model of colonization and sepsis. We propose that CbpG is a multifunctional surface protein that in the cell-attached or secreted form cleaves host extracellular matrix and in the cell-attached form participates in bacterial adherence. This is the first example of distinct functions in virulence that are dependent on natural variation in expression of a choline binding domain.
NontypeableHaemophilus influenzaeAdheres to Intercellular Adhesion Molecule 1 (ICAM-1) on Respiratory Epithelial Cells and Upregulates ICAM-1 Expression
Authors: Avadhanula Vasanthi , Rodriguez Carina A. , Ulett Glen C. , Bakaletz Lauren O. , Adderson Elisabeth E.
Published: January 2006

NontypeableHaemophilus influenzae(NTHI) is an important respiratory pathogen. NTHI initiates infection by adhering to the airway epithelium. Here, we report that NTHI interacts with intracellular adhesion molecule 1 (ICAM-1) expressed by respiratory epithelial cells. A fourfold-higher number of NTHI bacteria adhered to Chinese hamster ovary (CHO) cells transfected with human ICAM-1 (CHO-ICAM-1) than to control CHO cells (P≤ 0.005). Blocking cell surface ICAM-1 with specific antibody reduced the adhesion of NTHI to A549 respiratory epithelial cells by 37% (P= 0.001) and to CHO-ICAM-1 cells by 69% (P= 0.005). Preincubating the bacteria with recombinant ICAM-1 reduced adhesion by 69% (P= 0.003). The adherence to CHO-ICAM-1 cells of NTHI strains deficient in the adhesins P5, P2, HMW1/2, and Hap or expressing a truncated lipooligosaccharide was compared to that of parental strains. Only strain 1128f−, which lacks the outer membrane protein (OMP) P5-homologous adhesin (P5 fimbriae), adhered less well than its parental strain. The numbers of NTHI cells adhering to CHO-ICAM-1 cells were reduced by 67% (P= 0.009) following preincubation with anti-P5 antisera. Furthermore, recombinant ICAM bound to an OMP preparation from strain 1128f+, which expresses P5, but not to that from its P5-deficient mutant, confirming a specific interaction between ICAM-1 and P5 fimbriae. Incubation of respiratory epithelial cells with NTHI increased ICAM-1 expression fourfold (P= 0.001). Adhesion of NTHI to the respiratory epithelium, therefore, upregulates the expression of its own receptor. Blocking interactions between NTHI P5 fimbriae and ICAM-1 may reduce respiratory colonization by NTHI and limit the frequency and severity of NTHI infection.
Characterization of Enteropathogenic
Escherichia coli
Mutants That Fail To Disrupt Host Cell Spreading and Attachment to Substratum
Authors: Nadler Chen , Shifrin Yulia , Nov Shani , Kobi Simi , Rosenshine Ilan
Published: January 2006

Upon infection of host cells, enteropathogenic
Escherichia coli
(EPEC) delivers a set of effector proteins into the host cell cytoplasm via the type III secretion system (TTSS). The effectors subvert various host cell functions. We found that EPEC interferes with the spreading and ultimately with the attachment of suspended fibroblasts or epithelial cells, and we isolated mini-Tn
10
kan insertion mutants that failed to similarly affect host cells. In most mutants, the insertion sites were mapped to genes encoding TTSS components, including
cesD
,
escC
,
escJ
,
escV
,
espD
,
sepL
,
espB
, and
escF
. Other mutants contained insertions in
micC
or upstream of
bfpP
,
yehL
, or
ydeP
. The insertion upstream of
ydeP
was associated with a reduction in TTSS protein production and was studied further. To determine whether the apparent repression was due to constitutive expression of the downstream encoded genes,
ydeP
and
ydeO
expression vectors were constructed. Expression of recombinant YdeP, YdeO, or EvgA, a positive regulator of both
ydeP
and
ydeO
, repressed TTSS protein production. Our results suggest that upon activation of the EvgAS two-component system, EvgA (the response regulator) activates both
ydeP
and
ydeO
expression and that YdeP and YdeO act conjointly, directly or indirectly repressing expression of the TTSS genes.
Phospholipase A2Functions inPseudomonas aeruginosa- Induced Apoptosis
Authors: Kirschnek Susanne , Gulbins Erich
Published: January 2006

