Alexis Apostolos

Anti–programmed cell death protein 1 (PD-1) therapy provides long-term clinical benefits to patients with advanced melanoma. The composition of the gut microbiota correlates with anti–PD-1 efficacy in preclinical models and cancer patients. To investigate whether resistance to anti–PD-1 can be overcome by changing the gut microbiota, this clinical trial evaluated the safety and efficacy of responder-derived fecal microbiota transplantation (FMT) together with anti–PD-1 in patients with PD-1–refractory melanoma. This combination was well tolerated, provided clinical benefit in 6 of 15 patients, and induced rapid and durable microbiota perturbation. Responders exhibited increased abundance of taxa that were previously shown to be associated with response to anti–PD-1, increased CD8⁺ T cell activation, and decreased frequency of interleukin-8–expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Collectively, our findings show that FMT and anti–PD-1 changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti–PD-1 in a subset of PD-1 advanced melanoma.

Sorting out sortases: The bacterial transpeptidase sortase A has entered center stage as a versatile tool for protein bioconjugation and semisynthesis. Sortase A of Staphylococcus is widely used, but suffers from slow kinetics and a narrow specificity profile. Recent protein engineering campaigns aiming to improve these shortcomings have been successful and delivered enhanced sortase mutants with enhanced catalytic properties.

Abstract

The transpeptidase sortase A of Staphylococcus aureus (Sa‐SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in Gram‐positive bacteria. This reaction is exploited for sortase‐mediated ligation (SML), allowing the site‐specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa‐SrtA fueled the development of new biocatalysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa‐SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This minireview focuses on recent advances in the field of directed sortase evolution and applications of these tailor‐made enzymes in biochemistry.

Simple Secondary Amines Inhibit Growth of Gram-Negative Bacteria through Highly Selective Binding to Phenylalanyl-tRNA Synthetase.

J Am Chem Soc. 2021 Jan 07;:

Authors: Baidin V, Owens TW, Lazarus MB, Kahne D

Abstract
Antibiotics to treat drug-resistant Gram-negative infections are urgently needed but challenging to discover. Using a cell-based screen, we identified a simple secondary amine that inhibited the growth of wild-type Escherichia coli and Acinetobacter baumannii but not the growth of the Gram-positive organism Bacillus subtilis. Resistance mutations in E. coli and A. baumannii mapped exclusively to the aminoacyl-tRNA synthetase PheRS. We confirmed biochemically that the compound inhibited PheRS from these organisms and showed that it did not inhibit PheRS from B. subtilis or humans. To understand the basis for the compound's high selectivity for only some PheRS enzymes, we solved crystal structures of E. coli and A. baumannii PheRS complexed with the inhibitor. The structures showed that the compound's benzyl group mimics the benzyl of phenylalanine. The other amine substituent, a 2-(cyclohexen-1-yl)ethyl group, induces a hydrophobic pocket in which it binds. Through bioinformatic analysis and mutagenesis, we show that the ability to induce a complementary hydrophobic pocket that can accommodate the second substituent explains the high selectivity of this remarkably simple molecular scaffold for Gram-negative PheRS. Because this secondary amine scaffold is active against wild-type Gram-negative pathogens but is not cytotoxic to mammalian cells, we suggest that it may be possible to develop it for use in combination antibiotic therapy to treat Gram-negative infections.

PMID: 33411531 [PubMed - as supplied by publisher]

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post‐translational modification. Here we show that selenoproteins can be expressed in high yield and purity by cell‐free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent‐exposed selenocysteine residues was achieved in 10 minutes with 4‐chloro‐methylene‐dipicolinic acid (4Cl‐MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd(III)–Gd(III) distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl‐MDPA‐Gd(III) were indistinguishable from Gd(III)–Gd(III) distances measured of MBP containing cysteine reacted with 4Br‐MDPA tags.

Wide-open conformation of UDP-MurNc-tripeptide ligase revealed by the substrate-free structure of MurE from Acinetobacter baumannii.

