bluuebunny/crossref_metadata_2025 huggingface dataset:
Tn
5406
, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin
Authors: Haroche Julien , Allignet Jeanine , El Solh Névine
Published: July 2002

We characterized a new transposon, Tn
5406
(5,467 bp), in a clinical isolate of
Staphylococcus aureus
(BM3327). It carries a variant of
vgaA
, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn
554
. Like Tn
554
, Tn
5406
failed to generate target repeats. In BM3327, the single copy of Tn
5406
was inserted into the chromosomal
att554
site, which is the preferential insertion site of Tn
554
. In three other independent
S. aureus
clinical isolates, Tn
5406
was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn
5406
-carrying plasmids also contain two other genes,
vgaB
and
vatB
. The insertion sites of Tn
5406
in BM3252 were studied: one copy was in
att554
, and one copy was in the additional
SCCmec
element. Amplification experiments revealed circular forms of Tn
5406
, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.
Effects of Gender, AIDS, and Acetylator Status on Intrapulmonary Concentrations of Isoniazid
Authors: Conte John E. , Golden Jeffrey A. , McQuitty Mari , Kipps Juliana , Duncan Sheila , McKenna Elaine , Zurlinden Elisabeth
Published: July 2002

The objective of the present study was to evaluate the effects of gender, AIDS, and acetylator status on the steady-state concentrations of orally administered isoniazid in plasma and lungs. Isoniazid was administered at 300 mg once daily for 5 days to 80 adult volunteers. Subjects were assigned to eight blocks according to gender, presence or absence of AIDS, and acetylator status. Blood was obtained prior to administration of the first dose, 1 h after administration of the last dose, and at the completion of bronchoscopy and bronchoalveolar lavage (BAL), which was performed 4 h after administration of the last dose. The metabolism of caffeine was used to determine acetylator status. Standardized bronchoscopy was performed without systemic sedation. The volume of epithelial lining fluid (ELF) recovered was calculated by the urea dilution method. Isoniazid concentrations in plasma, BAL fluid, and alveolar cells (ACs) were measured by high-performance liquid chromatography. AIDS status or gender had no significant effect on the concentrations of isoniazid in plasma at 1 or 4 h. Concentrations in plasma at 4 h and concentrations in ELF were greater in slow acetylators than fast acetylators. The concentration in plasma (1.85 ± 1.60 μg/ml [mean ± standard deviation;n= 80]) at 1 h following administration of the last dose was not significantly different from that in ELF (2.25 ± 3.50 μg/ml) or ACs (2.61 ± 5.01 μg/ml). For the entire study group, concentrations in plasma at 1 h were less than 1.0, 2.0, and 3.0 μg/ml for 34.7, 60, and 82.7% of the subjects, respectively; concentrations in ELF were less than 1.0, 2.0, and 3.0 μg/ml in 30 (37.5%), 53 (66.0%), and 58 (72.5%) of the subjects, respectively; and concentrations in ACs were less than 1.0, 2.0, and 3.0 μg/ml in 43 (53.8%), 59 (73.8%), and 65 (81.3%) of the subjects, respectively. The concentrations of orally administered isoniazid in plasma were not affected by gender or the presence of AIDS. The concentrations in plasma at 4 h and the concentrations in ELF, but not the concentrations in ACs, were significantly greater in slow acetylators than fast acetylators. Concentrations in plasma and lungs were low compared to recommended therapeutic concentrations in plasma and published MICs of isoniazid forMycobacterium tuberculosis. The optimal concentrations of isoniazid in ACs and ELF are unknown, but these data suggest that the drug enters these compartments by passive diffusion and achieves concentrations similar to those found in plasma.
Characterization of a Novel Plasmid-Mediated Cephalosporinase (CMY-9) and Its Genetic Environment in anEscherichia coliClinical Isolate
Authors: Doi Yohei , Shibata Naohiro , Shibayama Keigo , Kamachi Kazunari , Kurokawa Hiroshi , Yokoyama Keiko , Yagi Tetsuya , Arakawa Yoshichika
Published: July 2002

