Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Mitochondrial DNA content, an inaccurate biomarker of mitochondrial alteration in human immunodeficiency virus-related lipodystrophy.

Lipoatrophy is a prevalent side effect of antiretroviral treatment of human immunodeficiency virus (HIV) infection. Its mechanisms are still disputed but include mitochondrial toxicity and, in particular, mitochondrial DNA (mtDNA) depletion induced by nucleoside reverse transcriptase inhibitors. To obtain an integrated evaluation of the mitochondrial alteration in lipoatrophy, we investigated the DNA, RNA, and protein levels in 15 samples of abdominal subcutaneous adipose tissue from HIV-infected patients with peripheral lipoatrophy and compared the results with those for 15 samples from age- and body mass index-matched controls. The DNA and RNA analyses used PCR-based techniques, while proteins were quantified with enzyme-linked immunosorbent assay and measurement of activities with spectrophotometric assays. Depletion of mtDNA and mtDNA-encoded MT-CO2 mRNA was present, but normal levels of mtDNA-dependent activity (cytochrome c oxidase) and protein (MT-CO2p) showed that it was compensated for. An increase in nuclear-DNA-dependent mitochondrial activities (citrate synthase and malate dehydrogenase) and protein (COX4I1p), as well as transcriptional up-regulation of nuclear-DNA-encoded mitochondrial genes (COX4I1 and UCP2), demonstrated increased mitochondrial biogenesis. However, the expression of the known transcription factors of mitochondrial biogenesis (TFAM, NRF1, GABPA, PPARGC1A, PPARGC1B, and PPRC1) was normal or decreased. Increased amounts of activated caspase 3 and of DDIT3 mRNA showed the induction of apoptosis and oxidative stress, respectively. The mtDNA content did not correlate with any other mitochondrial parameter. In conclusion, mtDNA content does not appear to be an accurate biomarker of mitochondrial alteration in lipoatrophic adipose tissue. The preservation of mtDNA-dependent mitochondrial functions occurred despite severe mtDNA depletion. The presence of significant oxidative stress and apoptosis did not correlate with the mtDNA content.

Complete Genome Sequence of the Polymyxin E (Colistin)-Producing Paenibacillus sp. Strain B-LR.

Paenibacillus bacteria are recovered from varied niches, including human lung, rhizosphere, marine sediments, and hemolymph. Paenibacilli can have plant growth-promoting activities and be antibiotic producers. They can produce exopolysaccharides and enzymes of industrial interest. Illumina and PacBio reads were used to produce a complete genome sequence of the colistin producer Paenibacillus sp. strain B-LR.

Novel nonribosomal peptide synthetase (NRPS) genes sequenced from intertidal mudflat bacteria.

Nonribosomal peptide synthetases (NRPS) are actively sought out, due to pharmacologically important activities of their metabolites. In marine environment, the most prevalent nonribosomal peptide antibiotic producers are sponges inhabiting microorganisms. Conversely, strains from marine sediments and more especially from intertidal mudflats have not been extensively screened for the presence of new NRPS. In this study, for the first time, a collection of one hundred intertidal mudflat bacterial isolates (Marennes-Oléron Bay, France) was assessed for (1) the presence of NRPS genes by degenerated PCR targeting conserved adenylation domains and (2) for their production of antimicrobial molecules. (1) Bacteria with adenylation domains (14 strains) were identified by 16S rRNA gene sequence analysis and grouped into Firmicutes (one strain) and Proteobacteria (13 strains). In silico analysis of the NRPS amino acid sequences (n = 7) showed 41-58% ID with sequences found in the NCBI database. Three new putative adenylation domain signatures were found. (2) The culture supernatant of one of these strains, identified as a Bacillus, was shown to strongly inhibit the growth of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. This study portends that the intertidal mudflat niche could be of interest for the discovery of new NRPS genes and antimicrobial producing strains.

A Novel Depsipeptide Produced by Paenibacillus alvei 32 Isolated from a Cystic fibrosis Patient.

The important viscosity of the respiratory tract mucus of Cystic fibrosis (CF) patients impairs the mucociliary transport system and allows the growth of numerous micro-organisms. Among them, Pseudomonas aeruginosa and Staphylococcus aureus are known to be responsible for pulmonary infections. We imagined that CF microflora could also harbour micro-organisms naturally equipped to compete with these pathogens. A method was developed to recover these antibiotic-producing strains within 20 CF sputum. Using this approach, we have isolated an unusual Gram-positive bacterium identified as Paenibacillus alvei by Api galleries and 16S rRNA gene sequence analysis. This strain has inhibited the growth of P. aeruginosa, S. aureus and Klebsiella pneumoniae, in co-cultures. A liquid mineral medium named MODT50 was designed and optimised for the production and the recovery of the antimicrobial compounds. The supernatant has inhibited the growth of all Gram-positive strains tested, even Methicillin-resistant S. aureus. One antimicrobial compound with a peptide structure (mainly active against S. aureus, Micrococcus luteus, and Pseudomonas stutzeri) has been purified and characterised by liquid chromatography-mass spectrometry. The new active molecule (m/z 786.6) named depsipeptide L possesses a 15-guanidino-3-hydroxypentadecanoic acid side chain (m/z 298) linked on a cyclic part of four amino acids residues (Ser, two Leu/Ile, Arg). This work reports for the first time the production of such a molecule by a P. alvei strain in a mineral medium. The CF lung microflora might represent a valuable source for the discovery of new antimicrobial-producing strains.

