ATP disrupts lipid-binding equilibrium to drive retrograde transport critical for bacterial outer membrane asymmetry

The hallmark of the gram-negative bacterial envelope is the presence of the outer membrane (OM). The OM is asymmetric, comprising lipopolysaccharides (LPS) in the outer leaflet and phospholipids (PLs) in the inner leaflet; this critical feature confers permeability barrier function against external insults, including antibiotics. To maintain OM lipid asymmetry, the OmpC-Mla system is believed to remove aberrantly localized PLs from the OM and transport them to the inner membrane (IM). Key to the system in driving lipid trafficking is the MlaFEDB ATP-binding cassette transporter complex in the IM, but mechanistic details, including transport directionality, remain enigmatic. Here, we develop a sensitive point-to-point in vitro lipid transfer assay that allows direct tracking of [¹⁴C]-labeled PLs between the periplasmic chaperone MlaC and MlaFEDB reconstituted into nanodiscs. We reveal that MlaC spontaneously transfers PLs to the IM transporter in an MlaD-dependent manner that can be further enhanced by coupled ATP hydrolysis. In addition, we show that MlaD is important for modulating productive coupling between ATP hydrolysis and such retrograde PL transfer. We further demonstrate that spontaneous PL transfer also occurs from MlaFEDB to MlaC, but such anterograde movement is instead abolished by ATP hydrolysis. Our work uncovers a model where PLs reversibly partition between two lipid-binding sites in MlaC and MlaFEDB, and ATP binding and/or hydrolysis shift this equilibrium to ultimately drive retrograde PL transport by the OmpC-Mla system. These mechanistic insights will inform future efforts toward discovering new antibiotics against gram-negative pathogens.

The cell envelope of gram-negative bacteria is composed of two lipid bilayers. The inner membrane (IM) is a phospholipid (PL) bilayer while the outer membrane (OM) contains both PLs and lipopolysaccharides (LPS). The OM is asymmetric with LPS residing in the outer leaflet and PLs in the inner leaflet; such lipid asymmetry allows the membrane to function as an effective barrier against the entry of toxic substances, including antibiotics (1, 2). Extensive efforts have contributed toward an improved understanding of the molecular mechanisms involved in OM assembly and homeostasis. We now know that the Lol, Bam, and Lpt pathways are responsible for the unidirectional transport and/or assembly of OM lipoproteins, β-barrel proteins, and LPS, respectively (3–5). However, mechanisms for PL transport (6), which occur in both directions for assembly and homeostasis of the OM (7–9), remain largely elusive.The first pathway implicated in bacterial PL transport is the OmpC-Mla system (10, 11). Cells lacking this pathway accumulate PLs in the outer leaflet of the OM, indicating a role in the maintenance of OM lipid asymmetry. The OmpC-Mla system is believed to mediate retrograde PL transport where the periplasmic MlaC chaperone shuttles PLs from the OmpC-MlaA complex in the OM to the MlaFEDB ATP-binding cassette (ABC) transporter in the IM. MlaA is an OM lipoprotein that interacts and works with trimeric osmoporin OmpC (11, 12) but forms a separate hydrophilic channel in the OM for transbilayer PL translocation (12, 13). While MlaA also interacts with OmpF, only removing OmpC, but not OmpF, results in OM lipid asymmetry defects in cells (11); therefore, OmpC is functionally important in the Mla system, but its exact role is unclear. MlaC is found to bind PLs with high affinity in a deep hydrophobic pocket (14–16), presumably enabling extraction of PLs from the OM via OmpC-MlaA. In the IM ABC transporter, MlaE and MlaF constitute the permease and nucleotide binding domains, respectively. MlaD is a substrate-binding protein containing a single transmembrane helix, and its periplasmic MCE domain (17) forms a hexamer with a hydrophobic pore and copurifies with PLs (14, 18). Recent structural studies of the MlaFEDB complex revealed that the MlaE permease domains complement the MlaD hydrophobic pore to form a contiguous cavity for binding and transporting PLs (19–23). MlaB is a cytoplasmic protein containing the STAS domain (24) and has been shown to be essential for the proper assembly and activity of the IM complex (18, 25). How ATP hydrolysis may drive PL movement within the MlaFEDB complex is not known.Apart from the lack of mechanistic details, there is still substantial controversy with regards to the directionality of lipid transport mediated by the OmpC-Mla system. Genetic studies in Escherichia coli and Acinetobacter baumannii are more consistent with the retrograde (OM-to-IM) model (10, 26). In particular, E. coli mla or ompC mutants accumulate outer leaflet PLs in the OM, a phenotype that can be corrected by overexpression of PldA, the OM phospholipase (10, 11). In addition, A. baumannii strains lacking lipooligosaccharides have enhanced growth and restored OM barrier function when the Mla system and PldA are removed, presumably because these cells need enough PLs to maintain the outer leaflet of the OM (26, 27). Nevertheless, these experiments in cells lack evidence that demonstrate the direct transport of PLs. While there was a report suggesting the system in A. baumannii may be involved in anterograde (IM-to-OM) PL transport (28), it has recently been shown that the mla mutant strains used had secondary mutations in them, thus rendering the claim invalid (29). A key experiment that sheds light on transport is when the overexpression of MlaFEDB together with MlaC partially rescued defects in retrograde transport of PLs, observed in E. coli strains lacking the Tol-Pal complex (30). Despite this, however, in vitro reconstitution demonstrated ATP-independent (anterograde) transfer of PLs from the MlaFEDB complex (in liposomes) to MlaC (31). These conflicting data throw into question the biological significance of ATP hydrolysis and argue for the need to provide additional in vitro evidence to truly define the directionality of lipid transport mediated by the OmpC-Mla system.In this study, we utilize [¹⁴C]-labeled lipids to directly track the point-to-point transfer of PLs between MlaC and nanodisc-reconstituted MlaFEDB complex. We show that PLs bound to MlaC can be spontaneously transferred to MlaFEDB in an MlaD-dependent manner. The presence of ATP further enhances such retrograde transfer; we demonstrate that ATP hydrolysis is coupled to PL transport, and this process too is modulated by MlaD. Interestingly, spontaneous PL transfer can also happen from MlaFEDB to MlaC, but ATP hydrolysis prevents this “anterograde” transfer, ultimately driving PL transport in the retrograde direction. Our work establishes that the OmpC-Mla system powers the retrograde transport of PLs via ATP hydrolysis to maintain OM lipid asymmetry.     

Disruption of lipid homeostasis in the Gram-negative cell envelope activates a novel cell death pathway

Gram-negative bacteria balance synthesis of the outer membrane (OM), cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and cell death in stationary phase. Surprisingly, single-cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the cell envelope by depleting the inner membrane of phospholipids. This novel cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative cell envelope.The Gram-negative bacterial cell envelope is a remarkably complex structure with critical functions for cellular growth and viability. It protects the cell from rapidly changing and potentially harmful environments and must do so while also allowing the selective import of nutrients and export of waste (1). Structurally, the Gram-negative cell envelope consists of an inner membrane (IM) and an outer membrane (OM) that delimit an aqueous compartment known as the periplasm (1, 2). Within the periplasmic space is a mesh-like network of peptide-crosslinked glycan chains, known as the peptidoglycan cell wall (1, 3, 4). This structure shapes the cell and provides mechanical resistance to turgor pressure-driven expansion (3). After inoculation into fresh medium, cells use nutrients in the medium to carry out processes essential to growth. Once these nutrients are depleted, cells enter stationary phase, during which they undergo gross morphological and physiological changes and stop growing (5). Throughout these growth phases and during septum formation and cytokinesis, synthesis of the various layers of the cell envelope must remain coordinated.The Escherichia coli OM is an asymmetric bilayer that contains phospholipids (PLs) in the inner leaflet and LPS in the outer leaflet (6). This structure functions as a robust, highly selective permeability barrier that protects the cell from harmful agents such as detergents, bile salts, and antibiotics (1). The effectiveness of the OM can be attributed to the hydrophobicity of and strong lateral interactions between LPS molecules (6); E. coli must properly synthesize and transport LPS to the outer leaflet of the OM to survive (7). Many proteins contribute to LPS biosynthesis and assembly (for a review, see refs. 8 and 9). By contrast with LPS, how lipids are transported to the OM is virtually unknown.When LPS biosynthetic or transport proteins are compromised, PLs are flipped from the inner to the outer leaflet of the OM to accommodate the reduction in LPS abundance (10). In the outer leaflet, it is thought that PLs form rafts (11), creating patches in the membrane that are more susceptible to the influx of hydrophobic, toxic molecules. To prevent damage resulting from surface-exposed PLs in wild-type E. coli cells, several mechanisms destroy or remove these PLs from the outer leaflet. The OM β-barrel protein PagP is a palmitoyltransferase that removes a palmitate from the sn-1 position of a surface-exposed PL and transfers it to lipid A or phosphatidylglycerol (12, 13). Another OM β-barrel phospholipase, PldA, removes both sn-1 and sn-2 palmitate moieties from PLs and lyso-PLs (14).The Mla (maintenance of lipid asymmetry) ABC transport system is a third mechanism for maintaining lipid asymmetry. Mla proteins are present in all compartments of the cell envelope and facilitate retrograde phospholipid transport from the OM back to the IM (15). MlaA is the lipoprotein component that interacts with OmpC in the OM (16) and is thought to remove PLs from the outer leaflet of the OM and shuttle them to MlaC, the soluble periplasmic component. MlaC delivers the PLs to the IM MlaFEDB complex, which is presumed to aid in the reintegration of PLs into the IM. Null mutations in any mla gene increase the permeability of the OM, rendering cells susceptible to detergent by an increase in surface-exposed PLs (15).Here we show that a dominant mutation in mlaA disrupts the lipid balance of the OM by a mechanism that does not require the other mla gene products but does require active PldA. Cells carrying this mutation are sensitized to the transition to stationary phase in medium with low divalent cation concentrations. This transition triggers an unexpected cell-death trajectory in starving cells in which death is correlated with increased OM blebbing at sites of cell division concomitant with decreased cytoplasmic volume and IM surface area, suggesting that lipid flow from the IM to the OM compensates for lipid loss by OM blebs and vesicles. Thus, our data may provide insights into the long-standing question of how lipids are transported to the OM.     

