bluuebunny/crossref_metadata_2025 huggingface dataset:
β-Lactamases of
Kluyvera ascorbata
, Probable Progenitors of Some Plasmid-Encoded CTX-M Types
Authors: Humeniuk Christel , Arlet Guillaume , Gautier Valerie , Grimont Patrick , Labia Roger , Philippon Alain
Published: October 2002

Kluyvera ascorbata
produces a β-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene,
bla
KLUA
, were obtained and were found to have minor variations (96 to 100%). Otherwise,
bla
KLUA
was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type β-lactamases. Finally, mobilization of
bla
KLUA
on a plasmid was found to be mediated probably by a genetic mobile element like IS
Ecp1
.
Antibiotic Susceptibilities of
Parachlamydia acanthamoeba
in Amoebae
Authors: Maurin M. , Bryskier A. , Raoult D.
Published: October 2002

Parachlamydia acanthamoeba
are intracellular bacteria of amoebae and are considered potential etiological agents of human pneumonia. We have determined the in vitro antibiotic susceptibilities of two strains (strain Bn
9
and Hall's coccus) in
Acanthamoeba polyphaga
. The two strains were susceptible to tetracyclines, macrolides, and rifampin, but resistant to fluoroquinolones.
Evaluation of the Genetic Structure of
Xylella fastidiosa
Populations from Different
Citrus sinensis
Varieties
Authors: Della Coletta-Filho Helvécio , Machado Marcos Antonio
Published: October 2002

Xylella fastidiosa
was isolated from sweet orange plants (
Citrus sinensis
) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of
C. sinensis
cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (

H
T

) values of
X. fastidiosa
were similar for both sets of strains; however,

H
T

RAPD
values were substantially lower than

H
T

VNTR
values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the
X. fastidiosa
strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of
X. fastidiosa
. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.
Culture-Based and Non-Growth-Dependent Detection of the
Burkholderia cepacia
Complex in Soil Environments
Authors: Miller Suzanne C. M. , LiPuma John J. , Parke Jennifer L.
Published: October 2002

Burkholderia cepacia
complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. We sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards, and gardens. A total of 91 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bcc isolates were recovered on
Burkholderia cepacia
selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22°C. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and
recA
gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bcc; 14 (15%) of 91 sampled sites yielded Bcc isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bcc genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bcc. The four soil samples from which these clones were generated did not yield isolates identified as Bcc. Based on PCR detection, Bcc appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bcc may underestimate environmental populations.
The
pepR
Gene of
Lactobacillus sakei
Is Positively Regulated by Anaerobiosis at the Transcriptional Level
Authors: Champomier-Vergès Marie-Christine , Marceau Anika , Méra Thérèse , Zagorec Monique
Published: October 2002

Lactobacillus sakei
is a lactic acid bacterium belonging to the natural flora of meat products. It constitutes the main flora of vacuum-packed meat and is largely used in western Europe as a starter for the manufacturing of fermented sausages. This species is able to grow both under aerobiosis and anaerobiosis. In many technological processes involving it, oxygen is scarce. The aim of this study was to identify the major proteins affected by growth under anaerobiosis. Using two-dimensional electrophoresis, we showed that one spot was 10-fold overexpressed when cells were grown under anaerobiosis. By N-terminal sequencing it was identified as a peptidase (PepR), and the
pepR
gene was cloned. Northern analysis revealed that
pepR
was expressed as a single 1.27-kb transcript induced under anaerobiosis. A mutant was constructed by single crossover in the
pepR
gene, and its growth and survival were not affected by anaerobiosis.
Genes Encoding the
N
-Acyl Homoserine Lactone-Degrading Enzyme Are Widespread in Many Subspecies of
Bacillus thuringiensis
Authors: Lee Sang Jun , Park Sun-Yang , Lee Jung-Ju , Yum Do-Young , Koo Bon-Tag , Lee Jung-Kee
Published: October 2002

Gram-negative bacteria can communicate with each other by
N
-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the
aiiA
gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from
Bacillus
sp. strain 240B1. During investigations in the course of the ongoing
Bacillus thuringiensis
subsp.
morrisoni
genome project, an
aiiA
homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in
B. thuringiensis. aiiA
homologue genes were found in 16 subspecies of
B. thuringiensis
, and their sequences were determined. Comparison of the
Bacillus
sp. strain 240B1
aiiA
gene with the
B. thuringiensis aiiA
homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of
B. thuringiensis
having an
aiiA
gene, the subspecies
aizawai
,
galleriae
,
kurstaki
,
kyushuensis
,
ostriniae
, and
subtoxicus
were shown to degrade AHL. It was observed that recombinant
Escherichia coli
producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of
Erwinia carotovora
. These results indicate that insecticidal
B. thuringiensis
strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.
Characterization of Virus Adsorption by Using DEAE-Sepharose and Octyl-Sepharose
Authors: Shields Patricia A. , Farrah Samuel R.
Published: October 2002

