bluuebunny/crossref_metadata_2025 huggingface dataset:
Anthrax Lethal Toxin Impairs Innate Immune Functions of Alveolar Macrophages and Facilitates
Bacillus anthracis
Survival
Authors: Ribot Wilson J. , Panchal Rekha G. , Brittingham Katherine C. , Ruthel Gordon , Kenny Tara A. , Lane Douglas , Curry Bob , Hoover Timothy A. , Friedlander Arthur M. , Bavari Sina
Published: August 2006

Alveolar macrophages (AM) are very important for pulmonary innate immune responses against invading inhaled pathogens because they directly kill the organisms and initiate a cascade of innate and adaptive immune responses. Although several factors contribute to inhalational anthrax, we hypothesized that unimpeded infection of
Bacillus anthracis
is directly linked to disabling the innate immune functions contributed by AM. Here, we investigated the effects of lethal toxin (LT), one of the binary complex virulence factors produced by
B. anthracis
, on freshly isolated nonhuman primate AM. Exposure of AM to doses of LT that killed susceptible macrophages had no effect on the viability of AM, despite complete MEK1 cleavage. Intoxicated AM remained fully capable of
B. anthracis
spore phagocytosis. However, pretreatment of AM with LT resulted in a significant decrease in the clearance of both the Sterne strain and the fully virulent Ames strain of
B. anthracis
, which may have been a result of impaired AM secretion of proinflammatory cytokines. Our data imply that cytolysis does not correlate with MEK1 cleavage, and this is the first report of LT-mediated impairment of nonhuman primate AM bactericidal activity against
B. anthracis
.
Opacity-Associated Adhesin Repertoire in Hyperinvasive
Neisseria meningitidis
Authors: Callaghan Martin J. , Jolley Keith A. , Maiden Martin C. J.
Published: August 2006

The opacity (Opa) proteins mediate a variety of interactions between the bacterium
Neisseria meningitidis
and its human host. These interactions are thought to be of central importance in both the asymptomatic colonization of the nasopharynx and the sporadic occurrence of meningococcal disease. The receptor specificities of a limited number of Opa protein variants have been explored, but the high level of amino acid sequence diversity among variants has complicated the assignment of specific roles to individual Opa variants or combinations of variants. In addition, the distribution of Opa protein variants among diverse meningococci, information that is potentially informative for studies of Opa function, is poorly understood. A systematic survey of the genetic diversity in the four
opa
gene loci in each of 77 meningococcal isolates was undertaken. These isolates were representative of the seven hyperinvasive meningococcal clonal complexes that caused the majority of meningococcal disease over the last 50 years. Consistent with previous studies, a high level of sequence diversity was observed among the
opa
genes and the proteins that they encoded; however, particular sets of Opa protein variants were consistently associated with each of the clonal complexes over time periods often spanning decades and during global spread. These observations were consistent with the postulate that particular combinations of Opa proteins confer fitness advantages to individual clonal complexes and have implications for studies of Opa function and the inclusion of Opa proteins in novel meningococcal vaccines.
Mechanisms of Resistance ofPorphyromonas gingivalisto Killing by Serum Complement
Authors: Slaney Jennifer M. , Gallagher Alexandra , Aduse-Opoku Joseph , Pell Keith , Curtis Michael A.
Published: August 2006

The complement system plays an important role in the host defense against infection, and the formation of the terminal complement complex on the bacterial surface has been shown to be particularly important in killing of gram-negative bacteria. The gram-negative periodontal pathogenPorphyromonas gingivalisis resistant to complement killing, and possible mechanisms suggested for this resistance include protease production and capsule formation. In this study,P. gingivalisArg- and Lys-gingipain deletion mutants and polysaccharide synthesis deletion mutants have been used to investigate these hypotheses. When Arg- and Lys-gingipain protease mutants were incubated in 20% normal human serum, deposition of complement components on the cell surface was significantly increased compared to that for the wild-type organism. However, despite the increased deposition, the protease mutants maintained resistance to killing and their viability was equal to that seen with heat-inactivated serum. Similar data were obtained when the wild-type organism was treated with gingipain protease inhibitors. K-antigen expression mutants were also resistant to killing. However, mutants which no longer synthesized a surface anionic polysaccharide (APS) (a phosphorylated branched mannan) were extremely sensitive to serum killing. These mutants lack the organized dense glycan surface layer present on the parent strain on the basis of electron microscopy. We conclude that the production of APS at the surface ofP. gingivalisrather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance inP. gingivalis.
Thioredoxin 1 Promotes Intracellular Replication and Virulence of
Salmonella enterica
Serovar Typhimurium
Authors: Bjur Eva , Eriksson-Ygberg Sofia , Åslund Fredrik , Rhen Mikael
Published: August 2006

