Sabine Clavel-Sérès

Structure of the Cell Envelope of Mycobacterium avium

In this report the cell wall of Mycobacterium avium is shown as a triple-layered structure where the outermost layer was stained by the ruthenium red staining for polysaccharides. The outermost layer hindered the diffusion of chemotherapeutic agents across the wall thus causing multiple drug-resistance by exclusion. The concerted electron microscopy and chemical analysis of chloroform-methanol and Triton X-100 extracts indicated that the outer layer was made of diverse amphiphil glycolipids (mycosides C, glycolipids, peptidolipids, phospholipids) that formed a matrix in which proteins were embedded. The examination of a spontaneous rough mutant indicated that mutations blocking the synthesis of one or several of the amphiphils must leave unsubstituted mycolic acid residues, thus causing surface hydrophobicity and roughness. Judging from our data, a model describing the overall cell envelope of M. avium was proposed. From the comparative analysis of M. avium, its spontaneous rough mutant, and its spheroplasts, some of the functions of the outermost layer were disclosed.

Alterations in the outer wall architecture caused by the inhibition of mycoside C biosynthesis inMycobacterium avium

Radiolabeled amino acids (l-U[C14]alanine,d-U[C14]alanine,l-U[C14]threonine, andl-U[C14]phenylalanine) were exponentially incorporated into the trichloroacetic acid (TCA)-insoluble material (whole cells) ofMycobacterium avium during the first 30–60 min of labeling. Bacteria labeled for 48 h were extracted with chloroform-methanol (2∶1 vol/vol). The thin layer chromatography (TLC) analysis of native lipids showed that mycoside C was labeled by the amino acids used.d-cycloserine (d-CS) and other amino acid analogs were examined as potential inhibitors of mycoside C biosynthesis. It was found thatd-CS caused about 27% inhibition, whereaso-,p-, andm-fluoro-dl-phenylalanine (Fl-phe) caused 80%–90% inhibition of the mycoside C biosynthesis. Judging from the data on inhibition experiments, it was concluded that the mycoside C biosynthesis started from the fatty acyl end and proceeded by the stepwise addition ofd-phenylalanine,d-allo-threonine, andd-alanine. Thed-alanyl-d-alanine peptidoglycan intermediate did not seem to serve as a donor ofd-alanine for mycoside C biosynthesis. Ultrastructural observation of the bacteria treated withd-CS showed only partial alteration of the outer wall layer, whereasm-Fl-phe treatment caused profound alterations. Successive transfers of the bacteria in growth medium supplemented withm-Fl-phe resulted in extensive disorganization of the outer layer.

Uptake of selected antibacterial agents in Mycobacterium avium.

The antibacterial action of 64 drugs against Mycobacterium avium ATCC 15769 was screened in Middlebrook 7H9 liquid medium. The most active drugs were ansamycin, rifampicin, clofazimine, and pristinamycin. The antibacterial action of the selected drugs was confirmed by testing clinical isolates on Middlebrook 7H10 agar medium. The antibacterial actions were not related with the hydrophobicities and molecular weights of the drugs. However, all the active drugs were highly hydrophobic molecules of low polarity. These drugs dissolved into the lipids forming the outer layers of the bacterial envelope and they appeared to interact with the surface amphiphils.

Transformation of distinct mycobacterial species by shuttle vectors derived from theMycobacterium fortuitum pAL5000 plasmid

Mycobacterium tuberculosis H37Ra,M. smegmatisATCC 607,M. smegmatis MC2155,M. aurum A +,M. aurum A11, and one representative strain ofM. flavescens were transformed by electroporation with plasmid pMY10 and cosmid pDC100. Plasmid pMY 10 contained the origin of replication of pAL5000, the origin of replication of pBR322, a kanamycin resistance gene, and the origin of transfer of the Inc plasmid RK2; the cosmid pDC100 contained the pHC79 SS cosmid, the origin of replication of pAL5000, and a kanamycin resistance gene. The efficiency of transformation varied with the recipient cells used and was in decreasing order: 7×105 forM. smegmatis MC2155, 6×103 forM. tuberculosis H37Ra, 103 forM. aurum, 50 forM. smegmatis ATCC 607, and 5 forM. flavescens. A rapid protocol for plasmid extraction from mycobacteria was developed.The satisfactory transformation of the nonvirulentM. tuberculosis strain H37Ra was of interest for future studies on cloning of virulence genes, while the satisfactory transformation ofM. aurum was of interest for future studies on the genetics of drug resistance because these bacteria are sensitive to drugs specifically used in the treatment of tuberculosis and leprosy. However, neither vector was stably maintained inM. smegmatis, indicating that further investigations are still necessary to resolve this difficulty.

