A novel family of defensin-like peptides from Hermetia illucens with antibacterial properties.

BACKGROUND: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. RESULTS: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. CONCLUSIONS: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.

A coating of lipoproteins provides a stabilizing environment on the inner membrane of Bacillus subtilis spores.

A new study by M. J. Flores, K. Duricy, S. Choudhary, M. Laue, and D. L. Popham (J Bacteriol 205:e00142-23, 2023, https://doi.org/10.1128/jb.00142-23) demonstrates a role for the YlaJ/YhcN family of lipoproteins in the immobilization of the spore's inner membrane. In the absence of these lipoproteins, membrane fluidity increases and membrane-associated proteins like the GerA receptor complexes are more exposed to inimical conditions. The role of these proteins in stabilizing the Bacillus spore inner membrane is now being explored.

An attacin antimicrobial peptide, Hill_BB_C10074, from Hermetia illucens with anti-Pseudomonas aeruginosa activity.

BACKGROUND: There is a global need to develop new therapies to treat infectious diseases and tackle the rise in antimicrobial resistance. To date, the larvae of the Black Solider Fly, Hermetia illucens, have the largest repertoire of antimicrobial peptides derived from insects. Antimicrobial peptides are of particular interest in the exploration of alternative antimicrobials due to their potent action and reduced propensity to induce resistance compared with more traditional antibiotics. RESULTS: The predicted attacin from H. illucens, Hill_BB_C10074, was first identified in the transcriptome of H. illucens populations that had been fed a plant-oil based diet. In this study, recombinant Hill_BB_C10074 (500 µg/mL), was found to possess potent antimicrobial activity against the serious Gram-negative pathogen, Pseudomonas aeruginosa. Sequence and structural homology modelling predicted that Hill_BB_C10074 formed a homotrimeric complex that may form pores in the Gram-negative bacterial outer membrane. In vitro experiments defined the antimicrobial action of Hill_BB_C10074 against P. aeruginosa and transmission electron microscopy and electrochemical impedance spectroscopy confirmed the outer membrane disruptive power of Hill_BB_C10074 which was greater than the clinically relevant antibiotic, polymyxin B. CONCLUSIONS: Combining predictive tools with in vitro approaches, we have characterised Hill_BB_C10074 as an important insect antimicrobial peptide and promising candidate for the future development of clinical antimicrobials.

Prophylactic immunization to Helicobacter pylori infection using spore vectored vaccines.

BACKGROUND: Helicobacter pylori infection remains a major public health threat leading to gastrointestinal illness and increased risk of gastric cancer. Mostly affecting populations in developing countries no vaccines are yet available and the disease is controlled by antimicrobials which, in turn, are driving the emergence of AMR. MATERIALS AND METHODS: We have engineered spores of Bacillus subtilis to display putative H. pylori protective antigens, urease subunit A (UreA) and subunit B (UreB) on the spore surface. Following oral dosing of mice with these spores, we evaluated immunity and colonization in animals challenged with H. pylori. RESULTS: Oral immunization with spores expressing either UreA or UreB showed antigen-specific mucosal responses (fecal sIgA) including seroconversion and hyperimmunity. Following challenge, colonization by H. pylori was significantly reduced by up to 1-log. CONCLUSIONS: This study demonstrates the utility of bacterial spores for mucosal vaccination to H. pylori infection. The heat stability and robustness of Bacillus spores coupled with their existing use as probiotics make them an attractive solution for either protection against H. pylori infection or potentially for therapy and control of active infection.

Zinc associated nanomaterials and their intervention in emerging respiratory viruses: Journey to the field of biomedicine and biomaterials.

Respiratory viruses represent a severe public health risk worldwide, and the research contribution to tackle the current pandemic caused by the SARS-CoV-2 is one of the main targets among the scientific community. In this regard, experts from different fields have gathered to confront this catastrophic pandemic. This review illustrates how nanotechnology intervention could be valuable in solving this difficult situation, and the state of the art of Zn-based nanostructures are discussed in detail. For virus detection, learning from the experience of other respiratory viruses such as influenza, the potential use of Zn nanomaterials as suitable sensing platforms to recognize the S1 spike protein in SARS-CoV-2 are shown. Furthermore, a discussion about the antiviral mechanisms reported for ZnO nanostructures is included, which can help develop surface disinfectants and protective coatings. At the same time, the properties of Zn-based materials as supplements for reducing viral activity and the recovery of infected patients are illustrated. Within the scope of noble adjuvants to improve the immune response, the ZnO NPs properties as immunomodulators are explained, and potential prototypes of nanoengineered particles with metallic cations (like Zn2+) are suggested. Therefore, using Zn-associated nanomaterials from detection to disinfection, supplementation, and immunomodulation opens a wide area of opportunities to combat these emerging respiratory viruses. Finally, the attractive properties of these nanomaterials can be extrapolated to new clinical challenges.

