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Journal of Agricultural and Food... Jun 2024Using as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30...
Using as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from and a mannanase (ManB, 40 kDa) from on the surface of WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in and anchored to the cell surface of . The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.
Topics: Bacillus subtilis; Bacterial Proteins; Glycoside Hydrolases; Peptidoglycan; Enzymes, Immobilized; Protein Domains; Lactobacillus plantarum; Chitin
PubMed: 38775266
DOI: 10.1021/acs.jafc.4c01938 -
Insect Science May 2024C-type lectins (CTLs) act as pattern recognition receptors (PRRs) to initiate the innate immune response in insects. A CTL with dual carbohydrate recognition domains...
C-type lectins (CTLs) act as pattern recognition receptors (PRRs) to initiate the innate immune response in insects. A CTL with dual carbohydrate recognition domains (CRDs) (named immulectin-4 [IML-4]) was selected from the Ostrinia furnacalis transcriptome dataset for functional studies. We cloned the full-length complementary DNA of O. furnacalis IML-4 (OfIML-4). It encodes a 328-residue protein with a Glu-Pro-Asn (EPN) and Gln-Pro-Asp (QPD) motifs in 2 CRDs, respectively. OfIML-4 messenger RNA levels increased significantly upon the bacterial and fungal infection. Recombinant OfIML-4 (rIML-4) and its individual CRDs (rCRD1 and rCRD2) exhibited the binding ability to various microorganisms including Escherichia coli, Micrococcus luteus, Pichia pastoris, and Beauveria bassiana, and the cell wall components including lipopolysaccharide from E. coli, peptidoglycan from M. luteus or Bacillus subtilis, and curdlan from Alcaligenes faecalis. The binding further induced the agglutination of E. coli, M. luteus, and B. bassiana in the presence of calcium, the phagocytosis of Staphylococcus aureus by the hemocytes, in vitro encapsulation and melanization of nickel-nitrilotriacetic acid beads, and a significant increase in phenoloxidase activity of plasma. In addition, rIML-4 significantly enhanced the phagocytosis, nodulation, and resistance of O. furnacalis to B. bassiana. Taken together, our results suggest that OfIML-4 potentially works as a PRR to recognize the invading microorganisms, and functions in the innate immune response in O. furnacalis.
PubMed: 38772748
DOI: 10.1111/1744-7917.13382 -
Acta Veterinaria Scandinavica May 2024Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials... (Review)
Review
Bacteriophage-encoded endolysins, peptidoglycan hydrolases breaking down the Gram-positive bacterial cell wall, represent a groundbreaking class of novel antimicrobials to revolutionize the veterinary medicine field. Wild-type endolysins exhibit a modular structure, consisting of enzymatically active and cell wall-binding domains, that enable genetic engineering strategies for the creation of chimeric fusion proteins or so-called 'engineered endolysins'. This biotechnological approach has yielded variants with modified lytic spectrums, introducing new possibilities in antimicrobial development. However, the discovery of highly similar endolysins by different groups has occasionally resulted in the assignment of different names that complicate a straightforward comparison. The aim of this review was to perform a homology-based comparison of the wild-type and engineered endolysins that have been characterized in the context of bovine mastitis-causing streptococci and staphylococci, grouping homologous endolysins with ≥ 95.0% protein sequence similarity. Literature is explored by homologous groups for the wild-type endolysins, followed by a chronological examination of engineered endolysins according to their year of publication. This review concludes that the wild-type endolysins encountered persistent challenges in raw milk and in vivo settings, causing a notable shift in the field towards the engineering of endolysins. Lead candidates that display robust lytic activity are nowadays selected from screening assays that are performed under these challenging conditions, often utilizing advanced high-throughput protein engineering methods. Overall, these recent advancements suggest that endolysins will integrate into the antibiotic arsenal over the next decade, thereby innovating antimicrobial treatment against bovine mastitis-causing streptococci and staphylococci.
Topics: Animals; Mastitis, Bovine; Cattle; Endopeptidases; Staphylococcus; Bacteriophages; Staphylococcal Infections; Streptococcus; Female; Streptococcal Infections; Anti-Bacterial Agents
PubMed: 38769566
DOI: 10.1186/s13028-024-00740-2 -
International Journal of Systematic and... May 2024A Gram-stain-positive actinomycete, designated REN17, was isolated from fermented grains of Baijiu collected from Sichuan, PR China. It exhibited branched substrate...