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and φX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included φX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.
The Diversity of
Phaseolus
-Nodulating Rhizobial Populations Is Altered by Liming of Acid Soils Planted with
Phaseolus vulgaris
L. in Brazil
Authors: Andrade D. S. , Murphy P. J. , Giller K. E.
Published: October 2002

PCR-mediated restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA internally transcribed spacer (ITS) region and the 16S rRNA gene indicated that the rhizobial populations isolated from common bean (
Phaseolus vulgaris
L.) nodules in the unlimed soil from a series of five lime rates applied 6 years previously to plots of an acidic oxisol had less diversity than those from plots with higher rates of liming. Isolates affiliated with
Rhizobium tropici
IIB and
Rhizobium leguminosarum
bv. phaseoli were predominant independent of lime application. An index of richness based on the number of ITS groups increased from 2.2 to 5.7 along the soil liming gradient, and the richness index based on “species” types determined by RFLP analysis of the 16S rRNA gene varied from 0.5 to 1.4. The Shannon index of diversity, based on the number of ITS groups, increased from 1.8 in unlimed soil to 2.8 in limed soil, and, based on RFLP analysis of the 16S rRNA gene, ranged from 0.9 to 1.4. In the limed soil, the subpopulation of
R. tropici
IIB pattern types contained the largest number of ITS groups. In contrast, there were more
R. leguminosarum
bv. phaseoli types in the unlimed soil with the lowest pH than in soils with the highest pH. The number of ITS (“strain”) groups within
R. leguminosarum
bv. phaseoli did not change with increased abundance of rhizobia in the soil, while with
R. tropici
IIB, the number of strain groups increased significantly. Some cultural and biochemical characteristics of
Phaseolus
-nodulating isolates were significantly related to changes in soil properties caused by liming, largely due to changes in the predominance of the rhizobial species groups.
Different Responses of Pyoverdine Genes to Autoinduction in
Pseudomonas aeruginosa
and the Group
Pseudomonas fluorescens-Pseudomonas putida
Authors: Ambrosi Cecilia , Leoni Livia , Visca Paolo
Published: October 2002

We investigated the regulation of the
psbA
and
pvdA
pyoverdine biosynthesis genes, which encode the
l
-ornithine
N
5
-oxygenase homologues in
Pseudomonas
strain B10 and
Pseudomonas aeruginosa
PAO1, respectively. We demonstrate that pyoverdine
B10
, as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in
Pseudomonas
strain B10. In
P. aeruginosa
PAO1, however, pyoverdine
PAO1
has no apparent role in the positive regulation of the
pvdA
gene.
Susceptibility of Interleukin-2-Deficient Mice toToxoplasma gondiiIs Associated with a Defect in the Production of Gamma Interferon
Authors: Villegas Eric N. , Lieberman Linda A. , Carding Simon R. , Hunter Christopher A.
Published: October 2002

Costimulation through the B7-CD28 interaction is an important second signal for T-cell activation, and previous studies have shown that CD28−/−mice infected withToxoplasma gondiigenerate suboptimal CD4+T-cell responses, associated with a defect in production of the T-cell growth factor interleukin-2 (IL-2). To address the role of IL-2 in the expansion of T cells during toxoplasmosis, IL-2−/−mice were infected withT. gondiiand their ability to generate a protective T-cell response was assessed. Although IL-2−/−mice produced normal levels of IL-12p40, they had reduced levels of gamma interferon (IFN-γ) in serum, had an increased parasite burden, and succumbed to infection withT. gondiiwithin 20 days. Fluorescence-activated cell sorter analysis revealed that, although uninfected IL-2−/−mice had an increased number of activated T cells compared with uninfected IL-2+/+mice, following infection they were unable to further upregulate this population. Examination of the ability of splenocytes from uninfected and infected mice to produce IFN-γ revealed that IL-2−/−mice were hyporesponsive to stimulation with anti-CD3 or parasite antigen compared with wild-type mice, and the addition of IL-2 alone or in combination with IL-12 or stimulation with phorbol myristate acetate and ionomycin did not restore the production of IFN-γ. Together, these studies reveal that IL-2−/−mice are unable to generate a protective IFN-γ response following infection withT. gondiiand suggest that IL-2−/−mice have an intrinsic defect in their ability to activate and expand IFN-γ-producing T cells required for resistance toT. gondii.
Comparison of Host Resistance to Primary and SecondaryListeria monocytogenesInfections in Mice by Intranasal and Intravenous Routes
Authors: Mizuki Mayuko , Nakane Akio , Sekikawa Kenji , Tagawa Yoh-ich , Iwakura Yoichiro
Published: October 2002

