Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Evidence for a direct link between stress and immunity in the mollusc Haliotis tuberculata

Stress is thought to cause increased disease outbreaks and mortality in a number of invertebrates but currently very little information is available on mechanisms linking physiological states of stress and reduced disease resistance in these organisms. In the present study, we examined the possibility that stress alters immune functions, the principal line of defense against pathogens, in a molluscan model, the abalone Haliotis turbeculata. Immune parameters were investigated in abalones subjected to a 15 min mechanical disturbance which, as indicated by noradrenaline and dopamine hemolymphatic levels, resulted in a transient state of physiological stress. During the application of the stressor, immune parameters such as the number of circulating hemocytes, the migratory activity, the phagocytic capacity and the respiratory burst responses of hemocytes, decreased significantly. All parameters returned to initial values within 15-30 min after the end of the disturbance and a transient period of immunostimulation occurred between 100 and 480 min after the stress for all immune parameters except intracellular superoxide anion production. These results indicate that in the abalone H. tuberculata, as in vertebrates, a link exists between stress and the immune system. This may begin to answer why stress and disease outbreaks are linked in shellfish.     

Status of the so-called African pygmy elephant (Loxodonta pumilio (NOACK 1906)): phylogeny of cytochrome b and mitochondrial control region sequences

Among the African elephants, it has been unanimously acknowledged that the forest elephants (cyclotis form) are peculiar, so that they have been elevated to the specific rank. The development of molecular analyses of extant Loxodonta has only focused on two forms yet: the savannah form (africana) and the forest form (cyclotis), disregarding the so-called pygmy elephants (pumilio or fransseni) the systematic status of which has been debated since their discovery. Therefore, we have sampled nine dwarfed-labelled specimens in collection and eight specimens of typical forest elephants that we compared to three savannah elephants and two Asian elephants. Because of the degraded nature of the nuclear DNA content in bone samples of old specimens, we assayed mitochondrial markers; 1961 bp of the mitochondrial genome were sequenced (over a continuous range spanning the cytochrome b gene, tRNA Thr, tRNA Pro, hypervariable region 1 and central conserved region of the control region). Pumilio and cyclotis are not sister-taxa: the phylogenetic analyses rather account for the inclusion of the so-called pygmy elephants within a monophyletic group of forest elephants sensu lato. The internal structure of this clade reveals to depend on isolation and remoteness between populations, characteristics that may have been extensively influenced by climatic variations during the Quaternary period. We conclude that the specific taxon Loxodonta pumilio (or Loxodonta fransseni) should be abandoned.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram-positive bacteria

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis. We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus. Sequence analysis of the S. aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B. subtilis, but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S. aureus ctsR and hrcA genes were cloned under the control of the PxylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B. subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S. aureus CtsR and HrcA proteins. DeltactsR, DeltahrcA and DeltactsRDeltahrcA mutants of S. aureus were constructed, indicating that the two repressors are not redundant but, instead, act together synergistically to maintain low basal levels of expression of the dnaK and groESL operons in the absence of stress. This novel regulatory mode appears to be specific to Staphylococci.     

Bone biochemistry in rat femoral diaphysis after space flight

The aim of this experiment was to identify the location of the biochemical changes associated with depressed mineralization during space flight. We carried out biochemical analysis of 4 sections of the femoral diaphyses from 107 day old male rats flown aboard Cosmos 2044 Biosatellite for 16 days. Control femurs were preflight, vivarium, synchronous for feed, cage and temperature exposure, and a flight simulation model. Distal sections in both the flight and synchronous femurs showed mineral deficits associated with reduced levels of the reducible cross-link product of type I collagen, dehydro-dihydroxylysinonorleucine (deH-DHLNL) (p<.05). Unloaded bones in the ground based flight simulation model showed changes in cross-links similar to flight and synchronous controls, but were not associated with the mineral deficit. Mean values of elements measured in each section of all groups revealed significant associations (p<.005) between the non-collagenous protein, osteocalcin and calcium (r=0.774), phosphorus (r=-.624) and deH-DHLNL/deH-HLNL (r=.883). The ratio of the nonreducible cross-link, pyridinoline, to its lysl analogue, deoxypyridinoline, was consistently lower in the distal than proximal sections of the groups tested. None of the changes during space flight were unique to flight bone. The most significant and extensive changes in bone composition, i.e. mineral deficits associated with changes in both osteocalcin and reducible cross-links, were located in the distal section of the diaphysis of the femur.     