The sacrificial adaptor protein Skp functions to remove stalled substrates from the β-barrel assembly machine

The biogenesis of integral β-barrel outer membrane proteins (OMPs) in gram-negative bacteria requires transport by molecular chaperones across the aqueous periplasmic space. Owing in part to the extensive functional redundancy within the periplasmic chaperone network, specific roles for molecular chaperones in OMP quality control and assembly have remained largely elusive. Here, by deliberately perturbing the OMP assembly process through use of multiple folding-defective substrates, we have identified a role for the periplasmic chaperone Skp in ensuring efficient folding of OMPs by the β-barrel assembly machine (Bam) complex. We find that β-barrel substrates that fail to integrate into the membrane in a timely manner are removed from the Bam complex by Skp, thereby allowing for clearance of stalled Bam–OMP complexes. Following the displacement of OMPs from the assembly machinery, Skp subsequently serves as a sacrificial adaptor protein to directly facilitate the degradation of defective OMP substrates by the periplasmic protease DegP. We conclude that Skp acts to ensure efficient β-barrel folding by directly mediating the displacement and degradation of assembly-compromised OMP substrates from the Bam complex.

The cell envelopes of gram-negative bacteria, mitochondria, and chloroplasts all contain an outer membrane (OM) consisting of integral transmembrane proteins that assume a β-barrel conformation (1, 2). In gram-negative bacteria such as Escherichia coli, β-barrel outer membrane proteins (OMPs) contribute to the selective permeability of the OM, protecting the cell from harmful molecules while still allowing for the uptake of nutrients (3). Structurally and functionally diverse OMPs serve a number of roles critical to cell viability, namely the selective passage of small molecules, efflux of toxins, insertion of lipopolysaccharide (LPS) onto the cell surface, and assembly of OMPs themselves (1, 4). Reflective of their importance in maintaining cellular integrity, defects in OMP biogenesis confer sensitivity to a wide array of toxic molecules including detergents, bile salts, and most importantly, antibiotics (5, 6). As such, considerable efforts have been made to identify agents that inhibit essential cellular processes performed by OMPs (7–12), with hopes of hastening the development of novel therapeutics to combat the ever-growing threat of antibiotic-resistant infections caused by gram-negative microbes (13, 14).Ensuring efficient OMP biogenesis is a particularly challenging cellular feat. Newly synthesized OMPs must traverse the aqueous, oxidizing periplasm in an unfolded state, avoid self-aggregation, and subsequently complete proper assembly, all in an environment devoid of cellular energy such as adenosine triphosphate (15). A multitude of molecular chaperones and proteases function to overcome this challenge by minimizing unfolded OMP accumulation and facilitating OMP transport to the OM assembly machinery (16). Although more than a dozen chaperones and proteases with clear implications in OMP biogenesis have been identified (16–18), the most well-characterized and predominant proteins in E. coli are the chaperones SurA and Skp, as well as the chaperone protease DegP. Numerous genetic, biochemical, and proteomic studies have demonstrated that SurA is the primary periplasmic chaperone that facilitates transport of the bulk mass of OMP substrates to the OM (19–24). Skp and DegP, on the other hand, comprise a secondary, partially redundant OMP biogenesis pathway that primarily serves to minimize accumulation of unfolded OMPs, either by rescuing their assembly or promoting their degradation (19, 20).Notably, Skp binds unfolded OMPs with dissociation constants in the low nanomolar range (25, 26), exceeding the binding affinities of either SurA or DegP (27–29), to form highly stable Skp–OMP complexes that display lifetimes on the order of hours (30). Given the substantial stability of Skp–OMP complexes, the precise mechanism of OMP release from Skp remains poorly understood. The rapid conformational dynamics of OMPs bound within the Skp cavity have been proposed to enable local substrate release that is ultimately driven by the recognition and folding of OMPs by the OM assembly machinery (30), thus coupling client release from Skp to the thermodynamic stability provided by OMP integration into a membrane (31). Indeed, substrate release and folding of OMPs from Skp–OMP complexes is enabled in vitro by incubation with OM folding machinery–containing liposomes (28, 32), demonstrating that Skp can facilitate productive OMP assembly. This mechanism of folding-driven substrate release has been similarly observed in genetic and biochemical studies indicating that Skp is capable of directly inserting OMPs into lipid bilayers in vitro (33), as well as the inner membrane in vivo (34), without assistance from the OM assembly machinery.Whether OMPs are capable of being removed from Skp within physiological timescales in the absence of coupled folding, however, is not entirely clear. Under conditions of periplasmic stress, in which the burden of unfolded OMPs exceeds the rate at which they can be assembled, the activities of both Skp and DegP become crucial (19, 20, 24, 35). Given that Skp not only binds substrates with a higher affinity than DegP (29) but also does so several orders of magnitude faster (36), how unfolded OMPs are transferred from Skp to DegP for degradation under stress conditions is not obvious. Indeed, direct transfer of an OMP from Skp to DegP has yet to be demonstrated, and intriguingly, the formation of Skp–DegP–OMP ternary complexes has been reported in such experiments (29, 36).Folding and insertion of nascent OMPs into the OM is catalyzed by the heteropentameric β-barrel assembly machine (Bam) complex, consisting of the BamA β-barrel and four accessory lipoproteins, BamBCDE (37, 38). Recent biochemical and structural studies have provided a relatively clear current model for the mechanism of β-barrel assembly. Following substrate recruitment to BamD (39), BamA catalyzes the sequential addition of β-hairpins in a C-to-N-terminal manner (40), with early folding occurring within the interior of the BamA barrel (41). Folding proceeds until membrane integration occurs, and subsequent stepwise hydrogen-bond formation between N and C substrate termini facilitates barrel closure and substrate release into the membrane (40).One outstanding question concerns the fate of OMP substrates that have stalled while folding on the Bam complex. Protein misfolding in the periplasm, translational error, or impaired Bam complex function can result in substrates arresting on the assembly machinery, a condition that can ultimately be lethal if left unchecked (42–44). Until recently, investigations of stalled OMP substrates have been largely impeded by a lack of structurally defined folding intermediates and the absence of an established general mechanism of OMP assembly. Several studies to date have utilized mutant alleles of the large β-barrel LptD to probe Bam complex assembly (39, 41, 45, 46), and multiple proteases that degrade assembly-compromised LptD within distinct stages of its folding regime have been identified (46, 47). It is unclear, however, whether these stringent quality control mechanisms monitoring assembly of LptD are exerted on all β-barrel substrates or whether LptD represents a unique case given its remarkably complex folding trajectory (48). Given that OMP assembly by the Bam complex has evolved to be incredibly efficient—so efficient that unfolded OMPs cannot be detected at steady state—it stands to reason that quality control mechanisms ensuring the efficient assembly of all β-barrel substrates exist. Recently, it has been shown that extracellular loop deletions within the C-terminal half of the BamA β-barrel cause early folding defects and thus render stalled BamA susceptible to proteolysis by DegP (40). How DegP actively disengages a partially folded, stalled substrate from its folding on BamA, given the relatively weak and slow nature of DegP binding, is not obvious.Here, we have utilized an assembly-defective variant of a slow-folding β-barrel OMP to investigate the fate of substrates that engage the OM assembly machinery but otherwise fail to undergo efficient folding and membrane integration. We identify a specific role for the periplasmic chaperone Skp in facilitating the degradation of defective OMP substrates by the protease DegP, thus imposing an active quality control mechanism that serves to remove assembly-compromised substrates from the Bam complex. Strikingly, we find that Skp is degraded alongside its bound substrate by DegP, thereby functioning as a sacrificial adaptor protein. By evaluating the requirement for Skp in degradation of a series of sequentially stalled β-barrel substrates, we find that Skp is only required to degrade substrates that have initiated folding on the Bam complex. Thus, β-barrel OMPs that have stalled during assembly specifically require Skp for their removal from the Bam complex and subsequent degradation by DegP. We conclude that Skp acts to ensure efficient β-barrel assembly by facilitating both the direct removal and degradation of stalled substrates from the Bam complex.     

High-throughput suppressor screen demonstrates that RcsF monitors outer membrane integrity and not Bam complex function

The regulator of capsule synthesis (Rcs) is a complex signaling cascade that monitors gram-negative cell envelope integrity. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. Elucidating whether RcsF/Bam or RcsF/OMP interactions are important for its sensing function is challenging because the Bam complex is essential, and partial loss-of-function mutations broadly compromise the OM biogenesis. Our recent discovery that, in the absence of nonessential component BamE, RcsF inhibits function of the central component BamA provided a genetic tool to select mutations that specifically prevent RcsF/BamA interactions. We employed a high-throughput suppressor screen to isolate a collection of such rcsF and bamA mutants and characterized their impact on RcsF/OMP assembly and Rcs signaling. Using these mutants and BamA inhibitors MRL-494 and darobactin, we provide multiple lines of evidence against the model in which RcsF senses Bam complex function. We show that Rcs activation in bam mutants results from secondary OM and lipopolysaccharide defects and that RcsF/OMP assembly is required for this activation, supporting an active role of RcsF/OMP complexes in sensing OM stress.