There have been no studies on the susceptibility and host immune responses to an intranasal infection withListeria monocytogenes. In this study, we compared the susceptibilities and cytokine responses between intranasal and intravenous infections withL. monocytogenesin mice. Moreover, we compared efficiency of acquisition of host resistance toL. monocytogenesinfection between intranasally and intravenously immunized mice because an intranasal immunization of vaccines is reportedly available for induction of adaptive immunity against various infectious pathogens. The susceptibility to an intranasal infection withL. monocytogeneswas markedly lower than that to the intravenous infection. The bacterial growth in the lungs, spleens, and livers was substantially similar between intranasally and intravenously infected mice. Titers of endogenous gamma interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) in the spleens, livers, and lungs were parallel to bacterial numbers in each organ of mice during intranasal infection and intravenous infection. IFN-γ-deficient mice and TNF-α-deficient mice were highly susceptible to intranasal infection as well as intravenous infection. Susceptibilities to intranasal and intravenousL. monocytogenesinfection were the same in these cytokine-deficient mice. These results suggest that both IFN-γ and TNF-α play critical roles in host resistance to intranasalL. monocytogenesinfection as well as the intravenous infection. Acquisition of host resistance to intravenous and intranasalL. monocytogenesinfection was induced in intranasally immunized mice as well as intravenously immunized mice, suggesting that intranasal immunization is effective for prevention of a systemic infection withL. monocytogenes.
Macrophage Plasma Membrane Cholesterol Contributes to
Brucella abortus
Infection of Mice
Authors: Watarai Masahisa , Makino Sou-ichi , Michikawa Makoto , Yanagisawa Katsuhiko , Murakami Shigeru , Shirahata Toshikazu
Published: October 2002

Brucella abortus
is a facultative intracellular bacterium capable of surviving inside macrophages. Intracellular replication of
B. abortus
requires the VirB complex, which is highly similar to conjugative DNA transfer systems. In this study, we show that plasma membrane cholesterol of macrophages is required for the VirB-dependent internalization of
B. abortus
and also contributes to the establishment of bacterial infection in mice. The internalization of
B. abortus
was accelerated by treating macrophages with acetylated low-density lipoprotein (acLDL). Treatment of acyl coenzyme A:cholesterol acyltransferase inhibitor, HL-004, to macrophages preloaded with acLDL accelerated the internalization of
B. abortus
. Ketoconazole, which inhibits cholesterol transport from lysosomes to the cell surface, inhibited the internalization and intracellular replication of
B. abortus
in macrophages. The Niemann-Pick C1 gene (NPC1), the gene for Niemann-Pick type C disease, characterized by an accumulation of cholesterol in most tissues, promoted
B. abortus
infection. NPC1-deficient mice were resistant to the bacterial infection. Molecules associated with cholesterol-rich microdomains, “lipid rafts,” accumulate in intracellular vesicles of macrophages isolated from NPC1-deficient mice, and the macrophages yielded no intracellular replication of
B. abortus
. Thus, trafficking of cholesterol-associated microdomains controlled by NPC1 is critical for the establishment of
B. abortus
infection.
Incorporation ofN-Acetylneuraminic Acid intoHaemophilus somnusLipooligosaccharide (LOS): Enhancement of Resistance to Serum and Reduction of LOS Antibody Binding
Authors: Inzana Thomas J. , Glindemann Gretchen , Cox Andrew D. , Wakarchuk Warren , Howard Michael D.
Published: October 2002