The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of
Salmonella enterica
serovar Typhimurium was evaluated by deleting the
trxA
,
trxB
, or
trxC
gene of the cellular thioredoxin system, the
grxA
or
gshA
gene of the glutathione/glutaredoxin system, or the
dsbC
gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the
gshA
mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the
trxA
mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the
trxA
mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in
trans
but not by expression of a catalytically inactive variant. These results clearly imply that in
S. enterica
serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione.
Legionella pneumophila
Mip, a Surface-Exposed Peptidylproline
cis-trans
-Isomerase, Promotes the Presence of Phospholipase C-Like Activity in Culture Supernatants
Authors: DebRoy Sruti , Aragon Virginia , Kurtz Sherry , Cianciotto Nicholas P.
Published: August 2006

The type II secretion system of
Legionella pneumophila
promotes pathogenesis. Among the
Legionella
type II-dependent exoenzymes is a
p
-nitrophenol phosphorylcholine (
p
-NPPC) hydrolase whose activity is only partially explained by the PlcA phospholipase C. In a screen to identify other factors that promote secreted hydrolase activity, we isolated a
mip
mutant.
L. pneumophila
Mip is a surface-exposed, FK506-binding protein that is needed for optimal infection and has peptidylproline
cis-trans
-isomerase (PPIase) activity. Since the molecular target of Mip was undefined, we investigated a possible relationship between Mip and the secreted
p-
NPPC hydrolase activity. In the
mip
mutant there was a 40 to 70% reduction in secreted activity that was successfully complemented by providing
mip
on a plasmid. A similar phenotype was observed when we examined four other independently derived
mip
mutants, and in all cases the defect was complemented by reintroduction of
mip
. Thus,
mip
promotes the presence of a
p
-NPPC hydrolase activity in culture supernatants. We also found that the C terminus of Mip is required for this effect. When supernatants were examined by anion-exchange chromatography, the
p
-NPPC hydrolase activity associated with Mip proved to be type II dependent but distinct from PlcA. This conclusion was supported by the phenotype of a newly constructed
mip plcA
double mutant. Thus, Mip promotes the elaboration of a new type II exoprotein. These data provide both the first evidence for a target for Mip and the first indication that a surface PPIase is involved in the secretion or activation of proteins beyond the outer membrane.
Identification of In Vivo-Induced Bacterial Protein Antigens during Human Infection with
Salmonella enterica
Serovar Typhi
Authors: Harris Jason B. , Baresch-Bernal Andrea , Rollins Sean M. , Alam Ashfaqul , LaRocque Regina C. , Bikowski Margaret , Peppercorn Amanda F. , Handfield Martin , Hillman Jeffery D. , Qadri Firdausi , Calderwood Stephen B. , Hohmann Elizabeth , Breiman Robert F. , Brooks W. Abdullah , Ryan Edward T.
Published: August 2006

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with
Salmonella enterica
serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of
S. enterica
. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in
S. enterica
serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses.
Dissecting the Contributions of
Clostridium perfringens
Type C Toxins to Lethality in the Mouse Intravenous Injection Model
Authors: Fisher Derek J. , Fernandez-Miyakawa Mariano E. , Sayeed Sameera , Poon Rachael , Adams Victoria , Rood Julian I. , Uzal Francisco A. , McClane Bruce A.
Published: August 2006

The gram-positive anaerobe
Clostridium perfringens
produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals.
C. perfringens
type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (
plc
), perfringolysin O (
pfoA
), beta toxin (
cpb
), and sometimes beta2 toxin (
cpb2
) and/or enterotoxin (
cpe
). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.
The GTPase Rab4 Interacts withChlamydia trachomatisInclusion Membrane Protein CT229
Authors: Rzomp K. A. , Moorhead A. R. , Scidmore M. A.
Published: August 2006

Chlamydiae, which are obligate intracellular bacteria, replicate in a nonlysosomal vacuole, termed an inclusion. Although neither the host nor the chlamydial proteins that mediate the intracellular trafficking of the inclusion have been clearly identified, several enhanced green fluorescent protein (GFP)-tagged Rab GTPases, including Rab4A, are recruited to chlamydial inclusions. GFP-Rab4A associates with inclusions in a species-independent fashion by 2 h postinfection by mechanisms that have not yet been elucidated. To test whether chlamydial inclusion membrane proteins (Incs) recruit Rab4 to the inclusion, we screened a collection of chlamydial Incs for their ability to interact with Rab4A by using a yeast two-hybrid assay. From our analysis, we identified a specific interaction between Rab4A andChlamydia trachomatisInc CT229, which is expressed during the initial stages of infection. CT229 interacts with only wild-type Rab4A and the constitutively active GTPase-deficient Rab4AQ67L but not with the dominant-negative GDP-restricted Rab4AS22N mutant. To confirm the interaction between CT229 and Rab4A, we demonstrated that DsRed-CT229 colocalized with GFP-Rab4A in HeLa cells and more importantly wild-type and constitutively active GFP-Rab4A colocalized with CT229 at the inclusion membrane inC. trachomatisserovar L2-infected HeLa cells. Taken together, these data suggest that CT229 interacts with and recruits Rab4A to the inclusion membrane and therefore may play a role in regulating the intracellular trafficking or fusogenicity of the chlamydial inclusion.
The Contribution of PspC to Pneumococcal Virulence Varies between Strains and Is Accomplished by Both Complement Evasion and Complement-Independent Mechanisms
Authors: Kerr Alison R. , Paterson Gavin K. , McCluskey Jackie , Iannelli Francesco , Oggioni Marco R. , Pozzi Gianni , Mitchell Tim J.
Published: August 2006

