bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of p38 Mitogen-Activated Protein Kinase Augments Lipopolysaccharide-Induced Cell Proliferation in CD14-Expressing Chinese Hamster Ovary Cells
Authors: Chakravortty Dipshikha , Kato Yutaka , Sugiyama Tsuyoshi , Koide Naoki , Mu Mya Mya , Yoshida Tomoaki , Yokochi Takashi
Published: July 2002

CD14-expressing Chinese hamster ovary (CD14-CHO) cells, established by transfection of human CD14 DNA, acquired high responsiveness to lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On the other hand, SB203580 exhibited no effect on epidermal growth factor-induced DNA synthesis in CD14-CHO cells, although PD98059 inhibited it significantly. The activation and inactivation of p38 MAP kinase with dominant negative and dominant positive mutants also suggested the participation of p38 MAP kinase in LPS-induced DNA synthesis. It was therefore suggested that the activation of p38 MAP kinase can negatively regulate LPS-induced cell proliferation in CD14-CHO cells.
Human Neutrophils and Their Products Induce Shiga Toxin Production by Enterohemorrhagic
Escherichia coli
Authors: Wagner Patrick L. , Acheson David W. K. , Waldor Matthew K.
Published: July 2002

The Shiga toxins (Stx) are critical virulence factors for
Escherichia coli
O157:H7 and other serotypes of enterohemorrhagic
E. coli
(EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H
2
O
2
), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H
2
O
2
and also human neutrophils, an important endogenous source of H
2
O
2
, induced Stx2 expression by an EHEC clinical isolate. Both H
2
O
2
and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.
Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding
Authors: Gauduchon Valérie , Werner Sandra , Prévost Gilles , Monteil Henri , Colin Didier A.
Published: July 2002

The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV. The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety. The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (
K

d

= 0.07 ± 0.02 nM,
n
= 5) and monocytes (

K
d

= 0.020 ± 0.003 nM,
n
= 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin. LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca
2+
at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn
2+
. The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation. The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.
Knockout of the Rodent Malaria Parasite Chitinase PbCHT1 Reduces Infectivity to Mosquitoes
Authors: Dessens Johannes T. , Mendoza Jacqui , Claudianos Charles , Vinetz Joseph M. , Khater Emad , Hassard Stuart , Ranawaka Gaya R. , Sinden Robert E.
Published: July 2002

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium.
Plasmodium
chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite,
Plasmodium berghei
. We show that the gene, named
PbCHT1
, is a structural ortholog of
PgCHT1
of the avian malaria parasite
Plasmodium gallinaceum
and a paralog of
PfCHT1
of the human malaria parasite
Plasmodium falciparum
. Targeted disruption of
PbCHT1
reduced parasite infectivity in
Anopheles stephensi
mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds—an artificial situation where midgut invasion occurs before PM formation—suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that
P. berghei
ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of
Plasmodium
-mosquito interactions.
Coiled-Coil Domain of Enteropathogenic
Escherichia coli
Type III Secreted Protein EspD Is Involved in EspA Filament-Mediated Cell Attachment and Hemolysis
Authors: Daniell Sarah J. , Delahay Robin M. , Shaw Robert K. , Hartland Elizabeth L. , Pallen Mark J. , Booy Frank , Ebel Frank , Knutton Stuart , Frankel Gad
Published: July 2002

Many animal and plant pathogens use type III secretion systems to secrete key virulence factors, some directly into the host cell cytosol. However, the basis for such protein translocation has yet to be fully elucidated for any type III secretion system. We have previously shown that in enteropathogenic and enterohemorrhagic
Escherichia coli
the type III secreted protein EspA is assembled into a filamentous organelle that attaches the bacterium to the plasma membrane of the host cell. Formation of EspA filaments is dependent on expression of another type III secreted protein, EspD. The carboxy terminus of EspD, a protein involved in formation of the translocation pore in the host cell membrane, is predicted to adopt a coiled-coil conformation with 99% probability. Here, we demonstrate EspD-EspD protein interaction using the yeast two-hybrid system and column overlays. Nonconservative triple amino acid substitutions of specific EspD carboxy-terminal residues generated an enteropathogenic
E. coli
mutant that was attenuated in its ability to induce attaching and effacing lesions on HEp-2 cells. Although the mutation had no effect on EspA filament biosynthesis, it also resulted in reduced binding to and reduced hemolysis of red blood cells. These results segregate, for the first time, functional domains of EspD that control EspA filament length from EspD-mediated cell attachment and pore formation.
Nitric Oxide Is Protective in Listeric Meningoencephalitis of Rats
Authors: Remer K. A. , Jungi T. W. , Fatzer R. , Täuber M. G. , Leib S. L.
Published: July 2002