The bacterial cell envelope is an essential structure, acting as a first line of defense against environmental assault. The gram-negative cell envelope is complex, consisting of an inner (IM) and outer (OM) membrane that encloses the cell wall in an aqueous periplasmic space (1). The OM is asymmetric, with phospholipids and lipopolysaccharides (LPS) in the inner and outer leaflets, respectively. The cation cross-bridged LPS molecules confer extreme resistance to detergents and many antibiotics (2).The regulator of capsule synthesis (Rcs) signaling cascade is one of several envelope stress responses that monitor envelope integrity and biogenesis (Fig. 1) (3). Rcs involves at least six proteins spanning all cellular compartments from the cell surface to the cytoplasm. At the core of this pathway is the RcsCDB Histidine-Aspartate phosphorelay complex consisting of the IM hybrid histidine protein kinase RcsC, the IM phosphotransferase protein RcsD, and the cytoplasmic DNA-binding response regulator RcsB (4–6). The activity of this Rcs phosphorelay is regulated by interactions with two upstream components, IgaA and RcsF. IgaA is a polytopic IM protein with a large periplasmic domain, and it inhibits the phosphorelay through RcsD (7, 8). The OM lipoprotein RcsF is a sensory component of the Rcs cascade, which activates downstream signaling in response to stress by releasing IgaA inhibition (8–12). However, sensing by RcsF and signal transduction to IgaA are poorly understood at a molecular level, in part because many distinct genetic and chemical stimuli can induce Rcs, including defects in lipoprotein biogenesis (13–15), cell wall biogenesis (12, 16–19), and the defects of LPS at the cell surface (as a result of Polymyxin B [PMB] treatment, for example) (19–22).Open in a separate windowFig. 1.Proposed mechanistic models for the Rcs stress response. Rcs components (orange) are shown in the context of the envelope structure and biogenesis pathways. The sensory lipoprotein RcsF and the negative regulator IgaA are central to the regulation of RcsCDB phosphorelay. RcsF is exported to the OM by the Lol pathway; the Bam complex assembles RcsF with partner OMPs, leading to a partially surface-exposed topology. Red arrows represent proposed signaling events in response to stress (red stars) that are not yet fully understood. (A) Proposed model for the OM/LPS sensing by RcsF. Cell surface localization of RcsF NTD enables RcsF to monitor the integrity of the outer leaflet. Upon LPS stress (e.g., PMB treatment), the signal is transduced to the periplasmic CTD through the conformational change in the RcsF/OMP complex stimulating downstream signaling. (B) Proposed model for the Bam complex sensing function of RcsF. Envelope stress by an unknown mechanism inhibits the Bam complex function; as a result, RcsF/BamA interaction is prevented, and RcsF is accumulated in the periplasmic-facing orientation stimulating downstream signaling.At the OM, RcsF forms a complex with β-barrel OM proteins (OMPs) such as OmpA, OmpC, and OmpF, adopting a transmembrane orientation in which RcsF is partially surface exposed (12, 23). The β-barrel assembly machinery (Bam complex) that assembles all OMPs also assembles RcsF/OMP complexes, and RcsF interacts with its central and essential component, BamA (12, 23).Defective lipoprotein biogenesis results in the retention of RcsF at the IM, promoting physical association with IgaA and the constitutive activation of signaling (12, 13). Two hypotheses have been proposed to explain how RcsF signals from the OM (Fig. 1 A and B): the first suggests that the surface-exposed N-terminal domain (NTD) of RcsF in an RcsF/OMP complex monitors the integrity of LPS at the outer leaflet, transmitting the signal to the periplasmic carboxyl-terminal domain (CTD) to induce downstream signaling (19, 23) (Fig. 1A); the second argues that the RcsF/OMP complex plays no active role in signal transduction, with stress signals altering the RcsF/BamA interaction to retain RcsF in a periplasm-facing orientation, allowing downstream signaling (12) (Fig. 1B). This altering of the RcsF/BamA interaction is thought to allow RcsF to monitor Bam complex activity (12). Testing these hypotheses has proven to be challenging, as the Bam complex is essential, and there was no clear path to identifying point mutations that specifically disrupt RcsF/OMP or RcsF/BamA interactions without compromising OMP biogenesis and OM integrity.The Bam complex consists of five components, A through E (24): BamA is a β-barrel with five periplasmic Potra domains that scaffold four regulatory lipoproteins, BamB through E. An essential lipoprotein, BamD, recruits OMP substrates to the Bam complex and activates BamA for OMP folding and insertion into the OM (25–29). Coordination of BamA and BamD activities is essential for the OMP assembly and is mediated by their direct interaction at the Potra 5 interface, for which the salt bridge between BamA E373 and BamD R197 is critically important (29, 30). Previously, we reported that the loss of the nonessential Bam component BamE results in a significant decrease in RcsF/OMP assembly (SI Appendix, Fig. S1) (19). The ΔbamE strain and an assembly-defective rcsF^A55Y mutant strain (SI Appendix, Table S1) are both significantly deficient in the detection of PMB-induced LPS stress, providing the first evidence to support an active role for RcsF/OMP in signaling under conditions of LPS stress (19). In the absence of BamE, BamA binds RcsF but is unable to engage with BamD to complete RcsF/OMP assembly (31) (SI Appendix, Fig. S1). As a result, RcsF accumulates on BamA, preventing it from functioning in OMP assembly (31, 32). This RcsF-dependent “jamming” of BamA is the reason for the synthetic lethal interaction of ΔbamE and various bam mutants, including a bamB null (SI Appendix, Table S1) (31, 32).We exploited the lethal interaction between BamA and RcsF in the bamE bamB double mutant to select for mutations that disrupt this RcsF/BamA interaction. Our characterization of the effects of the rcsF and bamA suppressor mutations identified on Rcs signaling and RcsF/OMP assembly demonstrates that assembly of the RcsF/OMP complex is required for Rcs signaling and argues against the model that proposes that RcsF monitors BamA activity. Moreover, our data suggest that the recently published RcsF/BamA structure corresponds to the "jammed" RcsF-BamA complex and not an assembly intermediate, as suggested (33).     

Outer membrane β-barrel protein folding is physically controlled by periplasmic lipid head groups and BamA

Outer membrane β-barrel proteins (OMPs) are crucial for numerous cellular processes in prokaryotes and eukaryotes. Despite extensive studies on OMP biogenesis, it is unclear why OMPs require assembly machineries to fold into their native outer membranes, as they are capable of folding quickly and efficiently through an intrinsic folding pathway in vitro. By investigating the folding of several bacterial OMPs using membranes with naturally occurring Escherichia coli lipids, we show that phosphoethanolamine and phosphoglycerol head groups impose a kinetic barrier to OMP folding. The kinetic retardation of OMP folding places a strong negative pressure against spontaneous incorporation of OMPs into inner bacterial membranes, which would dissipate the proton motive force and undoubtedly kill bacteria. We further show that prefolded β-barrel assembly machinery subunit A (BamA), the evolutionarily conserved, central subunit of the BAM complex, accelerates OMP folding by lowering the kinetic barrier imposed by phosphoethanolamine head groups. Our results suggest that OMP assembly machineries are required in vivo to enable physical control over the spontaneously occurring OMP folding reaction in the periplasm. Mechanistic studies further allowed us to derive a model for BamA function, which explains how OMP assembly can be conserved between prokaryotes and eukaryotes.Outer membrane β-barrel proteins (OMPs) are found in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts (1). The functions of OMPs are versatile and often essential as they include transport of metabolites and toxins as well as membrane biogenesis (2). Alterations of outer membranes and outer membrane proteins can lead to the development of antibiotic-resistance in pathogenic bacteria, and dysfunction of OMPs in outer membranes of mitochondria plays a role in diabetes and neurodegenerative diseases, among other life-threatening illnesses (3–7). How OMPs attain their native fold in their natural lipid environment is therefore a fundamental question in biological and biomedical research.The biological assembly of outer membrane proteins into bacterial outer membranes requires a functionally conserved protein complex, termed β-barrel assembly machinery (BAM) (8, 9). Previous work suggested that the main subunit of the BAM complex, the OMP BamA, carries out its essential function by providing a structural basis for OMP folding (10–12). However, it has been shown many times that OMPs are capable of spontaneously folding to their native state in model membranes in vitro through an intrinsic folding pathway in the absence of BamA (13–17). Neither the folding in vivo nor in vitro requires an external energy source such as ATP or a redox potential (18, 19).The observation that OMPs can fold to their native states in vitro raises the important question of why OMPs require assembly machineries such as BAM to fold into their cellular outer membranes. To address this question, we developed an experimental strategy that enabled us to monitor the folding kinetics of bacterial OMPs in the absence and presence of prefolded BamA under membrane conditions that mimicked the periplasmic lipid environment. We discovered that native lipid head groups impose a kinetic barrier to folding that is relieved by the catalytic action of BamA. Our findings explain many in vivo observations and allowed us to derive a biophysical model of OMP sorting to the correct cellular membrane followed by its folding into bacterial outer membranes.     

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy–entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Molecular chaperones are key cellular components that play fundamental roles in maintaining cellular proteostasis (1, 2). Essential activities of chaperones include the assistance of de novo protein folding, the stabilization of nonnative proteins in folding competent or unfolded states, and the rescue of misfolded and aggregated proteins (3–5). Chaperones are an integral part of a wide range of protein quality-control systems, and their activities are intimately coupled to the biogenesis networks that aid the structural and functional maturation of proteins from their site of cellular synthesis to their target cellular compartments.One network where chaperone activity is of particular relevance is the biogenesis of outer membrane proteins (OMPs) (6, 7). OMPs are a diverse group of β-barrel membrane proteins found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. They fulfill a plethora of functions in cell signaling, metabolism, and transport (8–10); are indispensable to the survival of bacteria (10, 11); and constitute important virulence factors and drug targets (12–14). The OMP biosynthesis pathway is highly complex and conserved across all kingdoms of life (15) and involves the coordinated action of a multicomponent protein machinery that aids in overcoming the many hurdles that these proteins have to surmount on their way to their target outer membrane (4, 7, 16).In Gram-negative bacteria, OMPs are translated in the cytoplasm, from where they are translocated across the inner bacterial membrane via the Sec machinery to the periplasmic space (17, 5). Within this aqueous compartment, OMPs are escorted to the outer membrane in an unfolded state (denoted as the uOMP state) with the aid of various chaperones that maintain the largely insoluble and aggregation-prone uOMP polypeptide chains in a protected, partially unfolded state (18, 19). At the outer membrane, uOMPs are transferred to the β-barrel assembly machinery (BAM), which facilitates their native folding and insertion into the membrane (20). Noteworthy, the periplasm is devoid of any known source of energy-providing molecules, such as adenosine triphosphate (ATP); hence, all chaperones as well as the entire folding machinery likely operate without the aid of external energy, following thermodynamic principles (21).Two chaperones which have been shown to be indispensable for the biogenesis of bacterial OMPs are the 17-kilodalton protein (Skp) (22) and survival factor A (SurA) (23). Skp and SurA, both located in the periplasm, exhibit antifolding activity (also known as holdase activity), whereby they sequester uOMP substrates to prevent aggregation until they reach the bacterial outer membrane (24–26). Their interaction with uOMPs is thermodynamically modulated due to the lack of energy-carrying molecules in the periplasm (27–29). Depletion studies of periplasmic chaperones identified SurA as an essential chaperone for OMP biogenesis, leading to a drastic decrease in OMP density in the outer membrane due to the loss of SurA (30). Skp depletion, on the other hand, led to an accumulation of misfolded OMPs and the activation of the cellular stress response (30), while OMP density in the outer membrane remained the same. Interestingly, recent studies suggest substrate selectivity among the two chaperones (31). Hence, it is of importance to understand the functional mechanisms underlying both Skp and SurA association with uOMPs.Structural studies of the eight β-stranded protein outer membrane protein X (OmpX) in the presence of Skp using NMR spectroscopy have found that unfolded OmpX (uOmpX) shows submillisecond backbone dynamics (32) in complex with Skp. For binding of SurA to unfolded outer membrane protein A (uOmpA), both fluid globular (32) and expanded states (31) have been proposed. Recent studies have located various interaction sites of SurA and uOmpX using cross-linking, suggesting that SurA-bound uOmpX populates multiple conformations (31, 33). Yet, long-range polypeptide chain dynamics and conformational heterogeneity of unfolded OMPs upon binding to chaperones remain elusive. In particular, it is unknown how SurA- and Skp-bound OMP dynamics and heterogeneities differ, given their differential roles in regulating protein folding in the periplasmic space. Dynamic aspects are hypothesized to be important for chaperone–uOMP interactions, particularly to fine-tune energetics of the binding reaction through a reduction of the entropic costs upon binding (28, 29, 34–37). Yet, the enthalpic and entropic changes that determine Skp–OMP or SurA–OMP interactions and affect the conformations of the denatured substrate proteins are largely undefined.In addition to the well-described holdase activities of Skp and SurA that protect OMPs from misfolding or aggregating, a recent study has suggested that Skp disaggregates oligomeric uOMP structures (38). While SurA has not been directly implicated as a disaggregase, modeling studies propose a synergistic interaction among these chaperones especially under conditions of stress (18, 39), thus raising the question of the role that both chaperones played in disassembling OMP aggregates.To gain insights into the multifaceted functionalities of Skp and SurA and their action mechanisms, we study here the conformational dynamics and thermodynamics of the eight β-stranded protein OmpX in the presence of the chaperones and explore their disaggregation activities. Using single-molecule Förster resonance energy transfer (smFRET), we resolve the heterogeneities, structural dynamics, and thermodynamics underlying the different states of uOmpX at near-native conditions. Strikingly, we find that both chaperones expand the unfolded polypeptide chain upon binding. The degree of expansion is concentration dependent for SurA, but not for Skp. Probing structural changes and chaperone interaction at different temperatures, we gain insights into the enthalpic and entropic contributions of complex formation and find that the interaction modes of both chaperones differ strongly and are dictated by entropy–enthalpy compensation. Finally, we use fluorescence correlation spectroscopy (FCS) to probe the disaggregation capabilities of Skp and SurA and find synergistic activity of both chaperones in the disassembly reaction of oligomeric OmpX aggregates. Our findings provide fundamental insights into the structural and energetic mechanisms underlying Skp and SurA chaperone–OMP interactions and their role in OMP biogenesis.     

