Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Functional analysis of gammaretroviral vector transduction by quantitative PCR.

BACKGROUND: In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. METHODS: We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. RESULTS: Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. CONCLUSIONS: The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.

Real-time reverse transcription-PCR analysis of expression of halobenzoate and salicylate catabolism-associated operons in two strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa JB2 can use 2-chlorobenzoate (2-CBa), 3-CBa, 2,3-dichlorobenzoate (2,3-DCBa), and 2,5-DCBa as sole carbon and energy sources, whereas strain 142 can only grow on 2-CBa and 2,4-DCBa. Both strains, however, harbor the same halobenzoate 1,2-dioxygenase (ohbAB) and chlorocatechol (clcABD) degradation genes necessary for the metabolism of ortho-CBas. In addition, the hybABCD operon, encoding a salicylate 5-hydroxylase, is also found in both strains. The expression of ohbAB, hybABCD, and clcABD operons was measured in cultures grown on different CBas as the sole carbon source and also in glucose-grown cells supplemented with CBas as inducers. A method to standardize real-time reverse transcription-PCR experimental data was used that allows the comparison of semiquantitative mRNA accumulation in different strains and culture conditions. In both strains, the ohb and hyb systems were induced in cells grown on 2-CBa or DCBas, whereas clc was induced only by DCBas. Repression by catabolite was observed both on ohb and clc systems in glucose-grown cells. Chlorocatechol 1,2-dioxygenase activity in JB2 was detected even in clc-repressed conditions, confirming the presence of additional isofunctional genes previously detected in P. aeruginosa 142. Although similar levels of induction of ohbAB were observed in strain JB2 grown on either benzoate, monochlorobenzoates, or DCBas, the ohbAB operon of strain 142 was only strongly induced by growth on 2-CBa and, to a lesser extent, on 2,4-DCBa. This observation suggests that regulation of the ohbAB operon may be different in both strains. The concomitant induction of ohb and hyb by CBas may allow the formation of hybrid halobenzoate dioxygenase(s) composed of Ohb/Hyb dioxygenase subunits and Hyb ferredoxin/ferredoxin reductase components.

Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10.

A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor. The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid as intermediates. Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion. Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels. Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose. Oxidation to [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity. Inhibition of TDG transformation in the presence of several alcohols is also shown.

MalR-mediated regulation of the Streptococcus pneumoniae malMP operon at promoter PM. Influence of a proximal divergent promoter region and competition between MalR and RNA polymerase proteins.

The Streptococcus pneumoniae mal regulon contains two operons, malXCD and malMP involved in the uptake and utilization of maltosaccharides. Both operons are transcribed from two divergent promoters, P(X) and P(M), and are negatively regulated by the MalR transcriptional repressor. Purified MalR protein binds to two DNA regions that encompasses both promoters, thus occupying its two operators, O(M) and O(X). However, the levels of occupation and repression were different, being higher when MalR was bound to O(M) than when it was anchored to O(X). Competition experiments between MalR and the Escherichia coli RNA polymerase on promoters P(M) and P(X) showed that the affinity of either protein for the promoter/operator DNA sequences was important to determine the frequency of transcription initiation. In addition to the control exerted by MalR, expression from promoter P(M) was affected by upstream sequences located within or close to P(X) promoter.

Induction of the halobenzoate catabolic pathway and cometabolism of ortho-chlorobenzoates in Pseudomonas aeruginosa 142 grown on glucose-supplemented media.

The aerobic cometabolism of ortho-substituted chlorobenzoates by Pseudomonas aeruginosa strain 142 growing on glucose-supplemented medium was analyzed. The strain, which can use 2-chlorobenzoate (2-CBA) and 2,4-dichlorobenzoate (2,4-DCBA) as sole carbon and energy sources, showed high rates of 2-CBA metabolism in glucose-fed cells. In contrast, 2,4-DCBA was metabolized only after extended incubation of the full grown culture and depletion of glucose. In addition to the ortho-dehalogenation (ohb142) genes encoding the alpha and beta subunits of the oxygenase component of a 2-halobenzoate dioxygenase, strain 142 harbours a closely related ohbABCDFG gene cluster previously identified in P. aeruginosa JB2 (ohbJB2). The genes for the chlorocatechol ortho-catabolic pathway were identified and sequenced in this strain, showing a near complete identity with the clcABD operon of the pAC27 plasmid. Relative quantification of mRNA by RT-PCR shows a preferential induction of ohb142 by 2-CBA, which is abolished in glucose-grown cultures. The alternate ohbJB2 and clc genes were expressed preferentially in 2,4-DCBA grown cultures. Only ohbJB2 appears to be expressed in the presence of the carbohydrate. Detection of chlorocatechol-1,2-dioxygenase activity in 2,4-DCBA plus glucose grown cultures suggests the presence of an alternate system for the ortho-cleavage of chlorobenzoates. The recruitment of elements from two halobenzoate dioxygenase systems with different induction patterns, together with a chlorocatechol degradative pathway not repressed by carbon catabolite, may allow P. aeruginosa 142 to cometabolize haloaromatics in carbohydrate grown cultures.