Characterization of Heterogeneity and Dynamics of Lysis of Single Bacillus subtilis Cells upon Prophage Induction During Spore Germination, Outgrowth, and Vegetative Growth Using Raman Tweezers and Live-Cell Phase-Contrast Microscopy.

A prophage comprises a bacteriophage genome that has integrated into a host bacterium's DNA, which generally permits the cell to grow and divide normally. However, the prophage can be induced by various stresses, or induction can occur spontaneously. After prophage induction, viral replication and production of endolysins begin until the cell lyses and phage particles are released. However, the heterogeneity of prophage induction and lysis of individual cells in a population and the dynamics of a cell undergoing lysis by prophage induction have not been fully characterized. Here, we used Raman tweezers and live-cell phase-contrast microscopy to characterize the Raman spectral and cell length changes that occur during the lysis of individual Bacillus subtilis cells from spores that carry PBSX prophage during spores' germination, outgrowth, and then vegetative growth. Major findings of this work are as follows: (i) After addition of xylose to trigger prophage induction, the intensities of Raman spectral bands associated with nucleic acids of single cells in induced cultures gradually fell to zero, in contrast to the much smaller changes in protein band intensities and no changes in nucleic acid bands in uninduced cultures; (ii) the nucleic acid band intensities from an individual induced cell exhibited a rapid decrease, following a long lag period; (iii) after the addition of nutrient-rich medium with xylose, single spores underwent a long period (228 ± 41.4 min) for germination, outgrowth, and vegetative growth, followed by a short period of cell burst in 1.5 ± 0.8 min at a cell length of 8.2 ± 5.5 µm; (iv) the latent time (Tlatent) between the addition of xylose and the start of cell burst was heterogeneous in cell populations; however, the period (ΔTburst) from the latent time to the completion of cell lysis was quite small; (v) in a poor medium with l-alanine alone, addition of xylose caused prophage induction following spore germination but with longer Tlatent and ΔTburst times and without cell elongation; (vi) spontaneous prophage induction and lysis of individual cells from spores in a minimal nutrient medium were observed without xylose addition, and cell length prior to cell lysis was ∼4.1 µm, but spontaneous prophage induction was not observed in a rich medium; (vii) in a rich medium, addition of xylose at a time well after spore germination and outgrowth significantly shortened the average Tlatent time. The results of this study provide new insights into the heterogeneity and dynamics of lysis of individual B. subtilis cells derived from spores upon prophage induction.

Recombinant expression of insoluble enzymes in Escherichia coli: a systematic review of experimental design and its manufacturing implications.

Recombinant enzyme expression in Escherichia coli is one of the most popular methods to produce bulk concentrations of protein product. However, this method is often limited by the inadvertent formation of inclusion bodies. Our analysis systematically reviews literature from 2010 to 2021 and details the methods and strategies researchers have utilized for expression of difficult to express (DtE), industrially relevant recombinant enzymes in E. coli expression strains. Our review identifies an absence of a coherent strategy with disparate practices being used to promote solubility. We discuss the potential to approach recombinant expression systematically, with the aid of modern bioinformatics, modelling, and 'omics' based systems-level analysis techniques to provide a structured, holistic approach. Our analysis also identifies potential gaps in the methods used to report metadata in publications and the impact on the reproducibility and growth of the research in this field.

Thrombolytic Enzymes of Microbial Origin: A Review.

Enzyme therapies are attracting significant attention as thrombolytic drugs during the current scenario owing to their great affinity, specificity, catalytic activity, and stability. Among various sources, the application of microbial-derived thrombolytic and fibrinolytic enzymes to prevent and treat vascular occlusion is promising due to their advantageous cost-benefit ratio and large-scale production. Thrombotic complications such as stroke, myocardial infarction, pulmonary embolism, deep venous thrombosis, and peripheral occlusive diseases resulting from blood vessel blockage are the major cause of poor prognosis and mortality. Given the ability of microbial thrombolytic enzymes to dissolve blood clots and prevent any adverse effects, their use as a potential thrombolytic therapy has attracted great interest. A better understanding of the hemostasis and fibrinolytic system may aid in improving the efficacy and safety of this treatment approach over classical thrombolytic agents. Here, we concisely discuss the physiological mechanism of thrombus formation, thrombo-, and fibrinolysis, thrombolytic and fibrinolytic agents isolated from bacteria, fungi, and algae along with their mode of action and the potential application of microbial enzymes in thrombosis therapy.

What's new and notable in bacterial spore killing!

Spores of many species of the orders Bacillales and Clostridiales can be vectors for food spoilage, human diseases and intoxications, and biological warfare. Many agents are used for spore killing, including moist heat in an autoclave, dry heat at elevated temperatures, UV radiation at 254 and more recently 222 and 400 nm, ionizing radiation of various types, high hydrostatic pressures and a host of chemical decontaminants. An alternative strategy is to trigger spore germination, as germinated spores are much easier to kill than the highly resistant dormant spores-the so called "germinate to eradicate" strategy. Factors important to consider in choosing methods for spore killing include the: (1) cost; (2) killing efficacy and kinetics; (3) ability to decontaminate large areas in buildings or outside; and (4) compatibility of killing regimens with the: (i) presence of people; (ii) food quality; (iii) presence of significant amounts of organic matter; and (iv) minimal damage to equipment in the decontamination zone. This review will summarize research on spore killing and point out some common flaws which can make results from spore killing research questionable.