FEBS Lett. 2020 Nov 23;:

Authors: Jung KH, Kim YG, Kim CM, Ha HJ, Lee CS, Lee JH, Park HH

Abstract
MurE ligase catalyzes the attachment of meso-diaminopimelic acid to the UDP-MurNAc-L -Ala-D -Glu using ATP and producing UDP-MurNAc-L -Ala-D -Glu-meso-A2 pm during bacterial cell wall biosynthesis. Owing to the critical role of this enzyme, MurE is considered an attractive target for antibacterial drugs. Despite extensive studies on MurE ligase, the structural dynamics of its conformational changes are still elusive. In this study, we present the substrate-free structure of MurE from Acinetobacter baumannii, which is an antibiotic-resistant superbacterium that has threatened global public health. The structure revealed that MurE has a wide-open conformation and undergoes wide-open, intermediately closed, and fully closed dynamic conformational transition. Unveiling structural dynamics of MurE will help to understand the working mechanism of this ligase and to design next-generation antibiotics targeting MurE.

PMID: 33230844 [PubMed - as supplied by publisher]

Synthesis of Bacterial-Derived Peptidoglycan Cross-Linked Fragments.

J Org Chem. 2020 Oct 27;:

Authors: Mashayekh S, Bersch KL, Ramsey J, Harmon T, Prather B, Genova LA, Grimes CL

Abstract
Peptidoglycan (PG) is the core structural motif of the bacterial cell wall. Fragments released from the PG serve as fundamental recognition elements for the immune system. The structure of the PG, however, encompasses a variety of chemical modifications among different bacterial species. Here, the applicability of organic synthetic methods to address this chemical diversity is explored, and the synthesis of cross-linked PG fragments, carrying biologically relevant amino acid modifications and peptide cross-linkages, is presented using solution and solid phase approaches.

PMID: 33108204 [PubMed - as supplied by publisher]

Cell morphology maintenance in Bacillus subtilis through balanced peptidoglycan synthesis and hydrolysis.

Sci Rep. 2020 Oct 21;10(1):17910

Authors: Sassine J, Sousa J, Lalk M, Daniel RA, Vollmer W

Abstract
The peptidoglycan layer is responsible for maintaining bacterial cell shape and permitting cell division. Cell wall growth is facilitated by peptidoglycan synthases and hydrolases and is potentially modulated by components of the central carbon metabolism. In Bacillus subtilis, UgtP synthesises the glucolipid precursor for lipoteichoic acid and has been suggested to function as a metabolic sensor governing cell size. Here we show that ugtP mutant cells have increased levels of cell wall precursors and changes in their peptidoglycan that suggest elevated DL-endopeptidase activity. The additional deletion of lytE, encoding a DL-endopeptidase important for cell elongation, in the ugtP mutant background produced cells with severe shape defects. Interestingly, the ugtP lytE mutant recovered normal rod-shape by acquiring mutations that decreased the expression of the peptidoglycan synthase PBP1. Together our results suggest that cells lacking ugtP must re-adjust the balance between peptidoglycan synthesis and hydrolysis to maintain proper cell morphology.

PMID: 33087775 [PubMed - in process]

Revisiting peptidoglycan sensing: interactions with host immunity and beyond.

Chem Commun (Camb). 2020 Oct 15;:

Authors: Crump GM, Zhou J, Mashayekh S, Grimes CL

Abstract
The interaction between host immunity and bacterial cells plays a pivotal role in a variety of human diseases. The bacterial cell wall component peptidoglycan (PG) is known to stimulate an immune response, which makes PG a distinctive recognition element for unveiling these complicated molecular interactions. Pattern recognition receptor (PRR) proteins are among the critical components of this system that initially recognize molecular patterns associated with microorganisms such as bacteria and fungi. These molecular patterns are mostly embedded in the bacterial or fungal cell wall structure and can be released and presented to the immune system in various situations. Nonetheless, detailed knowledge of this recognition is limited due to the diversity among the PG polymer and its fragments; the subsequent responses by multiple hosts add more complexity. Here, we discuss how our understanding of the role and molecular mechanisms of the well-studied PRR, the NOD-like receptors (NLRs), in the human immune system has evolved in recent years. We highlight the instances of other classes of proteins with similar behavior in the recognition of PG that have been identified in other microorganisms such as yeasts. These proteins are particularly interesting because a network of cellular interactions exists between human host cells, bacteria and yeast as a part of the normal human flora. To support our understanding of these interactions, we provide insight into the chemist's toolbox of peptidoglycan probes that aid in the investigations of the behaviors of these proteins and other biological contexts relevant to the sensing and recognition of peptidoglycan. The importance of these interactions in human health for the development of biomarkers and biotherapy is highlighted.

PMID: 33057506 [PubMed - as supplied by publisher]

Further insights on structural modifications of muramyl dipeptides to study the human NOD2 stimulating activity.

Chem Asian J. 2020 Sep 25;:

Authors: Cheng WC, You TY, Teo ZZ, Sayyad AA, Maharana J, Guo CW, Liang PH, Lin CS, Meng FC

Abstract
A series of muramyl dipeptide (MDP) analogues with structural modifications at the C4 position of MurNAc and on the d- iso -glutamine ( iso Gln) residue of the peptide part were synthesized. The C4-diversification of MurNAc was conveniently achieved by using CuAAc click strategy to conjugate an azido muramyl dipeptide precursor with structurally diverse alkynes. d-Glutamic acid (Glu), replaced with iso Gln, was applied for the structural diversity through esterification or amidation of the carboxylic acid. In total, 26 MDP analogues were synthesized and bio-evaluated for the study of human NOD2 stimulation activity in the innate immune response. Interestingly, MDP derivatives with an ester moiety are found to be more potent than reference compound MDP itself or MDP analogues containing an amide moiety. Among the varied lengths of the alkyl chain in ester derivatives, the MDP analogue bearing the d-glutamate dodecyl (C12) ester moiety showed the best NOD2 stimulation potency.

PMID: 32975372 [PubMed - as supplied by publisher]

Isoform‐specific targeting: The synthesis and comparison of selective, bioorthogonal, activity‐based probes for penicillin‐binding proteins (PBPs) is reported. We demonstrate the expanded functional utility of bioorthogonal probes compared to fluorescent analogues and explore design considerations for the development of bioorthogonal probes that are applicable beyond the probes described in this work.

Abstract

Penicillin‐binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell‐wall biosynthesis and the target of β‐lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal β‐lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity‐based probes for PBPs will facilitate the activity‐based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

Sci Adv. 2020 Sep;6(38):

Authors: Justen AM, Hodges HL, Kim LM, Sadecki PW, Porfirio S, Ultee E, Black I, Chung GS, Briegel A, Azadi P, Kiessling LL

Abstract
Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.

PMID: 32938674 [PubMed - in process]

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species—Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species—that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A_2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.

Mechanical penetration of β-lactam-resistant Gram-negative bacteria by programmable nanowires.

Sci Adv. 2020 Jul;6(27):

Authors: Liu L, Chen S, Zhang X, Xue Z, Cui S, Hua X, Yang B, Yan H, Liu C, Wang J, Zhang Z, Yu W, Wu F, Xu W, Lehto VP, Yue T, Liu Y, Yu Y, Wang T, Wang J

Abstract
β-Lactam-resistant (BLR) Gram-negative bacteria that are difficult or impossible to treat are causing a global health threat. However, the development of effective nanoantibiotics is limited by the poor understanding of changes in the physical nature of BLR Gram-negative bacteria. Here, we systematically explored the nanomechanical properties of a range of Gram-negative bacteria (Salmonella, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae) with different degrees of β-lactam resistance. Our observations indicated that the BLR bacteria had cell stiffness values almost 10× lower than that of β-lactam-susceptible bacteria, caused by reduced peptidoglycan biosynthesis. With the aid of numerical modeling and experimental measurements, we demonstrated that these stiffness findings can be used to develop programmable, stiffness-mediated antimicrobial nanowires that mechanically penetrate the BLR bacterial cell envelope. We anticipate that these stiffness-related findings will aid in the discovery and development of novel treatment strategies for BLR Gram-negative bacterial infections.