Pneumococcal surface protein C (PspC) is a virulence factor of
Streptococcus pneumoniae
previously shown to play a role in bacterial adherence, invasion, and evasion of complement. We investigated the role of this protein in our murine models of pneumococcal pneumonia with different pneumococcal strains. The deletion of
pspC
in strains of serotypes 2, 3, and 19F did not significantly alter host survival times in the pneumonia model. In contrast,
pspC
deletion significantly reduced the virulence of the serotype 4 strain, TIGR4, in both the pneumonia and bacteremia models. Therefore,
pspC
is a systemic and pulmonary virulence determinant for
S. pneumoniae
, but its effects are influenced by the pneumococcal strain. Finally, pneumonia infection of complement-deficient (C3
−/−
) mice enhanced
pspC
virulence, illustrating that PspC-mediated complement evasion contributes to virulence. However, other functions of PspC also contribute to virulence, as demonstrated by the finding that the
pspC
-deficient TIGR4 mutant was still attenuated relative to the wild-type parent, even in the absence of C3.
Extensive Genomic Plasticity in
Pseudomonas aeruginosa
Revealed by Identification and Distribution Studies of Novel Genes among Clinical Isolates
Authors: Shen Kai , Sayeed Sameera , Antalis Patricia , Gladitz John , Ahmed Azad , Dice Bethany , Janto Benjamin , Dopico Richard , Keefe Randy , Hayes Jay , Johnson Sandra , Yu Sujun , Ehrlich Nathan , Jocz Jennifer , Kropp Laura , Wong Ray , Wadowsky Robert M. , Slifkin Malcolm , Preston Robert A. , Erdos Geza , Post J. Christopher , Ehrlich Garth D. , Hu Fen Z.
Published: August 2006

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of
Pseudomonas aeruginosa
, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, ∼1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the
P. aeruginosa
prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in
P. aeruginosa
. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.
AttenuatedFrancisella novicidaTransposon Mutants Protect Mice against Wild-Type Challenge
Authors: Tempel Rebecca , Lai Xin-He , Crosa Lidia , Kozlowicz Briana , Heffron Fred
Published: August 2006

Francisella tularensisis the bacterial pathogen that causes tularemia in humans and a number of animals. To date, there is no approved vaccine for this widespread and life-threatening disease. The goal of this study was to identifyF. tularensismutants that can be used in the development of a live attenuated vaccine. We screenedF. novicidatransposon mutants to identify mutants that exhibited reduced growth in mouse macrophages, as these cells are the preferred host cells ofFrancisellaand an essential component of the innate immune system. This approach yielded 16F. novicidamutants that were 100-fold more attenuated for virulence in a mouse model than the wild-type parental strain. These mutants were then tested to determine their abilities to protect mice against challenge with high doses of wild-type bacteria. Five of the 16 attenuated mutants (with mutations corresponding todsbB, FTT0742,pdpB,fumA, andcarBin theF. tularensisSCHU S4 strain) provided mice with protection against challenge with high doses (>8 × 105CFU) of wild-typeF. novicida. We believe that these findings will be of use in the design of a vaccine against tularemia.
Roles of Cellular Activation and Sulfated Glycans inHaemophilus somnusAdherence to Bovine Brain Microvascular Endothelial Cells
Authors: Behling-Kelly E. , Vonderheid H. , Kim Kwang Sik , Corbeil L. B. , Czuprynski C. J.
Published: August 2006

