Kanglemycin A Can Overcome Rifamycin Resistance Caused by ADP-Ribosylation by Arr Protein.

Rifamycins, such as rifampicin (Rif), are potent inhibitors of bacterial RNA polymerase (RNAP) and are widely used antibiotics. Rifamycin resistance is usually associated with mutations in RNAP that preclude rifamycin binding. However, some bacteria have a type of ADP-ribosyl transferases, Arr, which ADP-ribosylate rifamycin molecules, thus inactivating their antimicrobial activity. Here, we directly show that ADP-ribosylation abolishes inhibition of transcription by rifampicin, the most widely used rifamycin antibiotic. We also show that a natural rifamycin, kanglemycin A (KglA), which has a unique sugar moiety at the ansa chain close to the Arr modification site, does not bind to Arr from Mycobacterium smegmatis and thus is not susceptible to inactivation. We, found, however, that kanglemycin A can still be ADP-ribosylated by the Arr of an emerging pathogen, Mycobacterium abscessus. Interestingly, the only part of Arr that exhibits no homology between the species is the part that sterically clashes with the sugar moiety of kanglemycin A in M. smegmatis Arr. This suggests that M. abscessus has encountered KglA or rifamycin with a similar sugar modification in the course of evolution. The results show that KglA could be an effective antimicrobial against some of the Arr-encoding bacteria.

Burning behaviour of rainscreen façades.

Four reduced-height (5 m) BS 8414-1 façade flammability tests were conducted, three having mineral-filled aluminium composite material (ACM-A2) with polyisocyanurate (PIR) and phenolic (PF) foam and stone wool (SW) insulation, the fourth having polyethylene-filled ACM (ACM-PE) with PIR insulation. Each façade was constructed from a commercial façade engineer's design, and built by practising façade installers. The ACM-PE/PIR façade burnt so ferociously it was extinguished after 13.5 min, for safety. The three ACM-A2 cladding panels lost their structural integrity, and melted away from the test wall, whereupon around 40% of both the combustible PIR and PF insulation burnt and contributed to the fire spread. This demonstrates why all façade products must be non-combustible, not just the outer panels. For the three ACM-A2 tests, while the temperature in front of the cavity was independent of the insulation, the temperatures within it varied greatly, depending on the insulation. The system using PF/A2 allowed fire to break through to the cavity first, as seen by a sharp increase in temperature after 17 min. For PIR/A2, the temperature increased sharply at 22 minutes, as the panel started to fall away from the wall. For SW/A2, no rapid temperature rise was observed.

Smoke toxicity of rainscreen façades.

The toxic smoke production of four rainscreen façade systems were compared during large-scale fire performance testing on a reduced height BS 8414 test wall. Systems comprising 'non-combustible' aluminium composite material (ACM) with polyisocyanurate (PIR), phenolic foam (PF) and stone wool (SW) insulation, and polyethylene-filled ACM with PIR insulation were tested. Smoke toxicity was measured by sampling gases at two points - the exhaust duct of the main test room and an additional 'kitchen vent', which connects the rainscreen cavity to an occupied area. Although the toxicity of the smoke was similar for the three insulation products with non-combustible ACM, the toxicity of the smoke flowing from the burning cavity through the kitchen vent was greater by factors of 40 and 17 for PIR and PF insulation respectively, when compared to SW. Occupants sheltering in a room connected to the vent are predicted to collapse, and then inhale a lethal concentration of asphyxiant gases. This is the first report quantifying fire conditions within the cavity and assessing smoke toxicity within a rainscreen façade cavity.

Ureidothiophene inhibits interaction of bacterial RNA polymerase with -10 promotor element.

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.

Mechanisms of antibiotics inhibiting bacterial RNA polymerase.

Transcription, the first phase of gene expression, is performed by the multi-subunit RNA polymerase (RNAP). Bacterial RNAP is a validated target for clinical antibiotics. Many natural and synthetic compounds are now known to target RNAP, inhibiting various stages of the transcription cycle. However, very few RNAP inhibitors are used clinically. A detailed knowledge of inhibitors and their mechanisms of action (MOA) is vital for the future development of efficacious antibiotics. Moreover, inhibitors of RNAP are often useful tools with which to dissect RNAP function. Here, we review the MOA of antimicrobial transcription inhibitors.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.