Peptidoglycan-associated lipoprotein contributes to the virulence of Acinetobacter baumannii and serves as a vaccine candidate.

The role of peptidoglycan-associated lipoprotein (Pal) in A. baumannii pathogenesis remains unclear. Here, we illustrated its role by constructing a pal deficient A. baumannii mutant and its complementary strain.Transcriptome analysis of the WT and pal mutant revealed a total of 596 differentially expressed genes. Gene Ontology analysis revealed that pal deficiency caused the downregulation of genes related to material transport and metabolic processes. The pal mutant showed a slower growth and was sensitive to detergent and serum killing compared to WT strain, whereas, the complemented pal mutant showed rescued phenotype. The pal mutant caused decreased mortality in mice pneumonia infection compared to WT strain, while the complemented pal mutant showed increased mortality. Mice immunized with recombinant Pal showed 40% protection against A. baumannii-mediated pneumonia. Collectively, these data indicate Pal is a virulence factor of A. baumannii and may serve as a potential target for preventive or therapeutic interventions.

Cytobacillus citreus sp. nov., isolated from citrus rhizosphere soil.

A Gram-stain-positive, rod-shaped and motile strain, designated FJAT-49705T, was isolated from the citrus rhizosphere soil sample. Strain FJAT-49705T grew at 20-40 °C (optimum, 30 °C) and pH 6.0-11.0 (optimum, pH 7.0) with 0-5 % (w/v) NaCl (optimum, 2 %). Strain FJAT-49705T showed high 16S rRNA gene sequence similarity to 'Bacillus dafuensis' FJAT-25496T (99.7 %) and Cytobacillus solani FJAT-18043T (98.0 %). In phylogenetic (based on 16S rRNA gene sequences) and phylogenomic trees (based on 71 bacterial single-copy genes), strain FJAT-49705T clustered with the members of the genus Cytobacillus. MK-7 was the only isoprenoid quinone present. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The genomic DNA G+C content was 36.9 %. The average nucleotide identity (ANI) values between FJAT-49705T and 'B. dafuensis' FJAT-25496T and C. solani FJAT-18043T were below the cut-off level (95-96 %) recommended as the ANI criterion for interspecies identity. Based on the above results, strain FJAT-49705T represents a novel species of the genus Cytobacillus, for which the name Cytobacillus citreus sp. nov. is proposed. The type strain is FJAT-49705T (=CCTCC AB 2019243T= LMG 31580T).

Autolysin-mediated peptidoglycan hydrolysis is required for the surface display of Staphylococcus aureus cell wall-anchored proteins.

Peptidoglycan hydrolases, or autolysins, play a critical role in cell wall remodeling and degradation, facilitating bacterial growth, cell division, and cell separation. In Staphylococcus aureus, the so-called "major" autolysin, Atl, has long been associated with host adhesion; however, the molecular basis underlying this phenomenon remains understudied. To investigate, we used the type V glycopeptide antibiotic complestatin, which binds to peptidoglycan and blocks the activity of autolysins, as a chemical probe of autolysin function. We also generated a chromosomally encoded, catalytically inactive variant of the Atl enzyme. Autolysin-mediated peptidoglycan hydrolysis, in particular Atl-mediated daughter cell separation, was shown to be critical for maintaining optimal surface levels of S. aureus cell wall-anchored proteins, including the fibronectin-binding proteins (FnBPs) and protein A (Spa). As such, disrupting autolysin function reduced the affinity of S. aureus for host cell ligands, and negatively impacted early stages of bacterial colonization in a systemic model of S. aureus infection. Phenotypic studies revealed that Spa was sequestered at the septum of complestatin-treated cells, highlighting that autolysins are required to liberate Spa during cell division. In summary, we reveal the hydrolytic activities of autolysins are associated with the surface display of S. aureus cell wall-anchored proteins. We demonstrate that by blocking autolysin function, type V glycopeptide antibiotics are promising antivirulence agents for the development of strategies to control S. aureus infections.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/ß-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Visualization of Peptidoglycan Structures of Escherichia coli by Quick-Freeze Deep-Etch Electron Microscopy.

