Objectives To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as ...a model organism. Methods Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above. Results When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in 3Htetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in 3Htetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump. Conclusions The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.
Both intrinsic and acquired multidrug resistance play an important role in the insurgence of tuberculosis. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug ...transport systems is required for the development of new antibiotics that are not extruded or of inhibitors that block the multidrug transporter and allow traditional antibiotics to be effective.
We have undertaken the inventory of the drug transporters subfamily, included in the major facilitator superfamily (MFS), encoded by the complete genome of Mycobacterium tuberculosis (MTB). These proteins were identified on the basis of their characteristic stretches of amino acids and transmembrane segments (TMS) number.
Genome analysis and searches of homology between the identified transporters and proteins characterized in other organisms revealed 16 open reading frames encoding putative drug efflux pumps belonging to MFS. In the case of two of them, we also have demonstrated that they function as drug efflux proteins.
A segment of DNA was isolated that complemented several poorly characterised sporulation-defective white-colony mutants of Streptomyces coelicolor A3(2) from an early collection (Hopwood et al., J ...Gen Microbiol 61: 397-408, 1970). Complementation was attributable to a gene, SCO4543, named whiJ, encoding a likely DNA-binding protein. Surprisingly, although some mutations in whiJ had a white colony phenotype, complete deletion of the wild-type or mutant gene gave a wild-type morphology. The whiJ gene is a member of a large paralogous set of S. coelicolor genes including abaAorfA, which regulates antibiotic production; and genes flanking whiJ are paralogues of other gene classes that are often associated with whiJ-like genes (Gehring et al., Proc Natl Acad Sci USA 97: 9642-9647, 2000). Thus, the small gene SCO4542 encodes a paralogue of the abaAorfD gene product, and SCO4544 encodes a paralogue of a family of likely anti-sigma factors (including the product of abaAorfB). Deletion of SCO4542 resulted in a medium-dependent bald- or white-colony phenotype, which could be completely suppressed by the simultaneous deletion of whiJ. A model is proposed in which WhiJ binds to operator sequences to repress developmental genes, with repression being released by interaction with the WhiJ-associated SCO4542 protein. It is suggested that this activity of SCO4542 protein is prevented by an unknown signal.
The spread of multidrug-resistant isolates of
requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine ...(SCN) and
-methyl-seconeolitsine (
-SCN), against
. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of
. Both SCN and
-SCN inhibited
growth at 1.95-15.6 μM, depending on the strain. In
this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of
. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography.
inhibition of MtbTopoI activity by SCN and
-SCN was tested using a plasmid relaxation assay. Both SCN and
-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 μM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated
in plasmid-containing cultures of
. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC
-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of
-SCN. Altogether, these results demonstrate that the
topoisomerase I enzyme is an attractive drug target, and that SCN and
-SCN are promising lead compounds for drug development.
Mycobacterium kansasii (Mkn) causes tuberculosis-like lung infection in both immunocompetent and immunocompromised patients. Current standard therapy against Mkn infection is lengthy and difficult to ...adhere to. Although β-lactams are the most important class of antibiotics, representing 65% of the global antibiotic market, they have been traditionally dismissed for the treatment of mycobacterial infections, as they were considered inactive against mycobacteria. A renewed interest in β-lactams as antimycobacterial agents has shown their activity against several mycobacterial species, including M. tuberculosis, M. ulcerans or M. abscessus; however, information against Mkn is lacking. In this study, we determined the in vitro activity of several β-lactams against Mkn. A selection of 32 agents including all β-lactam chemical classes (penicillins, cephalosporins, carbapenems and monobactams) with three β-lactamase inhibitors (clavulanate, tazobactam and avibactam) were evaluated against 22 Mkn strains by MIC assays. Penicillins plus clavulanate and first- and third-generation cephalosporins were the most active β-lactams against Mkn. Combinatorial time-kill assays revealed favorable interactions of amoxicillin-clavulanate and cefadroxil with first-line Mkn treatment. Amoxicillin-clavulanate and cefadroxil are oral medications that are readily available, and well tolerated with an excellent safety and pharmacokinetic profile that could constitute a promising alternative option for Mkn therapy.
The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the ...Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium. By Western blotting, P55 was located in the membrane fraction of M. bovis. When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.
The synthesis of high-affinity reversible competitive inhibitors of Mycobacterium tuberculosis type II dehydroquinase, an essential enzyme in Mycobacterium tuberculosis bacteria, is reported. The ...inhibitors reported here are mimics of the enol intermediate and the effect of substitution on C2 was studied. The crystal structures of Mycobacterium tuberculosis type II dehydroquinase in complex with three of the reported inhibitors are also described. The results show that an aromatic substituent on C2 prevents the closure of the active site by impeding the hydrogen-bonding interaction of Arg108 with the essential Tyr24 of the flexible loop, the residue that initiates catalysis. Chemical modifications of the reported acids were also carried out to improve internalization into Mycobacterium tuberculosis through an ester prodrug approach. Propyl esters proved to be the most efficient in achieving optimal in vitro activities.
This study evaluated the effect of the incorporation of seaweed on the physicochemical and technological quality of pasta. For this purpose, enriched wheat pastas from different seaweeds (sea ...lettuce—Ulva lactuca, nori—Porphyra tenera, and wakame—Undaria pinnatifida) were made and compared with durum wheat pasta as a control treatment. Firstly, optimal cooking times were established by visual and instrumental methods. Then, the technological properties of weight gain (WG), swelling index (SI), cooking losses (CL), and moisture (H%) were determined. Protein and fiber analyses, texture profile analysis (TPA), and color measurements were also performed to evaluate the physicochemical properties. Overall, enriched pasta with seaweed revealed slightly shorter optimal cooking times than control pasta. Texture properties were also modified, with a lower value of hardness, and higher values of adhesiveness and resilience. However, due to the low percentages of seaweed (3%), noticeable effects were not appreciated. Moreover, color variations of enriched pasta were relevant due to the difference among seaweeds. Nonetheless, these additions increased the protein content and soluble fiber in these foods. In conclusion, pasta enriched with marine ingredients improved this nutritional profile, and the changes in technological properties did not have a major impact on the product quality.
Back Cover: Bedaquiline is encapsulated in polypeptidic micelles, which are subsequently coated with sodium alginate. Encapsulated bedaquiline shows increased in vitro activity against Mycobacterium ...tuberculosis compared to free bedaquiline. A SEM picture of the bedaquiline loaded coated nanocarriers is shown in the background. This is reported by Héctor Soria‐Carrera, Ainhoa Lucía, Laura De Matteis, José A. Aínsa, Jesús M. de la Fuente, Rafael Martín‐Rapún in artictle 1800397.
PDF