Pseudomonas aeruginosa, a gram-negative, facultative pathogen, causes severe and often even lethal infections in immunocompromised patients, as well as cystic fibrosis patients. We show here that a variety ofP. aeruginosastrains activate phospholipase A2(PLA2), cultured epithelial cells, and fibroblasts, resulting in increased intracellular and extracellular arachidonic acid release. The use of different PLA2inhibitors revealed thatP. aeruginosa-induced arachidonic acid release is mediated by activation of cytosolic PLA2(cPLA2), whereas iPLA2or sPLA2do not seem to be involved in the response toP. aeruginosa. Likewise, the cPLA2-specific inhibitors MAFP and AACOCF3 prevented apoptosis of cultured epithelial cells uponP. aeruginosainfection, whereas inhibitors specific for iPLA2or sPLA2were without effect. The physiological significance of these findings is indicated by an inhibition of apoptosis in tracheal epithelial cells upon in vivo infection withP. aeruginosa. The data indicate that arachidonic acid generation by activation of cPLA2duringP. aeruginosainfection plays an important role in the induction of host cell death.
Deletion of the
Mycobacterium tuberculosis
α-Crystallin-Like
hspX
Gene Causes Increased Bacterial Growth In Vivo
Authors: Hu Yanmin , Movahedzadeh Farahnaz , Stoker Neil G. , Coates Anthony R. M.
Published: January 2006

Hypervirulent mutants of
Mycobacterium tuberculosis
, whose growth rates are higher in vivo, have now been reported to have mutations in both regulatory and structural genes, but the basis for this unusual phenotype is not understood. One hypervirulence gene,
dosR
(
devR
, Rv2031c), activates transcription of approximately 50 genes in this pathogen in response to hypoxia and nitric oxide stress. The most dramatic activation (∼80-fold) is activation of the
hspX
(
acr
, Rv2031c) gene, which encodes a 16-kDa α-crystallin-like protein that is a major antigen. In this study we found that a Δ
acr
mutant exhibited increased growth following infection of BALB/c mice in vivo and in both resting and activated macrophages in vitro (as measured by the number of CFU). The increased growth in macrophages was equal to that of a Δ
dosR
mutant, while introduction of a constitutively expressed
hspX
gene reduced the Δ
dosR
virulence to wild-type levels. These results suggest that the increased number of CFU of the Δ
dosR
mutant was largely due to loss of
hspX
expression. We also confirmed that constitutive expression of
hspX
slows growth in vitro, and we propose that
hspX
plays an active role in slowing the growth of
M. tuberculosis
in vivo immediately following infection.
Importance of Heat-Labile Enterotoxin in Colonization of the Adult Mouse Small Intestine by Human EnterotoxigenicEscherichia coliStrains
Authors: Allen Kenneth P. , Randolph Mildred M. , Fleckenstein James M.
Published: January 2006

EnterotoxigenicEscherichia coli(ETEC) infections are a significant cause of diarrheal disease and infant mortality in developing countries. Studies of ETEC pathogenesis relevant to vaccine development have been greatly hampered by the lack of a suitable small-animal model of infection with human ETEC strains. Here, we demonstrate that adult immunocompetent outbred mice can be effectively colonized with the prototypical human ETEC H10407 strain (colonization factor antigen I; heat-labile and heat-stable enterotoxin positive) and that production of heat-labile holotoxin provides a significant advantage in colonization of the small intestine in this model.
Sigma B Contributes toListeria monocytogenesGastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model
Authors: Garner M. R. , Njaa B. L. , Wiedmann M. , Boor K. J.
Published: January 2006