Haemophilus somnuscan cause a devastating fibrinopurulent meningitis with thrombotic vasculitis and encephalitis in cattle. The mechanisms used byH. somnusto migrate from the bloodstream into the central nervous system (CNS) are unknown. In this study, we demonstrate thatH. somnusadheres to, but does not invade, bovine brain endothelial cells (BBEC) in vitro. The number of adherentH. somnuswas significantly increased by prior activation of the BBEC with tumor necrosis factor alpha (TNF-α). Addition of exogenous glycosaminoglycans significantly reducedH. somnusadherence to resting and TNF-α-activated BBEC. Heparinase digestion of the endothelial cell's glycocalyx or sodium chlorate inhibition of endothelial cell sulfated glycan synthesis significantly reduced the number of adherentH. somnus. In contrast, addition of hyaluronic acid, a nonsulfated glycosaminoglycan, had no inhibitory effect. These findings suggest a critical role for both cellular activation and sulfated glycosaminoglycans in adherence ofH. somnusto BBEC. Using heparin-labeled agarose beads, we demonstrated a high-molecular-weight heparin-binding protein expressed byH. somnus. Heparin was also shown to bindH. somnusin a 4°C binding assay. These data suggest that heparin-binding proteins onH. somnuscould serve as initial adhesins to sulfated proteoglycans on the endothelial cell surface, thus contributing to the ability ofH. somnusto infect the bovine CNS.
Autocrine Role for Interleukin-8 in
Bartonella henselae
-Induced Angiogenesis
Authors: McCord Amy M. , Resto-Ruiz Sandra I. , Anderson Burt E.
Published: August 2006

The gram-negative bacterium
Bartonella henselae
is capable of causing angiogenic lesions as a result of infection. Previously, it has been shown that
B. henselae
infection can result in production of the chemokine interleukin-8 (IL-8). In this study, we demonstrated that monocytes, endothelial cells, and hepatocytes produce IL-8 in response to
B. henselae
infection. We also investigated the role of IL-8 in
B. henselae
-induced endothelial cell proliferation and capillary tube formation. Both in vitro angiogenesis assays were IL-8 dependent.
B. henselae
-mediated inhibition of apoptosis, as indicated by gene expression of
Bax
and
Bcl-2
, was also shown to be IL-8 dependent in endothelial cells. Furthermore, infection of endothelial cells with
B. henselae
stimulated upregulation of the IL-8 chemokine receptor CXCR2. Infection of human endothelial cells by
B. henselae
resulting in IL-8 production likely plays a central role in the ability of this organism to cause angiogenesis during infection.
Role of Capsule and Interleukin-6 in Long-Term Immune Control ofCryptococcus neoformansInfection by Specifically Activated Human Peripheral Blood Mononuclear Cells
Authors: Siddiqui Asna A. , Shattock Robin J. , Harrison Thomas S.
Published: August 2006

Cryptococcus neoformansis a frequent cause of meningoencephalitis in immunosuppressed individuals. To better understand the mechanisms of a protective immune response toC. neoformans, a long-term in vitro model of human immune control of cryptococcal infection was developed. Peripheral blood mononuclear cells (PBMC) prestimulated with heat-killedC. neoformanssignificantly restricted the growth ofC. neoformansafter a subsequent live infection compared to that with unstimulated PBMC. Live infection with encapsulatedC. neoformanswas controlled for as long as 10 days, while infection with acapsular organisms could sometimes be eradicated. During immune control, fungal cells were both intracellular and extracellular within aggregates of mononuclear phagocytes and lymphocytes. Optimal immune control depended on the presence of both CD4+and CD8+T cells. Immune control of cryptococcal growth was more effective following prestimulation with acapsular compared with encapsulated organisms. Prestimulation with acapsular organisms was associated with a significant and prolonged increase in interleukin-6 (IL-6) production compared with prestimulation with encapsulatedC. neoformans. Addition of IL-6 and depletion of CD25+T cells prior to prestimulation and infection with encapsulated organisms resulted in reductions in cryptococcal growth that reached borderline statistical significance. Depletion of CD25+T cells significantly reduced cryptococcal growth in wells with unstimulated PBMC. The results demonstrate an association between high levels of IL-6 and resistance to infection and, through suppression of IL-6 release, an additional mechanism whereby the cryptococcal capsule subverts a protective immune response. Further work is required to clarify the mechanism of action of IL-6 in this setting and any interaction with regulatory T cells.
Innate Immune Responses to Lung-Stage Helminth Infection Induce Alternatively Activated Alveolar Macrophages
Authors: Reece Joshua J. , Siracusa Mark C. , Scott Alan L.
Published: August 2006

While it is well established that infection with the rodent hookwormNippostrongylus brasiliensisinduces a strongly polarized Th2 immune response, little is known about the innate host-parasite interactions that lead to the development of this robust Th2 immunity. We exploited the transient pulmonary phase ofN. brasiliensisdevelopment to study the innate immune responses induced by this helminth parasite in wild-type (WT) and severe-combined immune deficient (SCID) BALB/c mice. Histological analysis demonstrated that the cellular infiltrates caused byN. brasiliensistransit through the lungs were quickly resolved in WT mice but not in SCID mice. Microarray-based gene expression analysis demonstrated that there was a rapid induction of genes encoding molecules that participate in innate immunity and in repair/remodeling during days 2 to 4 postinfection in the lungs of WT and SCID mice. Of particular note was the rapid upregulation in both WT and SCID mice of the genes encoding YM1, FIZZ1, and Arg1, indicating a role for alternatively activated macrophages (AAMs) in pulmonary innate immunity. Immunohistochemistry revealed that nearly all alveolar macrophages became YM1-producing AAMs as early as day 2 postinfection. While the innate responses induced during the lung phase ofN. brasiliensisinfection were similar in complexity and magnitude in WT and SCID mice, only mice with functional T cells were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. The induction of alternatively activated alveolar macrophages could be important for dampening the level of inflammation in the lungs and contribute to the long-term decrease in pulmonary inflammation that has been associated with helminth infections.
Construction of a Mutant and Characterization of the Role of the Vaccine Antigen P6 in Outer Membrane Integrity of NontypeableHaemophilus influenzae
Authors: Murphy Timothy F. , Kirkham Charmaine , Lesse Alan J.
Published: August 2006