The bacteriumListeria monocytogenescauses meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation withL. monocytogeneson survival and activity were noted; 104L. monocytogenesorganisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO⋅) production. Treatment with the spin-trapping agent α-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitorl-N6-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO⋅contribute toListeriagrowth control.
Down-Regulated Lymphoproliferation Coincides with Parasite Maturation and with the Collapse of Both Gamma Interferon and Interleukin-4 Responses in a Bovine Model of Onchocerciasis
Authors: Graham Simon P. , Trees Alexander J. , Collins Robert A. , Moore Davina M. , Guy Francis M. , Taylor Mark J. , Bianco Albert E.
Published: July 2002

Onchocerciasis is a debilitating parasitic infection caused by the filarial nematodeOnchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire ofO. volvulus, we have used the cattle parasiteOnchocerca ochengito investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n= 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-γ]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-γ and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patentOnchocercainfections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship ofO. ochengiin cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.
Haemophilus ducreyi
Inhibits Phagocytosis by U-937 Cells, a Human Macrophage-Like Cell Line
Authors: Wood Gwendolyn E. , Dutro Susan M. , Totten Patricia A.
Published: July 2002

Haemophilus ducreyi
is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live
H. ducreyi
can be isolated from these lesions. To analyze the interaction of
H. ducreyi
with macrophages, we investigated the ability of
H. ducreyi
strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although
H. ducreyi
strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that
H. ducreyi
avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that
H. ducreyi
inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to
H. ducreyi
, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine
H. ducreyi
strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which
H. ducreyi
avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes.
Chlamydia pneumoniae
Expresses Genes Required for DNA Replication but Not Cytokinesis during Persistent Infection of HEp-2 Cells
Authors: Byrne Gerald I. , Ouellette Scot P. , Wang Zhao , Rao J. P. , Lu Lin , Beatty Wandy L. , Hudson Alan P.
Published: July 2002

Chlamydia pneumoniae
causes community-acquired pneumonia and is associated with several chronic diseases, including asthma and atherosclerosis. The intracellular growth rate of
C. pneumoniae
slows dramatically during chronic infection, and such persistence leads to attenuated production of new elementary bodies, appearance of morphologically aberrant reticulate bodies, and altered expression of several chlamydial genes. We used an in vitro system to further characterize persistent
C. pneumoniae
infection, employing both ultrastructural and transcriptional activity measurements. HEp-2 cells were infected with
C. pneumoniae
(TW-183) at a multiplicity of infection of 3:1, and at 2 h postinfection gamma interferon (IFN-γ) was added to the medium at 0.15 or 0.50 ng/ml. Treated and untreated cultures were harvested at several times postinfection. RNA was isolated and reverse transcribed, and reverse transcription (RT)-PCR analyses targeting primary transcripts from chlamydial rRNA operons as well as
dnaA
,
polA
,
mutS
,
minD
,
ftsK
, and
ftsW
mRNA were done. Some cultures were fixed and stained for electron microscopic analysis, and a real-time PCR assay was used to assess relative chlamydial chromosome accumulation under each culture condition. The latter assays showed that bacterial chromosome copies accumulated severalfold during IFN-γ treatment of infected HEp-2 cells, although less accumulation was observed in cells treated with the higher dose. Electron microscopy demonstrated that high-dose IFN-γ treatment elicited aberrant forms of the bacterium. RT-PCR showed that chlamydial primary rRNA transcripts were present in all IFN-γ-treated and untreated cell cultures, indicating bacterial metabolic activity. Transcripts from
dnaA
,
polA
,
mutS
, and
minD
, all of which encode products for bacterial chromosome replication and partition, were expressed in IFN-γ-treated and untreated cells. In contrast,
ftsK
and
ftsW
, encoding products for bacterial cell division, were expressed in untreated cells, but expression was attenuated in cells treated with low-dose IFN-γ and absent in cells given the high dose of cytokine. Thus, the development of persistence included production of transcripts for DNA replication-related, but not cell division-related, genes. These results provide new insight regarding molecular activities that accompany persistence of
C. pneumoniae
, as well as suggesting requirements for reactivation from persistent to productive growth.
Strain-Dependent Differences in the Regulatory Roles of
sarA
and
agr
in
Staphylococcus aureus
Authors: Blevins Jon S. , Beenken Karen E. , Elasri Mohamed O. , Hurlburt Barry K. , Smeltzer Mark S.
Published: July 2002