Contributions of the alternative sigma factor σBtoListeria monocytogenesinfection were investigated using strains bearing null mutations insigB,prfA, orinlAor in selectedinlAorprfApromoter regions. The ΔP4inlAstrain, which has a deletion in the σB-dependent P4inlApromoter, and the ΔsigBstrain had significantly reduced invasion efficiencies relative to that of the wild-type strain in the Caco-2 human colorectal epithelial cell line, while the invasion efficiency of a strain bearing a deletion in the partially σBdependent P2prfApromoter region did not differ from that of the wild type. The virulence of the ΔsigBand ΔP4inlAstrains was attenuated in intragastrically inoculated guinea pigs, with the ΔsigBstrain showing greater attenuation, while the virulence capacity of the ΔP2prfAstrain was similar to that of the wild-type strain, suggesting that attenuation of virulence due to the ΔsigBmutation does not result from loss of σB-dependentprfAtranscription. Our results show that σB-dependent activation ofinlAis important for cell invasion and gastrointestinal infection and suggest that σB-regulated genes in addition toinlAappear to contribute to gastrointestinal infection. Interestingly, the virulence of the ΔsigBstrain was not attenuated in intravenously infected guinea pigs. We conclude that (i)L. monocytogenesσBplays a critical role in invasion of human host cells, (ii) σB-mediated contributions to invasion are, in part, due to direct effects oninlAtranscription but not onprfAtranscription, and (iii) σBplays a critical role during the gastrointestinal stage of listeriosis in the guinea pig but is not important for systemic spread of the organism.
Phenotypic Switching in a
Cryptococcus neoformans
Variety
gattii
Strain Is Associated with Changes in Virulence and Promotes Dissemination to the Central Nervous System
Authors: Jain N. , Li Li , McFadden D. C. , Banarjee U. , Wang X. , Cook E. , Fries B. C.
Published: January 2006

This is the first report of a
Cryptococcus neoformans
var.
gattii
strain (serotype B) that switches reversibly between its parent mucoid (NP1-MC) colony morphology and a smooth (NP1-SM) colony morphology. Similar to
C. neoformans
var.
grubii
and
C. neoformans
var.
neoformans
strains, the switch is associated with changes in the polysaccharide capsule and virulence in animal models. In murine infection models, NP1-MC is significantly more virulent than NP1-SM (
P
< 0.021). In contrast to the serotype A and D strains, the serotype B strain switches in vivo reversibly between both colony morphologies. The polysaccharide of NP1-MC exhibits a thicker capsule, and thus NP1-MC exhibits enhanced intracellular survival in macrophages. Consistent with this finding, switching to the mucoid variant is observed in pulmonary infection with NP1-SM. In contrast, the thin polysaccharide capsule of NP1-SM permits better crossing of the blood-brain barrier. In this regard, only smooth colonies were grown from brain homogenates of NP1-MC-infected mice. Our findings have important implications for the pathogenesis of cryptococcosis and suggest that phenotypic switching affects host-pathogen interactions in the local microenvironment. This altered interaction then selects for specific colony variants to arise in a pathogen population.
Entamoeba histolyticaInfection in Children and Protection from Subsequent Amebiasis
Authors: Haque Rashidul , Mondal Dinesh , Duggal Priya , Kabir Mamun , Roy Shantanu , Farr Barry M. , Sack R. Bradley , Petri William A.
Published: January 2006

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh.Entamoeba histolyticainfection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child ofE. histolytica-associated diarrhea and 0.03 episodes/child ofE. histolytica-associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.
N
-Acylhomoserine Lactones Antagonize Virulence Gene Expression and Quorum Sensing in
Staphylococcus aureus
Authors: Qazi Saara , Middleton Barry , Muharram Siti Hanna , Cockayne Alan , Hill Philip , O'Shea Paul , Chhabra Siri Ram , Cámara Miguel , Williams Paul
Published: January 2006

