The Costa lab
We aim to understand the structural basis for assembly, regulation and function of bacterial double-membrane spanning molecular machines. To fully understand how these macromolecular machines work, we use a multi-disciplinary approach including, bacterial genetics (gene disruption and site-specific mutagenesis), membrane proteins biochemistry, cutting-edge single particle cryo-EM, X-ray crystallography and protein cross-linking coupled with mass spectrometry (XL-MS).
Conjugative Molecular Machines
The process of conjugation involves the transfer of genomic and plasmid DNA between bacteria. Plasmids and mobile genetic elements are vectors of antimicrobial resistance (AMR) genes; thus, conjugation is the major process behind the spread of AMR genes by horizontal gene transfer (HGT) in bacteria. HGT is also the major driver of bacterial evolution. Therefore, conjugation also plays a major role in bacterial adaptation to the environment, including the gut microbiome.
Central to conjugation is the type IV secretion systems (T4SS) found in both Gram-positive and Gram-negative bacteria. This widespread secretion nanomachine is crucial for the transfer of genetic material between bacteria and virulence factors from bacteria to their eukaryotic cell targets. Conjugative T4SSs are organised into three main subassemblies, an outer-membrane core complex (OMCC), an inner membrane complex (IMC) and an extracellular pilus.
Previously, we solved the structure of the iconic bacterial F-pilus at near-atomic resolution by cryo-EM, which provides the first atomic view of a bacterial extracellular appendage made of a protein-lipid complex and possibly the conduit via which the DNA is transported to the bacterial recipient cell. (Costa et al, Cell, 2016).
Now, we solved the structure of an intact outer-membrane core complex OMCC derived from the iconic F-plasmid. The structure provides the first atomic view of the complete distal portion of a conjugative T4SS; unveils novel structural features unseen so far, that could explain the dynamic properties of the T4SS during F-pilus extension and retraction; and importantly, its central role as a driving force for the spread of AMR. (Amin et al, Nat Comms, 2021)
By understand the atomic details behind the mechanism of DNA transport during conjugation, we hope to provide the molecular basis for the development of novel therapeutic strategies that will help combating AMR.
Amin, H., Ilangovan, A., Costa, T.R.D.* (2021) Architecture of the outer-membrane core complex from a conjugative type IV secretion system. Nat Communications Nature Communications 12, 6834 PMID: 34824240
Zheng, W., Pena, A., Ilangovan, A., Clark, J.N., Frankel, G., Egelman, E.H., and Costa, T.R.D.* (2021). Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament. Proc Natl Acad Sci 118 (2 ):e2022826118 PMID: 33397726
Sgro G.S.#, Costa, T.R.D.#, Cenens, W., Souza, D.P., Cassago, A., Oliveira, L.C., Salinas, R.K., Portugal, R.V., Farah, C.S., Waksman, G. (2018) Cryo-EM structure of the bacteria killing type IV secretion system core complex from Xanthomonas citri. Nature Microbiology doi: 10.1038/s41564-018-0262-z PMID: 30349081
Costa, T.R.D., Ilangovan, A., Ukleja, M., Redzej, A., Santini, J.M., Smith, T.K., Egelman, E.H., and Waksman, G. (2016). Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex. Cell 166, 1436-1444 e1410.PMID:27610568
Costa, T.R.D., Felisberto-Rodrigues, C., Meir, A., Prevost, M.S., Redzej, A., Trokter, M., and Waksman, G. (2015). Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nature Reviews Microbiology 13, 343-359. PMID:25978706