Structural basis of antibacterial peptide transport across membranes

Microcins are gene encoded antibacterial peptides secreted by enterobacteria in the gastrointestinal tract and play an important role in the control of bacterial populations. They present an attractive prospect in our effort to minimize the problem of bacterial drug resistance. Microcin J25 (MccJ25) is a 2 kDa plasmid encoded, ribosomally synthesized antimicrobial peptide comprised of 21 amino acid residues. MccJ25 undergoes post-translational modification and has a unique lasso structure. McjD, an ABC exporter, confers immunity to the producing strains by exporting the mature MccJ25 out of the cell. Studies have been designed to look into the transport mechanism of this peptide, which uses the siderophore receptor FhuA and ABC transporter McjD. MccJ25 uses the Trojan horse strategy by hijacking the iron import machineries as a mode of transport into the cell and acts as a transcription inhibitor by binding to RNA polymerase. Iron is an important nutrient for bacteria cell survival. To date, there is limited structural evidence on the import and extrusion mechanism of this antimicrobial peptide in Gram-negative bacteria. We have obtained a high-resolution structure of MccJ25 with its outer membrane receptor FhuA at 2.3 Å. FhuA is monomeric 22-strand antiparallel ß-barrel protein with the N-terminal domain folded inside to form a plug domain. MccJ25 binds to FhuA with hydrogen bonds and hydrophobic interactions with the extracellular loops of FhuA and its plug domain. We have also identified key residues that might play a role in MccJ25 translocation. Overall the structure provides information on how MccJ25 hijacks the iron uptake pathway to get into bacteria. Ligand binding studies and biochemical analysis demonstrate the functionality of McjD and its interaction with its natural ligand, MccJ25. The high substrate specificity and known cavity make McjD an excellent model for interaction studies.Open Acces

An update of the general health status in the indigenous populations of Malaysia

Objective. Health scenarios are constantly evolving, particularly in developing countries but little is known regarding the health status of indigenous groups in Malaysia. This study aims to elucidate the current health status in four indigenous populations in the country, who by and large been left out of mainstream healthcare developments. Methods. Participants were recruited from the Temuan, Jehai, Kensiu and Bidayuh indigenous groups throughout Peninsula Malaysia and Sarawak. Health parameters including body mass index (BMI), blood pressure, casual blood glucose and, total cholesterol levels were measured using established methods. Malondialdehyde (MDA) and ferric-reducing antioxidant power (FRAP) levels were measured to assess oxidative stress status. Blood films were screened for evidence of microbial or parasitic infections and leukocyte differential counting was performed. Results. The Temuan and Bidayuh who are more urbanized, had significantly higher mean body weight, BMI, total cholesterol (pB0.05) and higher prevalence of obesity and hypercholesterolemia. Low cholesterol levels, elevated eosinophil counts and increased total IgE, indicative of immune responses to infection or allergy, were recorded in the rural Kensiu and Jehai. The Kensiu had higher levels of FRAP and lower levels of MDA, whereas the reverse was found in the Temuan. This suggests reduced oxidative stress in the Kensiu compared to the Temuan. Expected correlations between FRAP and MDA levels with age, were evident in Jehai. Conclusions. Our findings reflect a shifting health burden and an epidemiological transition, particularly in the Temuan and Bidayuh. These changes could be attributed to dietary habits, lifestyles and socio-economic factors brought about by urbanization

Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state

Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATPbinding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-angstrom resolution, which is to the authors' knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3-6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outwardoccluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters

Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up

Crystal structure and epitope analysis of house dust mite allergen Der f 21

10.1038/s41598-019-40879-xSCIENTIFIC REPORTS91complete

Structural basis for hijacking siderophore receptors by antimicrobial lasso peptides.

The lasso peptide microcin J25 is known to hijack the siderophore receptor FhuA for initiating internalization. Here, we provide what is to our knowledge the first structural evidence on the recognition mechanism, and our biochemical data show that another closely related lasso peptide cannot interact with FhuA. Our work provides an explanation on the narrow activity spectrum of lasso peptides and opens the path to the development of new antibacterials