Many gram-negative bacteria employ
N
-acylhomoserine lactone
(AHL)-mediated quorum sensing to control virulence. To determine
whether gram-positive bacteria such as
Staphylococcus aureus
respond to AHLs, we used a growth-dependent
lux
reporter
fusion. Exposure of
S. aureus
to different AHLs revealed that
3-oxo-substituted AHLs with C
10
to C
14
acyl
chains inhibited light output and growth in a concentration-dependent
manner, while short-chain AHLs had no effect.
N
-(3-Oxododecanoyl)-
l
-homoserine lactone
(3-oxo-C
12
-HSL) inhibited the production of exotoxins and
cell wall fibronectin-binding proteins but enhanced protein A
expression. Since these processes are reciprocally regulated via the
S. aureus agr
quorum-sensing system, which in turn, is
regulated via
sar
, we examined the effect of AHLs on
sarA
and
agr
. At sub-growth-inhibitory concentrations
of 3-oxo-C
12
-HSL, both
sarA
expression and
agr
expression were inhibited, indicating that the action of
3-oxo-C
12
-HSL is mediated at least in part through
antagonism of quorum sensing in
S. aureus
. Spent culture
supernatants from
Pseudomonas aeruginosa
, which produces both
3-oxo-C
12
-HSL and
N
-butanoyl-homoserine lactone
(C
4
-HSL), also inhibited
agr
expression, although
C
4
-HSL itself was inactive in this assay. Since quorum
sensing in
S. aureus
depends on the activities of
membrane-associated proteins, such as AgrB, AgrC, and AgrD, we
investigated whether AHLs perturbed
S. aureus
membrane
functionality by determining their influence on the membrane dipole
potential. From the binding curves obtained, a dissociation constant of
7 μM was obtained for 3-oxo-C
12
-HSL, indicating the
presence of a specific saturable receptor, whereas no binding was
observed for C
4
-HSL. These data demonstrate that long-chain
3-oxo-substituted AHLs, such as 3-oxo-C
12
-HSL, are capable
of interacting with the
S. aureus
cytoplasmic membrane in a
saturable, specific manner and at sub-growth-inhibitory concentrations,
down-regulating exotoxin production and both
sarA
and
agr
expression.
HpaA Is Essential forHelicobacter pyloriColonization in Mice
Authors: Carlsohn Elisabet , Nyström Johanna , Bölin Ingrid , Nilsson Carol L. , Svennerholm Ann-Mari
Published: January 2006

Infection with the human gastric pathogenHelicobacter pylorican give rise to chronic gastritis, peptic ulcer, and gastric cancer. AllH. pyloristrains express the surface-localized protein HpaA, a promising candidate for a vaccine againstH. pyloriinfection. To study the physiological importance of HpaA, a mutation of thehpaAgene was introduced into a mouse-adaptedH. pyloristrain. To justify that the interruption of thehpaAgene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavilyH. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization ofH. pyloriin mice.
Activation of the
Vibrio cholerae
SOS Response Is Not Required for Intestinal Cholera Toxin Production or Colonization
Authors: Quinones Mariam , Davis Brigid M. , Waldor Matthew K.
Published: January 2006

Cholera toxin, one of the main virulence factors of
Vibrio cholerae
, is encoded in the genome of CTXφ, a
V. cholerae
-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin,
ctxAB
, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXφ promoter,

P
rstA

. The
V. cholerae
SOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from

P
rstA

. Here, we investigated whether
ctxA
mRNA levels increase along with the levels of the transcripts for the other CTXφ genes following stimulation of the
V. cholerae
SOS response. Treatment of
V. cholerae
with the SOS-inducing agent mitomycin C increased the level of
ctxA
mRNA approximately sevenfold, apparently by augmenting the activity of

P
rstA

. However, using suckling mice as a model host, we found that intraintestinal
ctxA
transcription does not depend on