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.The outer membrane of gram-negative bacteria is decorated with a potent endotoxin (called lipid A), which plays a significant role in bacterial pathogenicity and immune evasion (1). It also acts as a physical barrier protecting the cell from chemical attack and represents a significant obstacle for the effective delivery of numerous antimicrobial agents (2, 3). The outer membrane is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet (4). Phospholipids consist of a glycerol molecule, a phosphate group, and two fatty acid moieties (except for cardiolipins) (5) (see reviews (5, 6) and SI Appendix for the biosynthesis and regulation of phospholipids). LPS, on the other hand, contains three distinct components: lipid A, core oligosaccharides, and O-antigen (7, 8). Lipid A is the sole essential component of LPS, and its biosynthesis involves nine enzyme-catalyzed reactions (8). The lipid A pathway has been widely investigated, and we recently produced a pathway model that incorporates all of the known regulatory mechanisms (9). Briefly, the first reaction step catalyzed by LpxA is highly unfavorable, which makes the proceeding enzyme, LpxC, the first committed enzyme (10). LpxC is regulated by the protease FtsH (11, 12), and we recently postulated that the negative feedback signal arises from lipid A disaccharide, the substrate for LpxK (9). Furthermore, FtsH regulates WaaA (formerly called KdtA), an enzyme downstream of LpxC (13). The exact rationale for WaaA regulation remains unknown.A wealth of research exists for either LPS or phospholipids biosynthesis; however, our current understanding on the crosstalk between both pathways is limited at the moment. Because both pathways are synchronized to ensure a proper balance of membrane components (11, 14), studies underpinning the underlying mechanisms would appear valuable. There are a number of experimental findings that indicate the existence of strong links between both biosynthetic pathways (11, 15, 16). Thus, in the context of outer membrane biogenesis, the role involving phospholipids cannot be ignored in the study of LPS regulation. Furthermore, during membrane synthesis, ∼20 million molecules of fatty acids are synthesized in Escherichia coli (8). Yu et al. (17) reconstituted an in vitro steady-state kinetic system of fatty acid biosynthesis using purified enzymes and observed that the maximum fatty acid production rate obtainable was 100 µM/min. This production rate falls far below the amount of fatty acids required by a cell in vivo [if one assumes a cell volume of 6.7 × 10⁻¹⁶ L (18) and a generation time of 30 min (19)]. Therefore, to test the consistency of reported in vitro parameters and investigate the role of the biosynthetic enzymes on fatty acids turnover rate, a “systems” approach is necessary. Similarly, ever since the regulation of WaaA by FtsH was first reported (13), no study has investigated the underlying regulatory mechanism to date. This would also appear important because under wild-type conditions, WaaA catalyzes a step that is required for the endotoxic activity of lipid A (20).In this work, we present a detailed picture of the crosstalk between the LPS and phospholipids biosynthetic machinery. Our work involves a computational kinetic model spanning 81 chemical reactions and involving 90 chemical species. Additionally, we used a series of E. coli fatty acid biosynthesis mutants to investigate the effect of substrate flux into the saturated and unsaturated fatty acid pathway on LpxC stability. Our complete model agrees qualitatively with published datasets and with our own experiments. Our results imply that the catalytic activation of LpxK is dependent on unsaturated fatty acids. Furthermore, our experimental investigations have implicated a secondary protease involved in LpxC regulation. Finally, we have provided experimental evidence to explain the rationale for WaaA regulation.     

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Antimicrobial resistance is a major worldwide threat to human health. The World Health Organization has classified Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii as the most concerning pathogens (“critical priority”) (1). All three pathogens are Gram-negative bacteria with the characteristic inner and outer membranes. The outer membrane is a stringent permeability barrier that restricts the entry of most molecules and therefore presents a major challenge for the development of urgently needed novel antibiotics (2–5).The outer membrane consists of an asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet and various outer-membrane proteins that are embedded in, or attached to, the lipid bilayer. LPS contains negatively charged phosphate and carboxylate groups that are cross-linked by divalent Mg²⁺ and Ca²⁺ cations, resulting in stable clusters of LPS molecules that reduce the permeation of small molecules by 10- to 100-fold compared to phospholipid bilayers (6). Some outer membrane proteins form water-filled channels (so-called porins) that facilitate translocation of molecules through the outer membrane (4, 5). Enterobacteriaceae have general “unspecific” porins that permit the entry of molecules with a size of up to 600 Da. By contrast, P. aeruginosa and A. baumannii have a large set of “specific” porins that permit the entry of only few molecules with sizes below 200 Da. In addition, all three pathogens have porins with mainly structural roles in stabilizing the link between outer membrane and the underlying peptidoglycan layer (OmpA and OprF). It has been proposed that a small fraction of these structural porin molecules form large unspecific pores that permit entry of larger molecules at low rates (7), but this model remains controversial.Antimicrobials and nutrients can penetrate the outer membrane by two different pathways, through the lipid bilayer or through porins. Hydrophobic molecules might predominantly use the lipid pathway, while hydrophilic molecules might prefer porins. However, the quantitative relevance of each pathway for outer-membrane permeability remains unknown (3, 8, 9). Even slow permeation pathways that mediate concentration-equilibration times in the order of minutes (instead of seconds) can yield relevant intracellular drug concentrations in bacteria with generation times of more than 20 min, unless drug-efflux pumps and/or hydrolases diminish drug levels (2).Translocation pathways and their selectivity for specific physicochemical properties of molecules are crucial for the rational improvement of drug entry into Gram-negative bacteria. The important contribution of large cation-selective porins such as OmpF and OmpC for outer-membrane translocation into Enterobacteriaceae enabled the establishment of rules for medicinal chemistry to improve whole-cell activities of antimicrobials against these bacteria (10–12). These porins have been extensively studied, and in particular OmpF has a major impact on susceptibility to various β-lactam antibiotics (13). However, an Escherichia coli ΔompC ΔompF double mutant retains substantial susceptibility to diverse other antibiotics (9), suggesting alternative translocation pathways.For P. aeruginosa, physicochemical parameters favoring translocation have been more difficult to identify (10, 14, 15). Both P. aeruginosa and A. baumannii have lower outer-membrane permeability than Enterobacteriaceae for hydrophilic molecules because they lack unspecific porins (16), making antimicrobial development particularly difficult for these critical pathogens. Specific porins might facilitate antibiotic entry into P. aeruginosa (17), but clear evidence for standard assay conditions is only available for penetration of carbapenems through OprD (18). Functional studies of individual porins in P. aeruginosa are hampered by the large diversity of specific porins that are thought to each enable uptake of a few nutrients (19). Phenotypes of inactivating one particular porin might be masked by the numerous remaining other porins. To circumvent these issues, individual porins have been purified and reconstituted in artificial membranes, or expressed in E. coli, to determine their substrate specificity. However, the results might not reflect porin functions in their native context because their channel properties differ depending on the lipid environment (20, 21).In this study, we overcame these difficulties using extensive mutagenesis. In contrast to previous assumptions, we show that wild-type P. aeruginosa PA14 and a PA14 Δ40 mutant that lacks all identifiable 40 porin genes have indistinguishable susceptibility to diverse antibiotics. Moreover, the Δ40 strain grew normally on rich media, and nutrient consumption assays revealed substantial porin-independent uptake of diverse hydrophilic nutrients. Bringing back individual porins accelerated uptake of some neutral/zwitterionic molecules and was essential for efficient consumption of negatively charged carboxylate-containing compounds. Instead of narrow substrate specificity, porins actually had broad overlapping substrate selectivity. These results demonstrate an unexpected but efficient porin-independent translocation pathway through the outer-membrane lipid bilayer for diverse hydrophilic compounds and all antipseudomonal antibiotics. A detailed understanding of this pathway will facilitate the development of novel antibiotics.     

A multidomain connector links the outer membrane and cell wall in phylogenetically deep-branching bacteria

Deinococcus radiodurans is a phylogenetically deep-branching extremophilic bacterium that is remarkably tolerant to numerous environmental stresses, including large doses of ultraviolet (UV) radiation and extreme temperatures. It can even survive in outer space for several years. This endurance of D. radiodurans has been partly ascribed to its atypical cell envelope comprising an inner membrane, a large periplasmic space with a thick peptidoglycan (PG) layer, and an outer membrane (OM) covered by a surface layer (S-layer). Despite intense research, molecular principles governing envelope organization and OM stabilization are unclear in D. radiodurans and related bacteria. Here, we report a electron cryomicroscopy (cryo-EM) structure of the abundant D. radiodurans OM protein SlpA, showing how its C-terminal segment forms homotrimers of 30-stranded β-barrels in the OM, whereas its N-terminal segment forms long, homotrimeric coiled coils linking the OM to the PG layer via S-layer homology (SLH) domains. Furthermore, using protein structure prediction and sequence-based bioinformatic analysis, we show that SlpA-like putative OM–PG connector proteins are widespread in phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures with fluorescence and electron microscopy of cell envelopes of wild-type and mutant bacterial strains, we report a model for the cell surface of D. radiodurans. Our results will have important implications for understanding the cell surface organization and hyperstability of D. radiodurans and related bacteria and the evolutionary transition between Gram-negative and Gram-positive bacteria.