Haemophilus somnusisolates from cases of thrombotic meningoencephalitis, pneumonia, and other disease sites are capable of undergoing a high rate of phase variation in the oligosaccharide component of their lipooligosaccharides (LOS). In contrast, the LOS of commensal strains isolated from the normal reproductive tract phase vary little or not at all. In addition, the LOS ofH. somnusshares conserved epitopes with LOS fromNeisseria gonorrhoeae,Haemophilus influenzae, and other species that can incorporate sialic acid into their LOS. We now report that growth of disease isolates ofH. somnuswith CMP-N-acetylneuraminic acid (CMP-NeuAc) or NeuAc added to the medium resulted in incorporation of NeuAc into the LOS. However, NeuAc was not incorporated into the LOS of commensal isolates and one disease isolate following growth in medium containing CMP-NeuAc or NeuAc. Sialylated LOS was detected by an increase in the molecular size or an increase in the amount of the largest-molecular-size LOS electrophoretic bands, which disappeared following treatment with neuraminidase. Sialylated LOS could also be detected by reactivity withLimax flavusagglutinin lectin, which is specific for sialylated species, by dot blot assay; this reactivity was also reversed by neuraminidase treatment.H. somnusstrain 2336 LOS was found to contain some sialic acid when grown in medium lacking CMP-NeuAc or NeuAc, although supplementation enhanced NeuAc incorporation. In contrast strain 738, an LOS phase variant of strain 2336, was less extensively sialylated when the growth medium was supplemented with CMP-NeuAc or NeuAc, as determined by electrophoretic profiles and electrospray mass spectrometry. The sialyltransferase ofH. somnusstrain 738 was confirmed to preferentially sialylate the Galβ-(1-3)-GlcNAc component of the lacto-N-tetraose structure by capillary electrophoresis assay. Enhanced sialylation of the strain 2336 LOS inhibited the binding of monoclonal antibodies to LOS by enzyme immunoassay and Western blotting. Furthermore, sialylation of the LOS enhanced the resistance ofH. somnusto the bactericidal action of antiserum to LOS. Sialylation and increased resistance to killing by normal serum also occurred in a deletion mutant that was deficient in the terminal Gal-GlcNAc disaccharide. LOS sialylation may therefore be an important virulence mechanism to protectH. somnusagainst the host immune system.
Protection from Group B Streptococcal Infection in Neonatal Mice by Maternal Immunization with Recombinant Sip Protein
Authors: Martin Denis , Rioux Stéphane , Gagnon Edith , Boyer Martine , Hamel Josée , Charland Nathalie , Brodeur Bernard R.
Published: October 2002

The protective potential of antibodies directed against group B streptococcus (GBS) Sip surface protein was determined by using the mouse neonatal infection model. Rabbit Sip-specific antibodies administered passively to pregnant mice protected their pups against a GBS lethal challenge. In addition, active immunization with purified recombinant Sip protein of female CD-1 mice induced the production of specific antibodies that also confer protection to the newborn pups against GBS strains of serotypes Ia/c, Ib, II, III, and V. These data confirm that Sip-specific antibodies can cross the placenta and conferred protective immunity against GBS infections.
The
Haemophilus influenzae
Hap Autotransporter Binds to Fibronectin, Laminin, and Collagen IV
Authors: Fink Doran L. , Green Bruce A. , St. Geme Joseph W.
Published: October 2002

Nontypeable
Haemophilus influenzae
(NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the
H. influenzae
Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap
s
, from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap
s
. Purified Hap
s
binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap
s
to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.
Prevalence and Boosting of Antibodies toPlasmodium falciparumGlycosylphosphatidylinositols and Evaluation of Their Association with Protection from Mild and Severe Clinical Malaria
Authors: de Souza J. Brian , Todd James , Krishegowda Gowdahalli , Gowda D. Channe , Kwiatkowski Dominic , Riley Eleanor M.
Published: October 2002

Glycosylphosphatidylinositols (GPIs), the anchor molecules of some membrane proteins ofPlasmodiumspecies, have been implicated in the induction of immunopathology during malaria infections. Hence, neutralization of GPIs by antibodies may reduce the severity of clinical attacks of malaria. To test this hypothesis, we have assessed the levels of anti-GPI antibodies in plasma from children and adults living in areas of seasonal malaria transmission in The Gambia. In a prospective study of susceptibility to clinical or asymptomatic infection, the levels of anti-GPI antibodies were measured before and after the transmission season. Samples were also obtained from children recruited into a hospital-based study of severe malaria. We find that in malaria-exposed individuals both the prevalence and the concentration of anti-GPI antibodies increase with age and that antibody levels are significantly higher at the end of the malaria transmission season than at the start of the season. Antibody levels are also higher in children with asymptomatic infections (i.e., those with a degree of clinical immunity) than in children who developed clinical malaria and high parasitemia, although this difference is not statistically significant. Importantly, antibodies appear to be rapidly boosted by clinical malaria infection, but children under the age of two years are seronegative for anti-GPI antibodies, even during an acute infection. While GPIs may be involved in the pathogenesis of human malaria, the data from this study do not provide any strong evidence to support the notion that anti-GPI antibodies confer resistance to mild or severe malarial disease. Further case-control studies, ideally of a prospective nature, are required to elucidate the role of antiglycolipid antibodies in protection from severe malaria.
Antibodies toPlasmodium falciparumGlycosylphosphatidylinositols: Inverse Association with Tolerance of Parasitemia in Papua New Guinean Children and Adults
Authors: Boutlis Craig S. , Gowda D. Channe , Naik Ramachandra S. , Maguire Graeme P. , Mgone Charles S. , Bockarie Moses J. , Lagog Moses , Ibam Erwin , Lorry Kerry , Anstey Nicholas M.
Published: October 2002

