Clinical Strains of Mycobacterium tuberculosis Representing Different Genotype Families Exhibit Distinct Propensities to Adopt the Differentially Culturable State.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

Amidation of glutamate residues in mycobacterial peptidoglycan is essential for cell wall cross-linking.

Introduction: Mycobacteria assemble a complex cell wall with cross-linked peptidoglycan (PG) which plays an essential role in maintenance of cell wall integrity and tolerance to osmotic pressure. We previously demonstrated that various hydrolytic enzymes are required to remodel PG during essential processes such as cell elongation and septal hydrolysis. Here, we explore the chemistry associated with PG cross-linking, specifically the requirement for amidation of the D-glutamate residue found in PG precursors. Methods: Synthetic fluorescent probes were used to assess PG remodelling dynamics in live bacteria. Fluorescence microscopy was used to assess protein localization in live bacteria and CRISPR-interference was used to construct targeted gene knockdown strains. Time-lapse microscopy was used to assess bacterial growth. Western blotting was used to assess protein phosphorylation. Results and discussion: In Mycobacterium smegmatis, we confirmed the essentiality for D-glutamate amidation in PG biosynthesis by labelling cells with synthetic fluorescent PG probes carrying amidation modifications. We also used CRISPRi targeted knockdown of genes encoding the MurT-GatD complex, previously implicated in D-glutamate amidation, and demonstrated that these genes are essential for mycobacterial growth. We show that MurT-rseGFP co-localizes with mRFP-GatD at the cell poles and septum, which are the sites of cell wall synthesis in mycobacteria. Furthermore, time-lapse microscopic analysis of MurT-rseGFP localization, in fluorescent D-amino acid (FDAA)-labelled mycobacterial cells during growth, demonstrated co-localization with maturing PG, suggestive of a role for PG amidation during PG remodelling and repair. Depletion of MurT and GatD caused reduced PG cross-linking and increased sensitivity to lysozyme and ß-lactam antibiotics. Cell growth inhibition was found to be the result of a shutdown of PG biosynthesis mediated by the serine/threonine protein kinase B (PknB) which senses uncross-linked PG. Collectively, these data demonstrate the essentiality of D-glutamate amidation in mycobacterial PG precursors and highlight the MurT-GatD complex as a novel drug target.

Structure-Activity Relationship Study of Cannabidiol-Based Analogs as Negative Allosteric Modulators of the µ-Opioid Receptor.

The US faces an unprecedented surge in fatal drug overdoses. Naloxone, the only antidote for opiate overdose, competes at the mu opioid receptor (µOR) orthosteric site. Naloxone struggles against fentanyl-class synthetic opioids that now cause ∼80% of deaths. Negative allosteric modulators (NAMs) targeting secondary sites may noncompetitively downregulate µOR activation. (-)-Cannabidiol ((-)-CBD) is a candidate µOR NAM. To explore its therapeutic potential, we evaluated the structure-activity relationships among CBD analogs to identify NAMs with increased potency. Using a cyclic AMP assay, we characterize reversal of µOR activation by 15 CBD analogs, several of which proved more potent than (-)-CBD. Comparative docking investigations suggest that potent compounds interact with a putative allosteric pocket to stabilize the inactive µOR conformation. Finally, these compounds enhance naloxone displacement of fentanyl from the orthosteric site. Our results suggest that CBD analogs offer considerable potential for the development of next-generation antidotes for opioid overdose.

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis.

Enterococcus faecalis virulence requires cell wall-associated proteins, including the sortase-assembled endocarditis and biofilm associated pilus (Ebp), important for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that sortases attach substrates to lipid II peptidoglycan (PG) precursors, prior to their incorporation into the growing cell wall. Contrary to prevailing dogma, by following the distribution of Ebp and PG throughout the E. faecalis cell cycle, we found that cell surface Ebp do not co-localize with newly synthesized PG. Instead, surface-exposed Ebp are localized to the older cell hemisphere and excluded from sites of new PG synthesis at the septum. Moreover, Ebp deposition on the younger hemisphere of the E. faecalis diplococcus appear as foci adjacent to the nascent septum. We propose a new model whereby sortase substrate deposition can occur on older PG rather than at sites of new cell wall synthesis. Consistent with this model, we demonstrate that sequestering lipid II to block PG synthesis via ramoplanin, does not impact new Ebp deposition at the cell surface. These data support an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto uncrosslinked cell wall, independent of new PG synthesis.