AnEscherichia colistrain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The β-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of β-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase ofAeromonas hydrophila. Asul1-type class 1 integron containing anaacA1-orfGgene cassette was identified upstream ofblaCMY-9and ended with a truncated 3′ conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except thatorf513encoding a putative transposase was identified instead oforf341due to addition of a single nucleotide.blaCMY-9was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation ofblaCMY-9from some environmental microorganisms such as aeromonads.
Simple Fibroblast-Based Assay for Screening of New Antimicrobial Drugs against
Mycobacterium tuberculosis
Authors: Takii Takemasa , Yamamoto Yoshifumi , Chiba Taku , Abe Chiyoji , Belisle John T. , Brennan Patrick J. , Onozaki Kikuo
Published: July 2002

In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimycobacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly sensitive to mycobacterial cytotoxicity, are killed by virulent
Mycobacterium tuberculosis
strain H
37
Rv bacilli in response to the viability of bacilli. Clinically used antimycobacterial drugs inhibited the mycobacterial cytotoxicity to MRC-5 cells in a dose-dependent manner. MICs of isoniazid, streptomycin, rifampin, and ethambutol determined by this SFA (0.428, 1.816, 0.013, and 3.465 μg/ml, respectively) were within 1 log of MICs determined by the broth dilution test (BDT) using Middlebrook 7H9 medium. The MIC of pyrazinamide, which exhibits bactericidal activity only at a high dose by BDT (1,231 μg/ml at pH 6.6 and 492 μg/ml at pH 5.8), was 3.847 μg/ml in the modified method of SFA. On the other hand, sodium azide, a toxic agent for both mammalian cells and bacteria, exhibited cytotoxicity to fibroblasts at a dose lower than that required to inhibit mycobacterial growth. Thus, this fibroblast-based method enabled us to evaluate both antibacterial activity of drugs and their cytotoxicity to human cells within a short period of time.
Efficacy of Proton Pump Inhibitor Drugs against
Plasmodium falciparum
In Vitro and Their Probable Pharmacophores
Authors: Riel Michael A. , Kyle Dennis E. , Bhattacharjee Apurba K. , Milhous Wilbur K.
Published: July 2002

The substituted benzimidazoles omeprazole, lansoprazole, rabeprazole, and pantoprazole were found to have in vitro activity against three different isolates of
Plasmodium falciparum
: D6 (which is chloroquine and pyrimethamine sensitive), W2 (chloroquine and pyrimethamine resistant), and TM91C235 (multidrug resistant). Lansoprazole and rabeprazole were the most effective against all three isolates, with a 50% inhibitory concentration (IC
50
) range of 7 to 11 μM. Omeprazole showed intermediate activity against D6 and W2 isolates, with IC
50
s of 27 to 28 μM, but had poor activity against TM91C235, with an IC
50
of 76 μM. Pantoprazole was the least effective, with IC
50
s of 73 μM against D6, 53 μM against W2, and 39 μM against TM91C235. A pharmacophore model describing the important features responsible for potent activity of the drugs was developed using computational techniques of semiempirical quantum chemical methods and the three-dimensional QSAR procedure of the CATALYST software. The important features of the pharmacophore, according to the findings based on the CATALYST procedures, are the hydrogen bond acceptor and donor sites at the benzimidine nitrogen atoms and the two aromatic hydrophobic sites in the molecules. AM1 quantum chemical calculations identified the electrostatic potential surface surrounding the sulfoxide atom as crucial for potent activity.
Role of Active Efflux in Association with Target Gene Mutations in Fluoroquinolone Resistance in Clinical Isolates of
Vibrio cholerae
Authors: Baranwal Somesh , Dey Keya , Ramamurthy T. , Nair G. Balakrish , Kundu Manikuntala
Published: July 2002

Quinolones are among the drugs of choice in the management of cholera caused by
Vibrio cholerae
. In this study, we demonstrate that, in addition to mutations detected in the target genes
gyrA
and
parC
, proton motive force-dependent efflux is involved in quinolone resistance in clinical isolates of
V. cholerae
.
Extrusion of Penem Antibiotics by Multicomponent Efflux Systems MexAB-OprM, MexCD-OprJ, and MexXY-OprM of
Pseudomonas aeruginosa
Authors: Okamoto Kiyomi , Gotoh Naomasa , Nishino Takeshi
Published: July 2002

