Outer Membrane Biogenesis Research Articles

Thanatin and vinyl sulfide analogues as narrow spectrum antimicrobial peptides that synergise with polymyxin B

Thanatin is a β-hairpin antimicrobial peptide cyclised by a single disulfide bond that has shown potent broad-spectrum activity towards bacterial and fungal pathogens. Towards Gram-negative species, thanatin acts both by forming trans-membranal pores and inhibiting outer membrane biogenesis by binding to LptA and blocking lipopolysaccharide (LPS) transport. Inspired by previous modifications of thanatin, an analogue was prepared which demonstrated potent but selective activity towards E. coli. Furthermore, this compound was shown to act in synergy with the highly potent FDA-approved lipopeptide antibiotic polymyxin B, which engages LPS at the cytoplasmic membrane. Four analogues of thanatin in which the disulfide was substituted for vinyl sulfide bridge mimetics were prepared, all of which retained similar secondary structures. Two of these retained substantial potency and selectivity towards E. coli. Importantly, synergy with polymyxin B was also maintained for the lead analogue. The vinyl sulfide potentially offers a facile replacement strategy for labile disulfide bonds and the selective activity and drug synergy of the reported thanatin analogues is promising for the development of narrow spectrum antimicrobials with reduced likelihood of resistance emerging in clinical settings.

Cell envelope diversity and evolution across the bacterial tree of life.

The bacterial cell envelope is a complex multilayered structure conserved across all bacterial phyla. It is categorized into two main types based on the number of membranes surrounding the cell. Monoderm bacteria are enclosed by a single membrane, whereas diderm cells are distinguished by the presence of a second, outer membrane (OM). An ancient divide in the bacterial domain has resulted in two major clades: the Gracilicutes, consisting strictly of diderm phyla; and the Terrabacteria, encompassing monoderm and diderm species with diverse cell envelope architectures. Recent structural and phylogenetic advancements have improved our understanding of the diversity and evolution of the OM across the bacterial tree of life. Here we discuss cell envelope variability within major bacterial phyla and focus on conserved features found in diderm lineages. Characterizing the mechanisms of OM biogenesis and the evolutionary gains and losses of the OM provides insights into the primordial cell and the last universal common ancestor from which all living organisms subsequently evolved.

Outer membrane protein assembly mediated by BAM-SurA complexes

The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.

Terrabacteria: redefining bacterial envelope diversity, biogenesis and evolution.

The bacterial envelope is one of the oldest and most essential cellular components and has been traditionally divided into Gram-positive (monoderm) and Gram-negative (diderm). Recent landmark studies have challenged a major paradigm in microbiology by inferring that the last bacterial common ancestor had a diderm envelope and that the outer membrane (OM) was lost repeatedly in evolution to give rise to monoderms. Intriguingly, OM losses appear to have occurred exclusively in the Terrabacteria, one of the two major clades of bacteria. In this Review, we present current knowledge about the Terrabacteria. We describe their diversity and phylogeny and then highlight the vast phenotypic diversity of the Terrabacteria cell envelopes, which display large deviations from the textbook examples of diderms and monoderms, challenging the classical Gram-positive-Gram-negative divide. We highlight the striking differences in the systems involved in OM biogenesis in Terrabacteria with respect to the classical diderm experimental models and how they provide novel insights into the diversity and biogenesis of the bacterial cell envelope. We also discuss the potential evolutionary steps that might have led to the multiple losses of the OM and speculate on how the very first OM might have emerged before the last bacterial common ancestor.

Leptospira interrogans encodes a canonical BamA and three novel noNterm Omp85 outer membrane protein paralogs

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three ‘noNterm’ Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

Periplasmic Chaperones: Outer Membrane Biogenesis and Envelope Stress.