Deinococcus radiodurans is an evolutionarily deep-branching bacterium with several distinctive characteristics (1). It is remarkably tolerant to large doses of ionizing and ultraviolet (UV) radiation, extreme temperatures, osmotic pressure, oxidative stress, and desiccation, primarily owing to its extensive DNA repair system (2), complex cell envelope (3), and antioxidation systems, such as the one involving the carotenoid deinoxanthin (4, 5). In fact, D. radiodurans can even survive for years in outer space (6). Due to its ability to survive under extreme environmental conditions and its deep position in the bacterial tree of life, D. radiodurans has been of tremendous interest for several synthetic biology and evolutionary studies (2).The cell envelope of D. radiodurans is atypical. While it stains Gram positive, its architecture resembles that of Gram-negative bacteria, containing an inner membrane (IM) covered by a peptidoglycan (PG) layer in a large periplasmic space (7–9) and an outer membrane (OM). However, this OM lacks lipopolysaccharide and common phospholipids typical of Gram-negative bacterial OMs, and instead has a lipid composition similar to the IM (10). The D. radiodurans OM is also covered by a regularly spaced, hexagonal surface layer or S-layer (11, 12). Previous studies have suggested that the S-layer is made of a protein called hexagonally packed intermediate-layer (HPI) surface protein (3, 8, 11, 13–17), while newer studies have suggested that a heterocomplex with gating properties, termed the S-layer deinoxanthin-binding complex (SDBC), forms a large part of the D. radiodurans cell envelope, including the S-layer (18, 19). A previously identified abundant protein called SlpA (UniProtKB {"type":"entrez-protein","attrs":{"text":"Q9RRB6","term_id":"81550587"}}Q9RRB6) is suggested to be the main component of this complex. Recently, an 11-Å resolution structure of this complex was reported using electron cryomicroscopy (cryo-EM), showing how it exhibits a triangular base partly embedded in the OM and a stalk departing orthogonally from the base, presumably away from the membrane (18). Deletion of slpA leads to substantial disruption of the D. radiodurans cell envelope, suggesting its important role in the maintenance of cell envelope integrity (20). Finally, it has been shown using biochemical experiments that the N-terminus of D. radiodurans SlpA binds to the PG-containing cell wall, demonstrating that at least the N-terminal segment of the molecule resides in the periplasmic space (21).In addition to the experimental observations introduced above, from an evolutionary perspective, an ortholog of D. radiodurans SlpA (UniProtKB {"type":"entrez-protein","attrs":{"text":"Q5SH37","term_id":"62287492"}}Q5SH37) has also been characterized from the closely related thermophilic model bacterium Thermus thermophilus (22, 23). In line with data from D. radiodurans, deletion or truncation of slpA from T. thermophilus leads to remarkable disruption of the cell envelope (20, 24), underpinning its importance in cell surface organization. At the sequence level, SlpA contains a signal peptide, an SLH domain, a long, predicted α-helical region, and a C-terminal β-strand–rich domain, which is thought to fold into an OM β-barrel (18, 19) (Fig. 1A). Due to the presence of the N-terminal SLH domain, which commonly attaches S-layer proteins (SLPs) (23, 25–28) of Gram-positive bacteria to PG-linked pyruvylated secondary cell wall polymers (SCWPs), it has been suggested that SlpA constitutes the S-layer. Conversely, in T. thermophilus, SlpA has been shown to interact with PG through its SLH domain, suggesting a role for it as a periplasmic spacer (29). The role of SlpA in organizing the cell envelope of D. radiodurans and related deep-branching bacteria such as T. thermophilus is, therefore, still enigmatic.Open in a separate windowFig. 1.Cryo-EM reconstruction of D. radiodurans SlpA. (A) The SlpA protein contains a tripartite structure, including an N-terminal SLH domain, which is connected to a C-terminal β-barrel by a long coiled-coil segment. (B) Two-dimensional class averages of the trimeric SlpA specimen used for cryo-EM structure determination. Characteristic top and side views are shown. (C) Density map of the SlpA trimer (contour level on the Lower Left) shown from the Top. The resolution of the OMBB portion of the map is 2.9 Å, and resolution decreases toward the N-terminus, with a global resolution of 3.3 Å. Two subunits are shown as blue ribbons inside white envelope outlines and one as gray density (model hidden). Distance measurement includes the micelle density. (D) An orthogonal view of C, with the SlpA trimer shown from the side. The extended coiled coil degrades in resolution toward the N-terminus (see also SI Appendix, Fig. S1), presumably due to flexibility of the long stalk. (Scale bars in B, 100 Å; in C and D, 25 Å.)In this study, we report the cryo-EM structure of the SlpA protein complex from D. radiodurans. Our structure shows that SlpA exhibits a tripartite organization, with its C-terminal part forming a homotrimeric 30-stranded OM β-barrel (OMBB), its central part forming a trimeric coiled coil that can traverse the large periplasmic space, and the extreme N-terminal part forming an SLH domain trimer that can interact with the PG layer. Our structure- and sequence-based bioinformatic analyses further show the presence of SlpA-like proteins in several phyla of phylogenetically deep-branching Gram-negative bacteria. Finally, combining our atomic structures and bioinformatic results with microscopy of wild-type and mutant cells, we report a model for the cell envelope of D. radiodurans, showing how this Gram-negative (diderm) bacterial SlpA protein shares several characteristics commonly found in Gram-positive (monoderm) SLPs, with connotations on prokaryotic evolution.     

Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin,an insect innate immune response molecule

Antimicrobial peptides are innate immune molecules playing essential roles in insects, which lack the adaptive immune system. Insects possess Toll9, the innate pattern-recognition receptor highly similar to the mammalian Toll-like receptor 4 (TLR4), which is involved in recognizing lipopolysaccharide (LPS). TLR4 is an important therapeutic target, as it causes uncontrolled immune response in sepsis; therefore, identification of TLR4-targeting molecules is imperative. Papiliocin, an insect cecropin derived from the larvae of the swallowtail butterfly, possesses potent antibacterial activities against gram-negative bacteria. We investigated the molecular mechanism underlying the TLR4-antagonistic and antiseptic activities of papiliocin. Binding analysis, docking simulation, and flow cytometry showed that papiliocin inhibited LPS-induced TLR4 signaling by directly binding to TLR4/MD-2 and causing rapid dissociation of LPS from the TLR4/MD-2 complex. R13 and R16 in the N-terminal helix, conserved in insect cecropins, were the key binding sites at the TLR4/MD-2 interface, along with the flexible hinge region, which promoted the interaction of the hydrophobic carboxyl-terminal helix with the MD-2 pocket to competitively inhibit the LPS–TLR4/MD-2 interaction. Papiliocin, an antiendotoxin molecule and TLR4 inhibitor, rescued the pathology of Escherichia coli–induced sepsis in mice more effectively and with lower nephrotoxicity compared to polymyxin B. Our results provide insight into the key structural components and mechanism underlying the TLR4-antagonistic activities of papiliocin, which is essential for the innate immune response of the insect against microbial infection. Papiliocin may be useful for developing a multifunctional alternative to polymyxin B for treating gram-negative sepsis.

Toll-like receptors (TLRs) are innate immune receptors that recognize pathogen-associated molecular patterns to protect the host from invading pathogens (1). TLR4 is one of the most critical pattern-recognition receptors in the TLR family that recognizes lipopolysaccharide (LPS) released from the outer membrane of gram-negative bacteria to elicit innate immune response (2). Subsequently, an LPS-binding protein attracts LPS and facilitates CD14-dependent transfer of LPS to TLR4 via the adaptor protein MD-2, resulting in dimerization of the TLR4/MD-2 complex. The dimer mediates translocation of nuclear factor-kappa B (NF-κB), ultimately resulting in the production of proinflammatory cytokines. Therefore, uncontrolled LPS-induced inflammatory TLR4 signaling can cause acute sepsis (3). Sepsis induced by multidrug-resistant gram-negative bacteria, such as carbapenem-resistant bacteria, is difficult to eradicate and causes serious health issues (4, 5), as carbapenems such as imipenem, doripenem, and meropenem are generally the final choices for treating infections caused by gram-negative bacteria. As management of carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae including Klebsiella pneumoniae and Escherichia coli is extremely difficult, the World Health Organization has prioritized the development of methods for effectively treating infections caused by these pathogens (6, 7). Research in this field has mainly focused on developing antiseptic molecules that can either clear bacterial LPS or competitively target the binding of LPS to TLR4/MD-2, resulting in inhibition of the TLR4 signaling pathway (8–10). Therefore, molecules with dual effects can be advantageous for inhibiting systemic TLR4-mediated inflammatory sepsis.Antimicrobial peptides (AMPs) are important natural components of the innate immune system in various organisms (11) and typically kill pathogens by permeabilizing their membranes or targeting intracellular components (12). In addition, AMPs can modulate the host immune system via multiple pathways (13). Therefore, AMPs have emerged as effective molecules against multidrug-resistant bacteria and potential alternatives to conventional antibiotics for treating gram-negative infections (14). Polymyxin B (PMB) and colistin are cyclic cationic AMPs, which are used as a last resort for treating gram-negative infections (15). PMB prevents gram-negative sepsis by removing bacterial LPS. However, increased resistance, nephrotoxicity, and neurotoxicity associated with PMB have limited its use in clinical practice (16).Insects are extremely resistant to microbial infections owing to their strong innate immune system, which includes the production of AMPs (17). In insects, Toll was initially identified in Drosophila melanogaster as an integral membrane receptor (18). Insect Toll is highly similar to mammalian TLR, and Toll/TLRs are considered key regulators of the innate immune system in both insects and mammals (19). A recent study showed that in the silkworm, Bombyx mori, Toll9 recognizes LPS by interacting with two MD-2–related lipid recognition domains, named Toll9/MD-2A or Toll9/MD-2B, indicating their functional and evolutionary similarity with mammalian TLR4/MD-2 proteins (20, 21). Insect Toll9 may be a pattern-recognition immune receptor similar to mammalian TLR4 in complex with MD-2 during LPS recognition and signaling (21). Cecropins are a group of widely studied AMPs that play important roles in the innate immune response of insects (22–24). In 1981, Steiner et al. reported that cecropins are produced from the hemolymph of bacterially challenged diapausing pupae of the giant silk moth, Hyalophora cecropia (25). Since then, several cecropin-like peptides have been identified in insects such as B. mori (26) and D. melanogaster (24). In B. mori, LPS can activate the expression of AMPs such as cecropin B, moricin, lebocin3, and attacin1 (21, 27). Some insect cecropins have shown potent antibacterial activities and in vivo antiseptic activities, confirming their therapeutic potential (23, 28–31).Papiliocin, an AMP belonging to the insect cecropin family, was isolated from the larvae of the swallowtail butterfly, Papilio xuthus (32). We previously showed that papiliocin has broad-spectrum antibacterial activity—particularly against gram-negative bacteria—in which it disrupts the bacterial membrane, similar to other insect cecropins (33–37). We determined the solution structure of papiliocin, which contains two α-helices: an amphipathic N-terminal helix from R1 to K21 and a hydrophobic carboxyl-terminal helix from A25 to V37, separated by a hinge region (33). Most insect cecropins share this helix–hinge–helix structure with high sequence homology. Furthermore, papiliocin inhibits nitric oxide (NO) production and may suppress tumor necrosis factor (TNF)-α via innate defense response mechanisms, which involve the TLR4 pathway in LPS-stimulated RAW 264.7 cells (33). We also found that aromatic residues (W2 and F5), as well as the amphipathic N-terminal helix, play important roles in the membrane permeabilization and anti-inflammatory activities of papiliocin (34, 35). Therefore, using the sequence of the N-terminal helix of papiliocin, short peptide antibiotics, such as the papiliocin–magainin hybrid peptide and a 12-mer peptide, which exhibited a diverse range of antimicrobial activities against gram-negative infections, were designed (36, 37). However, the detailed mechanism underlying TLR4 signaling inhibition and the role of the conserved hydrophobic carboxyl-terminal helix remain unclear.Considering the emerging role of TLR4 in the progression of gram-negative bacterial infections to sepsis and the urgent need to find safe antiseptic alternatives to PMB for clinical use, we investigated the molecular mechanism of action of papiliocin as a TLR4 inhibitor using binding analysis, docking simulation, saturation transfer difference (STD) NMR, and flow cytometry. This study demonstrates the role of the conserved structural components of insect cecropins involved in direct binding to TLR4/MD-2, preventing its dimerization and thereby inhibiting the LPS-stimulated TLR4 inflammatory signaling pathway. This study also provides insight into the mechanism underlying the human TLR4-antagonistic activities of papiliocin, which may be essential for understanding the functionally similar Toll9/MD-2–mediated insect innate immune response against microbial infection. The antiseptic effect and low nephrotoxicity of papiliocin were confirmed using in vivo sepsis models and compared to that of PMB, highlighting its potential as a safe alternative to PMB for treating gram-negative sepsis.     