D-loop mutations in mitochondrial DNA: link with mitochondrial DNA depletion?

Clinical presentation of the patients with mitochondrial DNA depletion is quite diverse and is suggestive of genetic heterogeneity. Autosomal recessive inheritance of the disease appears likely, thus implying the nuclear origin of the disease. This has been demonstrated recently in large families with neonatal presentation of the disease. Here, we report upon a family with one child having a late-onset disease associated with severe mitochondrial DNA depletion. The presence of mitochondrial alterations in the muscle of the patient's mother prompted us to extensively analyse the mitochondrial DNA in the family. We found mitochondrial DNA multiple deletions, but also three heteroplasmic point mutations of the D-loop region, two of which (T119C and T408A) affect conserved regions involved in the mtDNA replication process. These mutations were non-randomly distributed in the maternal lineage and, for one of them, among single muscle fibres. Involvement of the mitochondrial DNA in its own depletion appears therefore possible. It may act in close relationship with a hypothetical modified nuclear factor.

Quantitation of blood lymphocyte mitochondrial DNA for the monitoring of antiretroviral drug-induced mitochondrial DNA depletion.

OBJECTIVE: To investigate the impact of antiretroviral treatment on the mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients. DESIGN: As absolute mtDNA copy numbers widely differ between individuals, we performed a longitudinal analysis where the patient's first historical specimen was obtained as a baseline reference for relative comparison with subsequent samples from that patient. METHODS: mtDNA and nuclear DNA quantitation per cell (beta-globin gene copies) were both measured by real-time polymerase chain reaction analysis of whole DNA extracts of 361 serial live-cryopreserved PBMCs collected in former trials and clinical follow-ups from 60 individuals with established or recently acquired HIV-1 infections before and during administration of various antiviral combination therapies. RESULTS: mtDNA amounts were stable or increasing over years of natural HIV-1 infection in untreated patients (n = 7), consistent with our finding of a lack of differences in mtDNA copy numbers in patients with either a long established or recent lentivirus infection. Our quantitation system revealed significant changes in mtDNA copy number depending on the designated triple, quadruple, or quintuple anti-HIV drug combinations. Zidovudine + zalcitabine + ritonavir and zidovudine + lamivudine + didanosine regularly lead to mtDNA depletion in each of the treated patients, whereas none of 7 patients (and 35 cell specimens) receiving a stavudine + lamivudine + indinavir combination had any significant mtDNA content variations. In 7 patients, mtDNA copy numbers returned to pretreatment levels and/or higher levels without any interruption of the previously mtDNA-depleting antiretroviral drug combination. CONCLUSION: Our assay system allowed the detection of significant changes in the mtDNA content of PBMCs from HIV-1-infected patients taking antiretroviral drugs, as has been reported in the literature with other detection systems. Yet, mtDNA copy numbers regularly diminished during administration of some but not all nucleoside analog-containing combinations. This, plus the occasional finding that depleted mtDNA contents spontaneously increased to baseline levels and/or higher levels during uninterrupted treatment, should raise a note of caution about resorting to the PBMC mtDNA marker for monitoring of antiretroviral drug-related mitochondrial toxicities.

Mitochondrial damage associated with long-term antiretroviral treatment: associated alteration or causal disorder?

Combination of antiretroviral drugs has dramatically improved the prognosis of human HIV infection but is also associated with many adverse effects, the mitochondrial origin of which is discussed. In this study using extensive diagnostic procedures set up for inherited mitochondrial disorders, we analyzed HIV patients under active antiretroviral therapy who complained of severe adverse symptoms unexplained by HIV. All these patients had been treated for at least 5 years. They all had significant mitochondrial damage as evidenced by the diverse combination of lactate accumulation in blood or cerebrospinal fluid, mitochondrial morphologic alterations in muscle, and biochemical defects in muscle and liver, which designated mitochondrial DNA (mtDNA) as the main target of the toxic mechanisms. Southern blot and/or polymerase chain reaction -based analyses disclosed multiple deletions of the muscle mtDNA and reduction of the muscle and/or liver mtDNA copy number in a majority of the patients. In opposition to muscle and liver, blood mononuclear cells were devoid of significant biochemical or genetic alterations. Whether the mitochondrial toxicity is directly responsible for the patients' adverse symptoms remains disputable, because the investigations were transversal. Its severity argues for its clinical relevance, however. The skewed tissue distribution of mitochondrial alterations indicates potential pitfalls in the needed future prospective studies.