An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Structure and Mechanism of the Lipid Flippase MurJ.

Biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates the transport of lipid-linked oligosaccharides (LLO) across membranes from their cytosolic site of synthesis to their sites of utilization. Much of our current understanding of LLO transport comes from genetic, biochemical, and structural studies of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily protein MurJ, which flips the peptidoglycan precursor lipid II. MurJ plays a pivotal role in bacterial cell wall synthesis and is an emerging antibiotic target. Here, we review the mechanism of LLO flipping by MurJ, including the structural basis for lipid II flipping and ion coupling. We then discuss inhibition of MurJ by antibacterials, including humimycins and the phage M lysis protein, as well as how studies on MurJ could provide insight into other flippases, both within and beyond the MOP superfamily.

Ticks Resist Skin Commensals with Immune Factor of Bacterial Origin.

Ticks transmit a diverse array of microbes to vertebrate hosts, including human pathogens, which has led to a human-centric focus in this vector system. Far less is known about pathogens of ticks themselves. Here, we discover that a toxin in blacklegged ticks (Ixodes scapularis) horizontally acquired from bacteria-called domesticated amidase effector 2 (dae2)-has evolved to kill mammalian skin microbes with remarkable efficiency. Secreted into the saliva and gut of ticks, Dae2 limits skin-associated staphylococci in ticks while feeding. In contrast, Dae2 has no intrinsic ability to kill Borrelia burgdorferi, the tick-borne Lyme disease bacterial pathogen. These findings suggest ticks resist their own pathogens while tolerating symbionts. Thus, just as tick symbionts can be pathogenic to humans, mammalian commensals can be harmful to ticks. Our study underscores how virulence is context-dependent and bolsters the idea that "pathogen" is a status and not an identity.

Imaging Bacterial Cell Wall Biosynthesis.

Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis.

Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

Intrinsically disordered protein regions are required for cell wall homeostasis in Bacillus subtilis.

Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.

P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

Nephrocytes Remove Microbiota-Derived Peptidoglycan from Systemic Circulation to Maintain Immune Homeostasis.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Cell envelope growth of Gram-negative bacteria proceeds independently of cell wall synthesis.

All bacterial cells must expand their envelopes during growth. The main load-bearing and shape-determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram-negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion-up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram-negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall-cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.

Cell division protein FtsK coordinates bacterial chromosome segregation and daughter cell separation in Staphylococcus aureus.

Unregulated cell cycle progression may have lethal consequences and therefore, bacteria have various mechanisms in place for the precise spatiotemporal control of cell cycle events. We have uncovered a new link between chromosome replication/segregation and splitting of the division septum. We show that the DNA translocase domain-containing divisome protein FtsK regulates cellular levels of a peptidoglycan hydrolase Sle1, which is involved in cell separation in the bacterial pathogen Staphylococcus aureus. FtsK interacts with a chaperone (trigger factor, TF) and establishes a FtsK-dependent TF concentration gradient that is higher in the septal region. Trigger factor binds Sle1 and promotes its preferential export at the septal region, while also preventing Sle1 degradation by the ClpXP proteolytic machinery. Upon conditions that lead to paused septum synthesis, such as DNA damage or impaired DNA replication/segregation, TF gradient is dissipated and Sle1 levels are reduced, thus halting premature septum splitting.

Microbiota-Derived Lactate Activates Production of Reactive Oxygen Species by the Intestinal NADPH Oxidase Nox and Shortens Drosophila Lifespan.

Commensal microbes colonize the gut epithelia of virtually all animals and provide several benefits to their hosts. Changes in commensal populations can lead to dysbiosis, which is associated with numerous pathologies and decreased lifespan. Peptidoglycan recognition proteins (PGRPs) are important regulators of the commensal microbiota and intestinal homeostasis. Here, we found that a null mutation in Drosophila PGRP-SD was associated with overgrowth of Lactobacillus plantarum in the fly gut and a shortened lifespan. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and the generation of reactive oxygen species (ROS). In turn, ROS production promoted intestinal damage, increased proliferation of intestinal stem cells, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing a host-commensal metabolic crosstalk that is probably broadly conserved. Our findings outline a mechanism whereby host immune dysfunction leads to commensal dysbiosis that in turn promotes age-related pathologies.

Identification of a family of peptidoglycan transpeptidases reveals that Clostridioides difficile requires noncanonical cross-links for viability.

Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.

Lipopeptide antibiotics disrupt interactions of undecaprenyl phosphate with UptA.

