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International audience; A series consisting of new polyaminoisoprenyl derivatives were prepared in moderate to good chemical yields varying from 32 to 64% according two synthetic pathways: 1) using a titanium reductive amination reaction affording a 50/50 mixture of cis and trans isomers 2) a direct nucleophilic substitution leading to a stereoselective synthesis of the compounds of interest. These compounds were then successfully evaluated for their in vitro antibiotic enhancer properties against resistant Gram-negative bacteria of four antibiotics belonging to four different families. The mechanism of action against Enterobacter aerogenes of one of the most efficient of these chemosensitizing agents was precisely evaluated by using fluorescent dyes to measure outer-membrane permeability and to determine membrane depolarization. The weak cytotoxicity encountered led us to perform an in vivo experiment dealing with the treatment of mice infected with Salmonella Typhimurium and affording preliminary promising results in terms of tolerance and efficiency of the polyaminoisoprenyl derivative 5r / doxycycline combination.

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a β-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c554. Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c552 and c554, are from the parent species. Cytochrome c552 belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

Weathering of the arsenopyrite-rich tailings of the Carnoules abandoned mining site (Gard, France) results in the formation of acid mine waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters is precipitated within a few meters from the bottom of the tailing dam by microorganisms. From these effluents, we have isolated and characterized a Gram negative bacterium involved in this bioremediation process. This bacterium, which belongs to the Thiomonas genus, is very efficient in removing arsenite from contaminated acidic waters by oxidizing it to arsenate, which precipitates.

International audience

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate reagent (BOP) serves as an efficient and versatile coupling reagent for the design and synthesis of new polyamino geranic acid derivatives in moderate to good chemical yields varying from 47% to 83%. These compounds induced a significant decrease of antibiotic resistance in two Gram-negative bacterial MDR strains. Our data suggested that their mechanism of action is closely associated with the inhibition of the efflux pumps.

International audience; OBJECTIVES: Bacterial drug resistance is a worrying public health problem and there is an urgent need for research and development to provide new antibacterial molecules. Peptide deformylase (PDF) is now a well-described intracellular target selected for the design of a new antibiotic group, PDF inhibitors (PDFIs). The initial bacterial susceptibility to an inhibitor of a cytoplasmic target is directly associated with the diffusion of the compound through the membrane barrier of Gram-negative bacteria and with its cytosolic accumulation at the required concentration. METHODS: We have recently demonstrated that the activity of different PDFIs is strongly dependent on the accumulation of the active molecules by using permeabilizing agents, efflux inhibitors or efflux-mutated strains. In this work we assessed various combination protocols using different putative inhibitors (PDFIs, methionine aminopeptidase inhibitors etc.) to improve antibacterial activity against various resistant Gram-negative bacteria. RESULTS: The maximum effect was observed when combining actinonin with a dual inhibitor of methionine aminopeptidase and PDF, this molecule being also able to interact with the target while actinonin is bound to the PDF active site. CONCLUSIONS: Such a combination of inhibitors acting on two tightly associated metabolic steps results in a cooperative effect on bacterial cells and opens an original way to combat multidrug-resistant bacteria.

New series of acids and hydroxamic acids linked to five-membered heterocycles including furan, oxazole, 1,2,4- or 1,3,4-oxadiazole, and imidazole were synthesized and tested as inhibitors against the Fe(II) , Co(II) , and Mn(II) forms of E. coli methionine aminopeptidase (MetAP) and as antibacterial agents against wild-type and acrAB E. coli strains. 2-Aryloxazol-4-ylcarboxylic acids appeared as potent and selective inhibitors of the Co(II) MetAP form, with IC(50) values in the micromolar range, whereas 5-aryloxazol-2-ylcarboxylic acid regioisomers and 5-aryl-1,2,4-oxadiazol-3-ylcarboxylic acids were shown to be inefficient against all forms of EcMetAP. Regardless of the heterocycle, all the hydroxamic acids are highly potent inhibitors and are selective for the Mn(II) and Fe(II) forms, with IC(50) values between 1 and 2 μM. One indole hydroxamic acid that we previously reported as a potent inhibitor of E. coli peptide deformylase also demonstrated efficiency against EcMetAP. To gain insight into the positioning of the oxazole heterocycle with reversed substitutions at positions 2 and 5, X-ray crystal structures of EcMetAP-Mn complexed with two such oxazole hydroxamic acids were solved. Irrespective of the [metal]/[apo-MetAP] ratio, the active site consistently contains a dinuclear manganese center, with the hydroxamate as bridging ligand. Asp 97, which adopts a bidentate binding mode to the Mn2 site in the holoenzyme, is twisted in both structures toward the hydroxamate bridging ligand to favor the formation of a strong hydrogen bond. Most of the compounds show weak antibacterial activity against a wild-type E. coli strain. However, increased antibacterial activity was observed mainly for compounds with a 2-substituted phenyl group in the presence of the nonapeptide polymyxin B and phenylalanine-arginine-β-naphthylamide as permeabilizer and efflux pump blocker, respectively, which boost the intracellular uptake of the inhibitors.

