Pharmacokinetic Study of Rectal Artesunate in Children with Severe Malaria in Africa.

When severe malaria is suspected in children, the WHO recommends pretreatment with a single rectal dose of artesunate before referral to an appropriate facility. This was an individually randomized, open-label, 2-arm, crossover clinical trial in 82 Congolese children with severe falciparum malaria to characterize the pharmacokinetics of rectal artesunate. At admission, children received a single dose of rectal artesunate (10 mg/kg of body weight) followed 12 h later by intravenous artesunate (2.4 mg/kg) or the reverse order. All children also received standard doses of intravenous quinine. Artesunate and dihydroartemisinin were measured at 11 fixed intervals, following 0- and 12-h drug administrations. Clinical, laboratory, and parasitological parameters were measured. After rectal artesunate, artesunate and dihydroartemisinin showed large interindividual variability (peak concentrations of dihydroartemisinin ranged from 5.63 to 8,090 nM). The majority of patients, however, reached previously suggested in vivo IC50 and IC90 values (98.7% and 92.5%, respectively) of combined concentrations of artesunate and dihydroartemisinin between 15 and 30 min after drug administration. The median (interquartile range [IQR]) time above IC50 and IC90 was 5.68 h (2.90 to 6.08) and 2.74 h (1.52 to 3.75), respectively. The absolute rectal bioavailability (IQR) was 25.6% (11.7 to 54.5) for artesunate and 19.8% (10.3 to 35.3) for dihydroartemisinin. The initial 12-h parasite reduction ratio was comparable between rectal and intravenous artesunate: median (IQR), 84.3% (50.0 to 95.4) versus 69.2% (45.7 to 93.6), respectively (P = 0.49). Despite large interindividual variability, rectal artesunate can initiate and sustain rapid parasiticidal activity in most children with severe falciparum malaria while they are transferred to a facility where parenteral artesunate is available. (This study has been registered at ClinicalTrials.gov under identifier NCT02492178.).

Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens.

The Agrobacterium tumefaciens genome contains a cluster of genes that are predicted to encode Fe-S cluster assembly proteins, and this cluster is known as the sufS2BCDS1XA operon. sufS2 is the first gene in the operon, and it was inactivated to determine its physiological function. The sufS2 mutant exhibited a small colony phenotype, grew slower than the wild-type strain and was more sensitive to various oxidants including peroxide, organic hydroperoxide and superoxide. The sufS2 gene was negatively regulated by iron response regulator (Irr) and rhizobial iron regulator (RirA) under low and high iron conditions, respectively, and was inducible in response to oxidative stress. The oxidant-induced expression of sufS2 was controlled by Irr, RirA and an additional but not yet identified mechanism. sufS2 was required for RirA activity in the repression of a sufS2 promoter-lacZ fusion. RirA may use Fe-S as its cofactor. sufS2 disruption may cause a defect in the Fe-S supply and could thereby affect the RirA activity. The three conserved cysteine residues (C91, C99 and C105) in RirA were predicted to coordinate with the Fe-S cluster and were shown to be essential for RirA repression of the sufS2-lacZ fusion. These results suggested that sufS2 is important for the survival of A. tumefaciens.

Identification of amino acid residues important for the function of Agrobacterium tumefaciens Irr protein.

The key amino acid residues that influence the function of the Agrobacterium tumefaciens iron response regulator protein (Irr(At) ) were investigated. Several Irr(At) mutant proteins containing substitutions in amino acids corresponding to candidate metal- and haem-binding sites were constructed. The ability of the mutant proteins to repress the promoter of the membrane bound ferritin (mbfA) gene was investigated using a promoter-lacZ fusion assay. A single mutation at residue H94 significantly decreased the repressive activity of Irr(At) . Multiple mutation analysis revealed the importance of H45, H65, the HHH motif (H92, H93 and H94) and H127 for the repressor function of Irr(At) . H94 is essential for the iron responsiveness of Irr(At) . Furthermore, the Irr(At) mutant proteins showed differential abilities to complement the H(2) O(2) -hyper-resistant phenotype of an irr mutant.

Agrobacterium tumefaciens membrane-bound ferritin plays a role in protection against hydrogen peroxide toxicity and is negatively regulated by the iron response regulator.

An Agrobacterium tumefaciens membrane-bound ferritin (mbfA) mutant was generated to assess the physiological functions of mbfA in response to iron and hydrogen peroxide (H(2) O(2) ) stresses. Wild-type and the mbfA mutant strains showed similar growth under high- and low-iron conditions. The mbfA mutant was more sensitive to H(2) O(2) than wild-type strain. Expression of a functional mbfA gene could complement the H(2) O(2) -hypersensitive phenotype of the mbfA mutant and a rhizobial iron regulator (rirA) mutant, suggesting that MbfA protects cells from H(2) O(2) toxicity by sequestering intracellular free iron, thus preventing the Fenton reaction. The expression of mbfA could be induced in response to iron and to H(2) O(2) treatment. The iron response regulator (irr) also acted as a repressor of mbfA expression. An irr mutant had high constitutive expression of mbfA, which partly contributed to the H(2) O(2) -hyperresistant phenotype of the irr mutant. The data reported here demonstrate an important role of A. tumefaciens MbfA in the cellular defence against iron and H(2) O(2) stresses.