Peptidoglycan maturation controls outer membrane protein assembly

Created By: Mathias Wang, Jake Weinstein, Leon Xu, Marius Ygonia, Ziran Yin, Junyi Zhang, Amy Zhao, Ray Zhu

As one of the first two authors and equal contributor to Mamou, Federico Corona is a post-doctoral researcher at Newcastle University where he obtained a Marie-Curie fellowship. With a background in biology and a master's in cellular and molecular biology, Corona's research focuses on the biogenesis of the cell wall of Gram-negative bacteria and the BAM system. His goal is to understand how they work and potentially use them as targets in the development of antibacterial drugs (“Federico Corona”, n.d.).

As the other first author and equal contributor to Corona, Gideon Mamou is a post-doctoral researcher in the biochemistry department at Kellogg College at the University of Oxford where he received an EMBO fellowship to study the spatiotemporal organization of the bacterial outer membrane. His research focus is on bacterial biochemistry and cell biology, and he is currently seeking to elucidate how new proteins are introduced into the outer membrane of Gram-Negative bacteria and to explore the dynamic nature of the machinery that inserts them (“Gideon Mamou”, n.d.).

The last author, Waldemar Vollmer, is a seasoned professor of bacterial biochemistry at Newcastle University. His research aims to decipher the molecular mechanisms of peptidoglycan growth in the model bacterium E. coli as well as other Gram-negative and Gram-positive bacteria. Currently, his research group is focusing on the interactions of peptidoglycan enzymes, aiming to identify new members in the pathway, while also examining the spatiotemporal regulation of peptidoglycan growth and the mechanisms of cell shape generation in a variety of species (“Waldemar Vollmer”, n.d.).

Bacteria can be classified based on their colour after gram staining procedure. The difference in colour between gram-positive and gram-negative bacteria arises from their distinct cell envelope structures. Gram-positive bacteria have a thick peptidoglycan layer while gram-negative bacteria have a thin peptidoglycan layer and an extra outer membrane. Specifically, gram-negative bacteria have a three-layered cell envelope, consisting of an inner plasma membrane, a peptidoglycan cell wall, and an outer membrane. The inner plasma membrane is made up of a phospholipid bilayer, the peptidoglycan cell wall consists covalently cross-linked macromolecule, and the outer membrane is a bilayer with phospholipids in the inner leaflet and lipopolysaccharides in the outer leaflet. The cell envelope functions to maintain cell shape and withstand mechanical forces, such as turgor pressure. The mechanical load is balanced between the outer membrane and the peptidoglycan cell wall, which are both essential for the survival of the bacterium (Rojas et al., 2018).

Figure 1. Gram-negative bacteria three-layered cell envelope (Created in BioRender.com).

Outer membrane proteins (OMP) play a role in many essential biological pathways of gram-negative bacteria such as nutrient uptake, waste export, cell signaling and cell adhesion. As a class of transmembrane protein, outer membrane proteins adopt a β-barrel structure. Compared to the α-helical bundle structure of transmembrane proteins in cytoplasmic membrane and ER, the β-barrel structure of OMPs comprise many anti-parallel β-strands warped into a cylinder shape (Lee et al., 2019). OMPs are inserted into the outer membrane with short loops between b-strands on the periplasmic side and large oops on the extracellular side (Rollauer et al., 2015). The two essential OMPs for E coli. are LptD (Lipopolysaccharide transport complex has 7 essential protein LptA-G; LptD and LptE are found in outer membrane) and BamA, which are responsible for LPS insertion into the outer leaflet of outer membrane and the folding and insertion of OMPs respectively (Rollauer et al., 2015). OMP are produced by ribosomes inside the cytoplasm, and therefore how OMP are transported and inserted into the OM is important for bacteria to assemble its envelope (Rollauer et al., 2015). Previous research found that OMPs transverse the inner membrane through the porin of SecYEG translocon in an unfolded conformation, and the chaperon in periplasm prevents the misfolding of OMPs (Rollauer et al., 2015). Three most important periplasmic chaperons are survival protein A (SurA), Seventeen kilodalton protein (Skp) and DegP. OMPs are then delivered to the BAM complex at where they will get folded(Rollauer et al., 2015).

Figure 2. Outer membrane proteins in gram-negative bacteria (Created in BioRender.com).

The outer membrane in gram negative bacteria is crucial in providing an extra layer of protection and functions as a selective barrier. These bacteria are challenging to treat because antibiotics need to penetrate the cell envelope to reach intracellular processes. However, as seen in figure 3A, large antibiotics such as vancomycin cannot penetrate the outer membrane, and efflux pumps in gram-negative bacteria can remove antibiotics to the external environment (Delcour, 2009). OMPs are general diffusion porins for many hydrophilic antibiotics, and their structure and distribution could greatly affect the permeability of antibiotics. Many antibiotic resistant bacteria arise from modifications in their outer membrane lipid and protein composition. Studying the insertion of outer membrane proteins and cell envelope assembly can improve our understanding of antibiotic translocation. New antibiotics as shown in figure 3B targeting the BAM complex are also being developed. Since BamA is exposed to the extracellular space, it gives the opportunity to exhibit antibiotic function without penetrating the cell. This approach will bypass the antibiotic resistant mechanisms commonly seen in gram-negative bacteria. The BAM complex is crucial in folding and inserting the outer membrane proteins. Thus, by inhibiting BamA extracellularly, the normal function of outer membrane essential to bacterial survival is being disrupted, allowing the inhibition of BamA and OMP protein folding without cell penetration (Sousa, 2019).