Antibody WN1 222-5 mimics Toll-like receptor 4 binding in the recognition of LPS

Escherichia coli infections, a leading cause of septic shock, remain a major threat to human health because of the fatal action to endotoxin (LPS). Therapeutic attempts to neutralize endotoxin currently focus on inhibiting the interaction of the toxic component lipid A with myeloid differentiating factor 2, which forms a trimeric complex together with Toll-like receptor 4 to induce immune cell activation. The 1.73-Å resolution structure of the unique endotoxin-neutralizing protective antibody WN1 222-5 in complex with the core region shows that it recognizes LPS of all E. coli serovars in a manner similar to Toll-like receptor 4, revealing that protection can be achieved by targeting the inner core of LPS and that recognition of lipid A is not required. Such interference with Toll-like receptor complex formation opens new paths for antibody sepsis therapy independent of lipid A antagonists.LPS from Gram-negative bacteria is the major etiological agent of septic shock, which is a serious and often fatal dysregulation of the innate immune response that affects 750,000 people in the United States annually (1). Infection with Escherichia coli, together with Klebsiella, Neisseria, and Pseudomonas, are the most frequent isolates in septic shock (2). A key event initiating the shock cascade is the induction of the innate immune response by the complex formation of a symmetric “m”-shaped multimer composed of two copies of Toll-like receptor 4 (TLR4), myeloid differentiating factor 2 (MD-2), and LPS (3, 4). In a landmark publication, the structure of TLR4-MD-2 bound to LPS (3) was recently described.LPS is composed of an acylated glucosamine phosphate disaccharide (i.e., lipid A), which is the endotoxic principle of LPS, a core oligosaccharide (core-OS) and a distal O-polysaccharide (O-PS) often composed of repeating units (Fig. 1A). Whereas the O-PS is structurally heterogeneous, with more than 180 reported E. coli serotypes (5), the core region is composed of a more conserved structure commonly divided into the inner Kdo-heptose and outer hexose regions (6).Open in a separate windowFig. 1.Structures of LPS and the shape of the combining site. (A) Structure of E. coli R2 dodecasaccharide-P₄, representing the core and lipid A of the LPS from Enterobacteria commonly associated with septic shock. (B) Stereo views of electron density corresponding to 10 sugar residues of the core antigen (the lipid A moiety is disordered) contoured at 1.0 σ.Recognition of LPS leads to a paramount immunological defense reaction caused by the activation of a complex network of immunological mediators. Attempts to control the clinical development of sepsis by neutralizing the most important proinflammatory mediators have failed, including the recent withdrawal of recombinant activated protein C (Xigris). A promising antagonistic lipid candidate called Eritoran (E5564; Eisai) (7) also recently failed in clinical trials, and alternative treatments are urgently needed. The discovery of TLR4 as the principal receptor for endotoxins (8) has stimulated the development of drugs aiming at its down-regulation (9) through interference of LPS–TLR4–MD-2 complex formation (4, 10–12).Antisera specific for O-PS have been shown to protect against LPS lethality (13); however, the diversity of enterobacterial O-PS together with the rapid onset of septic shock have hindered their introduction into clinical practice (11).The hypothesis that mAbs specific to the conserved inner core region or lipid A would be protective against a wide range of serovars and even different species was put forward (14) after the discovery of structural similarities within their respective LPSs. WN1 222-5 is the only neutralizing antibody reported to date that displays specificity for an epitope within the structurally conserved region of LPS from a large number of pathogenic E. coli, Salmonella, Shigella, and Citrobacter serovars (15). Further, WN1 222-5 has been shown to inhibit the recognition and uptake of LPS by cells expressing coreceptor mCD14, likely by hindering the transfer of LPS to TLR4–MD-2 (16).WN1 222-5 has been shown to inhibit the inflammatory cascade in in vivo studies of septic shock, in which it prevents the pyrogenic response in rabbits, inhibits the Limulus amoebocyte lysate assay, and inhibits LPS-induced monokine secretion (15–17).The difficulties in growing crystals of antibodies in complex with carbohydrate antigens has led to relatively few reported structures (18–21), leading, for example, to increased use of structure prediction tools such as molecular dynamics modeling (22). Thus, in contrast to their great immunological significance during infectious disease, still relatively little is known about carbohydrate recognition by antibodies at the structural level. Whereas cavity- or groove-shaped antibody-combining sites have been observed in most cases, a unique mechanism of binding has been observed for the HIV-1 neutralizing antibody 2G12, binding clusters of carbohydrates from the silent face of gp120 by using “domain swapping” (19, 23, 24).The structural analysis of antibodies Se155-4 and S20-4 against O-PS of Salmonella enterica and Vibrio cholerae, respectively, have revealed structural insights into the high specificity for a particular serotype (20, 25). However, because of their specificity, antibodies against O-PS are of limited use for the treatment of infectious disease. Nevertheless, structures of antibodies in complex with large carbohydrate antigens have revealed critical insights for vaccine development. The protective antibody F22-4 in complex with an 11-sugar segment from the O-PS of Shigella flexneri serotype 2a (26) allowed the design of new immunogens.Most attempts in obtaining antibodies that are broadly reactive with a wide variety of LPSs from different Gram-negative bacteria have failed, and epitopes within the deeper core region of LPS have been regarded as not accessible to antibodies in WT LPSs of infectious bacteria. To provide detailed insight on a unique cross-reactive and neutralizing ability, the Fab from WN1 222-5 in complex with a complete core-OS of LPS from E. coli has been crystallized and its structure determined to 1.73-Å resolution.     

Olfactory regulation by dopamine and DRD2 receptor in the nose

Olfactory behavior is important for animal survival, and olfactory dysfunction is a common feature of several diseases. Despite the identification of neural circuits and circulating hormones in olfactory regulation, the peripheral targets for olfactory modulation remain relatively unexplored. In analyzing the single-cell RNA sequencing data from mouse and human olfactory mucosa (OM), we found that the mature olfactory sensory neurons (OSNs) express high levels of dopamine D2 receptor (Drd2) rather than other dopamine receptor subtypes. The DRD2 receptor is expressed in the cilia and somata of mature OSNs, while nasal dopamine is mainly released from the sympathetic nerve terminals, which innervate the mouse OM. Intriguingly, genetic ablation of Drd2 in mature OSNs or intranasal application with DRD2 antagonist significantly increased the OSN response to odorants and enhanced the olfactory sensitivity in mice. Mechanistic studies indicated that dopamine, acting through DRD2 receptor, could inhibit odor-induced cAMP signaling of olfactory receptors. Interestingly, the local dopamine synthesis in mouse OM is down-regulated during starvation, which leads to hunger-induced olfactory enhancement. Moreover, pharmacological inhibition of local dopamine synthesis in mouse OM is sufficient to enhance olfactory abilities. Altogether, these results reveal nasal dopamine and DRD2 receptor as the potential peripheral targets for olfactory modulation.

Olfactory behavior is important for food seeking and animal survival. On the other hand, olfactory dysfunction is a common feature of several diseases such as psychiatric disorders, neurodegeneration, and COVID-19 (1–3). Interestingly, the olfactory ability can be regulated by feeding status and external environments (4, 5). Recent studies have made progress in identifying the neural circuits and circulating hormones in olfactory regulation (6–11). However, the peripheral targets modulating olfactory ability remain relatively unexplored (12).Dopamine (DA) is a monoamine neurotransmitter (13, 14), which plays important roles in a variety of brain functions. DA is released by dopaminergic neurons in the central nervous system. In addition, DA can be released by sympathetic nerves in the peripheral tissues including the olfactory mucosa (OM) (15–18). The sympathetic innervation of rodent OM originates predominantly from the superior cervical ganglion (SCG) (17). Tyrosine hydroxylase (TH) is the rate-limiting enzyme for DA synthesis (19). Intriguingly, the Th mRNA is locally translated in the sympathetic nerve axons, which facilitates local DA synthesis (20, 21).There are two types of DA receptors based on sequence homology and function: The excitatory D1-like receptors (DRD1 and DRD5) and inhibitory D2-like receptors (DRD2–DRD4) (22). Activation of DRD2, a Gα_i/o-coupled receptor, can reduce the intracellular levels of cyclic adenosine monophosphate (cAMP). Drd2 is associated with several neuropsychiatric diseases and is the target of some antipsychotic drugs (23–28). In the central nervous system including the olfactory bulb (OB), DA-DRD2 signaling plays important roles in regulating synaptic transmission and plasticity (29–33). However, the function and regulation of DA-DRD2 signaling in the peripheral tissues are relatively less understood.Here we show that DRD2 is expressed in the cilia and somata of mature olfactory sensory neurons (OSNs) in mice. We provide evidence that DA-DRD2 signaling has a tonic inhibition on OSN activity and olfactory function in mice. Intriguingly, hunger greatly reduces the N4-acetylcytidine (ac⁴C) modification of Th mRNA and local DA synthesis in mouse OM, which causes the olfactory enhancement during starvation. We further show that inhibition of local DA synthesis or DRD2 receptor in mouse OM recapitulates enhanced olfactory abilities during starvation. Collectively, these results reveal nasal DA and DRD2 receptor as the potential peripheral targets for olfactory regulation.     