Outer membrane protein P6 is the subject of investigation as a vaccine antigen to prevent infections caused by nontypeableHaemophilus influenzae, which causes otitis media in children and respiratory tract infections in adults with chronic lung disease. P6 induces protective immune responses in animal models and is the target of potentially protective immune responses in humans. P6 is a 16-kDa lipoprotein that shares homology with the peptidoglycan-associated lipoproteins of gram-negative bacteria and is highly conserved among strains ofH. influenzae. To characterize the function of P6, an isogenic mutant was constructed by replacing the P6 gene with a chloramphenicol resistance cassette. The P6 mutant showed altered colony morphology and slower growth in vitro than that of the parent strain. By electron microscopy, the P6 mutant cells demonstrated increased size, variability in size, vesicle formation, and fragility compared to the parent cells. The P6 mutant showed hypersensitivity to selected antibiotics with different mechanisms of action, indicating increased accessibility of the agents to their targets. The P6 mutant was more sensitive to complement-mediated killing by normal human serum. Complementation of the mutation intranscompletely or partially restored the phenotypes. We concluded that P6 plays a structural role in maintaining the integrity of the outer membrane by anchoring the outer membrane to the cell wall. The observation that the absence of expression of P6 is detrimental to the cell is a highly desirable feature for a vaccine antigen, supporting further investigation of P6 as a vaccine candidate forH. influenzae.
Role of Intrachain Disulfides in the Activities of the CdtA and CdtC Subunits of the Cytolethal Distending Toxin ofActinobacillus actinomycetemcomitans
Authors: Cao Linsen , Volgina Alla , Korostoff Jonathan , DiRienzo Joseph M.
Published: August 2006

The cytolethal distending toxin (Cdt) ofActinobacillus actinomycetemcomitansis an atypical A-B-type toxin consisting of a heterotrimer composed of thecdtA,cdtB, andcdtCgene products. The CdtA and CdtC subunits form two heterogeneous ricin-like lectin domains which bind the holotoxin to the target cell. Point mutations were used to study CdtC structure and function. One (mutC216F97C) of eight single-amino-acid replacement mutants identified yielded a gene product that failed to form biologically active holotoxin. Based on the possibility that the F97C mutation destabilized a predicted disulfide, targeted mutagenesis was used to examine the contribution of each of four cysteine residues, in two predicted disulfides (C96/C107 and C135/C149), to CdtC activities. Cysteine replacement mutations in two predicted disulfides (C136/C149 and C178/C197) in CdtA were also characterized. Flow cytometry and CHO cell proliferation assays showed that changing either C96 or C149 in CdtC to alanine abolished the biological activity of holotoxin complexes. However, replacing C107 or C135 in CdtC and any of the four cysteines in CdtA with alanine or serine resulted in only partial or no loss of holotoxin activity. Changes in the biological activities of the mutant holotoxins correlated with altered subunit binding. In contrast to elimination of the B chain of ricin, the elimination of intrachain disulfides in CdtC and CdtA by genetic replacement of cysteines destabilizes these subunit proteins but not to the extent that cytotoxicity is lost. Reduction of the wild-type holotoxin did not affect cytotoxicity, and the reduced form of wild-type CdtA exhibited a statistically significant increase in binding to ligand. A diminished role for intrachain disulfides in stabilizing CdtA and CdtC may have clinical relevance for theA. actinomycetemcomitansCdt. Thecdtgene products secreted by this pathogen assemble and bind to target cells in periodontally involved sites, which are decidedly reduced environments in the human oral cavity.
Biochemical Activities of Three Pairs of
Ehrlichia chaffeensis
Two-Component Regulatory System Proteins Involved in Inhibition of Lysosomal Fusion
Authors: Kumagai Yumi , Cheng Zhihui , Lin Mingqun , Rikihisa Yasuko
Published: August 2006