The high intrinsic penem resistance of
Pseudomonas aeruginosa
is due to the interplay among the outer membrane barrier, the active efflux system MexAB-OprM, and AmpC β-lactamase. We studied the roles of two other efflux systems, MexCD-OprJ and MexXY-OprM, in penem resistance by overexpressing each system in an AmpC- and MexAB-OprM-deficient background and found that MexAB-OprM is the most important among the three efflux systems for extrusion of penems from the cell interior.
Characterization of a
Listeria monocytogenes
Scott A Isolate with High Tolerance towards High Hydrostatic Pressure
Authors: Karatzas Kimon A. G. , Bennik Marjon H. J.
Published: July 2002

An isolate of
L. monocytogenes
Scott A that is tolerant to high hydrostatic pressure (HHP), named AK01, was isolated upon a single pressurization treatment of 400 MPa for 20 min and was further characterized. The survival of exponential- and stationary-phase cells of AK01 in ACES [
N
-(2-acetamido)-2-aminoethanesulfonic acid] buffer was at least 2 log units higher than that of the wild type over a broad range of pressures (150 to 500 MPa), while both strains showed higher HHP tolerance (piezotolerance) in the stationary than in the exponential phase of growth. In semiskim milk, exponential-phase cells of both strains showed lower reductions upon pressurization than in buffer, but again, AK01 was more piezotolerant than the wild type. The piezotolerance of AK01 was retained for at least 40 generations in rich medium, suggesting a stable phenotype. Interestingly, cells of AK01 lacked flagella, were elongated, and showed slightly lower maximum specific growth rates than the wild type at 8, 22, and 30°C. Moreover, the piezotolerant strain AK01 showed increased resistance to heat, acid, and H
2
O
2
compared with the wild type. The difference in HHP tolerance between the piezotolerant strain and the wild-type strain could not be attributed to differences in membrane fluidity, since strain AK01 and the wild type had identical in situ lipid melting curves as determined by Fourier transform infrared spectroscopy. The demonstrated occurrence of a piezotolerant isolate of
L. monocytogenes
underscores the need to further investigate the mechanisms underlying HHP resistance of food-borne microorganisms, which in turn will contribute to the appropriate design of safe, accurate, and feasible HHP treatments.
Tightly Bound Binary Toxin in the Cell Wall ofBacillus sphaericus
Authors: Klein Daniela , Uspensky Igor , Braun Sergei
Published: July 2002

We have shown that urea-extracted cell wall of entomopathogenicBacillus sphaericus2297 and some other strains is a potent larvicide againstCulex pipiensmosquitoes, with 50% lethal concentrations comparable to that of the well-knownB. sphaericusbinary toxin, with which it acts synergistically. The wall toxicity develops inB. sphaericus2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall ofB. sphaericus2297, lacking P42, killsC. pipienslarvae when mixed withEscherichia colicells expressing P42. Thus, the cell wall toxicity in strongly toxicB. sphaericusstrains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.
Molecular Characterization and Expression of Pyruvate Formate-Lyase-Activating Enzyme in a Ruminal Bacterium,
Streptococcus bovis
Authors: Asanuma Narito , Hino Tsuneo
Published: July 2002

To clarify the significance of the activation of pyruvate formate-lyase (PFL) by PFL-activating enzyme (PFL-AE) in
Streptococcus bovis
, the molecular properties and gene expression of PFL-AE were investigated.
S. bovis
PFL-AE was deduced to consist of 261 amino acids with a molecular mass of 29.9 kDa and appeared to be a monomer protein. Similar to
Escherichia coli
PFL-AE,
S. bovis
PFL-AE required Fe
2+
for activity. The gene encoding PFL-AE (
act
) was found to be polycistronic, and the PFL gene (
pfl
) was not included. However, the
act
mRNA level changed in parallel with the
pfl
mRNA level, responding to growth conditions, and the change was contrary to the change in the lactate dehydrogenase (LDH) mRNA level. PFL-AE synthesis appeared to change in parallel with PFL synthesis. Introduction of a recombinant plasmid containing
S. bovis pfl
and the
pfl
promoter into
S. bovis
did not affect formate and lactate production, which suggests that the activity of the
pfl
promoter is low. When the
pfl
promoter was replaced by the
S. bovis ldh
promoter, PFL was overexpressed, which caused an increase in the formate-to-lactate ratio. However, when PFL-AE was overexpressed, the formate-to-lactate ratio did not change, suggesting that PFL-AE was present at a level that was high enough to activate PFL. When both PFL-AE and PFL were overexpressed, the formate-to-lactate ratio further increased. It is conceivable that LDH activity is much higher than PFL activity, which may explain why the formate-to-lactate ratio is usually low.
Binding Rather Than Metabolism May Explain the Interaction of Two Food-Grade
Lactobacillus
Strains with Zearalenone and Its Derivative ɑ́-Zearalenol
Authors: El-Nezami Hani , Polychronaki Nektaria , Salminen Seppo , Mykkänen Hannu
Published: July 2002