The peptidoglycan pathway represents one of the most successful antibacterial targets with the last critical step being the flipping of carrier lipid, undecaprenyl phosphate (C55-P), across the membrane to reenter the pathway. This translocation of C55-P is facilitated by DedA and DUF368 domain-containing family membrane proteins via unknown mechanisms. Here, we employ native mass spectrometry to investigate the interactions of UptA, a member of the DedA family of membrane protein from Bacillus subtilis, with C55-P, membrane phospholipids, and cell wall-targeting antibiotics. Our results show that UptA, expressed and purified in Escherichia coli, forms monomer-dimer equilibria, and binds to C55-P in a pH-dependent fashion. Specifically, we show that UptA interacts more favorably with C55-P over shorter-chain analogs and membrane phospholipids. Moreover, we demonstrate that lipopeptide antibiotics, amphomycin and aspartocin D, can directly inhibit UptA function by out-competing the substrate for the protein binding, in addition to their propensity to form complex with free C55-P. Overall, this study shows that UptA-mediated translocation of C55-P is potentially mediated by pH and anionic phospholipids and provides insights for future development of antibiotics targeting carrier lipid recycling.

Cell Wall Biology of Vibrio cholerae.

Most bacteria are protected from environmental offenses by a cell wall consisting of strong yet elastic peptidoglycan. The cell wall is essential for preserving bacterial morphology and viability, and thus the enzymes involved in the production and turnover of peptidoglycan have become preferred targets for many of our most successful antibiotics. In the past decades, Vibrio cholerae, the gram-negative pathogen causing the diarrheal disease cholera, has become a major model for understanding cell wall genetics, biochemistry, and physiology. More than 100 articles have shed light on novel cell wall genetic determinants, regulatory links, and adaptive mechanisms. Here we provide the first comprehensive review of V. cholerae's cell wall biology and genetics. Special emphasis is placed on the similarities and differences with Escherichia coli, the paradigm for understanding cell wall metabolism and chemical structure in gram-negative bacteria.

Growth and Division of the Peptidoglycan Matrix.

Most bacteria are surrounded by a peptidoglycan cell wall that defines their shape and protects them from osmotic lysis. The expansion and division of this structure therefore plays an integral role in bacterial growth and division. Additionally, the biogenesis of the peptidoglycan layer is the target of many of our most effective antibiotics. Thus, a better understanding of how the cell wall is built will enable the development of new therapies to combat the rise of drug-resistant bacterial infections. This review covers recent advances in defining the mechanisms involved in assembling the peptidoglycan layer with an emphasis on discoveries related to the function and regulation of the cell elongation and division machineries in the model organisms Escherichia coli and Bacillus subtilis.

Escherichia coli utilizes multiple peptidoglycan recycling permeases with distinct strategies of recycling.

Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the existing layer of PG is remodeled and PG fragments are released. Enterics such as Escherichia coli go to great lengths to internalize and reutilize PG fragments. E. coli is estimated to break down one-third of its cell wall, yet only loses ~0 to 5% of meso-diaminopimelic acid, a PG-specific amino acid, per generation. Two transporters were identified early on to possibly be the primary permease that facilitates PG fragment recycling, i) AmpG and ii) the Opp ATP binding cassette transporter in conjunction with a PG-specific periplasmic binding protein, MppA. The contribution of each transporter to PG recycling has been debated. Here, we have found that AmpG and MppA/Opp are differentially regulated by carbon source and growth phase. In addition, MppA/Opp is uniquely capable of high-affinity scavenging of muropeptides from growth media, demonstrating that AmpG and MppA/Opp allow for different strategies of recycling PG fragments. Altogether, this work clarifies environmental contexts under which E. coli utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.

Microbiota-induced active translocation of peptidoglycan across the intestinal barrier dictates its within-host dissemination.

Peptidoglycan, the major structural polymer forming the cell wall of bacteria, is an important mediator of physiological and behavioral effects in mammalian hosts. These effects are frequently linked to its translocation from the intestinal lumen to host tissues. However, the modality and regulation of this translocation across the gut barrier has not been precisely addressed. In this study, we characterized the absorption of peptidoglycan across the intestine and its systemic dissemination. We report that peptidoglycan has a distinct tropism for host organs when absorbed via the gut, most notably by favoring access to the brain. We demonstrate that intestinal translocation of peptidoglycan occurs through a microbiota-induced active process. This process is regulated by the parasympathetic pathway via the muscarinic acetylcholine receptors. Together, this study reveals fundamental parameters concerning the uptake of a major microbiota molecular signal from the steady-state gut.