Ehrlichia chaffeensis
, the etiologic agent of human monocytic ehrlichiosis, replicates in early endosomes by avoiding lysosomal fusion in monocytes and macrophages. In
E. chaffeensis
we predicted three pairs of putative two-component regulatory systems (TCSs) designated PleC-PleD, NtrY-NtrX, and CckA-CtrA based on amino acid sequence homology. In the present study to determine biochemical pairs and specificities of the TCSs, the recombinant proteins of the three putative histidine kinase (HK) kinase domains (rPleCHKD, rNtrYHKD, and MBP-rCckAHKD) and the full-length forms of three putative response regulators (RRs) (rPleD, rNtrX, and rCtrA) as well as the respective mutant recombinant proteins (rPleCHKDH244A, rNtrYHKDH498A, MBP-rCckAHKDH449A, rPleDD53A, rNtrXD59A, and rCtrAD53A) were expressed and purified as soluble proteins. The in vitro HK activity, the specific His residue-dependent autophosphorylation of the kinase domain, was demonstrated in the three HKs. The specific Asp residue-dependent in vitro phosphotransfer from the kinase domain to the putative cognate RR was demonstrated in each of the three RRs. Western blot analysis of
E. chaffeensis
membrane and soluble fractions using antibodies specific for each recombinant protein detected PleC and CckA in the membrane fraction, whereas it detected NtrY, NtrX, and PleD in the soluble fraction. CtrA was found in the two fractions at similar levels.
E. chaffeensis
was sensitive to closantel, an HK inhibitor. Closantel treatment induced lysosomal fusion of the
E. chaffeensis
inclusion in a human monocytic leukemia cell line, THP-1 cells, implying that functional TCSs are essential in preventing lysosomal fusion of the
E. chaffeensis
inclusion compartment.
N
-Acetyllactosamine Conjugated to Gold Nanoparticles Inhibits Enteropathogenic
Escherichia coli
Colonization of the Epithelium in Human Intestinal Biopsy Specimens
Authors: Hyland Romney M. , Beck Paul , Mulvey George L. , Kitov Pavel I. , Armstrong Glen D.
Published: August 2006

We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic
Escherichia coli
to HEp-2, T84, and Caco-2 cells is inhibited by
N
-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.
Different Consequences of
ACE2
and
SWI5
Gene Disruptions for Virulence of Pathogenic and Nonpathogenic Yeasts
Authors: MacCallum Donna M. , Findon Helen , Kenny Claire C. , Butler Geraldine , Haynes Ken , Odds Frank C.
Published: August 2006

Mutants of
Candida albicans
,
Candida glabrata
, and
Saccharomyces cerevisiae
with disruptions in the
ACE2
gene and
C. glabrata
and
S. cerevisiae swi5
disruption mutants were tested for virulence in a murine challenge model of disseminated yeast infection. All mutants showed a clumping phenotype, but clumping was minimized in challenge inocula by inclusion of chitinase in the growth medium. In animals rendered temporarily neutropenic by cyclophosphamide treatment, the
C. glabrata ace2
null mutant was confirmed as hypervirulent: it led to early terminal illness and kidney, brain, and lung fungal burdens substantially and significantly larger than those in controls. The
C. glabrata swi5
null mutant did not lead to terminal illness but generated significantly larger brain and lung burdens than those in controls. The
C. albicans ace2
null mutant was very slightly attenuated and the
S. cerevisiae ace2
and
swi5
null mutants were substantially attenuated relative to their parental control strains. The phenotype of aggressive hypervirulence, unique to disruption of the
C. glabrata ACE2
gene among the strains tested, was not seen when the
C. glabrata ace2
strain was tested in immunologically intact mice. The different effects seen with these mutants rule out the clumping phenotype as the explanation for hypervirulence in the
C. glabrata ace2
mutant. The absence of
C. glabrata ace2
hypervirulence in healthy mice may be a tool for definitive future study of host-parasite cross talk in microbial opportunism.
Acquisition of Hemozoin by Monocytes Down-Regulates Interleukin-12 p40 (IL-12p40) Transcripts and Circulating IL-12p70 through an IL-10-Dependent Mechanism: In Vivo and In Vitro Findings in Severe Malarial Anemia
Authors: Keller Christopher C. , Yamo Ouma , Ouma Collins , Ong'echa John Michael , Ounah David , Hittner James B. , Vulule John M. , Perkins Douglas J.
Published: August 2006

