Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Tunable force transduction through the Escherichia coli cell envelope.

The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the Escherichia coli TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.

Force-Generation by the Trans-Envelope Tol-Pal System.

The Tol-Pal system spans the cell envelope of Gram-negative bacteria, transducing the potential energy of the proton motive force (PMF) into dissociation of the TolB-Pal complex at the outer membrane (OM), freeing the lipoprotein Pal to bind the cell wall. The primary physiological role of Tol-Pal is to maintain OM integrity during cell division through accumulation of Pal molecules at division septa. How the protein complex couples the PMF at the inner membrane into work at the OM is unknown. The effectiveness of this trans-envelope energy transduction system is underscored by the fact that bacteriocins and bacteriophages co-opt Tol-Pal as part of their import/infection mechanisms. Mechanistic understanding of this process has been hindered by a lack of structural data for the inner membrane TolQ-TolR stator, of its complexes with peptidoglycan (PG) and TolA, and of how these elements combined power events at the OM. Recent studies on the homologous stators of Ton and Mot provide a starting point for understanding how Tol-Pal works. Here, we combine ab initio protein modeling with previous structural data on sub-complexes of Tol-Pal as well as mutagenesis, crosslinking, co-conservation analysis and functional data. Through this composite pooling of in silico, in vitro, and in vivo data, we propose a mechanism for force generation in which PMF-driven rotary motion within the stator drives conformational transitions within a long TolA helical hairpin domain, enabling it to reach the TolB-Pal complex at the OM.

Bacterial Competition Systems Share a Domain Required for Inner Membrane Transport of the Bacteriocin Pyocin G from Pseudomonas aeruginosa.

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. IMPORTANCE Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

The multifarious roles of Tol-Pal in Gram-negative bacteria.

In the 1960s several groups reported the isolation and preliminary genetic mapping of Escherichia coli strains tolerant towards the action of colicins. These pioneering studies kick-started two new fields in bacteriology; one centred on how bacteriocins like colicins exploit the Tol (or more commonly Tol-Pal) system to kill bacteria, the other on the physiological role of this cell envelope-spanning assembly. The following half century has seen significant advances in the first of these fields whereas the second has remained elusive, until recently. Here, we review work that begins to shed light on Tol-Pal function in Gram-negative bacteria. What emerges from these studies is that Tol-Pal is an energised system with fundamental, interlinked roles in cell division - coordinating the re-structuring of peptidoglycan at division sites and stabilising the connection between the outer membrane and underlying cell wall. This latter role is achieved by Tol-Pal exploiting the proton motive force to catalyse the accumulation of the outer membrane peptidoglycan associated lipoprotein Pal at division sites while simultaneously mobilising Pal molecules from around the cell. These studies begin to explain the diverse phenotypic outcomes of tol-pal mutations, point to other cell envelope roles Tol-Pal may have and raise many new questions.

Predicting pharmacokinetic behaviour of drug release from drug-eluting embolization beads using in vitro elution methods.

Drug-eluting Embolic Bead - Transarterial Chemoembolisation (DEB-TACE) is a minimally invasive embolising treatment for liver tumours that allows local release of chemotherapeutic drugs via ion exchange, following delivery into hepatic arterial vasculature. Thus far, no single in vitro model has been able to accurately predict the complete kinetics of drug release from DEB, due to heterogeneity of rate-controlling mechanisms throughout the process of DEB delivery. In this study, we describe two in vitro models capable of distinguishing between early phase and late phase drug release by mimicking in vivo features of each phase. First, a vascular flow system (VFS) was used to simulate the early phase by delivering DEB into a silicon vascular cast under high pulsatile flow. This yielded a burst release profile of drugs from DEB which related to the dose adjusted Cmax observed in pharmacokinetic plasma profiles from a preclinical swine model. Second, an open loop flow-through cell system was used to model late phase drug release by packing beads in a column with an ultra-low flow rate. DEB loaded with doxorubicin, irinotecan and vandetanib showed differential drug release rates due to their varying chemical properties and unique drug-bead interactions. Using more representative in vitro models to map discrete phases of DEB drug release will provide a better capability to predict the pharmacokinetics of developmental formulations, which has implications for treatment safety and efficacy.