Dynamics of plasmid-mediated niche invasion, immunity to invasion, and pheromone-inducible conjugation in the murine gastrointestinal tract.

Microbial communities provide protection to their hosts by resisting pathogenic invasion. Microbial residents of a host often exclude subsequent colonizers, but this protection is not well understood. The Enterococcus faecalis plasmid pCF10, whose conjugative transfer functions are induced by a peptide pheromone, efficiently transfers in the intestinal tract of mice. Here we show that an invading donor strain established in the gastrointestinal tract of mice harboring resident recipients, resulting in a stable, mixed population comprised of approximately 10% donors and 90% recipients. We also show that the plasmid-encoded surface protein PrgB (Aggregation Substance), enhanced donor invasion of resident recipients, and resistance of resident donors to invasion by recipients. Imaging of the gastrointestinal mucosa of mice infected with differentially labeled recipients and donors revealed pheromone induction within microcolonies harboring both strains in close proximity, suggesting that adherent microcolonies on the mucosal surface of the intestine comprise an important niche for cell-cell signaling and plasmid transfer.

The bacterial tyrosine kinase system CpsBCD governs the length of capsule polymers.

Many pathogenic bacteria are encased in a layer of capsular polysaccharide (CPS). This layer is important for virulence by masking surface antigens, preventing opsonophagocytosis, and avoiding mucus entrapment. The bacterial tyrosine kinase (BY-kinase) regulates capsule synthesis and helps bacterial pathogens to survive different host niches. BY-kinases autophosphorylate at the C-terminal tyrosine residues upon external stimuli, but the role of phosphorylation is still unclear. Here, we report that the BY-kinase CpsCD is required for growth in Streptococcus pneumoniae Cells lacking a functional cpsC or cpsD accumulated low molecular weight CPS and lysed because of the lethal sequestration of the lipid carrier undecaprenyl phosphate, resulting in inhibition of peptidoglycan (PG) synthesis. CpsC interacts with CpsD and the polymerase CpsH. CpsD phosphorylation reduces the length of CPS polymers presumably by controlling the activity of CpsC. Finally, pulse-chase experiments reveal the spatiotemporal coordination between CPS and PG synthesis. This coordination is dependent on CpsC and CpsD. Together, our study provides evidence that BY-kinases regulate capsule polymer length by fine-tuning CpsC activity through autophosphorylation.

Unipolar Peptidoglycan Synthesis in the Rhizobiales Requires an Essential Class A Penicillin-Binding Protein.

Members of the Rhizobiales are polarly growing bacteria that lack homologs of the canonical Rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant pathogen Agrobacterium tumefaciens. The development of fluorescent d-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an additional mode of cell wall synthesis that requires ld-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation. IMPORTANCE While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation. Yet the vast majority of our understanding regarding bacterial elongation is derived from model organisms exhibiting lateral elongation. Here, we explore the role of penicillin-binding proteins in unipolar elongation of Agrobacterium tumefaciens and related bacteria within the Rhizobiales. Our findings suggest that penicillin-binding protein 1a, along with a subset of ld-transpeptidases, drives unipolar growth. Thus, these enzymes may serve as attractive targets for biocontrol of pathogenic Rhizobiales.

Class A Penicillin-Binding Protein-Mediated Cell Wall Synthesis Promotes Structural Integrity during Peptidoglycan Endopeptidase Insufficiency in Vibrio cholerae.