The accessory gene regulator (
agr
) and the staphylococcal accessory regulator (
sar
) are central regulatory elements that control the production of
Staphylococcus aureus
virulence factors. To date, the functions of these loci have been defined almost exclusively using RN6390, which is representative of the laboratory strain 8325-4. However, RN6390 was recently shown to have a mutation in
rsbU
that results in a phenotype resembling that of a
sigB
mutant (I. Kullik et al., J. Bacteriol. 180:4814–4820, 1998). For that reason, it remains unclear whether the regulatory events defined in RN6390 are representative of the events that take place in clinical isolates of
S. aureus
. To address this issue, we generated mutations in the
sarA
and
agr
loci of three laboratory strains (RN6390, Newman, and S6C) and four clinical isolates (UAMS-1, UAMS-601, DB, and SC-1). Mutation of
sarA
in the
cna
-positive strains UAMS-1 and UAMS-601 resulted in an increased capacity to bind collagen, while mutation of
agr
had little impact. Northern blot analysis confirmed that the increase in collagen binding was due to increased
cna
transcription. Without exception, mutation of
sarA
resulted in increased production of proteases and a decreased capacity to bind fibronectin. Mutation of
agr
had the opposite effect. Although mutation of
sarA
resulted in a slight reduction in
fnbA
transcription, changes in the ability to bind fibronectin appeared to be more directly correlated with changes in protease activity. Lipase production was reduced in both
sarA
and
agr
mutants. While mutation of
sarA
in RN6390 resulted in reduced hemolytic activity, it had the opposite effect in all other strains. There appeared to be reduced levels of the
sarC
transcript in RN6390, but there was no difference in the overall pattern of
sar
transcription or the production of SarA. Although mutation of
sarA
resulted in decreased RNAIII transcription, this effect was not evident under all growth conditions. Taken together, these results suggest that studies defining the regulatory roles of
sarA
and
agr
by using RN6390 are not always representative of the events that occur in clinical isolates of
S. aureus
.
Increased Susceptibility of C1q-Deficient Mice toSalmonella entericaSerovar Typhimurium Infection
Authors: Warren Joanna , Mastroeni Pietro , Dougan Gordon , Noursadeghi Mahdad , Cohen Jonathan , Walport Mark J. , Botto Marina
Published: July 2002

The role of the complement system in host defense againstSalmonellainfection is poorly defined. Bacterial cell wall O-antigen polysaccharide can activate the alternative pathway in vitro. No studies, however, have elucidated the role of the classical pathway in immunity toSalmonellaspp. in vivo. C1q-deficient mice (C1qa−/−) on a 129/Sv genetic background and strain-matched controls were infected intraperitoneally and intravenously withSalmonella entericaserovar Typhimurium and monitored over a 14-day period. After inoculation by either route, theC1qa−/−mice were found to be significantly more susceptible toSalmonellainfection. Hepatic and splenic bacterial counts, performed at various time points, showed increased numbers of colonies in complement-deficient mice compared to controls. Analysis of blood clearance showed no difference between the two experimental groups during the first 15 min. However, after 20 min and until 6 h postinfection, numbers of circulating bacteria were significantly higher in complement-deficient mice. In vitro experiments using either resident or thioglycolate-elicited peritoneal macrophages showed a significant increase in the number of bacteria inside C1q-deficient macrophages compared to controls irrespective of the serum used for opsonizing the bacteria. These findings could not be explained either by an increased bacterial uptake, analyzed in vitro and in vivo using green fluorescent protein-tagged salmonellae, or by a defect in the respiratory burst or in NO production. The data presented here suggest the possibility of novel pathways by which C1q may modulate the pathogenesis of infectious diseases caused by intracellular pathogens.
Investigation of Role of Nitric Oxide in Protection fromBordetella pertussisRespiratory Challenge
Authors: Canthaboo C. , Xing D. , Wei X. Q. , Corbel M. J.
Published: July 2002