Characterization of bacterial strains able to grow on high molecular mass residues from crude oil processing.

Oil residues containing high molecular mass hydrocarbons, rich in polyaromatic compounds, are frequent end-products of crude oil processing and are poorly biodegradable. Their disposal poses an environmental problem. Through batch-enrichments from contaminated soils we have isolated and characterized seven bacterial strains that can use a residue from crude oil processing as a source of carbon and energy. The residue was a complex mixture of high molecular mass compounds, including saturated, aromatic and polycyclic aromatic hydrocarbons (PAHs). Analysis of the metabolic profiles of the strains isolated showed that they could all metabolize long-chain-length alkanes efficiently, but not PAHs. Strains degrading naphthalene, a simple PAH, did exist in the soil inocula used, but could be isolated only when enrichments were performed using pure naphthalene as the sole carbon source. All strains tested emulsified the oil residue and their ability to produce surfactants was studied.

Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid.

The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively.

The maltose/maltodextrin regulon of Streptococcus pneumoniae. Differential promoter regulation by the transcriptional repressor MalR.

The Streptococcus pneumoniae MalR protein regulates the transcription of two divergent operons, malXCD and malMP, involved in maltosaccharide uptake and utilization, respectively. MalR belongs to the LacI-GalR family of transcription repressors. The protein binds specifically to two operator sequences in the intergenic region between these operons. The affinity of MalR for the malMP binding sequence is higher than for the malXCD site. Results obtained in vivo using transcriptional fusions with reporter genes indicate low repression level of malXCD by MalR when compared with malMP. This behavior may be correlated with the existence of separate induction pathways for maltose, maltotriose, and maltotetraose. The similarities found at the operator sequences and binding domains for MalR and enterococcal repressor proteins suggest that the pneumococcal maltosaccharide regulation system is closely related to several Gram-negative metabolic pathways, but not to the structurally similar Escherichia coli maltose regulon.

Improved catalytic performance of a 2-haloacid dehalogenase from Azotobacter sp. by ion-exchange immobilisation.

The stability and catalytic efficacy of the L-2-haloacid dehalogenase isolated from Azotobacter sp. RC26 were studied after immobilisation on a DEAE Sephacel solid matrix. While the optimum temperature for the soluble dehalogenase falls in the range of 30-40 degrees C, the activity of the immobilised enzyme shows a four-fold increase at 60 degree C. Immobilisation on a plug-flow bioreactor extends the range of usable substrate concentration. The improved catalytic characteristics after immobilisation of the haloacid dehalogenase may be relevant for its possible utilization in biotechnological applications ranging from waste treatment to synthesis of stereoisomers.

Characterization of the Streptococcus pneumoniae maltosaccharide regulator MalR, a member of the LacI-GalR family of repressors displaying distinctive genetic features.

The gene encoding a transcriptional repressor of the maltosaccharide utilization operons of the Gram-positive bacterium Streptococcus pneumoniae (malR) has been cloned and sequenced. The genetic structure of the locus reveals the presence of an upstream gene necessary for growth on maltotetraose medium (malA). The phenotype of malR- and malA- mutants obtained by interruption of the coding regions suggests that both genes could belong to the same transcription unit. Two copies of a DNA motif consisting of three conserved regions of 59, 42, and 49 nucleotides were found located upstream and downstream of the malA-malR putative operon. These DNA structures are almost identical to the reported Box sequences associated with several genes of S. pneumoniae. The protein encoded by malR was visualized and partially purified after selective expression in Escherichia coli, whereas the product of malA was identified in vitro. The amino acid sequence of MalR displays similarities with the Lac and Gal family of repressors. The highest similarities were found when comparing MalR with the E. coli MalI repressor, which is related with an indirect induction pathway of the maltose regulon. The significance of these similarities is discussed in terms of the possible evolutionary pathways followed by structural and regulatory genes of sugar utilization systems in bacteria.

Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae. Correlation to the Escherichia coli maltose regulon.

The mechanism of induction of the maltose/maltodextrin regulon of the Gram-positive bacterium Streptococcus pneumoniae seems to be different to the positively controlled maltose regulons of the enteric bacteria Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. In this work, we report on the structure of the S. pneumoniae genes involved in maltodextrin uptake malX, malC and malD. Comparisons of the amino acid sequences encoded by these genes indicate that they are homologous to the E. coli MalE periplasmic maltose binding protein and the two maltose permeases MalG and MalF. The analysis of transcription start points indicates that malXCD could be transcribed from a single consensus promoter sequence. Northern analysis of the mRNA molecules pertaining to this region reveals that the transcript encompassing all these three genes is apparently cleaved at a large putative mRNA secondary structure, yielding two mRNA molecules. The smaller of these molecules would include only the malX gene while a larger fragment spans through malC and malD. The processing of mRNA has not been reported in the Gram-negative maltose regulons, and may suggest either a less evolved or a divergent system for the control of gene expression of this regulon in S. pneumoniae.

Expression of bacterial genes involved in maltose metabolism.