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by "autolytic" enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae, highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae, aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion.IMPORTANCE Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are necessary for proper cell elongation and division. aPBPs become essential for maintaining structural integrity during EP insufficiency, while the Rod system remains active but contributes little to cell expansion under these conditions. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the coordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

A Division of Labor in the Recruitment and Topological Organization of a Bacterial Morphogenic Complex.

Bacteria come in an array of shapes and sizes, but the mechanisms underlying diverse morphologies are poorly understood. The peptidoglycan (PG) cell wall is the primary determinant of cell shape. At the molecular level, morphological variation often results from the regulation of enzymes involved in cell elongation and division. These enzymes are spatially controlled by cytoskeletal scaffolding proteins, which both recruit and organize the PG synthesis complex. How then do cells define alternative morphogenic processes that are distinct from cell elongation and division? To address this, we have turned to the specific morphotype of Alphaproteobacterial stalks. Stalk synthesis is a specialized form of zonal growth, which requires PG synthesis in a spatially constrained zone to extend a thin cylindrical projection of the cell envelope. The morphogen SpmX defines the site of stalk PG synthesis, but SpmX is a PG hydrolase. How then does a non-cytoskeletal protein, SpmX, define and constrain PG synthesis to form stalks? Here, we report that SpmX and the bactofilin BacA act in concert to regulate stalk synthesis in Asticcacaulis biprosthecum. We show that SpmX recruits BacA to the site of stalk synthesis. BacA then serves as a stalk-specific topological organizer for PG synthesis activity, including its recruiter SpmX, at the base of the stalk. In the absence of BacA, cells produce "pseudostalks" that are the result of unconstrained PG synthesis. Therefore, the protein responsible for recruitment of a morphogenic PG remodeling complex, SpmX, is distinct from the protein that topologically organizes the complex, BacA.

The Pneumococcal Iron Uptake Protein A (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes.

Streptococcus pneumoniae (Spn) is an important Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Fe acquisition is a crucial virulence determinant in Spn; further, Spn relies on exogenous FeIII-siderophore scavenging to meet nutritional Fe needs. Recent studies suggest that the human catecholamine stress hormone, norepinephrine (NE), facilitates Fe acquisition in Spn under conditions of transferrin-mediated Fe starvation. Here we show that the solute binding lipoprotein PiuA from the piu Fe acquisition ABC transporter PiuBCDA, previously described as an Fe-hemin binding protein, binds tetradentate catechol FeIII complexes, including NE and the hydrolysis products of enterobactin. Two protein-derived ligands (H238, Y300) create a coordinately saturated FeIII complex, which parallel recent studies in the Gram-negative intestinal pathogen Campylobacter jejuni. Our in vitro studies using NMR spectroscopy and 54Fe LC-ICP-MS confirm the FeIII can move from transferrin to apo-PiuA in an NE-dependent manner. Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus.

Non-walled spherical Acinetobacter baumannii is an important type of persister upon ß-lactam antibiotic treatment.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. ß-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from ß-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of ß-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by ß-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during ß-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

Distinct cytoskeletal proteins define zones of enhanced cell wall synthesis in Helicobacter pylori.

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori, but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori. Quantitative 3D microscopy analysis of short pulses with either N-acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape.

Round spheres, straight rods, and twisting corkscrews, bacteria come in many different shapes. The shape of bacteria is dictated by their cell wall, the strong outer barrier of the cell. As bacteria grow and multiply, they must add to their cell wall while keeping the same basic shape. The cells walls are made from long chain-like molecules via processes that are guided by protein scaffolds within the cell. Many common antibiotics, including penicillin, stop bacterial infections by interrupting the growth of cell walls. Helicobacter pylori is a common bacterium that lives in the gut and, after many years, can cause stomach ulcers and stomach cancer. H. pylori are shaped in a twisting helix, much like a corkscrew. This shape helps H. pylori to take hold and colonize the stomach. It remains unclear how H. pylori creates and maintains its helical shape. The helix is much more curved than other bacteria, and H. pylori does not have the same helpful proteins that other curved bacteria do. If H. pylori grows asymmetrically, adding more material to the cell wall on its long outer side to create a twisting helix, what controls the process? To find out, Taylor et al. grew H. pylori cells and watched how the cell walls took shape. First, a fluorescent dye was attached to the building blocks of the cell wall or to underlying proteins that were thought to help direct its growth. The cells were then imaged in 3D, and images from hundreds of cells were reconstructed to analyze the growth patterns of the bacteria's cell wall. A protein called CcmA was found most often on the long side of the twisting H. pylori. When the CcmA protein was isolated in a dish, it spontaneously formed sheets and helical bundles, confirming its role as a structural scaffold for the cell wall. When CcmA was absent from the cell of H. pylori, Taylor et al. observed that the pattern of cell growth changed substantially. This work identifies a key component directing the growth of the cell wall of H. pylori and therefore, a new target for antibiotics. Its helical shape is essential for H. pylori to infect the gut, so blocking the action of the CcmA protein may interrupt cell wall growth and prevent stomach infections.