The interaction between two
Fusarium
mycotoxins, zearalenone (ZEN) and its derivative ά-zearalenol (ά-ZOL), with two food-grade strains of
Lactobacillus
was investigated. The mycotoxins (2 μg ml
−1
) were incubated with either
Lactobacillus rhamnosus
strain GG or
L. rhamnosus
strain LC705. A considerable proportion (38 to 46%) of both toxins was recovered from the bacterial pellet, and no degradation products of ZEN and ά-ZOL were detected in the high-performance liquid chromatograms of the supernatant of the culturing media and the methanol extract of the pellet. Both heat-treated and acid-treated bacteria were capable of removing the toxins, indicating that binding, not metabolism, is the mechanism by which the toxins are removed from the media. Binding of ZEN or ά-ZOL by lyophilized
L. rhamnosus
GG and
L. rhamnosus
LC705 was a rapid reaction: approximately 55% of the toxins were bound instantly after mixing with the bacteria. Binding was dependent on the bacterial concentration, and coincubation of ZEN with ά-ZOL significantly affected the percentage of the toxin bound, indicating that these toxins may share the same binding site on the bacterial surface. These results can be exploited in developing a new approach for detoxification of mycotoxins from foods and feeds.
Distribution of Microbes Producing Antimicrobial ε-Poly-
l
-Lysine Polymers in Soil Microflora Determined by a Novel Method
Authors: Nishikawa Masanobu , Ogawa Ken'ichi
Published: July 2002

We developed a simple and sensitive screening method to investigate the distribution of microbes producing an antimicrobial poly(amino acid), ε-poly-
l
-lysine (ε-PL), in microflora. An acidic dye, Poly R-478, incorporated in an agar plate detected ε-PL producers by electrostatic interaction with the secreted basic polymers. All ε-PL producers, isolated after careful and sufficient screening of soil microflora, belonged exclusively to two groups of bacteria of the family
Streptomycetaceae
and ergot fungi. They were characterized based on the density and diameter of the concentric zone formed by the secreted polymers. The density depended on each isolate. The increase in the diameter of the concentric zone per unit of time varied among isolates and was negatively correlated with the molecular weight. Although the distribution of ε-PL producers was extremely limited, their products were structurally varied. The molecular masses of the secreted polymers among the isolates ranged from 0.8 to 2.0 kDa. There were also isolates producing unknown polymers inconsistent with the correlation or producing a mixture of polymers with original and modified structures. A chemically modified polymer was an ε-PL derivative, as determined by mass spectrometry. Since the structural variations had no relation to the phylogenetic position of the isolates, it is possible that enzymes involved in the synthesis diversified after putative horizontal transfers of relevant genes.
Antigenicity of
Leishmania braziliensis
Histone H1 during Cutaneous Leishmaniasis: Localization of Antigenic Determinants
Authors: Carmelo Emma , Martínez Enrique , González Ana Cristina , Piñero José Enrique , Patarroyo Manuel E. , del Castillo Antonio , Valladares Basilio
Published: July 2002

The humoral immune response against
Leishmania braziliensis
histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that
L. braziliensis
histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the
L. braziliensis
histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant
L. braziliensis
histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.
Epigallocatechin Gallate, a Potential Immunomodulatory Agent of Tea Components, Diminishes Cigarette Smoke Condensate-Induced Suppression of Anti-Legionella pneumophilaActivity and Cytokine Responses of Alveolar Macrophages
Authors: Matsunaga Kazuto , Klein Thomas W. , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