Action of Colistin (Polymyxin E) on the Lytic Cycle of the Mycobacteriophage D29 in Mycobacterium tuberculosis

The antibiotic colistin (polymyxin E) inhibited the lytic cycle of the mycobacteriophage D29 in the tubercle bacilli, but not the D29 adsorption. The protein and nucleic acid synthesis in D29-infected bacteria were not affected significantly. The inhibitory activity was reversed by washing off the antibiotic, and by addition of Ca++, but not in media made iso-osmotic by addition of NaCl or sucrose. Transmission electron microscopy revealed an asymmetric to symmetric transition in the staining profile of the cytoplasmic membrane. Though no mature phage particles were ever observed in colistin-treated, D29-infected tubercle bacilli, loosely arranged aggregates resembling phage proheads were occasionally found. Judging from the above data, it was concluded that colistin inhibited D29 lytic cycle by causing molecular displacements in the inner leaflet of the cytoplasmic membrane, and consequently, the binding sites for D29 structural proteins were not available.

Characterization ofcar α,car Lep, andCrt I genes controlling the biosynthesis of carotenes inMycobacterium aurum

The genes controlling the biosynthesis of carotenes inMycobacterium aurum responsible for its yellow pigmentation were previously cloned (FEMS Microbiol Lett 1992, 90:239–244). In this study, the genescrt I, car α, andcar Lep, controlling respectively the formation of lycopene from phytoene (phytoene desaturase), α-carotene from lycopene, and leprotene from lycopene, were localized.

A simple and reliable EDDA method for mycobactin production in mycobacteria: Optimal conditions and use in mycobacterial speciation

Ethylenediamino-di-(0-hydroxyphenylacetic acid) (EDDA) was found to be a suitable iron chelator for investigating mycobactin production in mycobacteria. The optimal conditions of EDDA concentration and time of exposure were established and were used to develop a twostep method for mycobactin production. Applied to representative strains of selected species, the method was found to yield reliable results useful for identification and speciation of mycobacteria.

Partial physical and functional map of aMycobacterium aurum carotenogenesis operon

The genes controlling the biosynthesis of the carotenes inMycobacterium aurum were clustered in a 10.83-kb segment. Fragments generated by endonuclease digestions of the segment were cloned into a pHLD69 shuttle vector. The plasmids so constructed were used to transform a colorless (albino)M. aurum mutant (strain A11), a brick-red mutant accumulating large amounts of lycopene (strain NgR9), the buff-coloredMycobacterium smegmatis MC2-155, and the buffcoloredMycobacterium tuberculosis H37Ra. From the endonuclease digestion patterns and the phenotypes of the transformed strains, the partial physical and functional maps of a carotenogenesis operon were established. This investigation also showed that the genes controlling the conversion of lycopene into the xanthophylls were not located in the 10.83-kb segment.

Studies on clofazimine-resistance in mycobacteria: Is the inability to isolate drug-resistance mutants related to its mode of action?

This study showed that clofazimine was not radiomimetic, it was not a mutagenic compound, it was not an inducer of prophage-lambda, and it did not eliminate plasmids from appropriate host bacteria. The drug caused an effective inhibition of the growth of Mycobacterium aurum, and also inhibited the growth cycle of the mycobacteriophage D29. Cross-resistance between clofazimine, streptomycin and rifampicin could not be demonstrated. Drug-resistance mutants towards clofazimine could not be isolated, and it was found that the existing clofazimine-resistant strains of Mycobacterium tuberculosis were rather susceptible organisms requiring clofazimine in their growth medium to maintain their drug-resistance. Ultrastructural studies showed that clofazimine did not act by cell wall lysis, nor did it act on bacterial ribosomes. Higher concentrations of the drug resulted in bacterial plasmolysis. These findings are discussed in the light of its known properties and proposed mode of action.

Extraction and preliminary characterizaion of a mycobacteriolytic preparation (stazyme) from aStaphylococcus strain: mycobactericidal activity and its use in rapid extraction of mycobacterial DNA

The clear culture filtrate from 1-L culture of a laboratory contaminant ofStaphylococcus (coagulase− strain, designated Clavelis) was filtered, concentrated, dialyzed, and the proteins were precipitated. The precipitate was washed, concentrated, and aliquoted (about 4 mg of total proteins/ml, designated as “Stazyme”). The crude preparation was subjected to gel filtration on Sephadex G-75, and the fractions were screened for their lytic ability againstMycobacterium smegmatis. Native proteins in the “stazyme” were electrophoretically separated, electroeluted, and their lytic activity againstM. smegmatis was compared with parallel controls (partially purified proteins extracted from the same quantity of the uninoculated bacterial growth medium). Only “stazyme” preparations caused significant growth inhibition ofM. smegmatis, M. chelonae, M. xenopi, M. tuberculosis, andM. kansasii. “Stazyme” essentially possessed a lytic activity measured with purifiedM. smegmatis andMicrococcus lysodeikticus cell walls and showed high bactericidal activity againstM. smegmatis, M. chelonae, andM. tuberculosis. It was also able to rapidly lyse intactM. smegmatis organisms, permitting significant yield of mycobacterial DNA.