Peptidoglycan (PG) is an essential component of the bacterial cell wall that protects the cell from turgor pressure and maintains its shape. In diderm (gram-negative) bacteria, such as Escherichia coli, the PG layer is flexible with a thickness of a 2-6 nm, and its visualization is difficult due to the presence of the outer membrane. The quick-freeze deep-etch replica method has been widely used for the visualization of flexible structures in cell interior, such as cell organelles and membrane components. In this technique, a platinum replica on the surface of a specimen fixed by freezing is observed using a transmission electron microscope. In this chapter, we describe the application of this method for visualizing the E. coli PG layer. We expect that these methods will be useful for the visualization of the PG layer in diverse bacterial species.

The Chloroplast Envelope of Angiosperms Contains a Peptidoglycan Layer.

Plastids in plants are assumed to have evolved from cyanobacteria as they have maintained several bacterial features. Recently, peptidoglycans, as bacterial cell wall components, have been shown to exist in the envelopes of moss chloroplasts. Phylogenomic comparisons of bacterial and plant genomes have raised the question of whether such structures are also part of chloroplasts in angiosperms. To address this question, we visualized canonical amino acids of peptidoglycan around chloroplasts of Arabidopsis and Nicotiana via click chemistry and fluorescence microscopy. Additional detection by different peptidoglycan-binding proteins from bacteria and animals supported this observation. Further Arabidopsis experiments with D-cycloserine and AtMurE knock-out lines, both affecting putative peptidoglycan biosynthesis, revealed a central role of this pathway in plastid genesis and division. Taken together, these results indicate that peptidoglycans are integral parts of plastids in the whole plant lineage. Elucidating their biosynthesis and further roles in the function of these organelles is yet to be achieved.

Peptidoglycan recognition protein MsPGRP in largemouth bass (Micropterus salmoides) mediates immune functions with broad nonself recognition ability.

Peptidoglycan (PGN) recognition proteins (PGRPs) are important immune factors in innate immunity that function in recognising pathogens and activating the immune system. These ubiquitous proteins are conserved in invertebrates and vertebrates. In this study, a PGRP gene (MsPGRP) from largemouth bass (Micropterus salmoides) was identified and characterised, and its transcription distribution was explored. Recombinant protein (rMsPGRP) exhibited dose-dependent binding to PGN and glucan (GLU), but weak binding to lipopolysaccharide (LPS). MsPGRP exhibited agglutinating activity against several Gram-negative bacteria, Gram-positive bacteria and fungi, and it promoted phagocytosis activity of leukocytes against Micrococcus luteus and Aeromonas hydrophila. The protein also possessed amidase activity in the presence of Zn2+, degraded PGN, and disrupted the M. luteus cell wall. The results suggest that MsPGRP plays an important role in pathogen recognition, and acts as a opsonin during immune system responses and elimination of invading pathogens.

Identification and characterization of two long-type peptidoglycan recognition proteins, PGRP-L1 and PGRP-L2, in the orange-spotted grouper, Epinephelus coioides.

Peptidoglycan recognition proteins (PGRPs) play an important role in innate immunity by recognizing components of pathogenic bacteria (such as peptidoglycan, PGN) and are evolutionarily conserved pattern recognition receptors (PRRs) in both invertebrates and vertebrates. In the present study, two long-type PGRPs (designed as Eco-PGRP-L1 and Eco-PGRP-L2) were identified in orange-spotted grouper (Epinephelus coioides), which is a major economic species cultured in Asia. The predicted protein sequences of both Eco-PGRP-L1 and Eco-PGRP-L2 contain a typical PGRP domain. Eco-PGRP-L1 and Eco-PGRP-L2 exhibited organ/tissue-specific expression patterns. An abundant expression of Eco-PGRP-L1 was observed in pyloric caecum, stomach and gill, whereas a highest expression level of Eco-PGRP-L2 was found in head kidney, spleen, skin and heart. In addition, Eco-PGRP-L1 is distributed in the cytoplasm and nucleus, while Eco-PGRP-L2 is mainly localized in cytoplasm. Both Eco-PGRP-L1 and Eco-PGRP-L2 were induced following the stimulation of PGN and have PGN binding activity. In addition, functional analysis revealed that Eco-PGRP-L1 and Eco-PGRP-L2 possess antibacterial activity against Edwardsiella tarda. These results may contribute to understand the innate immune system of orange-spotted grouper.

Chloride Ions Are Required for Thermosipho africanus MurJ Function.

Most bacteria have a peptidoglycan cell wall that determines their cell shape and helps them resist osmotic lysis. Peptidoglycan synthesis depends on the translocation of the lipid-linked precursor lipid II across the cytoplasmic membrane by the MurJ flippase. Structure-function analyses of MurJ from Thermosipho africanus (MurJTa) and Escherichia coli (MurJEc) have revealed that MurJ adopts multiple conformations and utilizes an alternating-access mechanism to flip lipid II. MurJEc activity relies on membrane potential, but the specific counterion has not been identified. Crystal structures of MurJTa revealed a chloride ion bound to the N-lobe of the flippase and a sodium ion in its C-lobe, but the role of these ions in transport is unknown. Here, we investigated the effect of various ions on the function of MurJTa and MurJEc in vivo. We found that chloride, and not sodium, ions are necessary for MurJTa function, but neither ion is required for MurJEc function. We also showed that murJTa alleles encoding changes at the crystallographically identified sodium-binding site still complement the loss of native murJEc, although they decreased protein stability and/or function. Based on our data and previous work, we propose that chloride ions are necessary for the conformational change that resets MurJTa after lipid II translocation and suggest that MurJ orthologs may function similarly but differ in their requirements for counterions. IMPORTANCE The biosynthetic pathway of the peptidoglycan cell wall is one of the most favorable targets for antibiotic development. Lipid II, the lipid-linked PG precursor, is made in the inner leaflet of the cytoplasmic membrane and then transported by the MurJ flippase so that it can be used to build the peptidoglycan cell wall. MurJ functions using an alternating-access mechanism thought to depend on a yet-to-be-identified counterion. This study fills a gap in our understanding of MurJ's energy-coupling mechanism by showing that chloride ions are required for MurJ in some, but not all, organisms. Based on our data and prior studies, we propose that, while the general transport mechanism of MurJ may be conserved, its specific mechanistic details may differ across bacteria, as is common in transporters. These findings are important to understand MurJ function and its development as an antibiotic target.

An NlpC/P60 protein catalyzes a key step in peptidoglycan recycling at the intersection of energy recovery, cell division and immune evasion in the intracellular pathogen Chlamydia trachomatis.

The obligate intracellular Chlamydiaceae do not need to resist osmotic challenges and thus lost their cell wall in the course of evolution. Nevertheless, these pathogens maintain a rudimentary peptidoglycan machinery for cell division. They build a transient peptidoglycan ring, which is remodeled during the process of cell division and degraded afterwards. Uncontrolled degradation of peptidoglycan poses risks to the chlamydial cell, as essential building blocks might get lost or trigger host immune response upon release into the host cell. Here, we provide evidence that a primordial enzyme class prevents energy intensive de novo synthesis and uncontrolled release of immunogenic peptidoglycan subunits in Chlamydia trachomatis. Our data indicate that the homolog of a Bacillus NlpC/P60 protein is widely conserved among Chlamydiales. We show that the enzyme is tailored to hydrolyze peptidoglycan-derived peptides, does not interfere with peptidoglycan precursor biosynthesis, and is targeted by cysteine protease inhibitors in vitro and in cell culture. The peptidase plays a key role in the underexplored process of chlamydial peptidoglycan recycling. Our study suggests that chlamydiae orchestrate a closed-loop system of peptidoglycan ring biosynthesis, remodeling, and recycling to support cell division and maintain long-term residence inside the host. Operating at the intersection of energy recovery, cell division and immune evasion, the peptidoglycan recycling NlpC/P60 peptidase could be a promising target for the development of drugs that combine features of classical antibiotics and anti-virulence drugs.

