Glucose uptake by <i>M. chelonae</i> ATCC 35752 and its isogenic porin knock-out mutants.

<p>The accumulation of [U-¹⁴C]glucose by the strains over time was measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#s2" target="_blank">Materials and Methods</a>. Glucose uptake rates were calculated on the first 10 min of the reactions. Uptake experiments were performed in triplicates and are shown with their standard deviations.</p

Gene Replacement in <i>Mycobacterium chelonae</i>: Application to the Construction of Porin Knock-Out Mutants

<div><p><i>Mycobacterium chelonae</i> is a rapidly growing mycobacterial opportunistic pathogen closely related to <i>Mycobacterium abscessus</i> that causes cornea, skin and soft tissue infections in humans. Although <i>M. chelonae</i> and the emerging mycobacterial pathogen <i>M. abscessus</i> have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of <i>M. chelonae</i> and <i>M. abscessus</i> is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for <i>M. abscessus</i>, genetic tools are lacking for <i>M. chelonae</i>. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker <i>sacB</i>, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of <i>M. chelonae</i> ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.</p></div

Comparative efficiency of the <i>Ts-sacB</i> system using <i>zeo</i> and <i>kan</i> disrupted allelic exchange substrates in <i>M. chelonae</i> ATCC 35752.

<p>One to three transformants (T1, T2 and T3) were selected on plates upon transformation with the pPR27-derived plasmids, grown in 7H9-OADC broth at 30°C for 5 to 7 days, and finally plated onto 7H11-OADC containing Kan or Zeo and 10% sucrose at 37°C. The percentage of CFUs presenting the expected phenotype for allelic exchange mutants at the last selection step of the Ts-SacB procedure (sucrose resistant; Kan^R or Zeo^R and XylE⁻) is indicated for each construct. Four to ten candidate mutants were analyzed by PCR in each case and the number of double crossover mutants identified is indicated in the last column.</p

Comparative electrotransformation efficiency and spontaneous resistance to different antibiotics in <i>M. chelonae</i> strains ATCC 35752 and 9917, and <i>M. abscessus</i> ATCC 19977.

<p>Transformation efficiencies upon selection with the indicated antibiotics on 7H11-OADC agar are expressed as numbers of drug-resistant CFUs per µg of DNA electroporated. The percentage below each transformation efficiency value represents the percentage of Kan, Zeo or Gen-resistant CFUs confirmed to be actual transformants either by PCR (pOMK-zeo) or determination of their XylE phenotype (all other plasmids).</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Gene replacement at the <i>MCH_4689c</i>, <i>MCH_4690c</i> and <i>MCH_4691c</i> porin loci of <i>M. chelonae</i> ATCC 35752 using the <i>Ts-sacB</i> and recombineering systems.

<p>(A) Porin gene cluster of <i>M. chelonae</i> ATCC 35752. The positions of the primers used to generate the allelic exchange substrates and analyze the candidate mutants are indicated. IGR1 and IGR2 represent the intergenic regions. (B) Candidate mutants obtained for each of the porin genes using the <i>Ts-sacB</i> or the recombineering systems were analyzed by PCR as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#s2" target="_blank">Materials and Methods</a> and confirmed by sequencing the regions flanking the resistance cassette. The expected size of the PCR fragments is 3.3 kb for the wild-type parent strain and 3.8 kb for the knock-out mutants. MWM, molecular weight marker. WT, wild-type. (C) Immunoblot analysis of porin production in the wild-type, mutant and complemented mutant strains. Strains were grown in 7H9-OADC-Tween 80 broth at 30°C to mid-log phase (OD600 = 1) and porins were selectively extracted from whole cells at 100°C using 0.5% <i>n</i>-octylpolyoxyethylene as a detergent as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Heinz1" target="_blank">[44]</a>. Protein samples prepared from the same amount of cells for each strain were denatured by boiling in 80% DMSO followed by acetone precipitation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Stahl1" target="_blank">[23]</a>. Denatured proteins were loaded volume to volume, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and porins were detected using rabbit antiserum to purified MspA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Stahl1" target="_blank">[23]</a>. Immune complexes were detected by chemiluminescence (Pierce, ELC) and semi-quantified using the Image Lab software (Biorad).</p

Counterselection efficiency of the <i>Ts-sacB</i> system in <i>M. chelonae</i> ATCC 35752.

(a)<p>The experiment was conducted on three independent transformants and mean counterselection efficiencies +/− standard deviations are indicated.</p

Growth rates of wild-type <i>M. chelonae</i> ATCC 35752, its isogenic porin knock-out mutants and complemented <i>MCH_4691</i> mutant strains in 7H9-OADC-Tween 80 broth at 30°C.

<p>Shown are representative results of two to three independent experiments using different culture batches.</p

A Noncompetitive Inhibitor for <i>Mycobacterium tuberculosis</i>’s Class IIa Fructose 1,6-Bisphosphate Aldolase

Class II fructose 1,6-bisphosphate aldolase (FBA) is an enzyme critical for bacterial, fungal, and protozoan glycolysis/gluconeogenesis. Importantly, humans lack this type of aldolase, having instead a class I FBA that is structurally and mechanistically distinct from class II FBAs. As such, class II FBA is considered a putative pharmacological target for the development of novel antibiotics against pathogenic bacteria such as <i>Mycobacterium tuberculosis</i>, the causative agent for tuberculosis (TB). To date, several competitive class II FBA substrate mimic-styled inhibitors have been developed; however, they lack either specificity, potency, or properties that limit their potential as possible therapeutics. Recently, through the use of enzymatic and structure-based assisted screening, we identified 8-hydroxyquinoline carboxylic acid (HCA) that has an IC₅₀ of 10 ± 1 μM for the class II FBA present in <i>M. tuberculosis</i> (MtFBA). As opposed to previous inhibitors, HCA behaves in a noncompetitive manner, shows no inhibitory properties toward human and rabbit class I FBAs, and possesses anti-TB properties. Furthermore, we were able to determine the crystal structure of HCA bound to MtFBA to 2.1 Å. HCA also demonstrates inhibitory effects for other class II FBAs, including pathogenic bacteria such as methicillin-resistant <i>Staphylococcus aureus</i>. With its broad-spectrum potential, unique inhibitory characteristics, and flexibility of functionalization, the HCA scaffold likely represents an important advancement in the development of class II FBA inhibitors that can serve as viable preclinical candidates

Identification of a Novel Mycobacterial Arabinosyltransferase Activity Which Adds an Arabinosyl Residue to α‑d‑Mannosyl Residues

The arabinosyltransferases responsible for the biosynthesis of the arabinan domains of two abundant heteropolysaccharides of the cell envelope of all mycobacterial species, lipoarabinomannan and arabinogalactan, are validated drug targets. Using a cell envelope preparation from <i>Mycobacterium smegmatis</i> as the enzyme source and di- and trimannoside synthetic acceptors, we uncovered a previously undetected arabinosyltransferase activity. Thin layer chromatography, GC/MS, and LC/MS/MS analyses of the major enzymatic product are consistent with the transfer of an arabinose residue to the 6 position of the terminal mannosyl residue at the nonreducing end of the acceptors. The newly identified enzymatic activity is resistant to ethambutol and could correspond to the priming arabinosyl transfer reaction that occurs during lipoarabinomannan biosynthesis