PMID: 32937461 [PubMed - as supplied by publisher]

Revealing the in vivo growth and division patterns of mouse gut bacteria.

Sci Adv. 2020 Sep;6(36):

Authors: Lin L, Wu Q, Song J, Du Y, Gao J, Song Y, Wang W, Yang C

Abstract
Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid-based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid-based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut "dark matter."

PMID: 32917613 [PubMed - as supplied by publisher]

Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of Bacterial Response to Vancomycin Treatment.

Biochemistry. 2020 Sep 04;:

Authors: Zhao W, Liang Y, Yan X, Yang L, Wang Q

Abstract
Pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which bacterial cell wall is the most front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical D-amino acid (D-X) that was edited in a small proportion into the terminus acyl-D-Ala-D-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional HPLC-based methods. Newly synthesized azide-D-Leu and ketone-D-Met were used together with alkynyl-D-Ala for their metabolically assembly, and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu and azide-DOTA-Sm tags. This approach allows direct quantification of the D-X in-situ in the cell wall using 158Gd-, 153Eu- and 154Sm-species-unspecific isotope-dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex 'cell-broken' pretreatment procedures that might induce racemization of the D-X. The obtained site-specific and accurate in-situ information of the D-X enables quantitative monitoring of bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of the azide-D-Leu and ketone-D-Met assembled are more important after knowing the structure- and composition-dependent bacterial antibiotic resistance mechanisms. Besides, we found that the combined use of vancomycin and D-Ala recovers the efficacy of vancomycin, and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

PMID: 32885950 [PubMed - as supplied by publisher]

Synthesis of 9-Dechlorochrysophaentin A Enables Studies Revealing Bacterial Cell Wall Biosynthesis Inhibition Phenotype in B. subtilis.

J Am Chem Soc. 2020 Aug 31;:

Authors: Fullenkamp C, Hsu YP, Quardokus E, Zhao G, Bewley CA, VanNieuwenhze MS, Sulikowski GA

Abstract
Chrysophaentin A is an antimicrobial natural product isolated from the marine alga C. taylori in milligram quantity. Structurally, chrysophaentin A features a macrocyclic biaryl ether core incorporating two trisubstituted chloroalkenes at its periphery. A concise synthesis of iso- and 9-dechlorochrysophaentin A enabled by a Z-selective RCM cyclization followed by an oxygen to carbon ring contraction is described. Fluorescent microscopy studies revealed 9-dechlorochrysophaentins leads to inhibition of bacterial cell wall biosynthesis by disassembly of key divisome proteins, the cornerstone to bacterial cell wall biosynthesis and division.

PMID: 32866011 [PubMed - as supplied by publisher]

Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare.

Sci Adv. 2020 Jul;6(30):eabb5614

Authors: Le NH, Peters K, Espaillat A, Sheldon JR, Gray J, Di Venanzio G, Lopez J, Djahanschiri B, Mueller EA, Hennon SW, Levin PA, Ebersberger I, Skaar EP, Cava F, Vollmer W, Feldman MF

Abstract
Peptidoglycan (PG) is essential in most bacteria. Thus, it is often targeted by various assaults, including interbacterial attacks via the type VI secretion system (T6SS). Here, we report that the Gram-negative bacterium Acinetobacter baumannii strain ATCC 17978 produces, secretes, and incorporates the noncanonical d-amino acid d-lysine into its PG during stationary phase. We show that PG editing increases the competitiveness of A. baumannii during bacterial warfare by providing immunity against peptidoglycan-targeting T6SS effectors from various bacterial competitors. In contrast, we found that d-Lys production is detrimental to pathogenesis due, at least in part, to the activity of the human enzyme d-amino acid oxidase (DAO), which degrades d-Lys producing H2O2 toxic to bacteria. Phylogenetic analyses indicate that the last common ancestor of A. baumannii had the ability to produce d-Lys. However, this trait was independently lost multiple times, likely reflecting the evolution of A. baumannii as a human pathogen.

