Antibiotics Against Gram-Negative Bacteria

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Researchers all over the world work hard in the discovery of antibiotics, one such researcher is Dr. Kim Lewis, leader of the Lewis Lab at Northeastern University where he is a distinguished professor with expertise in molecular microbiology. Dr. Kim Lewis’ lab consists of the Lewis Lab which studies persister cells responsible for tolerance to antibiotics, uncultured bacteria of the environment and the microbiome. It also includes the antimicrobial discovery center which translates basic discoveries into novel antimicrobial therapies to combat conventional pathogen threats and biowarfare. Working under Dr. Kim Lewis are Dr. Yu Imai and Dr. Kirsten Meyer, who together studied the effects of many potential compounds and their antimicrobial effectiveness in hopes of discovering a viable antibiotic. Dr. Yu Imai started as postdoctoral research associate and became associate research scientist and now works as assistant professor at Shinshu University. He received his bachelor’s degree in the faculty of agriculture, then a master’s degree from the graduate school of agriculture to then receive a doctor of philosophy, PhD, at the Interdisciplinary Graduate School of Science and Technology, all at Shinshu university. Dr. Kirsten Meyer was a postdoctoral research associate in Lewis lab at Northeastern University and is now a postdoctoral fellow at the University of Toronto. She got a bachelor’s degree from Victoria University of Wellington in honours biomedical science, majoring in molecular pharmacology and medicinal chemistry to then go on and achieve a PhD in pharmacology from Johns Hopkins University School of Medicine. Her work now at the University of Toronto involves optimized antimicrobial combinations and their packaging in extracellular vesicles from soil bacteria, towards development of new antimicrobials to counter resistance. Together at Northeastern University, along with the collaborative efforts of other researchers, they worked to find new antibiotics that selectively kill Gram-negative pathogens as an effort against the emergence of antibacterial resistance.

A new potential antibiotic is darobactin which targets an outer membrane protein (OMP) of gram negative bacteria (Guo et al., 2022). The target of darobactin is an OMP called the β-barrel assembly machinery (BAM), which is a pentameric complex with BamA being the essential core and BamB, BamC, BamD, and BamE being the accessory lipoproteins to BamA (Guo et al., 2022; Noinaj et al., 2017; Pinto et al., 2018). This OMP is essential for bacterial physiology, pathogeny, and drug resistance (Pinto et al., 2018). The four accessory lipoproteins each carry a N-terminal post-translational lipid modification that signals them to attach to BamA (Noinaj et al., 2017). However, the component of interest is BamA which has been studied to be the target of darobactin (Guo et al., 2022). As the core of the BAM complex, BamA is found embedded within the outer membrane due to its amphiphilic nature, being hydrophobic on the outside to interact with the membrane and hydrophilic on the inside to interact with solvents (Noinaj et al., 2017). It has a N-terminal periplasmic domain that carries five polypeptide transport associated (POTRA) domains and a C-terminal domain that consists of a 16 stranded, anti-parallel, β-barrel domain (Noinaj et al., 2017). POTRA domains do not have a clear role in the OMP’s functions but it is suggested that they act as docking sites for BamB, BamC, BamD, and BamE onto BamA (Kim et al., 2012). In particular, β1 and β16 interact in a way that form a bent or kink towards the lumen of the domain which gives rise to a lateral gate with the ability to provide direct insertion of nascent OMPs into the outer membrane during biogenesis (Noinaj et al., 2017). The lateral gate has two proposed functions being that it’s involved in β-augmentation and that it releases the substrate into the bilayer due to the kink producing a slight destabilization (Pinto et al., 2018). β-augmentation occurs when β1 and β16 strands and the substrate protein interact to fold the substrate of interest (Pinto et al., 2018).

Other than POTRA5 which is known to be an essential domain, the other POTRA domains appear to have varying conclusions drawn in the literature (Browning et al., 2013). Some have been shown to be both essential and non-essential. Amongst the domains, there exists conservation with their folding structures but not with their sequences or function (Konovalova et al., 2017). This structure consists of three strand β-barrel covered by an antiparallel helix pair (Konovalova et al., 2017). In terms of their function, the POTRA domains allow for interactions between BamA and both other units in BAM and other proteins. POTRA1 allows for interactions with the chaperone SurA, POTRA3 allows for interactions with BamB, and POTRA5 allows for interactions with the BamCDE subcomplex through the formation of an interaction interface (Konovalova et al., 2017). Alongside the other parts of BAM, the POTRA domains form a ring-shaped structure oriented parallel to the plane of the membrane (Konovalova et al., 2017).

Figure X: Two views of the BAM complex as modeled using cryo-EM (Konovalova et al., 2017).

Due to the hydrogen bonds that form between the phospholipid heads of the membrane and the POTRA domains, there is a high affinity between the two where tryptophan residues, conserved in the POTRA domains, allow the domains to partition into the membrane (Konovalova et al., 2017).

The goal of BAM is to catalyze the proper folding and insertion of new β-barrel proteins such as BAM itself through a process called bacterial OMP biogenesis (Imai et al., 2019; Kim et al., 2012). Nascent OMPs are produced within the cytosol with a N-terminal signal sequence that directs it to the inner membrane to be transported to the periplasm through the Sec translocation system (Kim et al., 2012). During this time, the signal sequence is cleaved by signal peptidase I (SPase I) in order for the OMP to travel to the periplasm (Kim et al., 2012). Then, it will travel to the outer membrane through the SurA pathway or Skp/DegP pathway and finally integrate itself into the outer membrane as a β-barrel protein (Kim et al., 2012).