Individuals living in regions of intense malaria transmission exhibit natural immunity that facilitates persistence of parasitemia at controlled densities for much of the time without symptoms. This aspect of immunity has been referred to as malarial “tolerance” and is thought to partly involve inhibition of the chain of events initiated by a parasite toxin(s) that may otherwise result in cytokine release and symptoms such as fever. Antibodies to the candidatePlasmodium falciparumglycosylphosphatidylinositol (GPI) toxin have been viewed as likely mediators of such tolerance. In this study, the relationship between antibodies toP. falciparumGPIs, age, and parasitemia was determined in asymptomatic children and adults living in Madang, Papua New Guinea. The prevalence and intensity of antibody responses increased with age and were lowest in children 1 to 4 years old with the highest-density parasitemias. In children of this age group who were tolerant of parasitemia during the study, only 8.3% had detectable immunoglobulin G (IgG) and none had IgM antibodies to GPI. This suggests that anti-GPI antibodies are unlikely to be the sole mediator of malarial tolerance, especially in children younger than 5 years. Following antimalarial treatment, clearance of parasitemia led to a fall in anti-GPI IgG response in children and adolescents within 6 weeks. As anti-GPI antibodies potentially play a role in protecting against disease progression, our results caution against the treatment of asymptomatic parasitemia and suggest that generation of a sustained antibody response in children poses a challenge to novel antitoxic vaccination strategies.
Endogenous Interleukin-12 Is Critical for Controlling the Late but Not the Early Stage ofLeishmania mexicanaInfection in C57BL/6 Mice
Authors: Aguilar Torrentera Fabiola , Laman Jon D. , Van Meurs Marjan , Adorini Luciano , Muraille Eric , Carlier Y.
Published: October 2002

The role of interleukin-12 (IL-12) has been clearly established in the resistance of C57BL/6 (B6) mice toLeishmania majorinfection, but its involvement in the control ofLeishmania mexicanainfection remains to be determined. Here, we show the following. (i)L. mexicana, in contrast toL. major, induces the development of nonhealing lesions in B6 mice. (ii) Cells expressing IL-12p40, gamma interferon (IFN-γ), NOS2, and CD40L are numerous in the footpad lesion and/or the draining popliteal lymph node of animals infected for up to 14 weeks withL. mexicana. (iii) B6 mice, either IL-12p40 deficient or treated with IL-12p40-neutralizing antibodies, display a dramatic enhancement of primary and secondary lesions leading to death 10 weeks after inoculation withL. mexicana. (iv) Splenocytes harvested 4 and 8 weeks after infection of IL-12p40−/−B6 mice withL. mexicanaare unable to produce IFN-γ, but secrete IL-4, IL-10, and IL-18. Thus, the early control ofL. mexicanainfection by B6 mice is independent of IL-12, whereas IL-12 and Th1 responses play a key role in controlling the late stages ofL. mexicanainfection. However, they fail to resolve lesions, in contrast toL. majorinfection, emphasizing the different outcomes induced by these twoLeishmaniaspecies in B6 mice.
Ganglioside Mimicry ofCampylobacter jejuniLipopolysaccharides Determines Antiganglioside Specificity in Rabbits
Authors: Ang C. W. , Noordzij P. G. , de Klerk M. A. , Endtz H. P. , van Doorn P. A. , Laman J. D.
Published: October 2002

The core oligosaccharides ofCampylobacter jejunilipopolysaccharides (LPS) display molecular mimicry with gangliosides. Cross-reactive anti-LPS-antiganglioside antibodies have been implicated to show a crucial role in the pathogenesis of the Guillain-Barré and Miller Fisher syndrome. The specificity of the antiganglioside response is thought to depend on the oligosaccharide structure of the ganglioside mimic. To test this hypothesis and to investigate the potential of LPS fromCampylobacterstrains from enteritis patients to induce an antiganglioside response, we immunized rabbits with purified LPS from eightCampylobacter jejunireference strains with biochemically well-defined distinct ganglioside mimics and determined the presence of antiganglioside antibodies. All rabbits produced immunoglobulin G (IgM) and IgG anti-LPS antibodies, and the specificity of the cross-reactive antiganglioside response indeed corresponded with the biochemically defined mimic. Most rabbits also had antibody reactivity against additional gangliosides, and there were slight differences in the fine specificity of the antibody response between rabbits that had been immunized with LPS from the sameCampylobacterstrain. High anti-LPS and antiganglioside titers persisted over a 10-month period. In conclusion, the structure of the LPS only partly determines the antiganglioside specificity. Other strain-specific as well as host-related factors influence the induction and fine-specificity of the cross-reactive anti-LPS-antiganglioside response.
Fcγ Receptor I- and III-Mediated Macrophage Inflammatory Protein 1α Induction in Primary Human and Murine Microglia
Authors: Song Xianyuan , Shapiro Scott , Goldman David L. , Casadevall Arturo , Scharff Matthew , Lee Sunhee C.
Published: October 2002