Severe malarial anemia (SMA) is a primary cause of morbidity and mortality in immune-naïve infants and young children residing in areas of holoendemicPlasmodium falciparumtransmission. Although the immunopathogenesis of SMA is largely undefined, we have previously shown that systemic interleukin-12 (IL-12) production is suppressed during childhood blood-stage malaria. Since IL-10 and tumor necrosis factor alpha (TNF-α) are known to decrease IL-12 synthesis in a number of infectious diseases, altered transcriptional regulation of these inflammatory mediators was investigated as a potential mechanism for IL-12 down-regulation. Ingestion of naturally acquired malarial pigment (hemozoin [PfHz]) by monocytes promoted the overproduction of IL-10 and TNF-α relative to the production of IL-12, which correlated with an enhanced severity of malarial anemia. Experiments with cultured peripheral blood mononuclear cells (PBMC) and CD14+cells from malaria-naïve donors revealed that physiological concentrations of PfHz suppressed IL-12 and augmented IL-10 and TNF-α by altering the transcriptional kinetics of IL-12p40, IL-10, and TNF-α, respectively. IL-10 neutralizing antibodies, but not TNF-α antibodies, restored PfHz-induced suppression of IL-12. Blockade of IL-10 and the addition of recombinant IL-10 to cultured PBMC from children with SMA confirmed that IL-10 was responsible for malaria-induced suppression of IL-12. Taken together, these results demonstrate that PfHz-induced up-regulation of IL-10 is responsible for the suppression of IL-12 during malaria.
In Vivo Bioluminescence Imaging of the Murine Pathogen
Citrobacter rodentium
Authors: Wiles Siouxsie , Pickard Karen M. , Peng Katian , MacDonald Thomas T. , Frankel Gad
Published: August 2006

Citrobacter rodentium
is a natural mouse pathogen related to enteropathogenic and enterohemorrhagic
Escherichia coli
. We have previously utilized bioluminescence imaging (BLI) to determine the in vivo colonization dynamics of
C. rodentium
. However, due to the oxygen requirement of the bioluminescence system and the colonic localization of
C. rodentium
, in vivo localization studies were performed using harvested organs. Here, we report the detection of bioluminescent
C. rodentium
and commensal
E. coli
during colonization of the gastrointestinal tract in intact living animals. Bioluminescence was dependent on intact blood circulation, suggesting that the colonic environment is not anaerobic but nanaerobic. In addition, BLI revealed that
C. rodentium
colonizes the rectum, a site previously unreported for this pathogen.
Nucleotide Sequence Analysis of the Enteropathogenic
Escherichia coli
Adherence Factor Plasmid pMAR7
Authors: Brinkley Carl , Burland Valerie , Keller Rogéria , Rose Debra J. , Boutin Adam T. , Klink Sara A. , Blattner Frederick R. , Kaper James B.
Published: August 2006

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic
Escherichia coli
(EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator
per
. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The
tra
locus, a highly conserved 33-kb segment found in pMAR7, is similar to the
tra
(conjugation) region of the F plasmid. IS
Ec13
copies flanking the pMAR7
tra
region could potentially mobilize or delete the
tra
genes. Hybridization of 134 EPEC strains showed that a complete
tra
region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.
Interplay of Pneumococcal Hydrogen Peroxide and Host-Derived Nitric Oxide
Authors: Hoffmann Olaf , Zweigner Janine , Smith Shannon H. , Freyer Dorette , Mahrhofer Cordula , Dagand Emilie , Tuomanen Elaine I. , Weber Joerg R.
Published: August 2006

Reactive oxygen and nitrogen species are released by immune-competent cells and contribute to cellular damage. On the other hand, certain pathogens, includingStreptococcus pneumoniae, are known to produce hydrogen peroxide (H2O2), while production of nitrogen radicals by bacteria presumably occurs but has been poorly studied. We determined the relative contributions of bacterial versus host-derived oxygen and nitrogen radicals to cellular damage in pneumococcal infection. A special focus was placed on peroxynitrite as a hypothetical common product formed by the reaction of H2O2and NO. In microglial cultures, reduction of the formation of 3-nitrotyrosine and cellular damage required H2O2-deficient (ΔspxBor ΔcarB) pneumococci and inhibition of host NO synthesis with aminoguanidine. In infected C57BL/6 mice, neuronal loss and immunopositivity for nitrotyrosine in the dentate gyrus were markedly reduced with ΔspxBor ΔcarBbacterial mutants and in inducible nitric oxide synthase knockout mice. We conclude that although host and bacteria both produce oxygen and nitrogen radicals, the interplay of prokaryotic H2O2and eukaryotic NO is a major contributor to cellular damage in pneumococcal meningitis.
Brucella melitensisTriggers Time-Dependent Modulation of Apoptosis and Down-Regulation of Mitochondrion-Associated Gene Expression in Mouse Macrophages
Authors: He Yongqun , Reichow Sherry , Ramamoorthy Sheela , Ding Xicheng , Lathigra Raju , Craig Johanna C. , Sobral Bruno W. S. , Schurig Gerhardt G. , Sriranganathan Nammalwar , Boyle Stephen M.
Published: August 2006