The mechanism whereby whole-cell pertussis vaccines (WCV) confer protection againstBordetella pertussisis still not fully understood. We have previously reported that macrophage activation produced by vaccination with WCV is associated with induction of NO synthesis by macrophages in response to in vitro stimulation withB. pertussisantigens. To determine whether NO production is an effector of protection or simply a marker of activation, the susceptibility of inducible nitric oxide synthase (type II, iNOS) knockout mice to infection withB. pertussiswas examined. We showed that iNOS knockout mice were more susceptible toB. pertussisrespiratory challenge than wild-type mice. iNOS-deficient mice also developed a less effective protective response than wild-type mice after the same immunization with WCV. This suggests that NO plays an important role in effecting protection againstB. pertussischallenge.
Onchocerca volvulusGlycolytic Enzyme Fructose-1,6-Bisphosphate Aldolase as a Target for a Protective Immune Response in Humans
Authors: McCarthy James S. , Wieseman Moira , Tropea Joe , Kaslow David , Abraham David , Lustigman Sara , Tuan Rocky , Guderian Ronald H. , Nutman Thomas B.
Published: July 2002

To identify potential vaccine candidates for the prevention of infection with the filarial nematodeOnchocerca volvulus, we screened anO. volvulusL3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed toO. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models.
Gene Expression Profiling in AGS Cells Stimulated withHelicobacter pyloriIsogenic Strains (cagAPositive orcagANegative)
Authors: Bach Susanne , Makristathis Athanasios , Rotter Manfred , Hirschl Alexander M.
Published: July 2002

To study host response to CagA, human gastric cancer cell line AGS was infected with aHelicobacter pyloritype I wild-type or isogeniccagA-negative mutant. Differentially expressed genes were identified using cDNA array technology. By Northern blotting, downregulation of focal adhesion kinase and upregulation of LIM kinase mRNA in the presence of CagA were clearly verified. Furthermore, upregulation of LIM kinase, macrophage inflammatory protein-2, c-myc, and bone morphogenetic protein-1 and downregulation of transcription factor Y-box binding protein-1 and focal adhesion kinase mRNA in response toH. pyloritype I infection compared to the uninfected control could be shown by Northern blotting. Hence, these findings identified new targets for further functional studies onH. pylori-associated pathogenesis.
Immune Responses to Recombinant Pneumococcal PspA Antigen Delivered by Live Attenuated
Salmonella enterica
Serovar Typhimurium Vaccine
Authors: Kang Ho Young , Srinivasan Jay , Curtiss Roy
Published: July 2002

Attenuated
Salmonella enterica
serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and
Salmonella
antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the
Salmonella
vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic α-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the β-lactamase signal sequence in the multicopy Asd
+
pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd
+
vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The
S. enterica
serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Δ
crp-28
and Δ
asdA16
. Approximately 50% of the recombinant PspA (rPspA) expressed in a
Salmonella
strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant
Salmonella
. After a single oral immunization in BALB/c mice with 10
9
CFU of the recombinant
Salmonella
vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and
Salmonella
lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with
Streptococcus pneumoniae
WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with
Salmonella-
PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent
S. pneumoniae
WU2.
Susceptibility to Experimental Cerebral Malaria Induced byPlasmodium bergheiANKA in Inbred Mouse Strains Recently Derived from Wild Stock
Authors: Bagot S. , Idrissa Boubou M. , Campino S. , Behrschmidt C. , Gorgette O. , Guénet J.-L. , Penha-Gonçalves C. , Mazier D. , Pied S. , Cazenave P.-A.
Published: July 2002