Microglial cell phagocytic receptors may play important roles in the pathogenesis and treatment of several neurological diseases. We studied microglial Fc receptor (FcR) activation with respect to the specific FcγR types involved and the downstream signaling events by using monoclonal antibody (MAb)-coatedCryptococcus neoformansimmune complexes as the stimuli and macrophage inflammatory protein 1α (MIP-1α) production as the final outcome.C. neoformanscomplexed with murine immunoglobulin G (IgG) of γ1, γ2a, and γ3, but not γ2b isotype, was effective in inducing MIP-1α in human microglia. Since murine γ2b binds to human FcγRII (but not FcγRI or FcγRIII), these results indicate that FcγRI and/or FcγRIII is involved in MIP-1α production. Consistent with this, an antibody that blocks FcγRII (IV.3) failed to inhibit MIP-1α production, while an antibody that blocks FcγRIII (3G8) did. An anti-C. neoformansMAb, 18B7 (IgG1), but not its F(ab′)2, induced extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase kinase phosphorylation, and MIP-1α release was suppressed by the ERK inhibitor U0126.C. neoformansplus 18B7 also induced degradation of I-κBα, and MIP-1α release was suppressed by the antioxidant NF-κB inhibitor pyrrolidine dithiocarbamate. To confirm the role of FcR more directly, we isolated microglia from wild-type and various FcR-deficient mice and then challenged them withC. neoformansplus 18B7. While FcγRII-deficient microglia showed little difference from the wild-type microglia, both FcγRI α-chain- and FcγRIII α-chain-deficient microglia produced less MIP-1α, and the common Fc γ-chain-deficient microglia showed no MIP-1α release. Taken together, our results demonstrate a definitive role for FcγRI and FcγRIII in microglial chemokine induction and implicate ERK and NF-κB as the signaling components leading to MIP-1α expression. Our results delineate a new mechanism for microglial activation and may have implications for central nervous system inflammatory diseases.
Mechanism of Entry Determines the Ability ofToxoplasma gondiiTo Inhibit Macrophage Proinflammatory Cytokine Production
Authors: Butcher Barbara A. , Denkers Eric Y.
Published: October 2002

Macrophages (Mφ) infected with tachyzoites of the opportunistic protozoanToxoplasma gondiiare blocked in production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12) in response to lipopolysaccharide (LPS) triggering, and this is associated with parasite-induced inhibition of NFκB translocation. Here, we demonstrate a requirement for active invasion in the ability of the parasite to mediate suppression. Neither soluble tachyzoite antigen nor secreted products were suppressive, and heat-inactivated, antibody-coated tachyzoites, which efficiently entered the cell through receptor-mediated uptake, failed to inhibit LPS responses. Cytochalasin D, a drug blocking tachyzoite invasion of, but not adherence to, Mφ, severely curtailedToxoplasma-induced suppression. In addition, parasite-induced nonresponsiveness, as measured by TNF-α production, was reversed by treating infected cells with the toxoplasmastatic drugs pyrimethamine and 6-thioxanthine prior to LPS stimulation. A divergence in IL-12 and TNF-α responses was found during extended incubation of tachyzoites and Mφ in that 24 h of incubation of infected Mφ resulted in IL-12, but not TNF-α, secretion, and production of the latter cytokine remained suppressed when these cells were subjected to LPS triggering. Our results demonstrate that active invasion and survival of the parasite within the parasitophorous vacuole are required to induce and maintain Mφ cytokine-specific nonresponsiveness to LPS. They also show that the effects ofToxoplasmaon IL-12 and TNF-α production are nonidentical, with the parasite exerting a longer-lasting suppression of the latter.
Contribution of
Candida albicans ALS1
to the Pathogenesis of Experimental Oropharyngeal Candidiasis
Authors: Kamai Yasuki , Kubota Mikie , Kamai Yoko , Hosokawa Tsunemichi , Fukuoka Takashi , Filler Scott G.
Published: October 2002

We investigated the contribution of
Candida albicans ALS1
, which encodes a candidal adhesin, to the pathogenesis of experimental murine oropharyngeal candidiasis. Our results indicate that the
ALS1
gene product is important for the adherence of the organism to the oral mucosa during the early stage of the infection.
Lipopolysaccharide Desensitization of Macrophages Provides Protection againstYersinia enterocolitica-Induced Apoptosis
Authors: Ruckdeschel Klaus , Richter Kathleen
Published: October 2002