A Gram-stain-positive actinomycete, designated REN17, was isolated from fermented grains of Baijiu collected from Sichuan, PR China. It exhibited branched substrate mycelia and a sparse aerial mycelium. The optimal growth conditions for REN17 were determined to be 28 °C and pH 7, with a NaCl concentration of 0 % (w/v). ll-Diaminopimelic acid was the diagnostic amino acid of the cell-wall peptidoglycan and the polar lipids were composed of phosphatidylethanolamine, phosphatidylinositol, an unidentified phospholipid, two unidentified lipids and four unidentified glycolipids. The predominant menaquinone was MK-9 (H), MK-9 (H), MK-9 (H) and MK-9 (H). The major fatty acids were iso-C. The 16S rRNA sequence of REN17 was most closely related to those of SUN 51 (99.8 %), BH-SS-21 (99.6 %) and JCM 4521 (98.9 %). The digital DNA-DNA hybridization, average nucleotide identity and average amino acid identify values between REN17 and its closest replated strain, of SUN 51, were 35.9, 88.9 and 87.3 %, respectively. Therefore, REN17 represents a novel species within the genus , for which the name sp. nov. is proposed. The type strain is REN17 (=GDMCC 4.193=JCM 34712). While exploring the function of the strain, REN17 was found to possess the ability to transform major ginsenosides of (Burk.) F.H. Chen (Araliaceae) into minor ginsenoside through HPLC separation, which was due to the presence of β-glucosidase. The recombinant β-glucosidase was constructed and purified, which could produce minor ginsenosides of Rg3 and C-K. Finally, the enzymatic properties were characterized.
Topics: RNA, Ribosomal, 16S; Phylogeny; Fatty Acids; Streptomyces; Vitamin K 2; DNA, Bacterial; Nucleic Acid Hybridization; China; Sequence Analysis, DNA; Fermentation; Panax notoginseng; Bacterial Typing Techniques; Ginsenosides; Peptidoglycan; Edible Grain; Diaminopimelic Acid; Phospholipids; Base Composition
PubMed: 38767616
DOI: 10.1099/ijsem.0.006392 -
Vaccine May 2024Mucosal vaccination presents a promising complement to parenteral vaccination. Bacterium-like particles (BLPs), peptidoglycan structures prepared from lactic acid...
Mucosal vaccination presents a promising complement to parenteral vaccination. Bacterium-like particles (BLPs), peptidoglycan structures prepared from lactic acid bacteria, are explored as potential nasal vaccine adjuvants for respiratory infections. To date, studies on BLP-adjuvanted nasal vaccines against intestinal infections have remained limited. In this study, we demonstrated the efficacy of intranasal BLP-adjuvanted vaccination in controlling intestinal infections using the Citrobacter rodentium (C. rodentium) model in C57BL/6 mice. Intranasal vaccination of Intimin, an adhesin critical for intimate bacterial adhesion to colonic epithelial cells, combined with BLP (BLP+I) elicited robust Intimin-specific intestinal secretory IgA production, reduced bacterial load in feces and almost completely inhibited colonic hyperplasia, a characteristic symptom of C. rodentium infection in mice. Conversely, parenteral vaccination with Alhydrogel-adjuvanted Intimin failed to induce intestinal Intimin-specific IgA production, resulting in poor protection against C. rodentium infection. This underscores the pivotal role of mucosal IgA responses elicited by intranasal immunization in its protective efficacy. As this study did not delineate the precise protective mechanism conferred by BLP+I intranasal immunization against C. rodentium infection, further elucidation of the mechanisms underlying intranasal BLP+I immunization is required.
PubMed: 38763852
DOI: 10.1016/j.vaccine.2024.05.023 -
Systematic and Applied Microbiology May 2024The genus Natronospira is represented by a single species of extremely salt-tolerant aerobic alkaliphilic proteolytic bacterium, isolated from hypersaline soda lakes....
Natronospira bacteriovora sp. nov., and Natronospira elongata sp. nov., extremely salt-tolerant predatory proteolytic bacteria from soda lakes and proposal to classify the genus Natronospira into Natronospiraceae fam. nov., and Natronospirales ord. nov., within the class Gammaproteobacteria.
