bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protection against FatalPseudomonas aeruginosaPneumonia in Mice after Nasal Immunization with a Live, AttenuatedaroADeletion Mutant
Authors: Priebe Gregory P. , Meluleni Gloria J. , Coleman Fadie T. , Goldberg Joanna B. , Pier Gerald B. 
Published: February 2003

ABSTRACTStudies of immunity toPseudomonas aeruginosahave indicated that a variety of potential immunogens can elicit protection in animal models, utilizing both antibody- and cell-mediated immune effectors for protection. To attempt to optimize delivery of multiple protective antigens and elicit a broad range of immune effectors, we produced anaroAdeletion mutant of theP. aeruginosaserogroup O2/O5 strain PAO1, designated PAO1ΔaroA. Previously, we reported that this strain elicits high levels of opsonic antibody directed against many serogroup O2/O5 strains after nasal immunization of mice and rabbits. Here, we assessed the protective efficacy of immunization with PAO1ΔaroAagainst acute fatal pneumonia in mice. After active immunization, high levels of protection were achieved against an ExoU-expressing cytotoxic variant of the parental strain PAO1 at doses up to 1,000-fold greater than the 50% lethal dose. Significant protection against PAO1 and two of four other serogroup O2/O5 strains was also found, but there was no protection against serogroup-heterologous strains. The serogroup O2/O5 strains not protected against were killed in opsonophagocytic assays as efficiently as the strains with which protection was seen, indicating a lack of correlation of protection and opsonic killing within the serogroup. In passive immunization experiments using challenge with wild-type PAO1 or other noncytotoxic members of the O2/O5 serogroup, there was no protection despite the presence of high levels of opsonic antibody in the mouse sera. However, passive immunization did prevent mortality from pneumonia due to the cytotoxic PAO1 variant at low-challenge doses. These data suggest that a combination of humoral and cellular immunity is required for protection againstP. aeruginosalung infections, that such immunity can be elicited by usingaroAdeletion mutants, and that a multivalentP. aeruginosavaccine composed ofaroAdeletion mutants of multiple serogroups holds significant promise.
Amino Acid Sequence Requirements in the Hinge of Human Immunoglobulin A1 (IgA1) for Cleavage by Streptococcal IgA1 Proteases
Authors: Batten Margaret R. , Senior Bernard W. , Kilian Mogens , Woof Jenny M. 
Published: February 2003

ABSTRACTThe amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species ofStreptococcuswere investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcα receptors. However, the substitutions had a substantial effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease fromStreptococcus pneumoniae, which showed no absolute requirement for either proline or threonine at residues 227 to 228. By contrast, the IgA1 proteases ofStreptococcus oralis,Streptococcus sanguis, andStreptococcus mitishad an absolute requirement for proline at 227 but not for threonine at 228, which could be replaced by valine. There was evidence inS. mitisthat proteases from different strains may have different amino acid requirements for cleavage. Remarkably, some streptococcal proteases appeared able to cleave the hinge at a distant alternative site if substitution prevented efficient cleavage of the original site. Hence, this study has identified key residues required for the recognition of the IgA1 hinge as a substrate by streptococcal IgA1 proteases, and it marks a preliminary step towards development of specific enzyme inhibitors.
Venezuelan Equine Encephalitis Virus-Vectored Vaccines Protect Mice against Anthrax Spore Challenge
Authors: Lee John S. , Hadjipanayis Angela G. , Welkos Susan L. 
Published: February 2003

ABSTRACT

Anthrax, a disease usually associated with herbivores, is caused by the bacterium
Bacillus anthracis
. The current vaccine licensed for human use requires a six-dose primary series and yearly boosters and causes reactogenicity in up to 30% of vaccine recipients. A minimally reactogenic vaccine requiring fewer inoculations is warranted. Venezuelan equine encephalitis (VEE) virus has been configured for use as a vaccine vector for a wide variety of immunogens. The VEE vaccine vector is composed of a self-replicating RNA (replicon) containing all of the VEE virus nonstructural genes and a multiple-cloning site in place of the VEE structural genes. Four different anthrax vaccines were constructed by cloning the protective antigen (PA) gene from
B. anthracis
into the VEE vaccine vector. The anthrax vaccines were produced by assembling the vectors into propagation-deficient VEE replicon particles in vitro. A/J mice inoculated subcutaneously with three doses of the mature 83-kDa PA vaccine were completely protected from challenge with the Sterne strain of
B. anthracis
. Similar results were obtained with vaccines composed of the PA gene fused to either the
B. anthracis
secretory sequence or to a tissue plasminogen activator secretory sequence in three additional mouse strains. Mice were unprotected from challenge after inoculation with the carboxy-terminal 63-kDa PA vaccine. These results suggest that these VEE-vectored vaccines may be suitable as candidate vaccines against anthrax.
Role of Toll-Like Receptor Signaling in the Apoptotic Response of Macrophages toYersiniaInfection
Authors: Zhang Yue , Bliska James B. 
Published: February 2003