PathogenicYersiniaspp. uncouple an array of signal transduction pathways in macrophages to disrupt their response to infection. This compels the macrophage to undergo apoptosis. Our study shows that macrophages that had acquired tolerance toYersiniainfection by preexposure to lipopolysaccharide were considerably protected againstY. enterocolitica-induced apoptosis. The desensitization of macrophages by lipopolysaccharide, which is thought to be a self-protective, adaptive response to sustained bacterial stimulation, may represent an immune mechanism that aids in overcomingYersinia-mediated apoptosis and infection.
A Mutant ofBurkholderia pseudomallei, Auxotrophic in the Branched Chain Amino Acid Biosynthetic Pathway, Is Attenuated and Protective in a Murine Model of Melioidosis
Authors: Atkins T. , Prior R. G. , Mack K. , Russell P. , Nelson M. , Oyston P. C. F. , Dougan G. , Titball R. W.
Published: October 2002

Using a transposon mutagenesis approach, we have identified a mutant ofBurkholderia pseudomalleithat is auxotrophic for branched chain amino acids. The transposon was shown to have interrupted theilvIgene encoding the large subunit of the acetolactate synthase enzyme. Compared to the wild type, this mutant was significantly attenuated in a murine model of disease. Mice inoculated intraperitoneally with the auxotrophic mutant, 35 days prior to challenge, were protected against a challenge dose of 6,000 median lethal doses of wild-typeB. pseudomallei.
Short-Sequence Tandem and Nontandem DNA Repeats and Endogenous Hydrogen Peroxide Production Contribute to Genetic Instability ofStreptococcus pneumoniae
Authors: Pericone Christopher D. , Bae Deborah , Shchepetov Mikhail , McCool Tera , Weiser Jeffrey N.
Published: October 2002

Loss-of-function mutations in the following seven pneumococcal genes were detected and analyzed:pspA,spxB,xba,licD2,lytA,nanA, andatpC. Factors associated with these mutations included (i) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as 6 bases, (ii) deletions caused by recombinational events between nontandem direct repeats as short as 8 bases, and (iii) substitutions of guanine residues caused at an increased frequency by the high levels of hydrogen peroxide (>2 mM) typically generated by this species under aerobic growth conditions. The latter accounted for a frequency as high as 2.8 × 10−6for spontaneous mutation to resistance to optochin and was 10- to 200-fold lower in the absence of detectable levels of H2O2. Some of these mutations appear to have been selected for in vivo during pneumococcal infection, perhaps as a consequence of immune pressure or oxidative stress.
Genes of De Novo Pyrimidine Biosynthesis from the Hyperthermoacidophilic Crenarchaeote
Sulfolobus acidocaldarius
: Novel Organization in a Bipolar Operon
Authors: Thia-Toong Thia-Lin , Roovers Martine , Durbecq Virginie , Gigot Daniel , Glansdorff Nicolas , Charlier Daniel
Published: October 2002

Sequencing a 8,519-bp segment of the
Sulfolobus acidocaldarius
genome revealed the existence of a tightly packed bipolar pyrimidine gene cluster encoding the enzymes of de novo UMP synthesis. The G+C content of 35.3% is comparable to that of the entire genome, but intergenic regions exhibit a considerably lower percentage of strong base pairs. Coding regions harbor the classical excess of purines on the coding strand, whereas intergenic regions do not show this bias. Reverse transcription-PCR and primer extension experiments demonstrated the existence of two polycistronic messengers,
pyrEF-orf8
and
pyrBI-orf1-pyrCD-orf2-orf3-orf4
, initiated from a pair of divergent and partially overlapping promoters. The gene order and the grouping in two wings of a bipolar operon constitute a novel organization of
pyr
genes that also occurs in the recently determined genome sequences of
Sulfolobus solfataricus
P2 and
Sulfolobus tokodaii
strain 7; the configuration appears therefore characteristic of
Sulfolobus
. The quasi-leaderless
pyrE
and
pyrB
genes do not bear a Shine-Dalgarno sequence, whereas the initiation codon of promoter-distal genes is preceded at an appropriate distance by a sequence complementary to the 3′ end of 16S rRNA. The polycistronic nature of the
pyr
messengers and the existence of numerous overlaps between contiguous open reading frames suggests the existence of translational coupling.
pyrB
transcription was shown to be approximately twofold repressed in the presence of uracil. The mechanism underlying this modulation is as yet unknown, but it appears to be of a type different from the various attenuation-like mechanisms that regulate
pyrB
transcription in bacteria. In contrast, the
pyrE-pyrB
promoter/control region harbors direct repeats and imperfect palindromes reminiscent of target sites for the binding of a hypothetical regulatory protein(s).
ThenptAGene ofVibrio choleraeEncodes a Functional Sodium-Dependent Phosphate Cotransporter Homologous to the Type II Cotransporters of Eukaryotes
Authors: Lebens Michael , Lundquist Patrik , Söderlund Lars , Todorovic Mirjana , Carlin Nils I. A.
Published: October 2002