Envelope biogenesis and homeostasis in gram-negative bacteria are exceptionally intricate processes that require a multitude of periplasmic chaperones to ensure cellular survival. Remarkably, these chaperones perform diverse yet specialized functions entirely in the absence of external energy such as ATP, and as such have evolved sophisticated mechanisms by which their activities are regulated. In this article, we provide an overview of the predominant periplasmic chaperones that enable efficient outer membrane biogenesis and envelope homeostasis in Escherichia coli. We also discuss stress responses that act to combat unfolded protein stress within the cell envelope, highlighting the periplasmic chaperones involved and the mechanisms by which envelope homeostasis is restored.

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The nosocomial pathogen Acinetobacter baumannii is a major threat to human health. The sensor kinase-response regulator system, BfmS-BfmR, is essential to multidrug resistance and virulence in the bacterium and represents a potential antimicrobial target. Important questions remain about how the system controls resistance and pathogenesis. Although BfmR knockout alters expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls the regulator is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro phosphorylation to study the functions of phospho-BfmR. We show that phosphorylation is required for BfmR-mediated gene regulation, antibiotic resistance, and sepsis development in vivo. Consistent with activating the protein, phosphorylation induces dimerization and target DNA affinity. Integrated analysis of genome-wide binding and transcriptional profiles of BfmR led to additional key findings: (1) Phosphorylation dramatically expands the number of genomic sites BfmR binds; (2) DNA recognition involves a direct repeat motif widespread across promoters; (3) BfmR directly regulates 303 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (pilus biogenesis); (4) BfmR controls several non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii disease and disentangle the extensive pathogenic gene-regulatory network under its control.

Determining the functional role of the Gluconobacter oxydans GOX1969 protein as a BamB homolog.

Acetic acid bacteria are used in many industrial processes such as the production of vinegar, vitamin C, the antidiabetic drug miglitol, and various artificial flavorings. These industrially important reactions are primarily carried out by an arsenal of periplasmic-facing membrane-bound dehydrogenases that incompletely oxidize their substrates and shuttle electrons directly into the respiratory chain. Among these dehydrogenases, GOX1969 in Gluconobacter oxydans was predicted to be a pyrroloquinoline quinone-dependent dehydrogenase of unknown function. However, after multiple analysis by a number of labs, no dehydrogenase activity has been detected. Reanalysis of GOX1969 sequence and structure reveals similarities to Escherichia coli BamB, which functions as a subunit of the β-barrel assembly machinery complex that is responsible for the assembly of β-barrel outer membrane proteins in Gram-negative bacteria. To test if the physiological function of GOX1969 is similar to BamB in E. coli, we introduced the gox1969 gene into an E. coli ∆bamB mutant. Growth deficiencies in the ∆bamB mutant were restored when gox1969 was expressed on the plasmid pGox1969. Furthermore, increased membrane permeability conferred by bamB deletion was restored upon gox1969 expression, which suggests a direct link between GOX1969 and a role in maintaining outer membrane stability. Together, this evidence strongly suggests that GOX1969 is functionally acting as a BamB in G. oxydans. As such, functional information on uncharacterized genes will provide new insights that will allow for more accurate modeling of acetic acid bacterial metabolism and further efforts to design rational strains for industrial use.IMPORTANCEGluconobacter oxydans is an industrially important member of the acetic acid bacteria. Experimental characterization of putative genes is necessary to identify targets for further engineering of rational acetic acid bacteria strains that can be used in the production of vitamin C, antidiabetic compounds, artificial flavorings, or novel compounds. In this study, we have identified an undefined dehydrogenase GOX1969 with no known substrate and defined structural similarities to outer membrane biogenesis protein BamB in E. coli K12. Furthermore, we demonstrate that GOX1969 is capable of complementing bamB knockout phenotypes in E. coli K12. Taken together, these findings enhance our understanding of G. oxydans physiology and expand the list of potential targets for future industrial strain design.

SurA Is Required for Outer Membrane Biogenesis and Can Be Used as a New Molecular Target for Plague Therapy

SurA Is Required for Outer Membrane Biogenesis and Can Be Used as a New Molecular Target for Plague Therapy

Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.