PMID: 32832672 [PubMed - as supplied by publisher]

Peptidoglycan-binding Protein Metamaterials Mediated Enhanced and Selective Capturing of Gram-Positive Bacteria and their Specific, Ultra-sensitive, and Reproducible detection via SERS.

ACS Sens. 2020 Aug 11;:

Authors: Lee T, Lim J, Park K, Lim EK, Lee J

Abstract
Biological metamaterials with a specific size and spacing are necessary for developing highly sensitive and selective sensing systems to detect hazardous bacteria in complex solutions. Herein, the construction of peptidoglycan-binding protein (PGBP)-based metamaterials to selectively capture gram-positive cells with high efficacy is reported. Nanoimprint lithography was used to generate a nanohole pattern as a template, the inside of which was modified with nickel(II)-nitrilotriacetic acid (Ni-NTA). Then, PGBP metamaterials were fabricated by immobilizing PGBP via chelation between Ni-NTA and six histidines on PGBP. Compared to the flat and spread PGBP-covered bare substrates, the PGBP-based metamaterials enabled selective capturing of gram-positive bacteria with high efficacy, owing to enhanced interactions between the metamaterials and bacterial surface not shown in bulk materials. Thereafter, the specific strain and quantitative information of the captured bacteria was obtained by SERS mapping analysis in the 1 to 1 × 106 CFU/mL range within 30 min. It should be noted that no additional signal amplification process was required for lowly abundant bacteria, even at the single-bacterium level. The PGBP-based metamaterials could be regenerated multiple times with preserved sensing efficiency. Finally, this assay can detect specific gram-positive bacteria, such as Staphylococcus aureus, in human plasma.

PMID: 32786378 [PubMed - as supplied by publisher]

NOD1 and NOD2 in inflammatory and infectious diseases.

Immunol Rev. 2020 Jul 17;:

Authors: Trindade BC, Chen GY

Abstract
It has been long recognized that NOD1 and NOD2 are critical players in the host immune response, primarily by their sensing bacterial peptidoglycan-conserved motifs. Significant advances have been made from efforts that characterize their upstream activators, assembly of signaling complexes, and activation of downstream signaling pathways. Disruption in NOD1 and NOD2 signaling has also been associated with impaired host defense and resistance to the development of inflammatory diseases. In this review, we will describe how NOD1 and NOD2 sense microbes and cellular stress to regulate host responses that can affect disease pathogenesis and outcomes.

PMID: 32677123 [PubMed - as supplied by publisher]

Bacterial Peptidoglycan as a Driver of Chronic Brain Inflammation.

Trends Mol Med. 2020 Jul;26(7):670-682

Authors: Laman JD, 't Hart BA, Power C, Dziarski R

Abstract
Peptidoglycan (PGN) is a cell wall component of both Gram-positive and Gram-negative bacteria. Signature fragments of PGN are proinflammatory through engagement of pattern recognition receptors (PRR) on resident tissue cells and circulating leukocytes. Despite its abundance in the gut microbiota, there is limited recognition that PGN could contribute to chronic neuroinflammation. This review highlights current insights into the roles of PGN as a determinant of brain inflammation, notably in multiple sclerosis (MS) and its experimental autoimmune encephalomyelitis (EAE) models. Recent studies demonstrate PGN in blood of healthy adult humans. PGN amplifies autoimmune pathology via activation of innate immune cells. Novel uptake routes through (altered) gut mucosa by myeloid leukocyte subsets promote PGN transport to the brain.

PMID: 32589935 [PubMed - as supplied by publisher]

Anchoring of heterologous proteins in multiple Lactobacillus species using anchors derived from Lactobacillus plantarum.