ThenptAgene ofVibrio choleraehas significant protein sequence homology with type II sodium-dependent phosphate (Pi) cotransporters found in animals but not previously identified in prokaryotes. The phylogeny of known type II cotransporter sequences indicates thatnptAmay be either an ancestral gene or a gene acquired from a higher eukaryotic source. The gene was cloned into an expression vector under the control of an inducible promoter and expressed inEscherichia coli. The results demonstrate thatnptAencodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent Piuptake with comparable affinities for both sodium and phosphate ions. Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon. Piuptake activity, a component of which appeared to be sodium dependent, was increased inV. choleraeby phosphate starvation. However, it appears from the use of a reporter gene expressed from thenptApromoter that none of this activity is attributable to the induction of expression fromnptA. It is thus proposed that the physiological function of NptA protein may be the rapid uptake of Piin preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.
Proteolytic Cleavage of the FlhB Homologue YscU of
Yersinia pseudotuberculosis
Is Essential for Bacterial Survival but Not for Type III Secretion
Authors: Lavander Moa , Sundberg Lena , Edqvist Petra J. , Lloyd Scott A. , Wolf-Watz Hans , Forsberg Åke
Published: October 2002

Pathogenic
Yersinia
species employ a type III secretion system (TTSS) to target antihost factors, Yop proteins, into eukaryotic cells. The secretion machinery is constituted of ca. 20 Ysc proteins, nine of which show significant homology to components of the flagellar TTSS. A key event in flagellar assembly is the switch from secreting-assembling hook substrates to filament substrates, a switch regulated by FlhB and FliK. The focus of this study is the FlhB homologue YscU, a bacterial inner membrane protein with a large cytoplasmic C-terminal domain. Our results demonstrate that low levels of YscU were required for functional Yop secretion, whereas higher levels of YscU lowered both Yop secretion and expression. Like FlhB, YscU was cleaved into a 30-kDa N-terminal and a 10-kDa C-terminal part. Expression of the latter in a wild-type strain resulted in elevated Yop secretion. The site of cleavage was at a proline residue, within the strictly conserved amino acid sequence NPTH. A YscU protein with an in-frame deletion of NPTH was cleaved at a different position and was nonfunctional with respect to Yop secretion. Variants of YscU with single substitutions in the conserved NPTH sequence—i.e., N263A, P264A, or T265A—were not cleaved but retained function in Yop secretion. Elevated expression of these YscU variants did, however, result in severe growth inhibition. From this we conclude that YscU cleavage is not a prerequisite for Yop secretion but is rather required to maintain a nontoxic fold.
Simultaneous Coexpression ofBorrelia burgdorferiErp Proteins Occurs through a Specific,erpLocus-Directed Regulatory Mechanism
Authors: El-Hage Nazira , Stevenson Brian
Published: October 2002

An individualBorrelia burgdorferibacterium can encode as many as 13 different Erp (OspE/F-related) proteins from mono-and bicistronic loci that are carried on up to 10 separate plasmids. We demonstrate through multilabel immunofluorescence analyses that individual bacteria simultaneously coexpress their entire Erp protein repertoire. While it has been proposed thatB. burgdorfericontrols expression of Erp and other plasmid-encoded proteins through changes in DNA topology, we observed regulated Erp expression in the absence of detectable differences in DNA supercoiling. Likewise, inhibition of DNA gyrase had no detectable effect on Erp expression. Furthermore, expression of loci physically adjacent toerploci was observed to be independently regulated. It is concluded that Erp expression is regulated by a mechanism(s) directed aterploci and not by a global, plasmid-wide mechanism.
From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways
Authors: Gerdes Svetlana Y. , Scholle Michael D. , D'Souza Mark , Bernal Axel , Baev Mark V. , Farrell Michael , Kurnasov Oleg V. , Daugherty Matthew D. , Mseeh Faika , Polanuyer Boris M. , Campbell John W. , Anantha Shubha , Shatalin Konstantin Y. , Chowdhury Shamim A. K. , Fonstein Michael Y. , Osterman Andrei L.
Published: October 2002

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting inEscherichia colifollowed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability ofE. coliin rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of theE. coligenesnadD,coaD, andribF), are discussed in detail.
Functional Annotation of Class I Lysyl-tRNA Synthetase Phylogeny Indicates a Limited Role for Gene Transfer
Authors: Ambrogelly Alexandre , Korencic Dragana , Ibba Michael
Published: October 2002

Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms. Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS. In bacteria the class I LysRS is only found in the α-proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea. To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins. Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognizeEscherichia colitRNALysanticodon mutants. Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNALys(U36) and those that recognize both the second and third positions (U35 and U36). Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the α-proteobacteria. Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division. The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria.