Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria.

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

PurA is the main target of aurodox, a type III secretion system inhibitor.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer.

Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the genetic level and provide the foundation for future mechanistic studies.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

The Peptide Antibiotic Corramycin Adopts a ß-Hairpin-like Structure and Is Inactivated by the Kinase ComG.

The rapid development of antibiotic resistance, especially among difficult-to-treat Gram-negative bacteria, is recognized as a serious and urgent threat to public health. The detection and characterization of novel resistance mechanisms are essential to better predict the spread and evolution of antibiotic resistance. Corramycin is a novel and modified peptidic antibiotic with activity against several Gram-negative pathogens. We demonstrate that the kinase ComG, part of the corramycin biosynthetic gene cluster, phosphorylates and thereby inactivates corramycin, leading to the resistance of the host. Remarkably, we found that the closest structural homologues of ComG are aminoglycoside phosphotransferases; however, ComG shows no activity toward this class of antibiotics. The crystal structure of ComG in complex with corramycin reveals that corramycin adopts a ß-hairpin-like structure and allowed us to define the changes leading to a switch in substrate from sugar to peptide. Bioinformatic analyses suggest a limited occurrence of ComG-like proteins, which along with the absence of cross-resistance to clinically used drugs positions corramycin as an attractive antibiotic for further development.

Rhizoma Paridis saponins attenuate Gram-negative bacteria-induced inflammatory acne by binding to KEAP1 and modulating Nrf2 and MAPK pathways.

Acne vulgaris represents a chronic inflammatory condition, the pathogenesis of which is closely associated with the altered skin microbiome. Recent studies have implicated a profound role of Gram-negative bacteria in acne development, but there is a lack of antiacne agents targeting these bacteria. Polyphyllins are major components of Rhizoma Paridis with great anti-inflammatory potential. In this study, we aimed to evaluate the antiacne effects and the underlying mechanisms of PPH and a PPH-enriched Rhizoma Paridis extract (RPE) in treating the Gram-negative bacteria-induced acne. PPH and RPE treatments significantly suppressed the mRNA and protein expressions of interleukin (IL)-1ß and IL-6 in lipopolysaccharide (LPS)-induced RAW 264.7 and HaCaT cells, along with the intracellular reactive oxygen species (ROS) generation. Furthermore, PPH and RPE inhibited the nuclear translocation of nuclear factor kappa-B (NF-κB) P65 in LPS-induced RAW 264.7 cells. Based on molecular docking, PPH could bind to kelch-like ECH-associated protein 1 (KEAP1) protein. PPH and RPE treatments could activate nuclear factor erythroid 2-related factor 2 (NRF2) and upregulate haem oxygenase-1 (HO-1). Moreover, RPE suppressed the mitogen-activated protein kinase (MAPK) pathway. Therefore, PPH-enriched RPE showed anti-inflammatory and antioxidative effects in vitro, which is promising for alternative antiacne therapeutic.

The Name Is Barrel, ß-Barrel.

ß-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel ß-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are ß-barrel outer membrane proteins (OMPs). ß-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of ß-barrel OMPs, how they are folded into the membrane, and the future of ß-barrel OMP research and its applications.

Examining Protein Translocation by ß-Barrel Membrane Proteins Using Reconstituted Proteoliposomes.

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.

Stress-Based Screening for Compounds That Inhibit ß-Barrel Outer Membrane Protein Assembly in Gram-Negative Bacteria.

Biogenesis of the outer membrane (OM) of Gram-negative bacteria involves two processes essential for growth, that is, the insertion of ß-barrel outer membrane proteins (OMPs) by the Bam complex and the assembly of the LPS-containing outer leaflet of the OM by the LptD/E complex from the Lpt pathway. These processes have only recently gained attention as targets for antimicrobial drugs. Our laboratory has developed a simple screening tool to identify compounds that target processes that disrupt the biogenesis of the cell envelope, among which the activity of the Bam complex. The tool is based on the observation that such a disruption triggers cell envelope stress response systems, such as the σE, Rcs, and Cpx responses. In essence, specific stress-responsive promoters are fused to a gene encoding a bright fluorescent protein to serve as a panel of easy-to-monitor stress reporter plasmids. Using these plasmids, compounds triggering these stress systems and, therefore, putatively disrupting the biogenesis of the cell envelope can be identified by the nature and kinetics of the induced stress responses. We describe here the use of the stress reporter plasmids in high-throughput phenotypic screening using multi-well plates.

