Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, P Ms1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β′ subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.
Mycobacterium tuberculosis, the
etiologic agent of tuberculosis, is an intracellular pathogen of alveolar
macrophages. These cells avidly take up nanoparticles, even without
the use of specific targeting ligands, making the use of nanotherapeutics
ideal for the treatment of such infections. Methoxy poly(ethylene
oxide)-block-poly(ε-caprolactone) nanoparticles
of several different polymer blocks’ molecular weights and
sizes (20–110 nm) were developed and critically compared as
carriers for rifampicin, a cornerstone in tuberculosis therapy. The
polymeric nanoparticles’ uptake, consequent organelle targeting
and intracellular degradation were shown to be highly dependent on
the nanoparticles’ physicochemical properties (the cell uptake
half-lives 2.4–21 min, the degradation half-lives 51.6 min–ca.
20 h after the internalization). We show that the nanoparticles are
efficiently taken up by macrophages and are able to effectively neutralize
the persisting bacilli. Finally, we demonstrate, using a zebrafish
model of tuberculosis, that the nanoparticles are well tolerated,
have a curative effect, and are significantly more efficient compared
to a free form of rifampicin. Hence, these findings demonstrate that
this system shows great promise, both in vitro and in vivo, for the treatment of tuberculosis.
This real-time PCR method for the detection of the gene encoding the surface lipoprotein LipL32 is a reliable, sensitive, and rapid method for the detection of the acute form of leptospirosis.
In this work, levofloxacin (LVX), a third‐generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan‐g‐poly(methyl methacrylate) produced by self‐assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non‐crosslinked nanoparticles display a size of 29 nm and a zeta‐potential of +36 mV, while the crosslinked counterparts display 45 nm and +24 mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH‐S and the human bronchial epithelial cell line BEAS‐2B in vitro. Internalization events are detected after 10 min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX‐loaded nanoformulations (50% w/w drug content) is assessed in MH‐S and BEAS‐2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.
Aims. To examine biological materials (blood, urine, cerebrospinal fluid) of patients with suspected leptospirosis using real-time PCR for detecting the gene that codes the superficial LipL32 lipoprotein, and to evaluate the contribution of the real-time PCR method for the laboratory diagnosis of the acute form of leptospirosis. Material and Methods. During the monitored period of April 2010 -December 2011, a total of 340 biological materials samples were examined (177x blood plasma, 88x urine, 68x, cerebrospinal fluid, 6x bronchoalveolar lavage and 1x sputum) from 216 patients with suspected leptospirosis using real-time PCR LipL32 gene detection. Results. From the mentioned 216 patients suspected of leptospirosis, 8 patients were evaluated as being PCR LipL32 positive, from which 14 positive biological materials originated (9 x urine, 4x blood and 1x liquor). Conclusion. As demonstrated in the study, the real-time PCR method for detecting the gene for the superficial lipoprotein LipL32 is an appropriate, quick and reliable method for the diagnosis of the acute form of leptospirosis.
Apart from the SARS-CoV-2 virus, tuberculosis remains the leading cause of death from a single infectious agent according to the World Health Organization. As part of our longterm research, we prepared a series of hybrid compounds combining pyrazinamide, a first-line antitubercular agent, and 4aminosalicylic acid (PAS), a second-line agent. Compound 11 was found to be the most potent, with a broad spectrum of antimycobacterial activity and selectivity toward mycobacterial strains over other pathogens. It also retained its in vitro activity against multiple-drug-resistant mycobacterial strains. Several structural modifications were attempted to improve the in vitro antimycobacterial activity. The δ-lactone form of compound 11 (11′) had more potent in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv. Compound 11 was advanced for in vivo studies, where it was proved to be nontoxic in Galleria mellonella and zebrafish models, and it reduced the number of colonyforming units in spleens in the murine model of tuberculosis. Biochemical studies showed that compound 11 targets mycobacterial dihydrofolate reductases (DHFR). An in silico docking study combined with molecular dynamics identified a viable binding mode of compound 11 in mycobacterial DHFR. The lactone 11′ opens in human plasma to its parent compound 11 (t 1/2 = 21.4 min). Compound 11 was metabolized by human liver fraction by slow hydrolysis of the amidic bond (t 1/2 = 187 min) to yield PAS and its starting 6-chloropyrazinoic acid. The long t 1/2 of compound 11 overcomes the main drawback of PAS (short t 1/2 necessitating frequent administration of high doses of PAS).
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