Recent advances in the knowledge of the maltose regulons of enteric bacteria have increased the number and complexity of factors involved both in gene expression and metabolite uptake by the cell. The transcription activation performed by the MalT protein and the CRP-cAMP complex have been found to be connected with several regulation pathways implicated in sugar transport and adaptation ot changes in osmolarity in the cell environment. In contrast to the positive regulation in enteric bacteria, the control of the maltose system in the Gram-positive bacterium Streptococcus pneumoniae may represent a more rudimentary scheme in which a classical repressor protein, MalR, regulates expression of the maltose operons. Considering the different mechanisms of transcription regulation proposed for these homologous systems, the maltose regulon of S. pneumoniae appears to be specially useful as a model to study the changes that may have taken place, both in gene organization and control of gene expression, to lead to divergent mechanisms of transcription regulation.

Genetic and structural characterization of endA. A membrane-bound nuclease required for transformation of Streptococcus pneumoniae.

The endA gene encoding the membrane nuclease of Streptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying the endA+ gene complemented cells with endA- in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end. In vitro synthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity of endA and the effect of various insertions and deletions, it appears that endA is the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.

The exoA gene of Streptococcus pneumoniae and its product, a DNA exonuclease with apurinic endonuclease activity.

The gene encoding the major DNA exonuclease of Streptococcus pneumoniae, exoA, was cloned in a streptococcal host vector system. Its location was determined by subcloning and by insertion mutations. Transfer of a DNA segment containing the gene to an Escherichia coli expression vector showed that exoA was the structural gene for the enzyme and that it was adjacent to its promoter. DNA sequence determination indicated that the gene encoded a protein, ExoA, of molecular weight 31,263. Under hyperexpression conditions, the ExoA protein constituted 10% of total cellular protein. In addition to previously demonstrated 3' to 5' exonuclease and 3'-phosphatase activities, ExoA was shown to make single-strand breaks at apurinic sites in DNA. Its enzymatic activities are thus similar to those of exonuclease III of E. coli and other gram-negative bacteria. The nucleotide sequence of exoA revealed it to be homologous to xth of E. coli, with 26% identity of amino acid residues in the predicted proteins. So far, no null chromosomal mutants of exoA have been obtained, and the biological function of ExoA remains unknown.

Changes of Escherichia coli cell cycle parameters during fast growth and throughout growth with limiting amounts of thymine.

It is generally accepted that during fast growth of Escherichia coli, the time (D) between the end of a round of DNA replication and cell division is constant. This concept is not consistent with the fact that average cell mass of a culture is an exponential function of the growth rate, if it is also accepted that average cell mass per origin of DNA replication (Mi) changes with growth rate and negative exponential cell age distribution is taken into account. Data obtained from cell composition analysis of E. coli OV-2 have shown that not only Mi but also D varied with growth rate at generation times (tau) between 54 and 30 min. E. coli OV-2 is a thymine auxotroph in which the replication time (C) can be lengthened, without inducing changes in tau, by growth with limiting amounts of thymine. This property has been used to study the relationship between cell size and division from cell composition measurements during growth with different amounts of thymine. When C increased, average cell mass at the end of a round of DNA replication also increased while D decreased, but only the time lapse (d) between the end of a replication round and cell constriction initiation appeared to be affected because the constriction period remained fairly constant. We propose that the rate at which cells proceed to constriction initiation from the end of replication is regulated by cell mass at this event, big cells having shorter d times than small cells.

Identification of the origin and direction of replication of the broad-host-range plasmid pLS1.

The replication origin of the fully sequenced broad-host-range streptococcal plasmid pLS1 has been determined by the use of an in vitro replication system prepared from Escherichia coli, a host in which the plasmid can be established. Replicative intermediates were isolated from reaction mixtures that contained dideoxythymidine triphosphate, thus limiting the average extent of in vitro synthesis. Analysis of HinfI-cleaved intermediates demonstrated that the origin of replication is included within a 443-bp fragment. Replication proceeds unidirectionally in the same direction as transcription of plasmid mRNAs. Isolation of deletion derivatives allowed us to define the replication origin of pLS1 within a region of 284 bp. Replication of pLS1 occurs through single-stranded intermediates by a rolling circle mechanism. Cleavage of supercoiled plasmid DNAs with endonuclease S1 followed by restriction mapping, allowed the positioning of three major specific S1 sites in regions of high potential to form secondary structures. One of these inverted repeats is located in the region where the origin of replication of pLS1 has been defined.

Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1.

We have characterized a region in the streptococcal plasmid pLS1 located between nucleotides 4103 and 4218 which is a signal involved in the conversion of single stranded intermediates of replication to double stranded plasmid forms. This region has a large axis of dyad symmetry resulting in the formation of a secondary structure as revealed by the location of endonuclease S1-cleavage sites in supercoiled covalently closed circular pLS1 DNA. Deletions affecting this region caused a fivefold reduction in plasmid copy number, plasmid instability and the accumulation of single-stranded DNA intermediates. The conversion signal of pLS1 has homologues in other staphylococcal plasmids, sharing a consensus sequence located in the loop of the signal. Computer assisted analysis showed that the signal detected in pLS1 has a high degree of homology with the complementary strand origin of the Escherichia coli single stranded bacteriophages phi X174 and M13.