Brucellaspp. are facultative intracellular bacteria that cause brucellosis in humans and other animals.Brucellaspp. are taken up by macrophages, and the outcome of the macrophage-Brucellainteraction is a basis for establishment of a chronicBrucellainfection. Microarrays were used to analyze the transcriptional response of the murine macrophage-like J774.A1 cell line to infection with virulentBrucella melitensisstrain 16M. It was found that most significant changes in macrophage gene transcription happened early following infection, and global macrophage gene expression profiles returned to normal between 24 and 48 h postinfection. These findings support the observation that macrophages kill the majority ofBrucellacells at the early infection stage, but the survivingBrucellacells are able to avoid macrophage brucellacidal activity inside replicative phagosomes at the later infection stage. At 4 h postinfection, macrophage genes involved in cell growth, metabolism, and responses to endogenous stimuli were down-regulated, while the inflammatory response (e.g., tumor necrosis factor alpha and Toll-like receptor 2), the complement system, the responses to external stimuli, and other immune responses were up-regulated. It is likely that the most active brucellacidal activity happened between 0 and 4 h postinfection. Mitochondrion-associated gene expression, which is involved in protein synthesis and transport, electron transfer, and small-molecule transfer, and many other mitochondrial functions were significantly down-regulated at 4 h postinfection. Although there were both pro- and antiapoptosis effects,B. melitensis16M appears to inhibit apoptosis of macrophages by blocking release of cytochromecand production of reactive oxygen species in the mitochondria, thus preventing activation of caspase cascades.
Role of Polymorphonuclear Leukocyte-Derived Serine Proteinases in Defense againstActinobacillus actinomycetemcomitans
Authors: de Haar Susanne F. , Hiemstra Pieter S. , van Steenbergen Martijn T. J. M. , Everts Vincent , Beertsen Wouter
Published: August 2006

Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such asActinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense againstA. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity againstA. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to killA. actinomycetemcomitansin an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.
Critical Role of Type 1 Cytokines in Controlling Initial Infection withBurkholderia mallei
Authors: Rowland Caroline A. , Lertmemongkolchai Ganjana , Bancroft Alison , Haque Ashraful , Lever M. Stephen , Griffin Kate F. , Jackson Matthew C. , Nelson Michelle , O'Garra Anne , Grencis Richard , Bancroft Gregory J. , Lukaszewski Roman A.
Published: August 2006

Burkholderia malleiis a gram-negative bacterium which causes the potentially fatal disease glanders in humans; however, there is little information concerning cell-mediated immunity to this pathogen. The role of gamma interferon (IFN-γ) duringB. malleiinfection was investigated using a disease model in which infected BALB/c mice normally die between 40 and 60 days postinfection. IFN-γ knockout mice infected withB. malleidied within 2 to 3 days after infection, and there was uncontrolled bacterial replication in several organs, demonstrating the essential role of IFN-γ in the innate immune response to this pathogen. Increased levels of IFN-γ, interleukin-6 (IL-6), and monocyte chemoattractant protein 1 were detected in the sera of immunocompetent mice in response to infection, and splenic mRNA expression of IFN-γ, IL-6, IL-12p35, and IL-27 was elevated 24 h postinfection. The effects of IL-18, IL-27, and IL-12 on stimulation of the rapid IFN-γ production were investigated in vitro by analyzing IFN-γ production in the presence of heat-killedB. mallei. IL-12 was essential for IFN-γ production in vitro; IL-18 was also involved in induction of IFN-γ, but IL-27 was not required for IFN-γ production in response to heat-killedB. mallei. The main cellular sources of IFN-γ were identified in vitro as NK cells, CD8+T cells, and TCRγδ T cells. Our data show thatB. malleiis susceptible to cell-mediated immune responses which promote expression of type 1 cytokines. This suggests that development of effective vaccines against glanders should target the production of IFN-γ.
ARE WE AT THE END OF CMOS SCALING?
Authors: SHAHIDI GHAVAM G.
Published: August 2006

CMOS scaling enabled by advances in lithography has been behind the information revolution. Over the last 15 years, there has been a new CMOS technology node approximately every two years. The key feature of every node has been 2X density shrink and ~35% performance gain per technology node. At 90 nm node a number usual knobs that have enabled the scaling have approached their limits. Furthermore chip power (both active and stand-by) has been increasing rapidly, approaching air cool limit. Chip stand-by power, which was negligible a few years ago, is now about the same order of magnitude as the active power in high end microprocessors.

In this talk it will be argued that because of power density limitation of 90 nm, 65 nm, and beyond nodes, performance and ability to shrink are more than ever linked, and in fact if the performance gain would significantly slow down (for the designs that operate at the existing cooling limit). It is more than ever critical to come up with technology features that will enhance the performance, at a given device leakage.
FRONTIERS OF NANO-BIO SYSTEM
Authors: CHANG CHUN-YEN
Published: August 2006

In this article, high lights of researches at NCTU on the topics of quantum transport of electrons, phonons in nanostructures, nano-bio application in SARS diagnoses and vaccinations, new development of THz lasing, blue vertical cavity surface emitting laser, and applications and new development of nano-bio devices are presented.