Even though cigarette smoking has been shown to suppress immune responses in the lungs, little is known about the effect of cigarette smoke components on respiratory infections. In the present study, the effects of cigarette smoke condensate (CSC) on bacterial replication in alveolar macrophages and the immune responses of macrophages to infection were examined. Furthermore, a possible immunotherapeutic effect of epigallocatechin gallate (EGCg), a major form of tea catechins, on the CSC-induced suppression of antimicrobial activity and immune responses of alveolar macrophages was also determined. The treatment of murine alveolar macrophage cell line (MH-S) cells with CSC significantly enhanced the replication ofLegionella pneumophilain macrophages and selectively down-regulated the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) induced by bacterial infection. The treatment of macrophages with EGCg not only overcame the CSC-induced suppression of antimicrobial activity but also strengthened the resistance of macrophages to infection. EGCg also markedly up-regulated the CSC-suppressed IL-6 and TNF-α production by macrophages in response to infection. The results of exogenous TNF-α treatment and neutralization treatment with anti-TNF-α and anti-gamma-interferon (IFN-γ) antibodies and the determination of IFN-γ mRNA levels indicate that CSC-suppressed macrophages can be activated by EGCg to inhibitL. pneumophilagrowth by up-regulation of TNF-α and IFN-γ production. Thus, this study revealed that CSC selectively alters the immune responses of macrophages toL. pneumophilainfection and leads to an enhancement of bacterial replication in macrophages. In addition, the tea catechin EGCg can diminish such suppressive effects of CSC on alveolar macrophages.
Screening of Neonatal Calves for Persistent Infection with Bovine Viral Diarrhea Virus by Immunohistochemistry on Skin Biopsy Samples
Authors: Grooms Daniel L. , Keilen Eric D.
Published: July 2002

Detection and elimination of cattle that are persistently infected with bovine viral diarrhea virus (BVDV) is important for controlling the transmission of this virus. Colostrum-derived antibodies make the detection of persistently BVDV-infected neonatal calves cumbersome and expensive. The objective of this study was to evaluate the use of immunohistochemical staining of skin biopsy samples from neonatal calves as a method for the early detection of persistent BVDV infection. Three hundred thirty-two 1- to 4-week-old dairy calves were screened for BVDV as part of a routine control program. Formalin-fixed skin biopsy samples were stained for BVDV antigen by immunohistochemistry (IHC), and the results were compared to those of virus isolation (VI) from white blood cell preparations. Six calves were positive by both IHC and VI and remained positive for BVDV upon subsequent follow-up testing; thus, they were classified as persistently infected with BVDV. One calf was positive by VI but negative by IHC. On subsequent testing, the calf was negative by VI, suggesting that the initial VI result was due to an acute BVDV infection. One calf was positive by IHC but negative by VI. This calf remained negative by VI on follow-up testing. Immunohistochemical staining of skin biopsy samples is a reliable method for screening neonatal calves for persistent BVDV infection and would be a useful management tool as an aid for controlling and preventing BVDV infection.
In Vitro Assessment of Antiborrelial Activity of OspA Vaccine Sera
Authors: Fawcett Paul T. , Rose Carlos D. , Gibney Kathleen M.
Published: July 2002

Prevention of Lyme disease by the recombinant OspA-based vaccine reportedly works by preventing transmission of spirochetes from ticks to humans. We report on an in vitro microculture assay, which can be used to provide an indicator of the need for booster doses of vaccine.
QuickVue Influenza Test for Rapid Detection of Influenza A and B Viruses in a Pediatric Population
Authors: Quach Caroline , Newby Diane , Daoust Ghislaine , Rubin Earl , McDonald Jane
Published: July 2002

The performance of a lateral-flow immunoassay, the QuickVue Influenza Test, for detection of influenza A and B viruses in comparison with that of cell culture was evaluated by using nasopharyngeal aspirates, in viral transport medium, from children with respiratory tract infections. The sensitivity and specificity were 79.2 and 82.6%, respectively.
Microimmunoassay Using a Protein Chip: Optimizing Conditions for Protein Immobilization
Authors: Seong Seung-Yong
Published: July 2002

Optimizing conditions for the microarraying of protein antigens onto glass slides were studied. Various vendors, surface functional groups, buffers, and fixatives were evaluated to enhance protein binding. A total of 125 pg of human immunoglobulin was detectable with this assay system, suggesting that protein microarray can be applied for routine immunodiagnosis.
Cultivation of Pathogenic and Opportunistic Free-Living Amebas
Authors: Schuster Frederick L.
Published: July 2002