Growth Arrest of Staphylococcus aureus Induces Daptomycin Tolerance via Cell Wall Remodelling.

Almost all bactericidal drugs require bacterial replication and/or metabolic activity for their killing activity. When these processes are inhibited by bacteriostatic antibiotics, bacterial killing is significantly reduced. One notable exception is the lipopeptide antibiotic daptomycin, which has been reported to efficiently kill growth-arrested bacteria. However, these studies employed only short periods of growth arrest (<1 h), which may not fully represent the duration of growth arrest that can occur in vivo. We found that a growth inhibitory concentration of the protein synthesis inhibitor tetracycline led to a time-dependent induction of daptomycin tolerance in S. aureus, with an approximately 100,000-fold increase in survival after 16 h of growth arrest, relative to exponential-phase bacteria. Daptomycin tolerance required glucose and was associated with increased production of the cell wall polymers peptidoglycan and wall-teichoic acids. However, while the accumulation of peptidoglycan was required for daptomycin tolerance, only a low abundance of wall teichoic acid was necessary. Therefore, whereas tolerance to most antibiotics occurs passively due to a lack of metabolic activity and/or replication, daptomycin tolerance arises via active cell wall remodelling. IMPORTANCE Understanding why antibiotics sometimes fail to cure infections is fundamental to improving treatment outcomes. This is a major challenge when it comes to Staphylococcus aureus because this pathogen causes several different chronic or recurrent infections. Previous work has shown that a lack of replication, as often occurs during infection, makes bacteria tolerant of most bactericidal antibiotics. However, one antibiotic that has been reported to kill nonreplicating bacteria is daptomycin. In this work, we show that the growth arrest of S. aureus does in fact lead to daptomycin tolerance, but it requires time, nutrients, and biosynthetic pathways, making it distinct from other types of antibiotic tolerance that occur in nonreplicating bacteria.

Insights into the Roles of Lipoteichoic Acids and MprF in Bacillus subtilis.

Gram-positive bacterial cells are protected from the environment by a cell envelope that is comprised of a thick layer of peptidoglycan that maintains cell shape and teichoic acid polymers whose biological function remains unclear. In Bacillus subtilis, the loss of all class A penicillin-binding proteins (aPBPs), which function in peptidoglycan synthesis, is conditionally lethal. Here, we show that this lethality is associated with an alteration of lipoteichoic acids (LTAs) and the accumulation of the major autolysin LytE in the cell wall. Our analysis provides further evidence that the length and abundance of LTAs act to regulate the cellular level and activity of autolytic enzymes, specifically LytE. Importantly, we identify a novel function for the aminoacyl-phosphatidylglycerol synthase MprF in the modulation of LTA biosynthesis in both B. subtilis and Staphylococcus aureus. This finding has implications for our understanding of antimicrobial resistance (particularly to daptomycin) in clinically relevant bacteria and the involvement of MprF in the virulence of pathogens such as methicillin-resistant S. aureus (MRSA). IMPORTANCE In Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, the cell envelope is a structure that protects the cells from the environment but is also dynamic in that it must be modified in a controlled way to allow cell growth. In this study, we show that lipoteichoic acids (LTAs), which are anionic polymers attached to the membrane, have a direct role in modulating the cellular abundance of cell wall-degrading enzymes. We also find that the apparent length of the LTA is modulated by the activity of the enzyme MprF, previously implicated in modifications of the cell membrane leading to resistance to antimicrobial peptides. These findings are important contributions to our understanding of how bacteria balance cell wall synthesis and degradation to permit controlled growth and division. These results also have implications for the interpretation of antibiotic resistance, particularly for the clinical treatment of MRSA infections.

Regulation of peptidoglycan hydrolases: localization, abundance, and activity.

Most bacteria are surrounded by a cell wall composed of peptidoglycan (PG) that specifies shape and protects the cell from osmotic rupture. Growth, division, and morphogenesis are intimately linked to the synthesis of this exoskeleton but also its hydrolysis. The enzymes that cleave the PG meshwork require careful control to prevent aberrant hydrolysis and loss of envelope integrity. Bacteria employ diverse mechanisms to control the activity, localization, and abundance of these potentially autolytic enzymes. Here, we discuss four examples of how cells integrate these control mechanisms to finely tune cell wall hydrolysis. We highlight recent advances and exciting avenues for future investigation.

Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-ß-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens, while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N2-fixing efficiency of the peanut nodules is three times higher than that of S. flavescens. By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

In situ visualization of Braun's lipoprotein on E. coli sacculi.

Braun's lipoprotein (Lpp) plays a major role in stabilizing the integrity of the cell envelope in Escherichia coli, as it provides a covalent cross-link between the outer membrane and the peptidoglycan layer. An important challenge in elucidating the physiological role of Lpp lies in attaining a detailed understanding of its distribution on the peptidoglycan layer. Here, using atomic force microscopy, we visualized Lpp directly on peptidoglycan sacculi. Lpp is homogeneously distributed over the outer surface of the sacculus at a high density. However, it is absent at the constriction site during cell division, revealing its role in the cell division process with Pal, another cell envelope-associated protein. Collectively, we have established a framework to elucidate the distribution of Lpp and other peptidoglycan-bound proteins via a direct imaging modality.

Interaction of the periplasmic chaperone SurA with the inner membrane protein secretion (SEC) machinery.

Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the ß-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC - aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.

Common carp Peptidoglycan Recognition Protein 2 (CcPGRP2) plays a role in innate immunity for defense against bacterial infections.

PGRP is a family of pattern recognition molecules of the innate immune system. PGRPs are conserved from insects to mammals and have diverse functions in antimicrobial defense. Here we cloned a common carp PGRP ortholog, CcPGRP2 containing a conserved C-terminal PGRP domain. We tested the expression levels of CcPGRP2 in the liver, spleen, kidney, foregut, midgut, and hindgut of the highest level in the liver. The expression of CcPGRP2 upregulated in common carp infected with Aeromonas hydrophila (A. hydrophila) or Staphylococcus aureus (S. aureus). Recombinant CcPGRP2 protein expressed in Escherichia coli (E. coli) system and the purified CcPGRP2 could maintain the integrity of intestinal mucosa of common carp infected with A. hydrophila. In addition, CcPGRP2 could agglutinate or bind both gram-positive and gram-negative bacteria in a Zn2+-dependent manner. CcPGRP2 has a stronger agglutination and bacterial binding ability in gram-positive bacteria than in gram-negative bacteria. It is perhaps because CcPGRP2 could bind peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). And CcPGRP2 shows antimicrobial activities in the presence of Zn2+. Our results of CcPGRP2 provided new insight into the function of PGRP in the innate immunity of the common carp.

Deep Antimicrobial Activity and Stability Analysis Inform Lysin Sequence-Function Mapping.

Antibiotic-resistant infectious disease is a critical challenge to human health. Antimicrobial proteins offer a compelling solution if engineered for potency, selectivity, and physiological stability. Lysins, which lyse cells via degradation of cell wall peptidoglycans, have significant potential to fill this role. Yet, the functional complexity of antimicrobial activity has hindered high-throughput characterization for discovery and design. To dramatically expand knowledge of the sequence-function landscape of lysins, we developed a depletion-based assay for library-scale measurement of lysin inhibitory activity. We coupled this platform with a high-throughput proteolytic stability assay to assess the activity and stability of ∼5 × 104 lysin catalytic domain variants, resulting in the discovery of a variant with increased activity (70 ± 20%) and stability (7.2 ± 0.4 °C increased midpoint of thermal denaturation). Ridge regression of the resulting data set demonstrated that libraries with a higher average Hamming distance better informed pairwise models and that coupling activity and stability assays enabled better prediction of catalytically active lysins. The best models achieved Pearson's correlation coefficients of 0.87 ± 0.01 and 0.61 ± 0.04 for predicting catalytic domain stability and activity, respectively. Our work provides an efficient strategy for constructing protein sequence-function landscapes, drastically increases screening throughput for engineering lysins, and yields promising lysins for further development.

L-form conversion in Gram-positive bacteria enables escape from phage infection.

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

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.