ABSTRACTMacrophages encode several Toll-like receptors (TLRs) that recognize bacterial components, such as lipoproteins (TLR2) or lipopolysaccharides (TLR4), and activate multiple signaling pathways. Activation of transcription factor NF-κB by TLR2 or TLR4 signaling promotes proinflammatory and cell survival responses. Alternatively, TLR2 or TLR4 signaling can promote apoptosis if the activation of NF-κB is blocked. The gram-negative bacterial pathogenYersinia pseudotuberculosissecretes into macrophages a protease (YopJ) that inhibits the activation of NF-κB and promotes apoptosis. We show that primary macrophages expressing constitutively active inhibitor κB kinase β (IKKβ) are completely resistant to YopJ-dependent apoptosis, indicating that YopJ inhibits signaling upstream of IKKβ. Apoptosis is reduced two- to threefold in TLR4−/−macrophages infected withY. pseudotuberculosis, while the apoptotic response of TLR2−/−macrophages toY. pseudotuberculosisinfection is equivalent to that of wild-type macrophages. Therefore, TLR4 is the primary source of apoptotic signaling inYersinia-infected macrophages. Our results also show that a small percentage of macrophages can die as a result of an apoptotic process that is YopJ dependent but does not require TLR2 or TLR4 signaling.
Effects of Ectopically Expressed Neuronal Wiskott-Aldrich Syndrome Protein Domains onRickettsia rickettsiiActin-Based Motility
Authors: Harlander Ronald S. , Way Michael , Ren Qun , Howe Dale , Grieshaber Scott S. , Heinzen Robert A. 
Published: February 2003

ABSTRACTNeuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility inListeria monocytogenesrequire the Arp2/3 complex but bypasses N-WASP signaling. Motility ofShigella flexneriand vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility ofRickettsia rickettsiihave not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects onShigellaand vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected withR. rickettsiito assess their effects on rickettsial motility.S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize toR. rickettsiior their actin tails. Expression of N-WASP missing the VCA domain (for “verprolin homology, cofilin homology, and acidic domains”), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility ofS. flexneriwhile only moderately inhibiting motility ofR. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility ofS. flexneri(no motility observed in the majority of expressing cells) but only moderately inhibitedR. rickettsiimotility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility ofR. rickettsiiis independent of N-WASP and the Arp2/3 complex.
Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of
Aeromonas sobria
in Stimulating T84 Cells To Produce Cyclic AMP
Authors: Fujii Yoshio , Nomura Tomohiko , Yokoyama Ritsuko , Shinoda Sumio , Okamoto Keinosuke 
Published: February 2003

ABSTRACT

We previously reported that the aerolysin-like hemolysin of
Aeromonas sobria
stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.
Expression of Variant Surface Antigens by
Plasmodium falciparum
Parasites in the Peripheral Blood of Clinically Immune Pregnant Women Indicates Ongoing Placental Infection
Authors: Ofori Michael F. , Staalsoe Trine , Bam Victoria , Lundquist Maja , David Kim P. , Browne Edmund N. L. , Akanmori Bartholomew D. , Hviid Lars 
Published: February 2003

ABSTRACT

Placenta-sequestered
Plasmodium falciparum
parasites that cause pregnancy-associated malaria (PAM) in otherwise clinically immune women express distinct variant surface antigens (VSA
PAM
) not expressed by parasites in nonpregnant individuals. We report here that parasites from the peripheral blood of clinically immune pregnant women also express VSA
PAM
, making them a convenient source of VSA
PAM
expressors for PAM vaccine research.
Interleukin-12 Regulates Chemokine Gene Expression during the Early Immune Response toLeishmania major
Authors: Zaph Colby , Scott Phillip 
Published: February 2003

ABSTRACTFollowing infection withLeishmania major, the chemokines XCL1, CXCL10, and CCL2 were preferentially expressed in draining lymph nodes of resistant mice. Neutralization of interleukin 12 (IL-12) or gamma interferon in resistant mice resulted in decreased chemokine expression, while administration of IL-12 to susceptible mice resulted in an increase in the level of chemokine gene expression.
DsbA of
Pseudomonas aeruginosa
Is Essential for Multiple Virulence Factors
Authors: Ha Un-Hwan , Wang Yanping , Jin Shouguang 
Published: February 2003