The neurological syndrome caused byPlasmodium bergheiANKA in rodents partially mimics the human disease. Several rodent models of cerebral malaria (CM) exist for the study of the mechanisms that cause the disease. However, since common laboratory mouse strains have limited gene pools, the role of their phenotypic variations causing CM is restricted. This constitutes an obstacle for efficient genetic analysis relating to the pathogenesis of malaria. Most common laboratory mouse strains are susceptible to CM, and the same major histocompatibility complex (MHC) haplotype may exhibit different levels of susceptibility. We analyzed the influence of the MHC haplotype on overcoming CM by using MHC congenic mice with C57BL/10 and C3H backgrounds. No correlation was found between MHC molecules and the development of CM. New wild-derived mouse strains with wide genetic polymorphisms were then used to find new models of resistance to CM. Six of the twelve strains tested were resistant to CM. For two of them, F1progeny and backcrosses performed with the reference strain C57BL/6 showed a high level of heterogeneity in the number and characteristics of the genetic factors associated with resistance to CM.
Lipopolysaccharide-Induced Granule Mobilization and Priming of the Neutrophil Response toHelicobacter pyloriPeptide Hp(2-20), Which Activates Formyl Peptide Receptor-Like 1
Authors: Bylund Johan , Karlsson Anna , Boulay Francois , Dahlgren Claes
Published: July 2002

The cecropin-like bactericidal peptide Hp(2-20) fromHelicobacter pyloriinduces activation of the NADPH oxidase in human neutrophils via formyl peptide receptor-like 1 (FPRL1) (J. Bylund, T. Christophe, F. Boulay, T. Nyström, A. Karlsson, and C. Dahlgren, Antimicrob. Agents Chemother. 45:1700-1704, 2001). Here we investigated the ability of bacterial lipopolysaccharide (LPS) to prime this response. Neutrophils treated with LPS for 30 min at 37°C produced substantially more superoxide anion than control cells upon stimulation with Hp(2-20). Hence, LPS primed the cells for subsequent stimulation through FPRL1. To study the molecular background of this priming phenomenon, we measured the degrees of granule mobilization and concomitant receptor upregulation to the cell surface in LPS-treated cells. Exposure of complement receptors 1 and 3 as well as the formyl peptide receptor (FPR) was markedly increased after LPS treatment. Since approximately 60% of the gelatinase granules were mobilized while the specific granules were retained, we hypothesized that the gelatinase granules were potential stores of FPRL1. The presence of FPRL1 mainly in the gelatinase granules was confirmed by Western blotting of subcellular fractions of resting neutrophils. These results suggest that the mechanism behind the LPS-induced priming of FPRL1-mediated responses lies at the level of granule (receptor) mobilization.
Role of Interleukin-18 in Host Defense against DisseminatedCandida albicansInfection
Authors: Stuyt Rogier J. L. , Netea Mihai G. , Verschueren Ineke , Fantuzzi Giamila , Dinarello Charles A. , Van der Meer Jos W. M. , Kullberg Bart Jan
Published: July 2002

In mice injected intravenously withCandida albicans, administration of anti-interleukin-18 (IL-18) antibodies increased the yeast load in the kidneys. There was no effect on the organ load withCandidawhen gamma interferon (IFN-γ)-deficient mice were treated with anti-IL-18 antibodies, suggesting that the protective effect of IL-18 is mediated through endogenous IFN-γ.
The
Bacillus subtilis
GTP Binding Protein Obg and Regulators of the ς
B
Stress Response Transcription Factor Cofractionate with Ribosomes
Authors: Scott Janelle M. , Ju Jingliang , Mitchell Theresa , Haldenwang W. G.
Published: July 2002