Figure 3. BAM complex as a novel target to fight antibiotics resistance. (A) Common mechanisms of antibiotic resistance in gram-negative bacteria. (B) New antibiotics targeting BamA extracellularly (Created in BioRender.com).

The β-barrel assembly machinery (BAM) complex is 200kDa large and consists of 5 major components. The most significant component of the BAM complex is BamA. BamA is a Beta-Barrel protein that is 810 amino acids long. The BAM complex also consists of 4 accessory lipoproteins (BamB, BamC, BamD, and BamE). BamB is the longest accessory lipoprotein at 371 amino acids in length. BamB features many beta-sheets arranged in a ring-like structure. BamD is 218 amino acids and is made up of many alpha helices. BamC and BamE are 165 and 87 amino acids in length respectively.

Figure 4. E. coli BAM complex structure demonstration in PyMol (PDB: 5LJO). The five proteins are highlighted as BamA in green, BamB in blue, BamC in purple, BamD in yellow, BamE in pink.

The BAM complex is responsible for catalyzing folding and inserting the B-barrel OMPs into the outer membrane. As OMP are important for many cellular processes such as movement of essential nutrients, secretion, and signalling. Thus, the activity of the Bam complex is very important for the survival of the bacteria. OMPs are first directed to the SecYEG translocon when they are destined for the BAM complex (Sklar et al., 2007). Following export through SecYEG, two potential chaperone pathways—SurA and Skp-DegP, recruit the nascent OMPs, which are then transported through the periplasm to the outer membrane (Sklar et al., 2007). Through an envelope stress response, proteases like DegP are directed toward the destruction of extra amounts of unfolded OMPs in the periplasm (Sklar et al., 2007). The BAM complex is assumed to be involved in the folding and insertion of developing OMPs. This process can be seen in figure 5. Exactly how the BAM complex participates in OMP folding and insertion is yet unknown.

Figure 5. The Bam complex with its function facilitating the folding of the OMP into the lipid bilayer (Li et al., 2020).

BamA belongs to the Omp85 superfamily of Gram-negative bacteria outer membrane protein. This family of proteins are structurally conserved translocation proteins or assembly factors, consisting of an N-terminal domain riches in periplasmic polypeptide transport-associated (POTRA) domains and a β-barrel C-terminal fits inside the outer membrane (Gentle et al., 2005)(Heinz and Lithgow, 2014). As a component of the Bam complex, BamA is a core of the β-barrel outer membrane protein. This component is very important in the Bam complex because it is the main and central component while also being vital to the protein, meaning that it is evolutionarily conserved. As the main component of the Bam complex, it facilitates the folding of client outer membrane proteins into the surrounding lipid bilayer.

The in vitro mechanism of how BamA catalyzes the folding of OMPs has been well understood. In order for OMPs to be inserted into the outer membrane of bacteria, it needs to overcome the kinetic energy barrier imposed by phosphoethanolamine head groups of phospholipids that make up the membrane (Gessmann et al., 2014). BamA therefore accelerates this process by lowering the kinetic energy needed (Gessmann et al., 2014). This process is achieved when OMPs are targeted to the outer membrane and it interacts with the BamA β-barrel. The C-terminal β-strand of the OMP forms a β-seam with BamA β-1 resulting in an asymmetric hybrid barrel with the β-strands of the OMP (Doyle & Bernstein, 2019). Afterwards, the first β-strand of the OMP forms a low stability contact with BamA β15/16 (Doyle & Bernstein, 2019). The rotation of BamA β1-β8, disturbance of the lpid bilayer, and dynamicity of β16 “swings” the OMP into the membrane (Doyle & Bernstein, 2019). Then the asymmetry between the two interfaces favours the closure of the β-barrel of the OMP (Doyle & Bernstein, 2019). This process can be seen in figure 6.

Figure 6. BamA promoting partially folded β-barrels into the membrane by forming asymmetric hybrid with the client OMP and a ‘swing’ mechanism (Doyle & Bernstein, 2019).