The genus Natronospira is represented by a single species of extremely salt-tolerant aerobic alkaliphilic proteolytic bacterium, isolated from hypersaline soda lakes. When cells of Gram-positive cocci were used as a substrate instead of proteins at extremely haloalkaline conditions, two new members of this genus were enriched and isolated in pure culture from the same sites. Strains AB-CW1 and AB-CW4 are obligate aerobic heterotrophic proteolytic bacteria able to feed on both live and dead cells of staphylococci and a range of proteins and peptides. Similar to the type species, N. proteinivora, the isolates are extremely salt-tolerant obligate alkaliphiles. However, N. proteinivora was unable to use bacterial cells as a substrate. Electron microscopy showed direct contact between the prey and predator cells. Functional analysis of the AB-CW1 and AB-CW4 genomes identified two sets of genes coding for extracellular enzymes potentially involved in the predation and proteolysis, respectively. The first set includes several copies of lysozyme-like GH23 peptidoglycan-lyase and murein-specific M23 [Zn]-di-peptidase enabling the cell wall degradation. The second set features multiple copies of secreted serine and metallopeptidases apparently allowing for the strong proteolytic phenotype. Phylogenomic analysis placed the isolates into the genus Natronospira as two novel species members, and furthermore indicated that this genus forms a deep-branching lineage of a new family (Natronospiraceae) and order (Natronospirales) within the class Gammaproteobacteria. On the basis of distinct phenotypic and genomic properties, strain AB-CW1 (JCM 335396 = UQM 41579) is proposed to be classified as Natronospira elongata sp. nov., and AB-CW4 (JCM 335397 = UQM 41580) as Natronospira bacteriovora sp. nov.
PubMed: 38759530
DOI: 10.1016/j.syapm.2024.126519 -
Molecular Biology of the Cell Jul 2024Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the...
Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in . Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.
Topics: Caulobacter crescentus; Bacterial Proteins; Cytoskeletal Proteins; Guanosine Triphosphate; GTP Phosphohydrolases; Cell Division; Hydrolysis; Mutation
PubMed: 38758654
DOI: 10.1091/mbc.E23-09-0365 -
Scientific Reports May 2024Alterations in the vaginal microbiota, including both species composition and functional pathways, have been associated with HPV infection and progression of dysplasia...
Alterations in the vaginal microbiota, including both species composition and functional pathways, have been associated with HPV infection and progression of dysplasia to cervical cancer. To further explore this, shotgun metagenomic sequencing was used to taxonomically and functionally characterize the vaginal microbiota of women with and without cervical dysplasia. Women with histologically verified dysplasia (n = 177; low grade dysplasia (LSIL) n = 81, high-grade dysplasia (HSIL) n = 94, cancer n = 2) were compared with healthy controls recruited from the cervical screening programme (n = 177). Women with dysplasia had a higher vaginal microbial diversity, and higher abundances of Gardnerella vaginalis, Aerococcus christensenii, Peptoniphilus lacrimalis and Fannyhessea vaginae, while healthy controls had higher relative abundance of Lactobacillus crispatus. Genes involved in e.g. nucleotide biosynthesis and peptidoglycan biosynthesis were more abundant in women with dysplasia. Healthy controls showed higher abundance of genes important for e.g. amino acid biosynthesis, (especially L-lysine) and sugar degradation. These findings suggest that the microbiota may have a role in creating a pro-oncogenic environment in women with dysplasia. Its role and potential interactions with other components in the microenvironment deserve further exploration.
Topics: Humans; Female; Vagina; Microbiota; Adult; Uterine Cervical Dysplasia; Middle Aged; Case-Control Studies; Metagenomics; Bacteria
PubMed: 38755259
DOI: 10.1038/s41598-024-61942-2 -
Gut May 2024Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known...
OBJECTIVE
Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies.
DESIGN
We used the KPC mouse model () and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent coculture assays, mouse and zebrafish tumorigenesis studies, and database approaches were performed.
RESULTS
Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC.
CONCLUSIONS
This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.
PubMed: 38754953
DOI: 10.1136/gutjnl-2023-330995 -
MBio Jun 2024Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors...
UNLABELLED
Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in . A small membrane protein, Gp11, with inhibitory effects on cell division was identified. A bacterial two-hybrid library containing 345 essential genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gene has been deleted showed a significant increase in lipid II production in . Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections.
IMPORTANCE
Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in , and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in . Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.
Topics: Staphylococcus aureus; Peptidoglycan; Cell Division; Staphylococcus Phages; Viral Proteins; Bacterial Proteins; Cell Wall; Membrane Proteins
PubMed: 38752726
DOI: 10.1128/mbio.00679-24