Projects

Our work on HGT focuses mainly on studying Distributive conjugal transfer (DCT), a unique form of HGT in mycobacteria and our work on Abi systems aims to characterize the RexA-RexB system in gram-negative bacteria.

Bacterial conjugation artwork. , Photograph by Karsten Schneider.

Conjugative systems in mycobacteria

The proliferation of antibiotic resistance (AMR) through horizontal-gene-transfer (HGT) is a formidable health challenge, accounting for 700,000 deaths annually (1). Conjugation, wherein large plasmid-DNA is transferred unidirectionally from a donor to a recipient cell through a pilus and a type-4 secretion-system (T4SS) is responsible for 80% of all HGT. Because secretion-systems are the primary drivers of bacterial conjugation, understanding their function is fundamental to combating AMR. My work on conjugative T4SSs (2) and pilus from led to the elucidation of the overall T4SS architecture and insights into the mechanism of pilus biogenesis (2). Although T4SSs from E. coli have been widely researched, there exist other clinically important conjugative-systems that are poorly understood. Distributive-Conjugal-Transfer (DCT) is one such mechanism where HGT occurs in gram+ Mycobacteriaceae using ESX-1 and ESX-4 systems (3) (members of ESAT-6 (ESX) type-7 secretion-system (T7SS) family) (Fig. 1a).

Fig.1: a. Schematic of DCT, b. Overview of ESX-1and its components (Gray et al., 2018), c. Genetic organization of esx-1 operon. Mutating genes labelled as DNA- and PR- disrupt DCT and protein transport respectively.

DCT differs mechanistically from classical conjugation on two levels: (1) unlike T4SS-driven conjugation, DCT is mediated by interactions between two T7SS, (2) DCT transfers short double-stranded genomic-DNA fragments compared to long single-stranded plasmid-DNA transferred during conjugation. This genomic exchange between mycobacterial cells often leads to the generation of mosaic genomes, enabling bacteria to respond swiftly to environmental challenges by rapidly evolving their genetic diversity. This transfer of multiple DNA fragments allows DCT to disseminate AMR within mycobacteria more efficiently than conjugation. Despite these differences, both DCT and conjugation involve contact-dependent interactions and require membrane-integrated secretion-systems including cytoplasmic and membrane-associated ATPases for DNA pre-processing and substrate-recruitment. Remarkably, despite being ubiquitous in mycobacteria (9,10), mechanisms underlying DCT remains unknown.

Transposon mutagenesis on the M. smegmatis esx-1 operon which codes for the ESX-1 T7SS and other accessory proteins for this transport (Fig. 1b) revealed three distinct sites where mutations impede DNA-transfer while not affecting protein-transfer (Fig. 1c) (4). Furthermore, ESX-4 is dispensable in donor, while recipient necessitates both ESX-1/-4 systems for DNA transfer (5). It is believed that ESX-1 facilitates secretion of mating-identity proteins inducing ESX-4 expression in recipients which results in assembly of a channel through which DNA is internalised (3) (Fig. 1a). Although genomic exchange using DCT does not occur specifically in M. tuberculosis, recent research has implicated HGT mechanisms in facilitating saltational-evolution of pathogenic mycobacteria (M. avium (6), the monophyletic M. tuberculosis complex (MTBC)/M. canettii (7) and M. abscessus (8)) into obligate human pathogens like M. tuberculosis.

Our current research program seeks to understand the molecular underpinnings of DCT in M. smegmatis by addressing the following objectives:

(1) investigating the architecture of ESX-1 T7SS

(2) exploring mechanisms involved in ESX-1 mediated substrate pre-processing, recruitment, and translocation

(3) dissecting the mechanics of donor-DNA fragmentation and role of ESX-1/ESX-4 intercellular communication in mediating DCT

Methodology:

The proposed project will utilize techniques in biochemistry, biophysics, and structural-biology to address the specific aims. The structural characterisation of protein-complexes requires membrane-protein biochemistry, cryo-electron microscopy/tomography (cryo-EM/cryo-ET), X-ray crystallography (XRD), structure-determination, AL/ML-led molecular modelling, and structure analysis. Purification of large membrane-integrated T7SSs will implement membrane protein purification methodologies, solubilization and stabilisation procedures. Biophysical experiments (SEC-MALS, SPR, DLS, ITC) will also be conducted in conjunction with structural studies to address these research questions. We collaborate with Prof. Gurdyal Besra (School of Biosciences, University of Birmingham) and Dr. Brian Ho (University College London) to undertake functional in-vivo characterization and live-cell imaging of these systems.

David Goodsell @ Scripps ©

Phage-defense mechanisms: Abortive Infection

Research interest in bacterial-phage interactions has experienced a revival due to its importance in bacterial evolution, requirement of phage resistant bacterial strains in many industries and as gene editing tools (CRISPR-Cas) (11). Of particular interest are the recent efforts to apply phage therapies to treat bacterial infections worldwide and counter antibiotic resistance (12). Infection due to M. tuberculosis (M. tb) is one such example where multi-drug (MDR) and extreme drug resistance (XDR)-TB has proved challenging to treat because M. tb resides deep within granulomas evading classical antibiotics. Consequently, using natural and engineered bacteriophages to target such persistent pathogens has recently gained popularity. Although utilizing phage therapies to treat bacterial infections and counter antibiotic resistance is promising (12), there is a risk of promoting the emergence of phage resistant bacterial pathogens. Our awareness of the plethora of phage defence mechanisms is relatively recent and the molecular basis of most bacterial phage resistant mechanisms, even in well-known model bacteriophages is largely unexplored.