From the Cover: Canine sexual dimorphism in Ardipithecus ramidus was nearly human-like

Body and canine size dimorphism in fossils inform sociobehavioral hypotheses on human evolution and have been of interest since Darwin’s famous reflections on the subject. Here, we assemble a large dataset of fossil canines of the human clade, including all available Ardipithecus ramidus fossils recovered from the Middle Awash and Gona research areas in Ethiopia, and systematically examine canine dimorphism through evolutionary time. In particular, we apply a Bayesian probabilistic method that reduces bias when estimating weak and moderate levels of dimorphism. Our results show that Ar. ramidus canine dimorphism was significantly weaker than in the bonobo, the least dimorphic and behaviorally least aggressive among extant great apes. Average male-to-female size ratios of the canine in Ar. ramidus are estimated as 1.06 and 1.13 in the upper and lower canines, respectively, within modern human population ranges of variation. The slightly greater magnitude of canine size dimorphism in the lower than in the upper canines of Ar. ramidus appears to be shared with early Australopithecus, suggesting that male canine reduction was initially more advanced in the behaviorally important upper canine. The available fossil evidence suggests a drastic size reduction of the male canine prior to Ar. ramidus and the earliest known members of the human clade, with little change in canine dimorphism levels thereafter. This evolutionary pattern indicates a profound behavioral shift associated with comparatively weak levels of male aggression early in human evolution, a pattern that was subsequently shared by Australopithecus and Homo.

A small canine tooth with little sexual dimorphism is a well-known hallmark of the human condition. The small and relatively nonprojecting deciduous canine of the first known fossil of Australopithecus, the Taung child skull, was a key feature used by Raymond Dart for his inference that the fossil represented an early stage of human evolution (1). However, recovery of additional Australopithecus fossils led to the canine of Australopithecus africanus to be characterized as large (compared to that of humans or “robust australopithecines”) and its morphology primitive, based on a projecting main cusp and crown structures lacking or hardly expressed in Homo (2). Later, the perception of a large and primitive canine was enhanced by the discovery and recognition of Australopithecus afarensis and Australopithecus anamensis (3–8), the latter species extending back in time to 4.2 million years ago (Ma). Although assessments of canine size variation and sexual dimorphism in Au. afarensis were hampered by limited sample sizes (9, 10), some suggested that the species had a more dimorphic canine than do humans, equivalent in degree to the bonobo (11) or to chimpanzees and orangutans (12). Initially, Au. anamensis was suggested to express greater canine dimorphism than did Au. afarensis (13, 14). However, based on a somewhat larger sample size, this is now considered to be the case with the tooth root but not necessarily its crown (15–17).Throughout the 1990s and 2000s, a pre-Australopithecus record of fossils spanning >6.0 to 4.4 Ma revealed that the canines of these earlier forms did not necessarily exceed those of Au. afarensis or Au. anamensis in general size (18–28). However, all these taxa apparently possessed canine crowns on average about 30% larger than in modern humans, which makes moderately high levels of sexual dimorphism potentially possible. Canine sexual dimorphism, combined with features such as body size dimorphism, inform sociobehavioral and ecological adaptations of past and present primates, and therefore have been of considerable interest since Darwin’s 1871 considerations (29–57). In particular, the relationship of canine size dimorphism (and/or male and female relative canine sizes) with reproductive strategies and aggression/competition levels in primate species have been a continued focus of interest (14, 33, 35–45, 49–56). Conspecific-directed agonistic behavior in primates related to mate and/or resource competition can be particularly intense among males both within and between groups (14, 44, 57). It is widely recognized that a large canine functions as a weapon in intra- and intergroup incidences of occasional lethal aggression (45, 58–61), and a large, tall canine has been shown or inferred to significantly enhance male fitness (50, 56). Hence, canine size and dimorphism levels in fossil species provide otherwise unavailable insights into their adaptive strategies.Here, we apply a recently developed method of estimating sexual size dimorphism from fossil assemblages of unknown sex compositions, the posterior density peak (pdPeak) method (62), and reexamine canine sexual dimorphism in Ardipithecus ramidus at ∼4.5 Ma. We include newly available fossils recovered from the Middle Awash and Gona paleoanthropological research areas in the Afar Rift, Ethiopia (26, 63, 64) in order to obtain the most reliable dimorphism estimates currently possible. We apply the same method to Australopithecus, Homo, and selected fossil apes, and evaluate canine sexual dimorphism through evolutionary time.We operationally define canine sexual dimorphism as the ratio between male and female means of basal canine crown diameters (the m/f ratio). Because the canines of Ar. ramidus, Au. anamensis, and extant and fossil apes are variably asymmetric in crown shape, we examine the maximum basal dimension of the crown. This can be either the mesiodistal crown diameter or a maximum diameter taken from the distolingual to mesiobuccal crown base (7, 27, 65). In the chronologically later Au. afarensis and all other species of Australopithecus sensu lato and Homo, we examine the more widely available conventional metric of buccolingual breadth, which corresponds to or approximates the maximum basal crown diameter. In anthropoid primates, canine height is more informative than basal canine diameter as a functional indicator of aggression and/or related display (14, 41–44). We therefore also examine available unworn and minimally worn fossil canines with reliable crown heights.     

Intracellular Shigella remodels its LPS to dampen the innate immune recognition and evade inflammasome activation

LPS is a potent bacterial effector triggering the activation of the innate immune system following binding with the complex CD14, myeloid differentiation protein 2, and Toll-like receptor 4. The LPS of the enteropathogen Shigella flexneri is a hexa-acylated isoform possessing an optimal inflammatory activity. Symptoms of shigellosis are produced by severe inflammation caused by the invasion process of Shigella in colonic and rectal mucosa. Here we addressed the question of the role played by the Shigella LPS in eliciting a dysregulated inflammatory response of the host. We unveil that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria grown in laboratory medium differ in the number of acyl chains in lipid A, with iLPS being the hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of bacteria grown in laboratory medium; (iv) both LPS forms mainly signal through the Toll-like receptor 4/myeloid differentiation primary response gene 88 pathway; (v) iLPS down-regulates the inflammasome-mediated release of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime polymorfonuclear cells for an oxidative burst. We propose a working model whereby the two forms of LPS might govern different steps of the invasive process of Shigella. In the first phases, the bacteria, decorated with hypoacylated LPS, are able to lower the immune system surveillance, whereas, in the late phases, shigellae harboring immunopotent LPS are fully recognized by the immune system, which can then successfully resolve the infection.LPS is a glycolipid located in the outer membrane of Gram-negative bacteria. It is composed of three covalently linked domains: lipid A, which is embedded in the outer membrane; the oligosaccharide core; and the O-polysaccharide or O-antigen, which cover the bacterial surface. During infections sustained by Gram-negative bacteria, detection of LPS initiates an acute inflammatory response as LPS, mainly by the lipid A, which is the real pathogen-associated molecular pattern (PAMP), is sensed by the innate immune system, through the binding to the pattern recognition receptor (PRR) complex of myeloid differentiation protein 2 (MD-2) and Toll-like receptor (TLR) 4 (TLR4) (1–3). The downstream effects of LPS recognition elicit effector mechanisms aimed at pathogen eradication. However, LPS can also elicit an host reaction because it is a major mediator of pathologic processes (4). The strength of the innate immune response to LPS can be modulated by its chemical structure; specifically, a fine tuning of the lipid A structure can significantly affect the immunostimulatory properties of the whole LPS molecule (5, 6). There is a strong correlation between the number of acyl chains of lipid A and the immunological response via the TLR4 pathway. In general, hexaacylated lipid A species are agonists, whereas tetraacylated species are antagonists with a weak inflammatory potential (7). Gram-negative bacteria can synthesize a range of differentially acylated LPSs as a result of the LPS biosynthesis. Changes in lipid A acylation underlie the adaptation of pathogens to different hosts, such as Yersinia pestis (8), or to different phases of pathogenesis such as Salmonella typhimurium (9) or in the establishment of chronic infection such as Pseudomonas aeruginosa (10, 11).Shigella flexneri is a Gram-negative pathogen that infects humans. The ingestion of as few as 100 bacteria is sufficient to cause bacillary dysentery, a severe rectocolitis caused by the dramatic inflammatory reaction induced by Shigella invasion on the colonic and rectal mucosa (12). Shigella enters epithelial cells by injecting effectors via a type III secretion system (T3SS) (13), escapes from the phagocytic vacuole, and actively proliferates within the cytosol of infected cells (14, 15). Bacterial proliferation is a potent signal to initiate inflammation because intracellular shigellae activate NF-κB following recognition of peptidoglycan (PGN) by the PRR Nod1, leading to IL-8 production (16, 17). IL-8 attracts neutrophils that are required for the clearance of shigellae, but also participates in epithelial barrier destruction (18). In macrophages, Shigella is able to trigger the assembly of the inflammasome, an important defense mechanism that is part of the innate immune system (19). The inflammasome is a multiprotein complex that mediates activation of caspase-1, which promotes the secretion of the proinflammatory cytokines IL-1β and IL-18 as well as a cell death process called pyroptosis (20, 21). Different PRRs, i.e., TLRs and nucleotide-binding oligomerization domain-like receptors (NLRs) contribute to the inflammasome assembly (22). In Shigella-infected macrophages, the activation of the NLRC4-mediated inflammasome triggers cell death and release of IL-1β and IL-18 (19, 23). Indeed, production of IL-1β is a paradigm of shigellosis: the chief role of this cytokine has been highlighted in vivo in several studies (24–26).In tissues of animals and in ex vivo human samples infected with Shigella (27), a huge amount of LPS is usually observed, reflecting the presence of living bacteria and/or of processed molecules. However, whether, how, and at to what extent this mass of LPS present in Shigella-infected tissues could play a role in the inflammation remains largely unknown.In 2002, D’Hauteville et al. reported that, in S. flexneri, the lack of msbB genes, msbB1 and msbB2, both encoding the enzyme myristoyl transferase, reduces lipid A acylation degree along with TNF-α production and epithelial lining inflammatory destruction in a rabbit model of Shigella infection (28, 29). This study suggests that LPS composition can greatly influence the degree of inflammation induced by Shigella.In line with these issues, here we intend to contribute to the understanding of the role played by LPS in Shigella pathogenesis. Hence, we addressed the question of whether Shigella could adapt the LPS structure to the host thereby exploiting the mechanism of LPS modification to hijack the innate immune response. With this aim, we extracted, purified, and analyzed the LPS of shigellae resident in epithelial cells. We detailed the immunopotential of this structure and compared it to that of conventionally grown bacteria. Together our results point to a key role for LPS during the Shigella invasive process.We report that (i) Shigella is able to modify the LPS composition, e.g., the lipid A and core domains, during proliferation within epithelial cells; (ii) the LPS of intracellular bacteria (iLPS) and that of bacteria conventionally grown (aLPS) differ in the number of acyl chains in lipid A, with iLPS being hypoacylated; (iii) the immunopotential of iLPS is dramatically lower than that of aLPS; (iv) both LPS forms signal mainly through the TLR4/MyD88 pathway; (v) iLPS influences the inflammasome-mediated production of IL-1β in Shigella-infected macrophages; and (vi) iLPS exhibits a reduced capacity to prime PMNs for an oxidative burst.     