P
rstA

. In fact, the suckling mouse intestine does not appear to be a potent inducer of the
V. cholerae
SOS response. Furthermore, alleviation of LexA-mediated repression of the
V. cholerae
SOS regulon was not required for
V. cholerae
growth in the suckling mouse intestine. Our observations suggest that pathogenicity of
V. cholerae
does not depend on its SOS response.
Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (
lpd
), in
Mycoplasma gallisepticum
through In Vivo Screening of Transposon Mutants
Authors: Hudson P. , Gorton T. S. , Papazisi L. , Cecchini K. , Frasca S. , Geary S. J.
Published: January 2006

To effectively analyze
Mycoplasma gallisepticum
for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global
M. gallisepticum
mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn
4001
mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn
4001
mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated
M. gallisepticum
genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn
4001
mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the
M. gallisepticum
genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn
4001
mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (
lpd
). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.
Activation of Toll-Like Receptor 9 by DNA from Different Bacterial Species
Authors: Dalpke Alexander , Frank Jutta , Peter Mirjam , Heeg Klaus
Published: January 2006

Toll-like receptor 9 (TLR-9) recognizes unmethylated CpG dinucleotides which are abundant in prokaryotic DNA and yet are rare in eukaryotic DNA. Little is known about the significance of TLR-9 in terms of recognition of different bacterial DNA species. In this study HEK293 cells stably transfected with human TLR-9 were used to analyze the immunostimulatory properties of 15 bacterial DNA preparations. In addition, bacterial genome data were analyzed for the frequency of unmethylated cytosine-guanosine ([CG]) dinucleotides. We observed that DNA samples of different bacteria showed considerable differences in their potential to stimulate TLR-9. This correlated with the frequency of [CG] dinucleotides. Based upon data from our experiments the estimate of immunostimulatory bacterial DNA concentrations translated to as high as 109bacteria/ml. Application of the transfection reagent DOTAP resulted in a more efficient delivery of DNA into the cell, and this went along with increased TLR-9 activation. The data indicate that bacterial DNA preparations from different species differ in their capacity to activate TLR-9, which is dependent on the individual [CG] content. Moreover, increased intracellular delivery results in a marked enhancement of immunostimulation.
Interleukin 1α Increases the Susceptibility of Rabbits to Experimental Viridans Streptococcal Endocarditis
Authors: Dankert Jacob , van der Werff Janneke , Joldersma Willem , Zaat Sebastian A. J.
Published: January 2006

Major predisposing conditions for infective endocarditis (IE) are the presence of a cardiac platelet-fibrin vegetation and of circulating bacteria with relatively low susceptibility to microbicidal activity of blood platelets. The influence of proinflammatory conditions on development of IE is unknown. We studied the effects of the presence of a catheter, inserted to induce platelet-fibrin vegetations, and of the proinflammatory cytokine interleukin-1α in rabbit experimental IE. Leaving the catheter in place after challenge with viridans streptococci predisposed for experimental IE. IE susceptibility rapidly decreased between 0 to 6 h after catheter removal. The catheter did not predispose for IE by providing a site for bacterial adherence, as almost all explanted catheters were culture negative. To mimic the proinflammatory influence of the catheter, rabbits were injected with interleukin-1α at 24 h after catheter removal and at 0, 1, and 3 h before bacterial challenge. Interleukin-1α injected 3 h prior to challenge significantly increased IE incidence due to a platelet releasate-susceptible
Streptococcus oralis
strain, with rapidly increasing numbers of bacteria within the vegetations. IE due to the
Streptococcus sanguis
strain less susceptible to platelet releasate was not enhanced. We conclude that proinflammatory stimuli, either a catheter or interleukin-1α, enhanced susceptibility to IE due to the platelet releasate-susceptible
S. oralis
. As with rabbits, temporary intravascular proinflammatory conditions may predispose for IE in humans at risk for this serious infection.
Ancylostoma caninum
MTP-1, an Astacin-Like Metalloprotease Secreted by Infective Hookworm Larvae, Is Involved in Tissue Migration
Authors: Williamson Angela L. , Lustigman Sara , Oksov Yelena , Deumic Vehid , Plieskatt Jordan , Mendez Susana , Zhan Bin , Bottazzi Maria Elena , Hotez Peter J. , Loukas Alex
Published: January 2006

Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm
Ancylostoma caninum
secretes an astacin-like metalloprotease,
Ac
-MTP-1, upon activation in vitro with host serum. Recombinant
Ac
-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant
Ac
-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within
A. caninum
L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that
Ac
-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration.
Safety and Immunogenicity of an Oral Inactivated Whole-CellPseudomonas aeruginosaVaccine Administered to Healthy Human Subjects
Authors: Cripps Allan W. , Peek Keith , Dunkley Margaret , Vento Kevin , Marjason Joanne K. , McIntyre Madonna E. , Sizer Phil , Croft Duncan , Sedlak-Weinstein Lis
Published: January 2006

This study examines the safety and immunogenicity of an oral, whole-cellPseudomonas aeruginosavaccine administered to healthy volunteers. Thirty subjects received an oral dose of Pseudostat in two timed, measured doses with serological follow-up to 56 days postvaccination. Following vaccination, several individuals were identified as antibody responders for all three immunoglobulin (Ig) isotypes tested, specifically against whole-cellP. aeruginosaextract and outer membrane proteins F and I. The mean pooled lipopolysaccharide antigen-specific IgA showed the most significant and constant increases in titer postdose, with a similar increase in titer for whole-cellP. aeruginosaextract-specific IgA. The results demonstrated an increased phagocytic ability of the selected macrophage cell line in post vaccination sera. Furthermore a significant increase in intracellular macrophage killing of opsonizedP. aeruginosawas also demonstrated (82% on day 14 postdose) in the presence of the postvaccination sera. The safety component of the study did not show any vaccine-attributable adverse effects in any of the subjects, as documented by clinical evidence, hematology, and biochemistry profiles. We conclude that Pseudostat is safe and immunogenic in humans at this dose and that further studies to determine the appropriate dosage and efficacy are needed. In our study, we have shown that the most significant and sustained responses to oral vaccination in human adult volunteers were serum IgA levels and that pooled sera collected postimmunization have an increased capacity to promote opsonophagocytotic killing ofP. aeruginosa.
A Live-Attenuated
Pseudomonas aeruginosa
Vaccine Elicits Outer Membrane Protein-Specific Active and Passive Protection against Corneal Infection
Authors: Zaidi Tanweer S. , Priebe Gregory P. , Pier Gerald B.
Published: January 2006

Pseudomonas aeruginosa
can cause sight-threatening corneal infections in humans, particularly those who wear contact lenses. We have previously shown that a live-attenuated
P. aeruginosa
vaccine given intranasally protected mice against acute lethal pneumonia in a lipopolysaccharide (LPS) serogroup-specific manner. In the current study, we evaluated the protective and therapeutic efficacies, as well as the target antigens, of this vaccine in a murine corneal infection model. C3H/HeN mice were nasally immunized with the vaccine (an
aroA
deletion mutant of strain PAO1, designated PAO1Δ
aroA
) or with
Escherichia coli
as a control and were challenged 3 weeks later by inoculating the scratch-injured cornea with
P. aeruginosa
. For passive prophylaxis and therapy, we utilized a serum raised in rabbits nasally immunized with PAO1Δ
aroA
or
E. coli
. Outcome measures included corneal pathology scores and, in some experiments, reductions in total and internalized bacterial CFU. We found that both active and passive immunization reduced corneal pathology scores after challenge with a variety of
P. aeruginosa
strains, including several serogroup-heterologous strains. Even when given therapeutically starting as late as 24 h after infection, the rabbit antiserum to PAO1Δ
aroA
was effective at reducing corneal pathology scores. Immunotherapy of established infections also reduced the numbers of total and internalized corneal
P. aeruginosa
bacteria. Experiments using absorbed sera showed that the protective antibodies are specific to outer membrane proteins. Thus, live-attenuated
P. aeruginosa
vaccines delivered nasally protect against corneal infections in mice and potentially can be used to prepare passive therapy reagents for the treatment of established
P. aeruginosa
corneal infections caused by diverse LPS serogroups.
Transfer of Antigen-Pulsed Dendritic Cells Induces Specific T-Cell Proliferation and a Therapeutic Effect against Long-TermHelicobacter pyloriInfection in Mice
Authors: Otsu Satoshi , Gotoh Kazuyo , Yamashiro Tetsu , Yamagata Junpei , Shin Kouichirou , Fujioka Toshio , Nishizono Akira
Published: January 2006