Acyl Transfer Activity of an Amidase from Rhodococcus sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids

The enantioselective amidase from Rhodococcus sp. strain R312 was produced in Escherichia coli and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the K_mamide values (e.g., K_mamide = 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (k_cat) were obtained with linear aliphatic amides (e.g., k_cat = 333 s⁻¹ with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g., K_mNH2OH = 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be l-enantioselective towards α-hydroxy- and α-aminoamides.Many bacterial amidases (EC 3.5.1.4) have been described previously because of their amide hydrolysis activities. Wide-spectrum amidases from Rhodococcus sp. strain R312 (26) and Pseudomonas aeruginosa (1), which are very similar, hydrolyze only short-chain amides. These enzymes are made up of four and six identical subunits having molecular weights of about 45,000 and 35,000, respectively. Based on the results of experiments performed with inhibitors, they have been classified as belonging to a branch of sulfhydryl enzymes (1, 26). The other amidases, the enantioselective amidases from Pseudomonas chlororaphis B23 (5), Rhodococcus erythropolis MP50 (12, 27), Rhodococcus sp. strain R312 (20), Rhodococcus sp. strain N-774 (10), Rhodococcus sp. (21), and Rhodococcus rhodochrous J1 (14), belong to a group of amidases containing a GGSS signature in the amino acid sequence (4) and are made up of two (or eight) identical subunits. The corresponding genes are located in clusters containing genes encoding the two subunits of a nitrile hydratase (EC 4.2.1.84). These amidases were also previously classified as sulfhydryl enzymes (5, 15), but no active amino acid residue was identified in any of them. Recently, Kobayashi et al. (15) showed that the real active site residues of the amidase from R. rhodochrous J1 were Asp-191 and Ser-195 rather than the generally accepted Cys-203 residue. These authors showed that aspartic acid and serine residues of this enzyme were also present in the active site sequences of aspartic proteinases and suggested that there is an evolutionary relationship between amidases and aspartic proteinases.All of the different amidases also exhibit an acyl transfer activity in the presence of hydroxylamine: RCONH₂ + NH₂OH ↔ RCONHOH + NH₃. This kind of reaction was previously described for the wide-spectrum amidase from Rhodococcus sp. strain R312 (6), but there has been no detailed study examining the acyl transfer reaction of amidases belonging to the GGSS signature-containing group. The final reaction products (hydroxamic acids) are known to possess high chelating properties. Some of them (particularly α-aminohydroxamic acid derivatives) are potent inhibitors of matrix metalloproteases, a family of zinc endopeptidases involved in tissue remodelling (3). Some other hydroxamic acids (α-aminohydroxamic acids, synthetic siderophores, acetohydroxamic acid, etc.) have also been investigated as anti-human immunodeficiency virus agents or antimalarial agents or have been recommended for treatment of ureaplasma infections and anemia (2, 8, 13, 28). Moreover, some fatty hydroxamic acids have been studied as inhibitors of cylooxygenase and 5-lipooxygenase with potent antiinflammatory activity (9).Apart from these medical applications, some hydroxamic acids (particularly polymerizable unsaturated hydroxamic acids and mid-chain or long-chain hydroxamic acids) have also been extensively investigated in wastewater treatment and nuclear technology studies as a way to eliminate contaminating metal ions (11, 16, 18).In this paper we describe the formation of a wide range of hydroxamic acids with the enantioselective amidase (a 120,000-dalton homodimer) from Rhodococcus sp. strain R312, and we provide some additional information which enhanced our comprehension of the reaction mechanism of this amidase.     

Comparison of artificial light photobioreactors and other production systems using Porphyridium cruentum

A new type of preparative photobioreactor for high quality production of microalgae is developed for hatchery-nursery of marine animals, as well as for fine chemicals extraction. Of modular conception, two artificial light photobioreactors in plastic and stainless steel are designed so as to provide strictly controlled conditions in an attempt to increase quality and diminish cost prices. They are assessed for production of Porphyridum cruentum and compared to conventional transparent tanks and solar photobioreactors. The concentration and productivity obtained are ten-fold higher than with hatchery tanks, which leads to a significant drop in cost price of biomass. Comparison is also made with a 10 m² solar photobioreactor operated in the south of France, for which biomass cost price is half that of 1.5 m² artificial light photobioreactor. Extrapolations erasing size discrepancy show that the cost price of the two technologies are not very different. This revised version was published online in June 2006 with corrections to the Cover Date.     

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.