-
Proceedings of the National Academy of... Jun 2024Ovoid-shaped bacteria, such as (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing...
Ovoid-shaped bacteria, such as (pneumococcus), have two spatially separated peptidoglycan (PG) synthase nanomachines that locate zonally to the midcell of dividing cells. The septal PG synthase bPBP2x:FtsW closes the septum of dividing pneumococcal cells, whereas the elongasome located on the outer edge of the septal annulus synthesizes peripheral PG outward. We showed previously by sm-TIRFm that the septal PG synthase moves circumferentially at midcell, driven by PG synthesis and not by FtsZ treadmilling. The pneumococcal elongasome consists of the PG synthase bPBP2b:RodA, regulators MreC, MreD, and RodZ, but not MreB, and genetically associated proteins Class A aPBP1a and muramidase MpgA. Given its zonal location separate from FtsZ, it was of considerable interest to determine the dynamics of proteins in the pneumococcal elongasome. We found that bPBP2b, RodA, and MreC move circumferentially with the same velocities and durations at midcell, driven by PG synthesis. However, outside of the midcell zone, the majority of these elongasome proteins move diffusively over the entire surface of cells. Depletion of MreC resulted in loss of circumferential movement of bPBP2b, and bPBP2b and RodA require each other for localization and circumferential movement. Notably, a fraction of aPBP1a molecules also moved circumferentially at midcell with velocities similar to those of components of the core elongasome, but for shorter durations. Other aPBP1a molecules were static at midcell or diffusing over cell bodies. Last, MpgA displayed nonprocessive, subdiffusive motion that was largely confined to the midcell region and less frequently detected over the cell body.
Topics: Streptococcus pneumoniae; Bacterial Proteins; Penicillin-Binding Proteins; Peptidoglycan; Peptidoglycan Glycosyltransferase
PubMed: 38875147
DOI: 10.1073/pnas.2401831121 -
Fish & Shellfish Immunology Jun 2024Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal...
Myeloid differentiation factor-88 (MyD88) is a key adaptor of the toll-like receptor (TLR) signaling pathway and plays a crucial role in innate immune signal transduction in animals. However, the MyD88-mediated signal transduction mechanism in shellfish has not been well studied. In this study, a new MyD88 gene, CfMyD88-2, was identified in the Zhikong scallop, Chlamys farreri. The 1779 bp long open reading frame encodes 592 amino acids. The N-terminus of CfMyD88-2 contains a conserved death domain (DD), followed by a TIR (TLR/Interleukin-1 Receptor) domain. The results of the multi-sequence comparison showed that the TIR domain sequences were highly conserved. Phylogenetic analysis revealed that CfMyD88-2 was first associated with Mizuhopecten yessoensis MyD88-4 and Argopecten irradians MyD88-4. CfMyD88-2 mRNA was expressed in all scallop tissues, as detected by qRT-PCR, and the expression level was the highest in the mantle and hepatopancreas. In addition, CfMyD88-2 mRNA expression significantly increased after pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharide, peptidoglycan, or polyinosinic-polycytidylic acid) stimulation. The results of the co-immunoprecipitation experiments in HEK293T cells showed that both CfMyD88-1 and CfMyD88-2 interacted with the TLR protein of scallops, suggesting the existence of more than one functional TLR-MyD88 signaling axis in scallops. Dual luciferase reporter gene assays indicated that the overexpressed CfMyD88-2 in HEK293T cells activated interferon (IFN) α, IFN-β, IFN-γ, and NF-κB reporter genes, indicating that the protein has multiple functions. The results of the subcellular localization experiment uncovered that CfMyD88-2 was mainly localized in the cytoplasm of human cells. In summary, the novel identified CfMyD88-2 can respond to the challenge of PAMPs, participate in TLR immune signaling, and may activate downstream effector genes such as NF-κB gene. These research results will be useful in advancing the theory of innate immunity in invertebrates and provide a reference for the selection of disease-resistant scallops in the future.
PubMed: 38871139
DOI: 10.1016/j.fsi.2024.109697 -
Protein Science : a Publication of the... Jul 2024Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent...
Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (K < 0.5 μM) include PBPs (PBP1a, K = 0.07 μM; PBP5 = 0.4 μM); other lytic transglycosylases (SltB2, K = 0.09 μM; RlpA, K = 0.4 μM); a type VI secretion system effector (Tse5, K = 0.3 μM); and a regulatory protease for alginate biosynthesis (AlgO, K < 0.4 μM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.
Topics: Pseudomonas aeruginosa; Bacterial Proteins; Periplasm; Periplasmic Proteins; Glycosyltransferases; Peptidoglycan
PubMed: 38864725
DOI: 10.1002/pro.5038 -
Antimicrobial Agents and Chemotherapy Jun 2024
PubMed: 38864614
DOI: 10.1128/aac.00727-24 -
PLoS Pathogens Jun 2024Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune...
Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune response, resulting in improved survival upon reinfection. Here, we investigated the mechanistic basis and epidemiological consequences of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and sexually dimorphic response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.
Topics: Animals; Immunity, Innate; Drosophila melanogaster; Providencia; Drosophila Proteins; Female; Male; Enterobacteriaceae Infections; Antimicrobial Peptides
PubMed: 38857285
DOI: 10.1371/journal.ppat.1012308 -
ELife Jun 2024is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of...
is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). However, the functions of SagA in were unknown. Here, we report that deletion of impaired growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, Δ showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, the plasmid-based expression of SagA, but not its catalytically inactive mutant, restored Δ growth, production of active muropeptides, and NOD2 activation. SagA is, therefore, essential for growth, stress resistance, and activation of host immunity.
Topics: Enterococcus faecium; N-Acetylmuramoyl-L-alanine Amidase; Immune Checkpoint Inhibitors; Humans; Animals; Bacterial Proteins; Peptidoglycan; Mice
PubMed: 38857064
DOI: 10.7554/eLife.95297 -
FEBS Letters Jun 2024The FtsEX membrane complex constitutes an essential component of the ABC transporter superfamily, widely distributed among bacterial species. It governs peptidoglycan... (Review)
Review
The FtsEX membrane complex constitutes an essential component of the ABC transporter superfamily, widely distributed among bacterial species. It governs peptidoglycan degradation for cell division, acting as a signal transmitter rather than a substrate transporter. Through the ATPase activity of FtsE, it facilitates signal transmission from the cytosol across the membrane to the periplasm, activating associated peptidoglycan hydrolases. This review concentrates on the latest structural advancements elucidating the architecture of the FtsEX complex and its interplay with lytic enzymes or regulatory counterparts. The revealed three-dimensional structures unveil a landscape wherein a precise array of intermolecular interactions, preserved across diverse bacterial species, afford meticulous spatial and temporal control over the cell division process.
PubMed: 38849310
DOI: 10.1002/1873-3468.14953 -
Journal of Biotechnology Jun 2024Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to...
Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZ) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZ expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 10 U mL to 2.11 × 10 U mL in shake flask culture, and eventually reaching 2.05 × 10 U mL in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.
PubMed: 38848819
DOI: 10.1016/j.jbiotec.2024.05.011 -
Applied and Environmental Microbiology Jun 2024Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their... (Review)
Review
Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their ability to destroy bacteria from without makes them promising antimicrobial agents that can be used as alternatives or supplements to antibiotics. In this paper, we briefly summarize basic terms and concepts used to describe lysin sequences and delineate major lysin groups. More importantly, we describe the domain repertoire found in lysins and critically review bioinformatic tools or databases which are used in studies of these enzymes (with particular emphasis on the repositories of Hidden Markov models). Finally, we present a novel comprehensive, meticulously curated set of lysin-related family and domain models, sort them into clusters that reflect major families, and demonstrate that the selected models can be used to efficiently search for new lysins.
PubMed: 38842338
DOI: 10.1128/aem.02361-23 -
Frontiers in Microbiology 2024Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant...
Bacterium-like particles (BLP) are the peptidoglycan skeleton particles of lactic acid bacteria, which have high safety, mucosal delivery efficiency, and adjuvant effect. It has been widely used in recent years in the development of vaccines. Existing anchoring proteins for BLP surfaces are few in number, so screening and characterization of new anchoring proteins are necessary. In this research, we created the OACD (C-terminal domain of outer membrane protein A) to serve as an anchoring protein on the surface of BLP produced by the immunomodulatory bacteria 23017. We used red fluorescent protein (RFP) to demonstrate the novel surface display system's effectiveness, stability, and ability to be adapted to a wide range of lactic acid bacteria. Furthermore, this study employed this surface display method to develop a novel vaccine (called COB17) by using the multi-epitope antigen of as the model antigen. The vaccine can induce more than 50% protection rate against type A challenge in mice immunized with a single dose and has been tested through three routes. The vaccine yields protection rates of 75% for subcutaneous, 50% for intranasal, and 75% for oral immunization. Additionally, it elicits a strong mucosal immune response, markedly increasing levels of specific IgG, high-affinity IgG, specific IgA, and SIgA antibodies. Additionally, we used protein anchors (PA) and OACD simultaneous to show several antigens on the BLP surface. The discovery of novel BLP anchoring proteins may expand the possibilities for creating mucosal immunity subunit vaccines. Additionally, it may work in concert with PA to provide concepts for the creation of multivalent or multiple vaccines that may be used in clinical practice to treat complex illnesses.
PubMed: 38841059
DOI: 10.3389/fmicb.2024.1395837