Obg, an essential GTP binding protein of
Bacillus subtilis
, is necessary for stress activation of the ς
B
transcription factor. We investigated Obg's cellular associations by differential centrifugation of crude
B. subtilis
extracts, using an anti-Obg antibody as a probe to monitor Obg during the fractionation, and by fluorescent microscopy of a
B. subtilis
strain in which Obg was fused to green fluorescent protein. The results indicated that Obg is part of a large cytoplasmic complex. In subsequent analyses, Obg coeluted with ribosomal subunits during gel filtration of
B. subtilis
lysates on Sephacryl S-400 and specifically bound to ribosomal protein L13 in an affinity blot assay. Probing the gel filtration fractions with antibodies specific for ς
B
and its coexpressed regulators (Rsb proteins) revealed coincident elution of the upstream components of the ς
B
stress activation pathway (RsbR, -S, and -T) with Obg and the ribosomal subunits. The data implicate ribosome function as a possible mediator of the activity of Obg and the stress induction of ς
B
.
Secretion of Nucleoside Diphosphate Kinase by Mucoid
Pseudomonas aeruginosa
8821: Involvement of a Carboxy-Terminal Motif in Secretion
Authors: Kamath Shilpa , Chen M. L. , Chakrabarty A. M.
Published: July 2002

Nucleoside diphosphate kinase (Ndk) is a ubiquitous enzyme which functions in balancing the nucleotide pool of the cell. We have recently reported that in addition to being intracellular in both mucoid and nonmucoid
Pseudomonas aeruginosa
, Ndk is also secreted into the extracellular environment by mucoid
P. aeruginosa
cells. This secreted Ndk has biochemical activity similar to the intracellular Ndk and is 16 kDa in size. To demonstrate that Ndk is indeed secreted and to localize the secretion motif, we constructed an
ndk
knockout mutant, which lacks both intracellular and extracellular forms of Ndk. In this study, we report the construction of deletion derivatives made from the carboxy-terminal region of Ndk. These deletion derivatives were introduced into the
ndk
::Cm knockout mutant and were examined for the intracellular and extracellular presence of Ndk. It was observed that the carboxy-terminal 8-amino-acid region is required for the secretion of Ndk into the extracellular region. This region has the sequence DXXX, where X is a predominantly hydrophobic residue. Such sequences represent a conserved motif in proteins secreted by the type I secretory pathway in gram-negative microorganisms. To investigate the significance of this motif in the secretion of Ndk, we constructed a fusion protein of Ndk and the blue fluorescent protein (BFP) as well as a fusion protein of mutated Ndk (whose DTEV motif has been changed to AAAA) and the BFP. The presence of extracellular Ndk was detected only in the
ndk
::Cm knockout mutant harboring the wild-type BFP-Ndk protein fusion. We could not detect the presence of extracellular Ndk in the
ndk
::Cm knockout mutant containing the mutated BFP-Ndk protein fusion. In addition, we have also used immunofluorescence microscopy to localize the wild-type and mutated BFP-Ndk proteins in the cell. The significance of these observations is discussed.
Motility and Chemotaxis of Filamentous Cells of
Escherichia coli
Authors: Maki Nazli , Gestwicki Jason E. , Lake Ellen M. , Kiessling Laura L. , Adler Julius
Published: July 2002

Filamentous cells of
Escherichia coli
can be produced by treatment with the antibiotic cephalexin, which blocks cell division but allows cell growth. To explore the effect of cell size on chemotactic activity, we studied the motility and chemotaxis of filamentous cells. The filaments, up to 50 times the length of normal
E. coli
organisms, were motile and had flagella along their entire lengths. Despite their increased size, the motility and chemotaxis of filaments were very similar to those properties of normal-sized cells. Unstimulated filaments of chemotactically normal bacteria ran and stopped repeatedly (while normal-sized bacteria run and tumble repeatedly). Filaments responded to attractants by prolonged running (like normal-sized bacteria) and to repellents by prolonged stopping (unlike normal-sized bacteria, which tumble), until adaptation restored unstimulated behavior (as occurs with normal-sized cells). Chemotaxis mutants that always ran when they were normal sized always ran when they were filament sized, and those mutants that always tumbled when they were normal sized always stopped when they were filament sized. Chemoreceptors in filaments were localized to regions both at the poles and at intervals along the filament. We suggest that the location of the chemoreceptors enables the chemotactic responses observed in filaments. The implications of this work with regard to the cytoplasmic diffusion of chemotaxis components in normal-sized and filamentous
E. coli
are discussed.
Evidence for Multimerization of Neu Proteins Involved in Polysialic Acid Synthesis in
Escherichia coli
K1 Using Improved LexA-Based Vectors
Authors: Daines Dayle A. , Silver Richard P.
Published: July 2002

Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205–212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an
Escherichia coli
background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in
E. coli
K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.
Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication
Authors: Lai Ying-Ta , Masker Warren
Published: July 2002

An in vitro system based on extracts of
Escherichia coli
infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5′→3′ exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.
β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis
Authors: Choi Keum-Hwa , Heath Richard J. , Rock Charles O.
Published: July 2002

A universal set of genes encodes the components of the dissociated, type II, fatty acid synthase system that is responsible for producing the multitude of fatty acid structures found in bacterial membranes. We examined the biochemical basis for the production of branched-chain fatty acids by gram-positive bacteria. Two genes that were predicted to encode homologs of the β-ketoacyl-acyl carrier protein synthase III of
Escherichia coli
(eFabH) were identified in the
Bacillus subtilis
genome. Their protein products were expressed, purified, and biochemically characterized. Both
B. subtilis
FabH homologs, bFabH1 and bFabH2, carried out the initial condensation reaction of fatty acid biosynthesis with acetyl-coenzyme A (acetyl-CoA) as a primer, although they possessed lower specific activities than eFabH. bFabH1 and bFabH2 also utilized iso- and anteiso-branched-chain acyl-CoA primers as substrates. eFabH was not able to accept these CoA thioesters. Reconstitution of a complete round of fatty acid synthesis in vitro with purified
E. coli
proteins showed that eFabH was the only
E. coli
enzyme incapable of using branched-chain substrates. Expression of either bFabH1 or bFabH2 in
E. coli
resulted in the appearance of a branched-chain 17-carbon fatty acid. Thus, the substrate specificity of FabH is an important determinant of branched-chain fatty acid production.
Dynamic Aspects of the Structured Cell Population in a Swarming Colony of
Proteus mirabilis
Authors: Matsuyama Tohey , Takagi Yuko , Nakagawa Yoji , Itoh Hiroto , Wakita Junichi , Matsushita Mitsugu
Published: July 2002

Proteus mirabilis
forms a concentric-ring colony by undergoing periodic swarming. A colony in the process of such synchronized expansion was examined for its internal population structure. In alternating phases, i.e., swarming (active migration) and consolidation (growth without colony perimeter expansion), phase-specific distribution of cells differing in length, in situ mobility, and migration ability on an agar medium were recognized. In the consolidation phase, the distribution of mobile cells was restricted to the inner part of a new ring and a previous terrace. Cells composing the outer part of the ring were immobile in spite of their ordinary swimming ability in a viscous solution. A sectorial cell population having such an internal structure was replica printed on fresh agar medium. After printing, a transplant which was in the swarming phase continued its ongoing swarming while a transplanted consolidation front continued its scheduled consolidation. This shows that cessation of migration during the consolidation phase was not due to substances present in the underlying agar medium. The ongoing swarming schedule was modifiable by separative cutting of the swarming front or disruption of the ring pattern by random mixing of the pattern-forming cell population. The structured cell population seemed to play a role in characteristic colony growth. However, separation of a narrow consolidation front from a backward area did not induce disturbance in the ongoing swarming schedule. Thus, cells at the frontal part of consolidation area were independent of the internal cell population and destined to exert consolidation and swarming with the ongoing ordinary schedule.
Escherichia coli
DNA Polymerase III τ- and γ-Subunit Conserved Residues Required for Activity In Vivo and In Vitro
Authors: Walker James R. , Hervas Christine , Ross Julie D. , Blinkova Alexandra , Walbridge Michael J. , Pumarega Emilynn J. , Park Mi-Oak , Neely Harold R.
Published: July 2002