Helicobacter pyloricauses persistent infection of the stomach and results in chronic gastritis and peptic ulcers. Jaws II cells, derived from mouse bone marrow, were pulsed with live or formalin-killed or whole-cell sonicates (WCS) ofH. pylori. Representative cell surface molecules were expressed at substantial levels on Jaws II cells, indicating that appropriate maturation of the cells was achieved with the threeH. pyloriantigens without any significant differences.H. pyloriWCS-pulsed Jaws II cells secreted a significant amount of tumor necrosis factor alpha into the culture supernatant. The naïve T cells exposed to the WCS-pulsed Jaws II cells showed significant proliferation and gamma interferon (IFN-γ) and interleukin-10 (IL-10) production in vitro. A 2-log reduction in the number of colonizing bacteria was observed in the mice treated with the WCS-pulsed Jaws II cells; however, no significant reductions were achieved in mice treated with Jaws II cells pulsed with otherH. pyloriantigens. Up-regulated production of IFN-γ and IL-10 was observed in the stomachs of the mice treated with the WCS-pulsed Jaws II cells, which is consistent with the result obtained in vitro. There were no differences in gastritis scores orH. pylori-specific antibody titers among the mice treated with Jaws II cells pulsed with the three differentH. pyloriantigens. The results suggest that Th1 cell-mediated immunity in combination with Th2 cell-mediated immunity plays a role in reducing colonizing bacterial numbers in mice with chronicH. pyloriinfections.
Increased vascular biosynthesis of tetrahydrobiopterin in apolipoprotein E-deficient mice
Authors: d'Uscio Livius V. , Katusic Zvonimir S.
Published: January 2006

Previous studies suggested that loss of tetrahydrobiopterin (BH4) may play an important role in the pathogenesis of vascular endothelial dysfunction induced by diabetes and hypertension. In contrast, controversial results have been reported regarding BH4metabolism in experimental models of atherosclerosis. Therefore, the present study was designed to characterize the expression and activity of GTP-cyclohydrolase I, a rate-limiting enzyme in biosynthesis of BH4, during atherogenesis. BH4levels were significantly increased in atherosclerotic aortas of apolipoprotein E (apoE)-deficient mice as compared with wild-type mice after 5 mo of Western diet treatment. This increase was further significantly enhanced in apoE-deficient mice fed for 9 and 14 mo. Removal of the endothelium almost eliminated BH4in wild-type mice but not in apoE-deficient mice, suggesting that a major component of increased BH4synthesis is localized in the vascular media of apoE-deficient mice. Oxidative products of BH4were low and did not differ between wild-type and apoE-deficient mice over the course of this study. Increased protein expression and enzymatic activity of GTP-cyclohydrolase I were detected in aortas of apoE-deficient mice ( P < 0.05), providing molecular mechanisms responsible for elevation of vascular BH4. In contrast to aortas, we did not detect any change in levels of BH4and in GTP-cyclohydrolase I expression in the brain. Our results demonstrate selective increase of intracellular BH4levels via elevation of GTP-cyclohydrolase I activity in vascular tissue of apoE-deficient mice.