International audience; BACKGROUND : Multi-drug resistant (MDR) bacteria have become a major concern in hospitals worldwide and urgently require the development of new antibacterial molecules. Peptide deformylase is an intracellular target now well-recognized for the design of new antibiotics. The bacterial susceptibility to such a cytoplasmic target primarily depends on the capacity of the compound to reach and accumulate in the cytosol. METHODOLOGY/PRINCIPAL FINDINGS : To determine the respective involvement of penetration (influx) and pumping out (efflux) mechanisms to peptide deformylase inhibitors (PDF-I) activity, the potency of various series was determined using various genetic contexts (efflux overproducers or efflux-deleted strains) and membrane permeabilizers. Depending on the structure of the tested molecules, two behaviors could be observed: (i) for actinonin the first PDF-I characterized, the AcrAB efflux system was the main parameter involved in the bacterial susceptibility, and (ii), for the latest PDF-Is such as the derivatives of 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide, the penetration through the membrane was a important limiting step CONCLUSIONS/SIGNIFICANCE : Our results clearly show that the bacterial membrane plays a key role in modulating the antibacterial activity of PDF-Is. The bacterial susceptibility for these new antibacterial molecules can be improved by two unrelated ways in MDR strains: by collapsing the Acr efflux activity or by increasing the uptake rate through the bacterial membrane. The efficiency of the second method is associated with the nature of the compound.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth., Author Summary Microorganisms play a crucial role in nutrient biogeochemical cycles. Arsenic is found throughout the environment from both natural and anthropogenic sources. Its inorganic forms are highly toxic and impair the physiology of most higher organisms. Arsenic contamination of groundwater supplies is giving rise to increasingly severe human health problems in both developing and industrial countries. In the present work, we investigated the metabolism of this metalloid in Herminiimonas arsenicoxydans, a representative organism of a novel bacterial genus widespread in aquatic environments. Examination of the genome sequence and experimental evidence revealed that it is remarkably capable of coping with arsenic. Our observations support the existence of multiple strategies allowing arsenic-metabolizing microbes to efficiently colonize toxic environments. In particular, arsenic oxidation and scavenging may have played a crucial role in the development of early stages of life on Earth. Such mechanisms may one day be exploited as part of a potential bioremediation strategy in toxic environments.

The Arabidopsis thaliana AtHMA3 protein belongs to the P(1B)-adenosine triphosphatase (ATPase) transporter family, involved in heavy metal transport. Functional expression of AtHMA3 phenotypically complements the Cd/Pb-hypersensitive yeast strain Deltaycf1, but not the Zn-hypersensitive mutant Deltazrc1. AtHMA3-complemented Deltaycf1 cells accumulate the same amount of cadmium as YCF1-complemented Deltaycf1 cells or wild-type cells, suggesting that AtHMA3 carries out an intracellular sequestration of Cd. A mutant of AtHMA3 altered in the P-ATPase phosphorylation domain did not complement Deltaycf1, suggesting that metal transport rather than chelation is involved. The fusion protein AtHMA3::green fluorescent protein (GFP) is localized at the vacuole, consistent with a role in the influx of cadmium into the vacuolar compartment. In A. thaliana, the mRNA of AtHMA3 was detected mainly in roots, old rosette leaves and cauline leaves. The expression levels were not affected by cadmium or zinc treatments.