A role for the Gram-negative outer membrane in bacterial shape determination.

The cell envelope of Gram-negative bacteria consists of three distinct layers: the cytoplasmic membrane, a cell wall made of peptidoglycan (PG), and an asymmetric outer membrane (OM) composed of phospholipid in the inner leaflet and lipopolysaccharide (LPS) glycolipid in the outer leaflet. The PG layer has long been thought to be the major structural component of the envelope protecting cells from osmotic lysis and providing them with their characteristic shape. In recent years, the OM has also been shown to be a load-bearing layer of the cell surface that fortifies cells against internal turgor pressure. However, whether the OM also plays a role in morphogenesis has remained unclear. Here, we report that changes in LPS synthesis or modification predicted to strengthen the OM can suppress the growth and shape defects of Escherichia coli mutants with reduced activity in a conserved PG synthesis machine called the Rod complex (elongasome) that is responsible for cell elongation and shape determination. Evidence is presented that OM fortification in the shape mutants restores the ability of MreB cytoskeletal filaments to properly orient the synthesis of new cell wall material by the Rod complex. Our results are therefore consistent with a role for the OM in the propagation of rod shape during growth in addition to its well-known function as a diffusion barrier promoting the intrinsic antibiotic resistance of Gram-negative bacteria.

Cross-talk between phospholipid synthesis and peptidoglycan expansion by a cell wall hydrolase.

The Gram-negative bacterial cell envelope is a complex multilayered structure comprising a bilayered phospholipid (PL) membrane that surrounds the cytoplasm (inner membrane or IM) and an asymmetric outer membrane (OM) with PLs in the inner leaflet and lipopolysaccharides in the outer leaflet. Between these two layers is the periplasmic space, which contains a highly cross-linked mesh-like glycan polymer, peptidoglycan (PG). During cell expansion, coordinated synthesis of each of these components is required to maintain the integrity of the cell envelope; however, it is currently not clear how such coordination is achieved. In this study, we show that a cross-link-specific PG hydrolase couples the expansion of PG sacculus with that of PL synthesis in the Gram-negative model bacterium, Escherichia coli. We find that unregulated activity of a PG hydrolytic enzyme, MepS is detrimental for growth of E. coli during fatty acid (FA)-limiting conditions. Further genetic and biochemical analyses revealed that cellular availability of FA or PL alters the post-translational stability of MepS by modulating the proteolytic activity of a periplasmic adaptor-protease complex, NlpI-Prc toward MepS. Our results indicate that loss of OM lipid asymmetry caused by alterations in PL abundance leads to the generation of a signal to the NlpI-Prc complex for the stabilization of MepS, which subsequently cleaves the cross-links to facilitate expansion of PG. In summary, our study shows the existence of a molecular cross-talk that enables coordinated expansion of the PG sacculus with that of membrane synthesis for balanced cell-envelope biogenesis.

Mechanistic insights into the regulation of cell wall hydrolysis by FtsEX and EnvC at the bacterial division site.

The peptidoglycan (PG) cell wall produced by the bacterial division machinery is initially shared between the daughters and must be split to promote cell separation and complete division. In gram-negative bacteria, enzymes that cleave PG called amidases play major roles in the separation process. To prevent spurious cell wall cleavage that can lead to cell lysis, amidases like AmiB are autoinhibited by a regulatory helix. Autoinhibition is relieved at the division site by the activator EnvC, which is in turn regulated by the ATP-binding cassette (ABC) transporter-like complex called FtsEX. EnvC is also known to be autoinhibited by a regulatory helix (RH), but how its activity is modulated by FtsEX and the mechanism by which it activates the amidases have remained unclear. Here, we investigated this regulation by determining the structure of Pseudomonas aeruginosa FtsEX alone with or without bound ATP, in complex with EnvC, and in a FtsEX-EnvC-AmiB supercomplex. In combination with biochemical studies, the structures reveal that ATP binding is likely to activate FtsEX-EnvC and promote its association with AmiB. Furthermore, the AmiB activation mechanism is shown to involve a RH rearrangement. In the activated state of the complex, the inhibitory helix of EnvC is released, freeing it to associate with the RH of AmiB, which liberates its active site for PG cleavage. These regulatory helices are found in many EnvC proteins and amidases throughout gram-negative bacteria, suggesting that the activation mechanism is broadly conserved and a potential target for lysis-inducing antibiotics that misregulate the complex.