Free-living amebas are widely distributed in soil and water, particularly members of the generaAcanthamoebaandNaegleria. Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts.Naegleriais the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis;Acanthamoebais responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis.Balamuthiasp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples.NaegleriaandAcanthamoebacan be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source.Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba,Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.
Cultivation of Clinically Significant Hemoflagellates
Authors: Schuster Frederick L. , Sullivan James J.
Published: July 2002

The hemoflagellates,
Trypanosoma
spp. and
Leishmania
spp., are causal agents of a number of parasitic diseases having a major impact on humans and domestic animals over vast areas of the globe. Among the diseases are some of the most pernicious and deadly of human afflictions: African sleeping sickness, Chagas' disease, kala-azar, and Oriental sore. The organisms have complex, pleomorphic life cycles typically involving a vertebrate and an invertebrate host, the latter serving as a vector. In the vertebrate host, they are primarily blood and tissue parasites. In their transition from one host to another, the hemoflagellates undergo morphological, physiological, and biochemical changes that facilitate their growth and subsequent transmission. A major goal in the study of the hemoflagellates has been the cultivation in vitro of both vertebrate and invertebrate stages of the organisms. The first types of media used in their cultivation, and still useful for establishment of cultures, were undefined and contained a complex of ingredients. These gave way to semidefined formulations which included tissue culture media as a base and, as a next step, addition of tissue culture cells as a feeder layer to promote parasite growth. More recently developed media are completely defined, having replaced the feeder cells with various supplements. Serum, a sometimes-variable component of the media, can be replaced by various serum substitutes. This review focuses on the hemoflagellates that infect humans, describing stages in the development of media leading to the fully defined formulations that are now available for the cultivation of many of these organisms.
Rickettsia rickettsiiInfection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression
Authors: Rydkina Elena , Sahni Abha , Silverman David J. , Sahni Sanjeev K.
Published: July 2002

Existing evidence suggests that oxidative insults and antioxidant defense mechanisms play a critical role in the host cell response during infection of endothelial cells byRickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Heme oxygenase (HO), a rate-limiting enzyme in the pathway for heme catabolism, protects against oxidant damage in a variety of stress situations. Here, we report on the expression of the inducible and constitutive HO isozymes, HO-1 and HO-2, duringR. rickettsiiinfection of endothelial cells. Steady-state levels for HO-1 mRNA were increased two- to threefold, as early as 4 h postinfection, whereas HO-2 mRNA was not affected. Induction of HO-1 mRNA was dependent on the dose of infection and occurred in a time-dependent manner, reaching maximal levels at 4 to 7 h. The increase in HO-1 mRNA occurred at the level of trancription as it was blocked by the transcriptional inhibitors, actinomycin D and α-amanitin. The eukaryotic protein synthesis inhibitor, cycloheximide, caused a >50% reduction in the infection-induced increase in HO-1 mRNA level, suggesting its dependence on de novo protein synthesis of host cell. The uptake of viable organisms appeared to be necessary, since inactivation ofR. rickettsiiby heat or formalin fixation, or incubation of cells with cytochalasin B to prevent entry resulted in marked inhibition of HO-1 response.N-Acetyl-l-cysteine, a known oxidant scavenger, inhibited the HO-1 induction byR. rickettsii.Finally, Western analysis with a specific monoclonal antibody revealed higher levels of HO-1 protein (∼32 kDa), confirming that changes in HO-1 mRNA levels were followed by increases in the levels of protein. The findings indicate thatR. rickettsiiinfection induces HO-1 expression in host endothelial cells and suggest an important role for this enzyme in cellular response to infection, possibly by serving a protective function against oxidative injury.
Eighty-Kilodalton N-Terminal Moiety of
Bordetella pertussis
Filamentous Hemagglutinin: Adherence, Immunogenicity, and Protective Role
Authors: Alonso Sylvie , Reveneau Nathalie , Pethe Kévin , Locht Camille
Published: July 2002