Computational Study of Designed Peptide Analogs against Pseudomonas aeruginosa as a Possible Inhibitor of LptD Membrane Protein

Background: Pseudomonas aeruginosa is a common cause of nosocomial infection worldwide and is responsible for 18-61% of deaths alone. The organism has gained resistance to many known antibiotics and evolved into a multidrug-resistant strain. The LptD outer membrane protein of P. aeruginosa is a special target of interest due to its role in outer membrane biogenesis. Objective: The study aimed to gain an insight into how mutations affect the overall properties of antimicrobial peptides and to identify novel peptide analogs against P. aeruginosa. Methods: The peptide analogs were designed and shortlisted based on physicochemical parameters and estimated free energy change in the current study. The docking studies for wild type and shortlisted peptides were performed against LptD protein of P. aeruginosa. The toxicity, allergenicity, and solubility analyses of peptide analogs with high binding affinity to LptD were also conducted. Results: The molecular docking results indicated that peptide analogs 523M26, 523M29, 523M34, and 523M35 for AP00523 (wild type); 608M12, 608M13, 608M19, 608M31, 608M32, 608M39, and 608M43 for AP00608 (wild type); and 2858M25 for AP02858 (wild type) bound effectively with LptD membrane than their wild types. The toxicity, allergenicity, and solubility analyses revealed all these peptide analogs to be nontoxic, non-allergen, and have good water solubility. Conclusion: The binding energies predicted 523M26, 608M39, and 2858M25 bind effectively to LptD proteins than their wild type. Based on docking analysis, it was further predicted that 608M39 has an estimated binding affinity greater than L27-11, which is a known peptidomimetic inhibitor of the LptD protein.

Detergents with Scalable Properties Identify Noncanonical Lipopolysaccharide Binding to Bacterial Inner Membrane Proteins.

Lipopolysaccharide (LPS) is vital for maintaining the outer membrane barrier in Gram-negative bacteria. LPS is also frequently obtained in complex with the inner membrane proteins after detergent purification. The question of whether or not LPS binding to inner membrane proteins not involved in outer membrane biogenesis reflects native lipid environments remains unclear. Here, we leverage the control of the hydrophilic-lipophilic balance and packing parameter concepts to chemically tune detergents that can be used to qualitatively differentiate the degree to which proteins copurify with phospholipids (PLs) and/or LPS. Given the scalable properties of these detergents, we demonstrate a detergent fine-tuning that enables the facile investigation of intact proteins and their complexes with lipids by native mass spectrometry (nMS). We conclude that LPS, a lipid that is believed to be important for outer membranes, can also affect the activity of membrane proteins that are currently not assigned to be involved in outer membrane biogenesis. Our results deliver a scalable detergent chemistry for a streamlined biophysical characterization of protein-lipid interactions, provide a rationale for the high affinity of LPS-protein binding, and identify noncanonical associations between LPS and inner membrane proteins with relevance for membrane biology and antibiotic research.

Structural Insights into the Lipopolysaccharide Transport (Lpt) System as a Novel Antibiotic Target.

Lipopolysaccharide (LPS) is a critical component of the extracellular leaflet within the bacterial outer membrane, forming an effective physical barrier against environmental threats in Gram-negative bacteria. After LPS is synthesized and matured in the bacterial cytoplasm and the inner membrane (IM), LPS is inserted into the outer membrane (OM) through the ATP-driven LPS transport (Lpt) pathway, which is an energy-intensive process. A trans-envelope complex that contains seven Lpt proteins (LptA-LptG) is crucial for extracting LPS from the IM and transporting it across the periplasm to the OM. The last step in LPS transport involves the mediation of the LptDE complex, facilitating the insertion of LPS into the outer leaflet of the OM. As the Lpt system plays an essential role in maintaining the impermeability of the OM via LPS decoration, the interactions between these interconnected subunits, which are meticulously regulated, may be potential targets for the development of new antibiotics to combat multidrug-resistant Gram-negative bacteria. In this review, we aimed to provide an overview of current research concerning the structural interactions within the Lpt system and their implications to clarify the function and regulation of LPS transport in the overall process of OM biogenesis. Additionally, we explored studies on the development of therapeutic inhibitors of LPS transport, the factors that limit success, and future prospects.