Sci Rep. 2020 Jun 15;10(1):9640

Authors: Mathiesen G, Øverland L, Kuczkowska K, Eijsink VGH

Abstract
Members of the genus Lactobacillus have a long history in food applications and are considered as promising and safe hosts for delivery of medically interesting proteins. We have assessed multiple surface anchors derived from Lactobacillus plantarum for protein surface display in multiple Lactobacillus species, using a Mycobacterium tuberculosis hybrid antigen as test protein. The anchors tested were a lipoprotein anchor and two cell wall anchors, one non-covalent (LysM domain) and one covalent (sortase-based anchoring using the LPXTG motif). Thus, three different expression vectors for surface-anchoring were tested in eight Lactobacillus species. When using the LPXTG and LysM cell wall anchors, surface display, as assessed by flow cytometry and fluorescence microscopy, was observed in all species except Lactobacillus acidophilus. Use of the cell membrane anchor revealed more variation in the apparent degree of surface-exposure among the various lactobacilli. Overproduction of the secreted and anchored antigen impaired bacterial growth rate to extents that varied among the lactobacilli and were dependent on the type of anchor. Overall, these results show that surface anchors derived from L. plantarum are promising candidates for efficient anchoring of medically interesting proteins in other food grade Lactobacillus species.

PMID: 32541679 [PubMed - in process]

Differential Peptidoglycan Recognition Assay Using Varied Surface Presentations.

J Am Chem Soc. 2020 Jun 10;:

Authors: D'Ambrosio EA, Bersch KL, Lauro ML, Grimes CL

Abstract
Bacterial peptidoglycan (PG) is recognized by the human innate immune system to generate an appropriate downstream response. In order to gain an appreciation of how this essential polymer is sensed, a surface plasmon resonance (SPR) assay using varied PG surface presentation was developed. PG derivatives were synthesized and immobilized on the surface at different positions on the molecule to assess effects of ligand orientation on the binding affinities of NOD-like receptors (NLRs). NLRP1 and NOD2 are cytosolic innate immune proteins known to generate an immune response to PG. Both possess conserved leucine rich repeat domains (LRR) as proposed site of molecular recognition, though limited biochemical evidence exists regarding the mechanisms of PG recognition. Here we show direct biochemical evidence for the association of PG fragments to NOD2 and NLRP1 with nanomolar affinity. The orientations in which the fragments were presented on the SPR surface greatly influenced the strength of PG recognition by both NLRs. This assay displays fundamental differences in binding preferences for PG by innate immune receptors and reveals unique recognition mechanisms between the LRRs. Each receptor uses specific ligand structural features to achieve optimal binding, which will be critical information to manipulate these responses and combat diseases. This assay will be valuable in teasing apart subtle but critical structural features of a variety of receptors with therapeutic potential.

PMID: 32520538 [PubMed - as supplied by publisher]

Characterization of the sortase A from Lactobacillus acidophilus ATCC 4356 involved in adherence to intestinal cells.

Future Microbiol. 2020 Jun 01;:

Authors: Wu Z, Wu J, Lang F, Cai Z, Zeng X, Guo Y, Liu X, Pan D

Abstract
Aim: Confirmation of the enzymatic activity of Class A sortase (SrtA) in probiotic strain Lactobacillus acidophilus associated with the adhesion properties. Materials & methods: SrtA from L. acidophilus ATCC 4356 was purified and its enzymatic properties was investigated by site-directed mutagenesis approach and the sensitivity to metal ions was also detected. Results: SrtA of L. acidophilus ATCC4356 can recognize LPxTG and LPxTD sorting motifs. The active sites of SrtA include His137, Cys198 and Arg205. Furthermore, acacetin can increase the activity of SrtA, while phenyl vinyl sulfone could effectively inhibit the activity of SrtA with an IC50 of 143.32 μg/ml. The adhesion ability of L. acidophilus was also decreased resulting from the inhibition of SrtA activity. Conclusion: The unique properties of SrtA of L. acidophilus can provide some insights into the development of high adhesion lactobacillus strains in the GI tract.

PMID: 32476478 [PubMed - as supplied by publisher]