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana. HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Plant innate immunity activation relies on detection of invasion pattern (IP) molecules that are perceived by plant cells (1, 2). Non–self-recognition IPs include essential components of whole classes of microorganisms, such as fragments of flagellin, peptidoglycans, mc-3-OH-FAs from bacteria or fragments of chitin, and β-glucans from fungi and oomycetes, respectively (3, 4). Apoplastic IPs are sensed by plant plasma membrane–localized receptor kinases (RKs) or receptor-like proteins (RLPs) that function as pattern recognition receptors (PRRs) (5, 6). Activation of the immune response requires the recruitment of regulatory receptor kinases and receptor-like cytoplasmic kinases (RLCKs) by PRRs (7). Early cellular immune signaling of pattern-triggered immunity (PTI) includes ion-flux changes at the plasma membrane, rise in cytosolic Ca²⁺ levels, production of extracellular reactive oxygen species (ROS), and activation of mitogen-activated protein kinases (MAPKs) and/or Ca²⁺-dependent protein kinases (3, 8–10). Biosynthesis and mobilization of plant hormones, including salicylic acid, jasmonic acid, ethylene, abscisic acid and brassinosteroids, ultimately modulate plant resistance to phytopathogens (11–14).Rhamnolipids (RLs) are extracellular amphiphilic metabolites produced by several bacteria, especially Pseudomonas and Burkholderia species (15–17). Acting as wetting agents, RLs are essential for bacterial surface dissemination called swarming motility and for normal biofilm development (18–20). These glycolipids are produced from l-rhamnose and 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) precursors (15, 21). HAAs are synthesized by dimerization of (R)-3-hydroxyalkanoyl-CoA in Pseudomonas, forming congeners through the RhlA enzyme (21). The opportunistic plant pathogen Pseudomonas aeruginosa and the phytopathogen Pseudomonas syringae produce extracellular HAAs (16, 22–24). In P. syringae, HAA synthesis is coordinately regulated with the late-stage flagellar gene encoding flagellin (22). HAA and RL production is finely tuned and modulates the behavior of swarming migrating bacterial cells by acting as self-produced negative and positive chemotactic-like stimuli (25). RLs contribute to the alteration of the bacterial outer membrane composition, by shedding flagellin from the flagella (26) and by releasing lipopolysaccharides (LPS), resulting in an increased hydrophobicity of the bacterial cell surface (27). In mammalian cells, RLs produced by Burkholderia plantarii exhibit endotoxin-like properties similar to LPS, leading to the production of proinflammatory cytokines in human mononuclear cells (28, 29). They also subvert the host innate immune response through manipulation of the human beta-defensin-2 expression (30). Moreover, RLs from Burkholderia pseudomallei induce interferon gamma (IFN-γ)–dependent host immune response in goat (31).In plants, RLs induce defense responses and resistance to biotrophic and necrotrophic pathogens (32, 33). They also contribute to the biocontrol activity of the plant beneficial bacterium P. aeruginosa PNA1 against oomycetes (17). Recently, it was reported that the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29) mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) sensing in Arabidopsis thaliana (hereafter, Arabidopsis) and that bacterial compounds comprising mc-3-OH-acyl building blocks including LPS and RLs do not stimulate LORE-dependent responses (34).Here, we show that the lipidic secretome produced by P. aeruginosa (RL secretome), mostly composed of RLs and HAAs, induces Arabidopsis immunity. HAAs are perceived through the RK LORE. We demonstrate that, albeit not being sensed by LORE, RLs trigger an immune response characterized by an atypical defense signature. Altogether, our results demonstrate that RLs and their precursors produced by Pseudomonas bacteria stimulate the plant immune response by two distinct mechanisms.     

Role of internal loop dynamics in antibiotic permeability of outer membrane porins

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open–closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF β-barrel. Mutation of key residues important for the loop’s conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Antibiotic resistance is a major concern in the treatment of bacterial infections (1–3). Design of antibiotics targeting Gram-negative bacteria is particularly challenging due to the presence of an outer membrane (OM) containing lipopolysaccharide glycolipids. The OM provides a major permeation barrier against the uptake of various substrates, including antibiotics (2, 4–11). Due to the low permeability of the OM, essential nutrients for the bacteria typically diffuse into the cell through a variety of general diffusion, β-barrel OM porins (6). Several OM porins have been shown to also be the main pathways for penetration of antibiotics into Gram-negative bacteria (6, 12).The general diffusion OM porins are typically organized into trimeric β-barrel structures with multiple loops connecting individual β-strands in each monomer. Of particular functional interest is a long internal loop (L3) that folds into the lumen of the monomeric β-barrels and forms a constriction region (CR) within the porin. The CR significantly narrows the pore (Fig. 1A) and acts as a major permeation barrier (13). Additionally, the CR contains many charged and polar residues, including acidic residues on the L3 loop and a cluster of tyrosine and basic residues on two opposite sides of the barrel wall (termed here as Y and B face, respectively) (Fig. 1B). The nature and organization of the B-face and Y-face residues will determine their interaction with the L3 loop and therefore, are expected to influence the dynamics of L3 and thus, the permeation properties of the porin.Open in a separate windowFig. 1.Structural features of OmpF in E. coli. (A) The monomeric radius profile [calculated using the program HOLE (31)] of the crystal structure of OmpF [PDB code 3POX (26)]. A molecular representation of a single monomer of OmpF is shown highlighting the internal loop L3 (orange) that constricts the pore. (B) A top-down view of OmpF showing that two acidic residues of L3 (D121 and E117) form hydrogen bonds with residues in the Y face. A cluster of basic residues (B face) on the opposite side of the Y face is hypothesized to facilitate the movement of L3 to further narrow the pore.A remarkable feature of many OM porins is their ability to undergo spontaneous conformational transitions between macroscopically distinct “open” and “closed” states. Functionally relevant conformational transitions in OM transport proteins are often active processes, coupled to and driven by an external energy input, such as the transmembrane voltage change (14–18). However, in OM porins, thermal fluctuations seem to be the main source for such conformational changes (19–24). The most abundant OM porin in Escherichia coli, OmpF, is an ideal model to study such conformational changes. OmpF is known for spontaneously fluctuating between highly stable conducting (open) and less stable nonconducting (closed) states, as observed in electrophysiological measurements under a low electric voltage (21, 22, 25). Notably, spontaneous fluctuations between conducting and nonconducting states have also been observed at 0 mV for OmpC, a close homolog of OmpF, where a KCl concentration gradient was used to drive ion diffusion (19). The asymmetric voltage-dependent inactivation of OmpC was not affected by mutations in these experiments, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. However, the molecular mechanism by which OmpF undergoes these transitions is still not well understood.We expect that the internal loop, L3, might play a direct role in the open–closed transitions in OmpF due to its location within the CR. Several of the acidic residues in L3, including E117 and D121, interact with tyrosine residues of the Y face in the crystal structure of OmpF (Protein Data Bank [PDB] code 3POX) (Fig. 1A) (26). It has been observed in a previous molecular dynamics (MD) study that L3 leaves the Y face to transiently interact with the B face, thereby narrowing the pore (27). These results led to the hypothesis that L3 movement could be responsible for controlling open–closed transitions of the pore. However, due to the short duration of the simulations and the nonphysiological conditions (vacuum) used in that computational study, structural support for this hypothesis remained lacking. The role of L3 conformational dynamics in permeability of OM porins remains an open subject. Notably, it is reported that the movement of L3 may not be critical to “voltage gating” of OM porins, a process occurring at voltages of 100 mV or more (28–31). However, this does not exclude the possibility that L3 can be involved in the open–closed transition at low or zero external voltage.As a support for the latter hypothesis, mutations to disrupt the hydrogen bonds between L3 and Y face in OmpC, a homologous protein to OmpF, have been reported to increase closed-state visiting frequency of the porin under no voltage conditions in electrophysiology experiments (19). Furthermore, mutation of B-face residues to uncharged or negatively charged residues in OmpF showed an increase in substrate permeation (32–34), possibly as a result of reduced attraction of L3 to the B face, shifting the equilibrium toward the open state. Moreover, the crystal structure of a clinically relevant mutant of OmpF, G119D (35), suggested that adding a negative charge to L3 can potentiate its attraction toward the B face, thereby further reducing the pore size (SI Appendix, Fig. S1) as compared with the wild type (WT) and therefore, shifting the porin toward the closed state. This shift resulted in decreased permeability of the mutant to substrates such as carbohydrates and antibiotics (35). Strikingly, this mutant porin was shown to confer colicin N resistance to clinical strains of E. coli (35).The aim of this study is to provide atomistic insight into the mechanisms controlling the equilibrium between open and closed states of OmpF using extended MD simulations, which are used to construct a Markov-state model (MSM) for the process. MSMs are a class of model used to describe the long-timescale dynamics of molecular systems and to obtain the thermodynamic and kinetic information about dynamic processes from MD simulations (36). Using MSMs, we find that large-scale motion of L3 controls the equilibrium between conducting and nonconducting states of OmpF. Further analysis using the transition path theory (TPT) (37) revealed that transitions between open and closed states occur in two steps: 1) movement of E117 to the B face to initiate the transition from the open state and 2) movement of D121 to the B face that drives a large-scale movement of L3 to mediate complete closure of the pore.The significant effect of pore narrowing in the closed state on OmpF permeability is examined by electrophysiology simulations, in which ionic currents are shown to be substantially reduced in the closed state, and by free energy calculations indicating the presence of a higher-energy barrier against permeation of an antibiotic. Furthermore, simulations of charge-reversal mutants of B-face residues key in our proposed mechanism show a significant decrease in the probability of the closed state. This agrees with the increased accumulation observed for several antibiotics in our whole-cell assays, in which we expressed the mutant porins. Furthermore, previous experiments also report an increase in substrate permeability of these mutants (32–34). According to our model, these mutations reduce the attraction of E117 or D121 to the B face, thereby reducing the closed-state probability. Overall, our results provide mechanistic details on how thermal motion of an internal loop controls a dynamic equilibrium between open and closed states, thereby regulating permeability of OM porins.