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria.

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions.

Silencing gram-negative bacteria binding protein 1 decreases the immunity of Tribolium castaneum against bacteria.

Gram-negative bacteria binding proteins (GNBPs) have the ability to recognize molecular patterns associated with microbial pathogens (PAMPs), leading to the activation of immune responses downstream. In the genome of Tribolium castaneum, three GNBP genes have been identified; however, their immunological roles remain unexplored. In our study, a GNBP1, designated as TcGNBP1, were identified from the cDNA library of T. castaneum. The coding sequence of TcGNBP1 consisted of 1137 bps and resulted in the synthesis of a protein comprising 378 amino acids. This protein encompasses a signal peptide, a low-complexity region, and a glycoside hydrolase 16 domain. TcGNBP1 was strongly expressed in early adult stages, and mainly distributed in hemolymph and gut. Upon being challenged with Escherichia coli or Staphylococcus aureus, the transcript levels of TcGNBP1 were significantly changed at different time points. Through molecular docking and ELISA analysis, it was observed that TcGNBP1 has the ability to interact with lipopolysaccharides, peptidoglycan, and ß-1, 3-glucan. Based on these findings, it was further discovered that recombinant TcGNBP1 can directly bind to five different bacteria in a Ca2+-dependent manner. After knockdown of TcGNBP1 with RNA interference, expression of antimicrobial peptide genes and prophenoloxidase (proPO) activity were suppressed, the susceptibility of T. castaneum to E. coli or S. aureus infection was enhanced, leading to low survival rate. These results suggest a regulatory mechanism of TcGNBP1 in innate immunity of T. castaneum and provide a potential molecular target for dsRNA-based insect pest management.

Оn the aggregation of polycationic photosensitizer upon binding to Gram-negative bacteria.

Polycationic photosensitizers (PS) are not susceptible to aggregation in solutions, but their high local concentrations in Gram-negative bacteria can be sufficient for aggregation and reduced effectiveness of antibacterial photodynamic treatment. By measuring fluorescence spectra and kinetics we were able to evaluate the degree of aggregation of polycationic PS ZnPcChol8in Gram-negative bacteria E.coliK12 TG1. Binding of ZnPcChol8toE.coliK12 TG1 leads to an appearance of groups of molecules with shorter PS fluorescence lifetime, a decrease in fluorescence intensity and a shift in the fluorescence spectral maximum. However, we evaluated that about 88% of the fluorescing PS molecules in the bacteria were in an unaggregated state, which indicates only a small reduction in the generation of reactive oxygen species.

Gram-negative endolysins: overcoming the outer membrane obstacle.

Our ability to control the growth of Gram-negative bacterial pathogens is challenged by rising antimicrobial resistance and requires new approaches. Endolysins are phage-derived enzymes that degrade peptidoglycan and therefore offer potential as antimicrobial agents. However, the outer membrane (OM) of Gram-negative bacteria impedes the access of externally applied endolysins to peptidoglycan. This review highlights recent advances in the discovery and characterization of natural endolysins that can breach the OM, as well as chemical and engineering approaches that increase antimicrobial efficacy of endolysins against Gram-negative pathogens.

Characterization of a Myeloid Differentiation Factor 2-Derived Peptide that Facilitates THP-1 Macrophage-Mediated Phagocytosis of Gram-Negative Bacteria.