Bordetella pertussis
, the etiological agent of whooping cough, produces a number of factors, such as toxins and adhesins, that are required for full expression of virulence. Filamentous hemagglutinin (FHA) is the major adhesin of
B. pertussis
. It is a protein of approximately 220 kDa, found both associated at the bacterial cell surface and secreted into the extracellular milieu. Despite its importance in
B. pertussis
pathogenesis and its inclusion in most acellular pertussis vaccines, little is known about the functional importance of individual domains in infection and in the induction of protective immunity. In this study, we analyzed the role of the approximately 80-kDa N-terminal domain of FHA, designated Fha44, in
B. pertussis
adherence, colonization, and immunogenicity. Although Fha44 contains the complete heparan sulfate-binding domain, it is not sufficient for adherence to epithelial cells or macrophages. It also cannot replace FHA during colonization of the mouse respiratory tract. Infection with a
B. pertussis
strain producing Fha44 instead of FHA does not induce anti-FHA antibodies, whereas such antibodies can readily be induced by intranasal administration of purified Fha44. In addition, mice immunized with purified Fha44 were protected against challenge with wild-type
B. pertussis
, indicating that Fha44 contains protective epitopes. Compared to FHA, Fha44 is much smaller and much more soluble and is therefore easier to purify and to store. These advantages may perhaps warrant considering Fha44 for inclusion in acellular pertussis vaccines.
Yersiniabactin Production Requires the Thioesterase Domain of HMWP2 and YbtD, a Putative Phosphopantetheinylate Transferase
Authors: Bobrov Alexander G. , Geoffroy Valerie A. , Perry Robert D.
Published: July 2002

One requirement for the pathogenesis of
Yersinia pestis
, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the
pgm
locus of the
Y. pestis
chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the
pgm
locus. We have identified a gene,
ybtD
, encoded outside the HPI and
pgm
locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of
Escherichia coli.
A deletion within
ybtD
yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37°C but could be cross-fed by culture supernatants from Ybt-producing strains of
Y. pestis.
The promoter region of
ybtD
was fused to
lacZ
; β-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the
ybt
system. The
ybtD
mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other
ybt
biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any
ybt
regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.
Orientia tsutsugamushiInhibits Apoptosis of Macrophages by Retarding Intracellular Calcium Release
Authors: Kim Mee-Kyung , Seong Seung-Yong , Seoh Ju-Young , Han Tae-Hee , Song Hyeon-Je , Lee Ji-Eun , Shin Jung-Hoon , Lim Byung-Uk , Kang Jae-Seung
Published: July 2002

Orientia tsutsugamushishows both pro- and antiapoptotic activities in infected vertebrate cells. Apoptosis of THP-1 cells induced by beauvericin was inhibited byO. tsutsugamushiinfection. Beauvericin-induced calcium redistribution was significantly reduced and retarded in cells infected withO. tsutsugamushi. Antiapoptotic activities ofO. tsutsugamushiin infected cells are most probably due to inhibition of the increase in the cytosolic calcium concentration.
Vibrio Pathogenicity Island and Cholera Toxin Genetic Element-Associated Virulence Genes and Their Expression in Non-O1 Non-O139 Strains of
Vibrio cholerae
Authors: Sarkar Amit , Nandy Ranjan K. , Nair G. Balakrish , Ghose Asoke C.
Published: July 2002

A non-O1 non-O139
Vibrio cholerae
strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX. While the nucleotide sequence of the strain 10259
tcpA
gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (
aldA
,
tagA
,
tcpP/H
,
toxT
,
acfB/C
, and
int
) and intergenic regions (
tcpF
to
toxT
and
tcpH
to
tcpA
) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains. Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains. Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30°C and pH 6.5) led to transcriptional activation of
tcpP/H
,
toxR
,
toxT
, and
tcpA
genes, but not of
ctxA
, as determined by reverse transcription-PCR (RT-PCR). Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of
ctxAB
. Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a
V. cholerae
O1 classical biotype strain (O395) that contained seven such repeats. The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of
toxT
,
ctxA
, and
tcpA
when grown under the ToxR inducing conditions. Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly (∼1,000-fold) higher level of CT in vitro compared to that of the parent strain. Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay. One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium. However, the other strain, V2, produced RT-PCR-detectable transcripts of
toxT
,
ctxA
, or
tcpA
genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium. These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable. The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of
V. cholerae
strains belonging to non-O1 serogroups.
Evidence from Terminal Recombination Gradients that FtsK Uses Replichore Polarity To Control Chromosome Terminus Positioning at Division inEscherichia coli
Authors: Corre Jacqueline , Louarn Jean-Michel
Published: July 2002