The
Escherichia coli
DNA polymerase III τ and γ subunits are single-strand DNA-dependent ATPases (the latter requires the δ and δ′ subunits for significant ATPase activity) involved in loading processivity clamp β. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic τ/γ homologs and 1 eukaryotic τ/γ homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.
Different Processing of an mRNA Species in
Bacillus subtilis
and
Escherichia coli
Authors: Persson Martin , Glatz Elisabeth , Rutberg Blanka
Published: July 2002

Expression of the
Bacillus subtilis glpD
gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of
glpD
contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds to
glpD
leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of the
glpD
leader result in GlpP-independent, temperature-sensitive (TS) expression of
glpD
. The TS phenotype is due to temperature-dependent degradation of the
glpD
mRNA. In the presence of GlpP, the
glpD
mRNA is stabilized.
glpD
leader-
lacZ
fusions were integrated into the chromosomes of
B. subtilis
and
Escherichia coli
. Determination of steady-state levels of fusion mRNA in
B. subtilis
showed that the stability of the fusion mRNA is determined by the
glpD
leader part. Comparison of steady-state levels and half-lives of
glpD
leader-
lacZ
fusion mRNA in
B. subtilis
and
E. coli
revealed significant differences. A
glpD
leader-
lacZ
fusion transcript that was unstable in
B. subtilis
was considerably more stable in
E. coli
. GlpP, which stabilizes the transcript in
B. subtilis
, did not affect its stability in
E. coli
. Primer extension analysis showed that the
glpD
leader-
lacZ
fusion transcript is processed differently in
B. subtilis
and in
E. coli
. The dominating cleavage site in
E. coli
was barely detectable in
B. subtilis
. This site was shown to be a target of
E. coli
RNase III.
Regulation of the
cnr
Cobalt and Nickel Resistance Determinant from
Ralstonia
sp. Strain CH34
Authors: Grass Gregor , Große Cornelia , Nies Dietrich H.
Published: July 2002

Ralstonia
sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the
cnr
determinant located on plasmid pMOL28. The
cnr
genes are organized in two clusters,
cnrYXH
and
cnrCBA
. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the
cnrY
and
cnrC
genes. These two promoters exhibit conserved sequences at the −10 (CCGTATA) and −35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni
2+
. Half-maximal activation from the
cnrCBA
operon was determined by using appropriate
lacZ
gene fusions and was shown to occur at an Ni
2+
concentration of about 50 μM.
Distribution of Intervening Sequences in the Genes for 23S rRNA and rRNA Fragmentation among Strains of the
Salmonella
Reference Collection B (SARB) and SARC Sets
Authors: Pabbaraju Kanti , Miller Wayne L. , Sanderson Kenneth E.
Published: July 2002

Intervening sequences (IVSs) occur sporadically in several bacterial genera in the genes for 23S rRNA at relatively conserved locations. They are cleaved after transcription and lead to the presence of fragmented rRNA, which is incorporated into the ribosomes without religation but is nevertheless functional. The fragmentation of rRNA and the number of IVSs in all 72 strains of the
Salmonella
Reference Collection B set and 16 strains of the
Salmonella
Reference Collection C set, which have been established on the basis of multilocus enzyme electrophoresis (MLEE), were analyzed in the present study. Fragmentation of 23S rRNA was restricted to conserved cleavage sites located at bp 550 (helix 25) and bp 1170 (helix 45), locations where IVSs have been reported. Random cleavage at sites where IVSs could not be detected was not seen. Uncleaved IVSs were not detected in any case; thus, the IVSs invariably led to rRNA fragmentation, indicating a strong selection for maintenance of RNase III cleavage sites. The distribution of the number of IVSs carried by the different strains in the seven
rrl
genes is diverse, and the pattern of IVS possession could not be related to the MLEE pattern among the various
Salmonella
strains tested; this indicates that the IVSs are frequently exchanged between strains by lateral transfer. All eight subspecies of the genus
Salmonella
, including subspecies V represented by
Salmonella bongori
, have IVSs in both helix 25 and helix 45; this indicates that IVSs entered the genus after its divergence from
Escherichia coli
(more than 100 million years ago) but before separation of the genus
Salmonella
into many forms or that they were in the ancestor but have been lost from
Escherichia
.