Mechanisms of Incorporation for D-Amino Acid Probes That Target Peptidoglycan Biosynthesis.

Bacteria exhibit a myriad of different morphologies, through the synthesis and modification of their essential peptidoglycan (PG) cell wall. Our discovery of a fluorescent D-amino acid (FDAA)-based PG labeling approach provided a powerful method for observing how these morphological changes occur. Given that PG is unique to bacterial cells and a common target for antibiotics, understanding the precise mechanism(s) for incorporation of (F)DAA-based probes is a crucial determinant in understanding the role of PG synthesis in bacterial cell biology and could provide a valuable tool in the development of new antimicrobials to treat drug-resistant antibacterial infections. Here, we systematically investigate the mechanisms of FDAA probe incorporation into PG using two model organisms Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive). Our in vitro and in vivo data unequivocally demonstrate that these bacteria incorporate FDAAs using two extracytoplasmic pathways: through activity of their D,D-transpeptidases, and, if present, by their L,D-transpeptidases and not via cytoplasmic incorporation into a D-Ala-D-Ala dipeptide precursor. Our data also revealed the unprecedented finding that the DAA-drug, D-cycloserine, can be incorporated into peptide stems by each of these transpeptidases, in addition to its known inhibitory activity against D-alanine racemase and D-Ala-D-Ala ligase. These mechanistic findings enabled development of a new, FDAA-based, in vitro labeling approach that reports on subcellular distribution of muropeptides, an especially important attribute to enable the study of bacteria with poorly defined growth modes. An improved understanding of the incorporation mechanisms utilized by DAA-based probes is essential when interpreting results from high resolution experiments and highlights the antimicrobial potential of synthetic DAAs.

d-Amino Acid Derivatives as in Situ Probes for Visualizing Bacterial Peptidoglycan Biosynthesis.

The bacterial cell wall is composed of membrane layers and a rigid yet flexible scaffold called peptidoglycan (PG). PG provides mechanical strength to enable bacteria to resist damage from the environment and lysis due to high internal turgor. PG also has a critical role in dictating bacterial cell morphology. The essential nature of PG for bacterial propagation, as well as its value as an antibiotic target, has led to renewed interest in the study of peptidoglycan biosynthesis. However, significant knowledge gaps remain that must be addressed before a clear understanding of peptidoglycan synthesis and dynamics is realized. For example, the enzymes involved in the PG biosynthesis pathway have not been fully characterized. Our understanding of PG biosynthesis has been frequently revamped by the discovery of novel enzymes or newly characterized functions of known enzymes. In addition, we do not clearly know how the respective activities of these enzymes are coordinated with each other and how they control the spatial and temporal dynamics of PG synthesis. The emergence of molecular probes and imaging techniques has significantly advanced the study PG synthesis and modification. Prior efforts utilized the specificity of PG-targeting antibiotics and proteins to develop PG-specific probes, such as fluorescent vancomycin and fluorescent wheat germ agglutinin. However, these probes suffer from limitations due to toxic effects toward bacterial cells and poor membrane permeability. To address these issues, we designed and introduced a family of novel molecular probes, fluorescent d-amino acids (FDAAs), which are covalently incorporated into PG through the activities of endogenous bacterial transpeptidases. Their high biocompatibility and PG specificity have made them powerful tools for labeling peptidoglycan. In addition, their enzyme-mediated incorporation faithfully reflects the activity of PG synthases, providing a direct in situ method for studying PG formation during the bacterial life cycle. In this Account, we describe our efforts directed at the development of FDAAs and their derivatives. These probes have enabled for the first time the ability to visualize PG synthesis in live bacterial cells and in real time. We summarize experimental evidence for FDAA incorporation into PG and the enzyme-mediated incorporation pathway. We demonstrate various applications of FDAAs, including bacterial morphology analyses, PG growth model studies, investigation of PG-enzyme correlation, in vitro PG synthase activity assays, and antibiotic inhibition tests. Finally, we discuss the current limitations of the probes and our ongoing efforts to improve them. We are confident that these probes will prove to be valuable tools that will enable the discovery of new antibiotic targets and expand the available arsenal directed at the public health threat posed by antibiotic resistance.