Coming back to the β-barrel at the C-terminus of BamA, there are various loops (L) and turns (T) connecting the 16 β-strands (Browning et al., 2013). All loops are extracellular while all turns are periplasmic. The loops are especially important in the function of BamA where insertions in some loops may cause large issues in protein function (Browning et al., 2013). Specially, loops L2, L5, and L8 appear to be conserved quite well amongst orthologues while contrarily L3, L4, and L7 are not (Browning et al., 2013). L1 is conserved well enough that mutations are quite rare to observe (Browning et al., 2013). This implies the possibility of functional/structural importance being linked to the amino acid sequences in L2, L5, and L8 (Browning et al., 2013). Experimentation has been that L4, L6, L7, and L8 cause major issues upon deletion, but deletion of L3 appears to have no major problems in the protein (Browning et al., 2013). Due to this, it was inferred that L3 instead has steric effects on the protein (Browning et al., 2013).

Figure Y: A models BamA using a collection of information from various studies; B is a topological model of the β-barrel constructed through predictions using bioinformatics (Browning et al., 2013).

Based on a homology with FhaC, another outer membrane protein, L6 shows importance being an essential structure in the functioning of FhaC (Browning et al., 2013). There exists a conserved RGF motif on L6, being the notable portion that allows for L6 to be essential (Browning et al., 2013). It is notable that the RGF motif cannot be deleted while retaining normal BamA function (Browning et al., 2013). Unlike the other loops which exist outside of the β-barrel, L6 instead is found inside the β-barrel (Browning et al., 2013). It has been hypothesized that the strands of the β-barrel and L6 undergo some conformational change during the protein folding process where the other loops aid via stabilization through the stages of movement (Browning et al., 2013).

Figure x. The predicted chemical structure of the antibiotic darobactin. Image from Imai et al. 2019.

Nematodes have similar requirements for antibacterials as humans (Tobias et al. 2018). The digestive system of the worms is lined with bacteria similar to E. coli; these bacteria are released by a nematode as it invades an insect larva to feed (Crawford and Clardy 2011). The antimicrobial substances prevent other microorganisms from feeding on the larvae (Tobias et al. 2018). Some of these environmental microorganisms are Gram-negative bacteria (Tambong, 2013). The antibiotics must harm the bacteria without harming the nematode as they are in close proximity (Imai et al. 2019). The fact that the nematode microbiome can kill these bacteria so effectively means there could be a candidate human antibiotic substance produced by one of the commensal bacteria (Imai et al. 2019).

28 species of bacteria from the nematode microbiome were screened for production of antimicrobial against E. coli (Imai et al. 2019). Very few candidates produced zones of inhibition, the antibacterials are not produced in high enough quantities in vitro (Imai et al. 2019). Concentrated extracts of the bacteria were prepared and an extract from ​​Photorhabdus khanii HGB1456 effectively produced a zone of inhibition and served as a potential antibiotic candidate (Imai et al. 2019). The compound was isolated and identified to have a molar mass of 966.41047, suggesting that the molecular formula would be C47H56O12N11+ (Imai et al. 2019). This compound was checked against a natural products database to ensure that it was novel (Imai et al. 2019). Once it was confirmed to be a new substance, the research group named it darobactin (Imai et al 2019).

Figure x. The predicted chemical structure of the antibiotic darobactin. Image from Imai et al. 2019.

Darobactin can effectively inhibit the growth of many Gram-negative bacteria and it has little effect against Gram-positive bacteria (Imai et al. 2019). There is also little activity against the Bacteroides that compose much of our commensal gut microbiome (Imai et al. 2019), Zafar et al. 2021). This led the research group to believe that the compound might inhibit lipopolysaccharide, the main component of the outer membrane of bacteria (Bertani et al. 2019). Tests of darobactin against purified LPS found that there was no inhibition of activity, meaning darobactin does not target LPS directly (Imai et al. 2019). It must target a different component of the outer membrane. A potential candidate would be BamA which is a large, crucial outer membrane protein (Kaur et al. 2021). To identify the target, a ligand-protection thermal proteome analysis was performed, this analysis showed that chaperone proteins were significantly upregulated, whereas outer membrane proteins were decreased (Imai et al. 2019). Mutant E. coli cultured in high quantities of darobactin developed resistance to the drug (Imai et al. 2019). The sequences of the mutant bacteria were observed to determine which mutated genes would confer darobactin resistance, each mutant bacteria had multiple mutations in the bamA which codes for the BamA protein (Imai et al. 2019). This proved that BamA was the likely target of darobactin (Imai et al. 2019).

Further studies were performed to determine how resistance affected the virulence and infectivity of the mutated bacteria (Imai et al. 2019). Mice injected with wild-type E. coli had a mortality rate of 60% within 24 hours of infection, whereas the bacteria injected with the resistant mutants had no deaths within 24h (Imai et al. 2019) The mutations that confer darobactin resistance significantly reduced the virulence of the bacteria (Imai et al. 2019).

Darobactin is an exciting drug candidate that targets the outer membrane protein BamA (Imai et al 2019). If approved for humans, it would be the first drug developed from the microbiome of another animal species (Candanosa, 2022).

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