Chromosome dimers inEscherichia coliare resolved at thediflocus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein. Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires thedifresolution site to be located at the junction between the two polarized chromosome arms or replichores. Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination). We had proposed thatdifsites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes. To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in anftsKmutant. The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on axerCmutant chromosome with respect to intensity, the region affected, and response to polarity distortion. The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localizedifat the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material.
The Glycolytic Flux in
Escherichia coli
Is Controlled by the Demand for ATP
Authors: Koebmann Brian J. , Westerhoff Hans V. , Snoep Jacky L. , Nilsson Dan , Jensen Peter R.
Published: July 2002

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F
1
part of the membrane-bound (F
1
F
0
) H
+
-ATP synthase were expressed in steadily growing
Escherichia coli
cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of
E. coli
. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly “carbon” limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.
Group I Self-Splicing Intron in the
recA
Gene of
Bacillus anthracis
Authors: Ko Minsu , Choi Hyang , Park Chankyu
Published: July 2002

Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the
recA
gene of
Bacillus anthracis
which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the
recA
precursor mRNA for RecA that was being translated in
E. coli
. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of
B. anthracis
expressed in
E. coli
was functional in its ability to complement a
recA
defect. When
recA
-negative
E. coli
cells were irradiated with UV, the
Bacillus
RecA reduced the UV susceptibility of the
recA
mutant, regardless of the presence of intron.
Export of
l
-Isoleucine from
Corynebacterium glutamicum
: a Two-Gene-Encoded Member of a New Translocator Family
Authors: Kennerknecht Nicole , Sahm Hermann , Yen Ming-Ren , Pátek Miroslav , Saier, Milton H. , Eggeling Lothar
Published: July 2002

Bacteria possess amino acid export systems, and
Corynebacterium glutamicum
excretes
l
-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for
l
-isoleucine export, we have used transposon mutagenesis to isolate mutants of
C. glutamicum
sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated
brnF
encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices.
brnE
is located downstream of
brnF
and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports
l
-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of
l
-leucine and
l
-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of
brnFE
and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in α-proteobacteria but not in other prokaryotes analyzed.
The Gonococcal Fur Regulon: Identification of Additional Genes Involved in Major Catabolic, Recombination, and Secretory Pathways
Authors: Sebastian Shite , Agarwal Sarika , Murphy John R. , Genco Caroline Attardo
Published: July 2002

In this study, we have characterized the in vitro binding of
Neisseria gonorrhoeae
Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in
Escherichia coli
HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including
tonB
,
fur
,
recN
,
secY
,
sodB
,
hemO
,
hmbR
,
fumC
, a hypothetical gene (Fe-S homolog), and the
opa
family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of
tonB
,
fur
,
recN
,
secY
,
sodB
,
hemO
,
hmbR
,
fumC
, the Fe-S homolog gene, and the
opa
gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11
opa
genes (
opaA
through -
K
), we did not detect binding of purified
E. coli
Fur with 8 of the 11
opa
members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the
opa
family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.
Differential Spectrum of Mutations That Activate the
Escherichia coli bgl
Operon in an
rpoS
Genetic Background
Authors: Moorthy Sudha , Mahadevan S.
Published: July 2002

The
bgl
promoter is silent in wild-type
Escherichia coli
under standard laboratory conditions, and as a result, cells exhibit a β-glucoside-negative (Bgl
−
) phenotype. Silencing is brought about by negative elements that flank the promoter and include DNA structural elements and sequences that interact with the nucleoid-associated protein H-NS. Mutations that confer a Bgl
+
phenotype arise spontaneously at a detectable frequency. Transposition of DNA insertion elements within the regulatory locus,
bglR
, constitutes the major class of activating mutations identified in laboratory cultures. The
rpoS
-encoded σ
S
, the stationary-phase sigma factor, is involved in both physiological as well as genetic changes that occur in the cell under stationary-state conditions. In an attempt to see if the
rpoS
status of the cell influences the nature of the mutations that activate the
bgl
promoter, we analyzed spontaneously arising Bgl
+
mutants in

rpoS
+

and
rpoS
genetic backgrounds. We show that the spectrum of activating mutations in
rpoS
cells is different from that in

rpoS
+

cells. Unlike

rpoS
+

cells, where insertions in
bglR
are the predominant activating mutations, mutations in
hns
make up the majority in
rpoS
cells. The physiological significance of these differences is discussed in the context of survival of natural populations of
E. coli
.