ABSTRACT

DsbA is a periplasmic thiol:disulfide oxidoreductase which contributes to the process of protein folding by catalyzing the formation of disulfide bonds. In this study, we demonstrate that the
dsbA
gene is required for the expression of the type III secretion system under low-calcium inducing conditions, intracellular survival of
P. aeruginosa
upon infection of HeLa cells, and twitching motility. The diverse phenotypes of the
dsbA
mutant are likely due to its defect in the folding of proteins that are involved in various biological processes, such as signal sensing, protein secretion, and defense against host clearing. In light of its effect on various virulence factors, DsbA could be an important target for the control of
P. aeruginosa
infections.
Effect of Vaccination with Refined Components of the Organism on Infection of Mice with
Mycobacterium leprae
Authors: Ngamying Maeya , Sawanpanyalert Pathom , Butraporn Raywadee , Nikasri Junjira , Cho Sang-Nae , Levy Louis , Brennan Patrick J. 
Published: February 2003

ABSTRACT

Only native products of
Mycobacterium leprae
, whether cell wall, cytosol, or membrane derived, can confer protective immunity against challenge in the mouse footpad. Previously, recombinant proteins were shown to be ineffective. The cell wall skeleton—the mycolyl-arabinogalactan-peptidoglycan complex—devoid of proteins is not protective.
CheZ-Mediated Dephosphorylation of the
Escherichia coli
Chemotaxis Response Regulator CheY: Role for CheY Glutamate 89
Authors: Silversmith Ruth E. , Guanga Gerald P. , Betts Laurie , Chu Carolyn , Zhao Rui , Bourret Robert B. 
Published: February 2003

ABSTRACT

The swimming behavior of
Escherichia coli
at any moment is dictated by the intracellular concentration of the phosphorylated form of the chemotaxis response regulator CheY, which binds to the base of the flagellar motor. CheY is phosphorylated on Asp57 by the sensor kinase CheA and dephosphorylated by CheZ. Previous work (Silversmith et al., J. Biol. Chem. 276:18478, 2001) demonstrated that replacement of CheY Asn59 with arginine resulted in extreme resistance to dephosphorylation by CheZ despite proficient binding to CheZ. Here we present the X-ray crystal structure of CheYN59R in a complex with Mn
2+
and the stable phosphoryl analogue BeF
3
−
. The overall folding and active site architecture are nearly identical to those of the analogous complex containing wild-type CheY. The notable exception is the introduction of a salt bridge between Arg59 (on the β3α3 loop) and Glu89 (on the β4α4 loop). Modeling this structure into the (CheY-BeF
3
−
-Mg
2+
)
2
CheZ
2
structure demonstrated that the conformation of Arg59 should not obstruct entry of the CheZ catalytic residue Gln147 into the active site of CheY, eliminating steric interference as a mechanism for CheZ resistance. However, both CheYE89A and CheYE89Q, like CheYN59R, conferred clockwise flagellar rotation phenotypes in strains which lacked wild-type CheY and displayed considerable (≈40-fold) resistance to dephosphorylation by CheZ. CheYE89A and CheYE89Q had autophosphorylation and autodephosphorylation properties similar to those of wild-type CheY and could bind to CheZ with wild-type affinity. Therefore, removal of Glu89 resulted specifically in CheZ resistance, suggesting that CheY Glu89 plays a role in CheZ-mediated dephosphorylation. The CheZ resistance of CheYN59R can thus be largely explained by the formation of the salt bridge between Arg59 and Glu89, which prevents Glu89 from executing its role in catalysis.
A Two-Component Regulator of Universal Stress Protein Expression and Adaptation to Oxygen Starvation inMycobacterium smegmatis
Authors: O'Toole Ronan , Smeulders Marjan J. , Blokpoel Marian C. , Kay Emily J. , Lougheed Kathryn , Williams Huw D. 
Published: February 2003

ABSTRACTWe identified a response regulator inMycobacterium smegmatiswhich plays an important role in adaptation to oxygen-starved stationary phase. The regulator exhibits strong sequence similarity to DevR/Rv3133c ofM. tuberculosis. The structural gene is present on a multigene locus, which also encodes a sensor kinase. AdevRmutant ofM. smegmatiswas adept at surviving growth arrest initiated by either carbon or nitrogen starvation. However, its culturability decreased several orders of magnitude below that of the wild type under oxygen-starved stationary-phase conditions. Two-dimensional gel analysis revealed that a number of oxygen starvation-inducible proteins were not expressed in thedevRmutant. Three of these proteins are universal stress proteins, one of which is encoded directly upstream ofdevR. Another protein closely resembles a proposed nitroreductase, while a fifth protein corresponds to the α-crystallin (HspX) orthologue ofM. smegmatis. None of the three universal stress proteins or nitroreductase, and a considerably lower amount of HspX was detected in carbon-starved wild-type cultures. A fusion of thehspXpromoter togfpdemonstrated that DevR directs gene expression whenM. smegmatisenters stationary phase brought about, in particular, by oxygen starvation. To our knowledge, this is the first time a role for a two-component response regulator in the control of universal stress protein expression has been shown. Notably, thedevRmutant was 104-fold more sensitive than wild type to heat stress. We conclude that DevR is a stationary-phase regulator required for adaptation to oxygen starvation and resistance to heat stress inM. smegmatis.
Glyceraldehyde-3-Phosphate Dehydrogenase Has No Control over Glycolytic Flux in
Lactococcus lactis
MG1363
Authors: Solem Christian , Koebmann Brian J. , Jensen Peter R. 
Published: February 2003

ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has previously been suggested to have almost absolute control over the glycolytic flux in
Lactococcus lactis
(B. Poolman, B. Bosman, J. Kiers, and W. N. Konings, J. Bacteriol. 169:5887-5890, 1987). Those studies were based on inhibitor titrations with iodoacetate, which specifically inhibits GAPDH, and the data suggested that it should be possible to increase the glycolytic flux by overproducing GAPDH activity. To test this hypothesis, we constructed a series of mutants with GAPDH activities from 14 to 210% of that of the reference strain MG1363. We found that the glycolytic flux was unchanged in the mutants overproducing GAPDH. Also, a decrease in the GAPDH activity had very little effect on the growth rate and the glycolytic flux until 25% activity was reached. Below this activity level, the glycolytic flux decreased proportionally with decreasing GAPDH activity. These data show that GAPDH activity has no control over the glycolytic flux (flux control coefficient = 0.0) at the wild-type enzyme level and that the enzyme is present in excess capacity by a factor of 3 to 4. The early experiments by Poolman and coworkers were performed with cells resuspended in buffer, i.e., nongrowing cells, and we therefore analyzed the control by GAPDH under similar conditions. We found that the glycolytic flux in resting cells was even more insensitive to changes in the GAPDH activity; in this case GAPDH was also present in a large excess and had no control over the glycolytic flux.
Multiple Light Inputs Control Phototaxis in
Synechocystis
sp. Strain PCC6803
Authors: Ng Wing-On , Grossman Arthur R. , Bhaya Devaki 
Published: February 2003

ABSTRACT

The phototactic behavior of individual cells of the cyanobacterium
Synechocystis
sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a
taxD1
mutant (Cyanobase accession no.
sll0041
; also called
pisJ1
) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A Δ
taxD1
mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in
Synechocystis
sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.
Chromosomal Expression of the
Haemophilus influenzae
Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis
Authors: Fink Doran L. , St. Geme Joseph W. 
Published: February 2003

ABSTRACT

The
Haemophilus influenzae
Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hap
β
and a 110-kDa extracellular passenger domain called Hap
S
. All adhesive activity resides within Hap
S
, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in
H. influenzae
clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap.
Structural Model for 12-Helix Transporters Belonging to the Major Facilitator Superfamily
Authors: Hirai Teruhisa , Heymann Jürgen A. W. , Maloney Peter C. , Subramaniam Sriram 
Published: February 2003

ABSTRACTThe major facilitator superfamily includes a large collection of evolutionarily related proteins that have been implicated in the transport of a variety of solutes and metabolites across the membranes of organisms ranging from bacteria to humans. We have recently reported the three-dimensional structure, at 6.5 Å resolution, of the oxalate transporter, OxlT, a representative member of this superfamily. In the oxalate-bound state, 12 helices surround a central cavity to form a remarkably symmetrical structure that displays a well-defined pseudo twofold axis perpendicular to the plane of the membrane as well as two less pronounced, mutually perpendicular pseudo twofold axes in the plane of the membrane. Here, we combined this structural information with sequence information from other members of this protein family to arrive at models for the arrangement of helices in this superfamily of transport proteins. Our analysis narrows down the number of helix arrangements from about a billion starting possibilities to a single probable model for the relative spatial arrangement for the 12 helices, consistent both with our structural findings and with the majority of previous biochemical studies on members of this superfamily.