Spheroplast-Mediated Carbapenem Tolerance in Gram-Negative Pathogens.

Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen Vibrio cholerae can tolerate exposure to the typically bactericidal ß-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread ß-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae, but not Escherichia coli, exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.

SEDS-bPBP pairs direct lateral and septal peptidoglycan synthesis in Staphylococcus aureus.

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure that is essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely, through the localization of late-stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in elongation and division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 probably constitute cognate pairs of interacting proteins. A lack of RodA-PBP3 resulted in more spherical cells due to deficient sidewall PGN synthesis, whereas depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilizing the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings or arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate sidewall and septal PGN incorporation, respectively, and that their activity must be balanced to maintain coccoid morphology.

Scalable Synthesis of Orthogonally Protected ß-Methyllanthionines by Indium(III)-Mediated Ring Opening of Aziridines.

Lantibiotics are a class of peptide antibiotics with activity against most Gram-positive bacteria. Lanthionine (Lan) and ß-MeLan are unusual thioether-bridged, non-proteinogenic amino acids, which are characteristic features of lantibiotics. In this paper, we report the facile stereoselective synthesis of ß-methyllanthionines with orthogonal protection by nucleophilic ring opening of aziridines. This method leads to an expedient access to ß-methyllanthionines and allows production of over 30 g of ß-methyllanthionine in a single batch.

An Acinetobacter baumannii, Zinc-Regulated Peptidase Maintains Cell Wall Integrity during Immune-Mediated Nutrient Sequestration.

Acinetobacter baumannii is an important nosocomial pathogen capable of causing wound infections, pneumonia, and bacteremia. During infection, A. baumannii must acquire Zn to survive and colonize the host. Vertebrates have evolved mechanisms to sequester Zn from invading pathogens by a process termed nutritional immunity. One of the most upregulated genes during Zn starvation encodes a putative cell wall-modifying enzyme which we named ZrlA. We found that inactivation of zrlA diminished growth of A. baumannii during Zn starvation. Additionally, this mutant strain displays increased cell envelope permeability, decreased membrane barrier function, and aberrant peptidoglycan muropeptide abundances. This altered envelope increases antibiotic efficacy both in vitro and in an animal model of A. baumannii pneumonia. These results establish ZrlA as a crucial link between nutrient metal uptake and cell envelope homeostasis during A. baumannii pathogenesis, which could be targeted for therapeutic development.

Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays.

Peptidoglycan is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions, which construct peptide crosslinkages between polysaccharide chains to endow mechanical strength. However, tracking the transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis and application of rotor-fluorogenic D-amino acids (RfDAAs), enabling real-time, continuous tracking of transpeptidation reactions. These probes allow peptidoglycan biosynthesis to be monitored in real time by visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D- and L,D-transpeptidases and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into peptidoglycan provides a powerful new tool to study peptidoglycan biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of peptidoglycan biosynthesis.