Bacteria resist phage infections by several strategies, interestingly as a measure of last resort, by committing suicide upon phage attack by abortive infection (Abi) (13). Although many Abi systems are known (Fig 2), the RexA/RexB system (14) from E. coli is the most extensively characterized system where a λ-lysogenic prophage encoded RexAB complex inactivates the infection of other phages namely T4, T5 and T7 (Fig. 3a). Despite being the first identified Abi system, their mode of action remains enigmatic. The RexA protein is thought to sense a poorly characterized T4-phage protein DNA complex and two copies of RexA activate one copy of RexB, an inner membrane ion channel resulting in severe loss of membrane potential and a drop in ATP-levels, eventually leading to cell death (Fig. 3a). RexAB-like systems have also been identified in Mycobacteriaceae. In mycobacteria, although mycobacteriophages have already been clinically shown to treat mycobacterial infections, the existence of phage resistance mechanisms remains a problem. The most interesting case is that of Sbash, a prophage that colludes with its host (M. smegmatis) to confer highly specific defence against infection by a mycobacteriophage Crossroads using a mechanism analogous to RexAB system (Fig. 3b) (15).

The overarching vision of this research project is to characterize the molecular processes associated with abortive infection in bacteria with the eventual aim of designing robust mycobacteriophage based treatments against drug-resistant TB. This involves characterizing the RexA/RexB from E. coli and Sbash systems from mycobacteria using a combination of biophysical and structural methods.

The key questions in this project are

(1) How does RexA sense phage-DNA-protein complexes?

(2) How does RexA-phageDNA-protein complex activate membrane-integrated RexB?

(3) How RexB causes pore formation, cation efflux and membrane depolarization?

Methodology:

In addition to implementing membrane protein biochemistry, biophysical chemistry (MALS, SPR, DLS, ITC) and structural (X-ray crystallography, cryo-EM and cryo-ET) techniques to achieve the research objectives, this project will also use techniques used for characterizing protein-DNA complexes like DNA-foot printing, electrophoretic mobility shift assay (EMSA) and co-immunoprecipitation to identify, stabilize and purify associated complexes. Functional studies involving site-directed mutagenesis, in-vitro liposome-based assays, patch clamp electrophysiology, radioactive or fluorescently labelled ion flux assays and live-cell imaging experiments is also expected to provide insights into the overall functioning of the system. We are collaborating with Prof. Martha Clokie (Department of Genetics and Genome Biology (GGB) and National Centre for Phage Research, UoL) for functional in-vivo characterizations of these systems.

Fig.2: Mechanisms of action of a selected set of phage abortive infection systems (Lopatina et al., Ann. Rev, 2020). (a) RexAB system and cell lysis; (b) Stp-PrrC and protein-synthesis inhibition; (c) PacK-Stk2 and protein autophosphorylation; (d) RnlAB and RNA-cleavage.

Fig.3: (a) Mode of action of the E. coli RexAB system, and (b) the mycobacterial Sbash system

References

IACG report on Antimicrobial Resistance, 2019.
Macé K* & Vadakkepat* AK et al. Cryo-EM structure of a type IV secretion system. Nature. 2022, 607:191-196. (* equal contribution).
Gray TA, Derbyshire KM. Blending genomes: distributive conjugal transfer in mycobacteria, a sexier form of HGT. Mol Microbiol. 2018, 108:601-613.
Coros A, Callahan B, Battaglioli E, Derbyshire KM. The specialized secretory apparatus ESX-1 is essential for DNA transfer in M. smeg. Mol Microbiol. 2008, 69:794-808.
Gray TA, Clark RR, Boucher N, Lapierre P, Smith C, Derbyshire KM. Intercellular communication mediated by ESX secretion systems in mycobacteria. Science. 2016, 21:347-350.
Uchiya KI, Tomida S, Nakagawa T, Asahi S, Nikai T, Ogawa K. Comparative genome analyses of M. avium reveal genomic features of its subspecies and strains that cause progression of pulmonary disease, Sci Rep., 2017, 7:39750.
Blouin Y et al. Progenitor “Mycobacterium canettii” clone responsible for lymph node tuberculosis epidemic, Djibouti. Emerg Infect Dis. 2014, 20:21-8.
Bryant JM et al. Stepwise pathogenic evolution of M. abscessus. Science. 2021, 372:eabb8699.
Sapriel G et al. Genome-wide mosaicism within M. abscessus. BMC Genomics. 2016, 17:118.
Boritsch EC et al., Key experimental evidence of chromosomal DNA transfer among selected tb-causing mycobacteria. PNAS. 2016, 113:9876-81.
Foss DV et al., CRISPR-based genome editing & diagnostics. Transfusion. 2019, 59:1389-1399.
Dedrick RM et al., Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. 2019, 25:730-733.
Lopatina A, Sorek R. Abortive Infection: An Antiviral Immune Strategy. Ann. Rev Virol. 2020, 7:371-384.
Parma DH et al., The Rex system of phage lambda: tolerance & cell death. Genes Dev. 1992, 6:497-510.
Gentile GM et al., More Evidence of Collusion: a New Prophage-Mediated Viral Defence System Encoded by Mycobacteriophage Sbash. mBio. 2019, 10:e00196-19.