Development of a sensor for disulfide bond formation in diverse bacteria.

Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

Outer-Membrane Permeabilization, LPS Transport Inhibition: Activity, Interactions, and Structures of Thanatin Derived Antimicrobial Peptides.

Currently, viable antibiotics available to mitigate infections caused by drug-resistant Gram-negative bacteria are highly limited. Thanatin, a 21-residue-long insect-derived antimicrobial peptide (AMP), is a promising lead molecule for the potential development of novel antibiotics. Thanatin is extremely potent, particularly against the Enterobacter group of Gram-negative pathogens, e.g., E. coli and K. pneumoniae. As a mode of action, cationic thanatin efficiently permeabilizes the LPS-outer membrane and binds to the periplasmic protein LptAm to inhibit outer membrane biogenesis. Here, we have utilized N-terminal truncated 16- and 14-residue peptide fragments of thanatin and investigated structure, activity, and selectivity with correlating modes of action. A designed 16-residue peptide containing D-Lys (dk) named VF16 (V1PIIYCNRRT-dk-KCQRF16) demonstrated killing activity in Gram-negative bacteria. The VF16 peptide did not show any detectable toxicity to the HEK 293T cell line and kidney cell line Hep G2. As a mode of action, VF16 interacted with LPS, permeabilizing the outer membrane and binding to LptAm with high affinity. Atomic-resolution structures of VF16 in complex with LPS revealed cationic and aromatic surfaces involved in outer membrane interactions and permeabilization. Further, analyses of an inactive 14-residue native thanatin peptide (IM14: IIYCNRRTGKCQRM) delineated the requirement of the β-sheet structure in activity and target interactions. Taken together, this work would pave the way for the designing of short analogs of thanatin-based antimicrobials.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

SurA is required for outer membrane biogenesis and can be used as a new molecular target for plague therapy

SurA is required for outer membrane biogenesis and can be used as a new molecular target for plague therapy

Monitoring the Interaction of the Peptidoglycan with the Bacterial β-Barrel Assembly Machinery.

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential β-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

Targeting Proteus mirabilis BAM Complex Proteins for Development of Novel Antibiotics

Urinary tract infections (UTIs) are frequent hospital-acquired infection, with Escherichia coli and Proteus mirabilis accounting for 90% of complicated UTIs. Emergence of multidrug-resistant (MDR) bacteria have complicated the treatment UTIs. P. mirabilis related UTIs has been associated with the production of urinary stones and long-term infections in patients with catheters. P. mirabilis and other uropathogens constitute a largely unexplored pathogen group. The pathogen is resistant to most antibiotics as a result of its impermeable outer membrane (OM). The β-barrel assemble machinery folds and inserts outer membrane proteins; however, there are no antibiotics targeting the OM assemble in clinical use currently. Therefore, this study seeks to identify drugs that will inhibit the activity of P. mirabilis B complex proteins and also determine their effects on P. mirabilis OM biogenesis. This would be achieved by screening approved drugs against the P. mirabilis Bam complex using computer-based in silico screening and cellular-based assays. First, the binding effects of drugs on P. mirabilis B complex proteins will be determined using docking algorithms. The antimicrobial and antivirulence activity of selected drugs from in silico analysis will be screened against MDR P. mirabilis. Finally, the effect of active drug(s) on the OM biogenesis of wild-type P. mirabilis and mutant P. mirabilis will be determined using peptide nucleic acids (PNA). Western lot analysis will be used to determine the abundance of proteins involved in OM biogenesis. Successful completion of this study will lead to the identification of novel antibiotics against MDR P. mirabilis and associated mechanisms while providing the foundation for future research endeavours on other uropathogens.