But the mechanisms of how BamA mediates the OMP biogenesis in the membranes of live bacteria is less known. There has been research that suggest that OMPs are inserted preferentially in the mid-cell region but other research suggest that OMPs are inserted throughout the cell membrane. Evidence that that OMPs are inserted preferentially mid-cell are from studies that support this theory. Specifically, a research article by Rassam et al. labelled old OMPs red and new OMPs green. This can be seen in figure 7, where the new OMPs in green are clustered around the mid-cell region and absent at the poles whereas the old OMPs in red can be found in the poles. These results suggest that OMP biogenesis occurs in the mid-cell and and migrate to the poles (Rassam et al., 2015). This process is called binary partitioning, where old OMPs are displaced to the poles of growing cells as new OMPs take their place (Rassam et al., 2015).

Figure 7. Growing E. coli JM83 with old OMP labelled ColE9TMR (red) and new OMP labelled ColE9AF488 (green) (Rassam et al., 2015).

However, a recent study has found using super-resolution microscopy studies in fixed cells that BamA was clustered throughout the outer membrane (Gunasinghe et al., 2018). This can be seen in figure 8 below. Because the Bam complex is involved in inserting OMPs into the outer membrane, if they are scattered throughout the membrane, then it would suggest that OMPs are also inserted throughout the membrane. However, this would contrast the results found in the study above by Rassam et al. where they found that OMP biogenesis occurs in the mid-cell region.

Figure 8: Study using direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ revealed BamA clusters across the outer membrane (Gunasinghe et al., 2018).

The WD40 protein BamB mediates BAM-BAM interactions to localize BAM complexes into groups, known as assembly precincts, in the bacterial outer membrane (Gunasinghe et al., 2018) Through in situ direct stochastic optical reconstruction microscopy (dSTORM), single-cell analysis of E. coli showed that these precincts have a nearest neighbour distance of about 200 nm (Gunasinghe et al., 2018). BamB's role in precinct formation is to bind to BamA and BamB of neighbouring complexes (Gunasinghe et al., 2018). Evidence of this binding is found in Figure 9, which was adapted from Figure 4D in the paper by Gunasinghe et al.

Figure 9: Evidence of in situ crosslinking between BamB and BamA.

Kim et al. reported the first crystal structure of BamD in complex with the N-terminal half of BamC. This N-terminal half has a stabilizing effect on BamD (Kim et al., 2011). The BamCD complex is held together by hydrogen bonds and salt bridges (Kim et al., 2011). Furthermore, BamD was found to have a significantly different conformation compared to its monomeric structure, which indicates that BamC may play a role in its activation (Kim et al., 2011).

They concluded that the N-terminal half of BamC interacts with BamD to form the BamCD complex through a series of truncations (Kim et al., 2011). In Figure 10, adapted from Figure 1B from the paper by Kim et al., they found that BamC would only interact with BamD if they either kept BamC in its entirety or left its N-terminal intact (Kim et al., 2011). Other truncations were unable to form a complex (Kim et al., 2011).

Figure 10: Only truncations containing the N-terminal half of BamC could interact with BamD to form a complex.

Figure 11, adapted from Figure 2B in the paper by Kim et al., shows BamC inserting itself along the longitudinal axis of BamD to form a complex. The N-terminal half of BamC interacts directly with all five tetratricopeptide repeats (TPR) motifs of BamD (Kim et al., 2011). When overlapping the BamD in the BamCD complex over BamD in its monomeric form, it can be seen that there is a significant conformational change in BamD (Kim et al., 2011).

Figure 11: BamC interacts directly with BamD's TPR motifs as it inserts itself along BamD's longitudinal axis.

Out of BAM's lipoproteins, BamD is essential to folding and inserting outer membrane proteins (Sandoval et al., 2011). Sandoval et al. studied R. marinus BamD, dubbed rmBamD. Its crystal structure revealed five TPR motifs; three in the N-terminal domain having similarity to proteins that recognize signals, and two in the C-terminal domain for interacting with other Bam components (Sandoval et al., 2011). These Bam components are BamC and BamE, which bind to BamD to help stabilize its complex with BamA, the main functional component of the BAM complex (Sandoval et al., 2011). This conclusion is supported by C-terminal truncations of BamD leading to impaired binding to BamA, C, and E (Sandoval et al., 2011).

Interestingly, BamD has a negatively charged groove between its N- and C-terminal domains that is about 24 Angstroms in diameter (Sandoval et al., 2011). This may be responsible for its interactions with BAM components.

BamE exists in two forms, but the native periplasmic state is the more important one when it comes to BAM function (Knowles et al., 2011). It accumulates in the periplasmic space (Knowles et al., 2011). NMR data showed that BamE binds specifically to phosphatidylglycerol, which can be found on the outer membrane (Knowles et al., 2011). Therefore, its role may be to anchor the complex to lipids (Knowles et al., 2011).

Figure 12, adapted from Figure 3 in the paper by Knowles et al., shows the absence of peaks in NMR data when in the presence of dihexanoyl phosphatidylglycerol (DHPG) to conclude that BamE binds to phosphatidylglycerol.

Figure 12: Left shows NMR data when BamE is in the absence of DHPG, while right shows NMR data when BamE is in the presence of DHPG.

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— Ygonia,Marius 2023/01/19 14:58