Myeloid differentiation factor 2 (MD2), the TLR4 coreceptor, has been shown to possess opsonic activity and has been implicated in phagocytosis and intracellular killing of Gram-negative bacteria. However, any MD2 protein segment involved in phagocytosis of Gram-negative bacteria is not yet known. A short synthetic MD2 segment, MD54 (amino acid regions 54 to 69), was shown to interact with a Gram-negative bacterial outer membrane component, LPS, earlier. Furthermore, the MD54 peptide induced aggregation of LPS and facilitated its internalization in THP-1 cells. Currently, it has been investigated if MD2-derived MD54 possesses any opsonic property and role in phagocytosis of Gram-negative bacteria. Remarkably, we observed that MD54 facilitated agglutination of Gram-negative bacteria, Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC BAA-427), but not of Gram-positive bacteria, Bacillus subtilis (ATCC 6633) and Staphylococcus aureus (ATCC 25923). The MD54-opsonized Gram-negative bacteria internalized within PMA-treated THP-1 cells and were killed over a longer incubation period. However, both internalization and intracellular killing of the MD54-opsonized Gram-negative bacteria within THP-1 phagocytes were appreciably inhibited in the presence of a phagocytosis inhibitor, cytochalasin D. Furthermore, MD54 facilitated the clearance of Gram-negative bacteria E. coli (ATCC 25922) and P. aeruginosa (ATCC BAA-427) from the infected BALB/c mice whereas an MD54 analog, MMD54, was inactive. Overall, for the first time, the results revealed that a short MD2-derived peptide can specifically agglutinate Gram-negative bacteria, act as an opsonin for these bacteria, and facilitate their phagocytosis by THP-1 phagocytes. The results suggest that the MD54 segment could have a crucial role in MD2-mediated host-pathogen interaction involving the Gram-negative bacteria.

Quorum sensing regulation in Microcystis aeruginosa: Insights into AHL-mediated physiological processes and MC-LR production.

Quorum sensing (QS) is a widespread regulatory mechanism in Gram-negative bacteria, primarily involving the secretion of N-acyl homoserine lactone (AHL) to facilitate population density sensing. However, the existence of QS in blue-green algae, a subset of photoautotrophic Gram-negative bacteria forming high-density communities in water blooms, remains elusive. This study delves into the unexplored realm of QS in Microcystis aeruginosa (M. aeruginosa) by investigating AHL-related regulatory mechanisms and their impact on various physiological processes. Utilizing high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and biosensors, a hitherto unknown long-chain AHL exhibiting a mass-to-charge ratio of 318 was identified in sterile M. aeruginosa cultures. Our investigation focused on discerning correlations between AHL activity fluctuations and key parameters such as microcystin (MC-LR) production, algal density, photosynthesis, buoyancy, and aggregation. Furthermore, the AHL extract was introduced during the logarithmic stage of M. aeruginosa cultures to observe the response in physiological processes. The results revealed that AHL, functioning as an autoinducer (AI), positively influenced algal growth and photosynthesis, as evidenced by the upregulated photosynthetic conversion efficiency of PSI and chlorophyll synthesis gene (psbA). AI also played a crucial role in altering surface characteristics through the synthesis of polysaccharides and proteins in EPS, subsequently promoting cell aggregation. Concomitantly, AI upregulated mcyD, enhancing the synthesis of MC-LR. Notably, our investigation pinpointed the initiation of QS in Microcystis at a density of approximately 1.22 × 10^7 cells/mL. This groundbreaking evidence underscores the regulatory role of AI in governing the physiological processes of growth, aggregation, buoyancy, and MC-LR production by activating pertinent gene expressions. This study significantly expands the understanding of QS in AHL, providing crucial insights into the regulatory networks operating in blue-green algae.

Rapid Biodistribution of Fluorescent Outer-Membrane Vesicles from the Intestine to Distant Organs via the Blood in Mice.

A cell's ability to secrete extracellular vesicles (EVs) for communication is present in all three domains of life. Notably, Gram-negative bacteria produce a specific type of EVs called outer membrane vesicles (OMVs). We previously observed the presence of OMVs in human blood, which could represent a means of communication from the microbiota to the host. Here, in order to investigate the possible translocation of OMVs from the intestine to other organs, the mouse was used as an animal model after OMVs administration. To achieve this, we first optimized the signal of OMVs containing the fluorescent protein miRFP713 associated with the outer membrane anchoring peptide OmpA by adding biliverdin, a fluorescence cofactor, to the cultures. The miRFP713-expressing OMVs produced in E. coli REL606 strain were then characterized according to their diameter and protein composition. Native- and miRFP713-expressing OMVs were found to produce homogenous populations of vesicles. Finally, in vivo and ex vivo fluorescence imaging was used to monitor the distribution of miRFP713-OMVs in mice in various organs whether by intravenous injection or oral gavage. The relative stability of the fluorescence signals up to 3 days post-injection/gavage paves the way to future studies investigating the OMV-based communication established between the different microbiotas and their host.

Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

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 ß-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.

Unveiling the mechanism of bactericidal activity of a cecropin A-fused endolysin LNT113.

Endolysins are lytic enzymes produced by bacteriophages at the end of their lytic cycle and degrade the peptidoglycan layer of the bacterial cell wall. Thus, they have been extensively explored as a promising antibacterial agent to replace or supplement current antibiotics. Gram-negative bacteria, however, are prone to resist exogenous endolysins owing to their protective outer membrane. We previously engineered endolysin EC340, encoded by the Escherichia coli phage PBEC131, by substituting its seven amino acids and fusing an antimicrobial peptide cecropin A at its N-terminus. The engineered endolysin LNT113 exerted superior activity to its intrinsic form. This study investigated how cecropin A fusion facilitated the bactericidal activity of LNT113 toward Gram-negative bacteria. Cecropin A of LNT113 markedly increased the interaction with lipopolysaccharides, while the E. coli defective in the core oligosaccharide was less susceptible to endolysins, implicating the interaction between the core oligosaccharide and endolysins. In fact, E. coli with compromised lipid A construction was more vulnerable to LNT113 treatment, suggesting that the integrity of the lipid A layer was important to resist the internalization of LNT113 across the outer membrane. Cecropin A fusion further accelerated the inner membrane destabilization, thereby enabling LNT113 to deconstruct it promptly. Owing to the increased membrane permeability, LNT113 could inactivate some Gram-positive bacteria as well. This study demonstrates that cecropin A fusion is a feasible method to improve the membrane permeability of endolysins in both Gram-negative and Gram-positive bacteria.

Genetic analysis reveals a robust and hierarchical recruitment of the LolA chaperone to the LolCDE lipoprotein transporter.

The outer membrane (OM) is an essential organelle of Gram-negative bacteria. Lipoproteins are key to building the OM, performing essential functions in several OM assembly machines. Lipoproteins mature in the inner membrane (IM) and are then trafficked to the OM. In Escherichia coli, the LolCDE transporter is needed to extract lipoproteins from the IM to begin trafficking. Lipoproteins are then transferred from LolCDE to the periplasmic chaperone LolA which ferries them to the OM for insertion by LolB. LolA recruitment by LolC is an essential trafficking step. Structural and biochemical studies suggested that two regions (termed Hook and Pad) within a periplasmic loop of LolC worked in tandem to recruit LolA, leading to a bipartite model for recruitment. Here, we genetically examine the LolC periplasmic loop in vivo using E. coli. Our findings challenge the bipartite interaction model. We show that while the Hook is essential for lipoprotein trafficking in vivo, lipoproteins are still efficiently trafficked when the Pad residues are inactivated. We show with AlphaFold2 multimer modeling that Hook:LolA interactions are likely universal among diverse Gram-negative bacteria. Conversely, Pad:LolA interactions vary across phyla. Our in vivo data redefine LolC:LolA recruitment into a hierarchical interaction model. We propose that the Hook is the major player in LolA recruitment, while the Pad plays an ancillary role that is important for efficiency but is ultimately dispensable. Our findings expand the understanding of a fundamental step in essential lipoprotein trafficking and have implications for efforts to develop new antibacterials that target LolCDE.IMPORTANCEResistance to current antibiotics is increasingly common. New antibiotics that target essential processes are needed to expand clinical options. For Gram-negative bacteria, their cell surface-the outer membrane (OM)-is an essential organelle and antibiotic barrier that is an attractive target for new antibacterials. Lipoproteins are key to building the OM. The LolCDE transporter is needed to supply the OM with lipoproteins and has been a focus of recent antibiotic discovery. In vitro evidence recently proposed a two-part interaction of LolC with LolA lipoprotein chaperone (which traffics lipoproteins to the OM) via "Hook" and "Pad" regions. We show that this model does not reflect lipoprotein trafficking in vivo. Only the Hook is essential for lipoprotein trafficking and is remarkably robust to mutational changes. The Pad is non-essential for lipoprotein trafficking but plays an ancillary role, contributing to trafficking efficiency. These insights inform ongoing efforts to drug LolCDE.