DNA Nanostructures for Targeted Antimicrobial Delivery.

We report the use of DNA origami nanostructures, functionalized with aptamers, as a vehicle for delivering the antibacterial enzyme lysozyme in a specific and efficient manner. We test the system against Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) targets. We use direct stochastic optical reconstruction microscopy (dSTORM) and atomic force microscopy (AFM) to characterize the DNA origami nanostructures and structured illumination microscopy (SIM) to assess the binding of the origami to the bacteria. We show that treatment with lysozyme-functionalized origami slows bacterial growth more effectively than treatment with free lysozyme. Our study introduces DNA origami as a tool in the fight against antibiotic resistance, and our results demonstrate the specificity and efficiency of the nanostructure as a drug delivery vehicle.

Biochemical and mutational analysis of spore cortex-lytic enzymes in the food spoiler Bacillus licheniformis.

Bacillus licheniformis is frequently associated with food spoilage due to its ability to form highly resistant endospores. The present study reveals that B. licheniformis spore peptidoglycan shares a similar structure to spores of other species of Bacillus. Two enzymatic activities associated with depolymerisation of the cortical peptidoglycan, which represents a crucial step in spore germination, were detected by muropeptide analysis. These include lytic transglycosylase and N-acetylglucosaminidase activity, with non-lytic epimerase activity also being detected. The role of various putative cortex-lytic enzymes that account for the aforementioned activity was investigated by mutational analysis. These analyses indicate that SleB is the major lysin involved in cortex depolymerisation in B. licheniformis spores, with CwlJ and SleL having lesser roles. Collectively, the results of this work indicate that B. licheniformis spores employ a similar approach for cortical depolymerisation during germination as spores of other Bacillus species.

Orthologues of Bacillus subtilis Spore Crust Proteins Have a Structural Role in the Bacillus megaterium QM B1551 Spore Exosporium.

The exosporium of Bacillus megaterium QM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of the Bacillus megaterium exosporium. Here, we provide evidence that pBM600-encoded orthologues of the Bacillus subtilis CotW and CotX proteins, which form the crust layer in spores of that species, are structural components of the Bacillus megaterium QM B1551 spore exosporium. The introduction of plasmid-borne cotW and orthologous cotX genes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in the Bacillus megaterium QM B1551 exosporium.IMPORTANCE When starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, in Bacillus megaterium QM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenic Bacillus cereus family. In this work, we provide evidence that structurally important protein constituents of the Bacillus megaterium exosporium are different from those in the Bacillus cereus family. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

Proteins Encoded by the gerP Operon Are Localized to the Inner Coat in Bacillus cereus Spores and Are Dependent on GerPA and SafA for Assembly.

The germination of Bacillus spores is triggered by certain amino acids and sugar molecules which permeate the outermost layers of the spore to interact with receptor complexes that reside in the inner membrane. Previous studies have shown that mutations in the hexacistronic gerP locus reduce the rate of spore germination, with experimental evidence indicating that the defect stems from reduced permeability of the spore coat to germinant molecules. Here, we use the ellipsoid localization microscopy technique to reveal that all six Bacillus cereus GerP proteins share proximity with cortex-lytic enzymes within the inner coat. We also reveal that the GerPA protein alone can localize in the absence of all other GerP proteins and that it has an essential role for the localization of all other GerP proteins within the spore. Its essential role is also demonstrated to be dependent on SafA, but not CotE, for localization, which is consistent with an inner coat location. GerP-null spores are shown also to have reduced permeability to fluorescently labeled dextran molecules compared to wild-type spores. Overall, the results support the hypothesis that the GerP proteins have a structural role within the spore associated with coat permeability.IMPORTANCE The bacterial spore coat comprises a multilayered proteinaceous structure that influences the distribution, survival, and germination properties of spores in the environment. The results from the current study are significant since they increase our understanding of coat assembly and architecture while adding detail to existing models of germination. We demonstrate also that the ellipsoid localization microscopy (ELM) image analysis technique can be used as a novel tool to provide direct quantitative measurements of spore coat permeability. Progress in all of these areas should ultimately facilitate improved methods of spore control in a range of industrial, health care, and environmental sectors.

Paramagnetism in Bacillus spores: Opportunities for novel biotechnological applications.

Spores of Bacillus megaterium, Bacillus cereus, and Bacillus subtilis were found to exhibit intrinsic paramagnetic properties as a result of the accumulation of manganese ions. All three Bacillus species displayed strong yet distinctive magnetic properties arising from differences in manganese quantity and valency. Manganese ions were found to accumulate both within the spore core as well as being associated with the surface of the spore. Bacillus megaterium spores accumulated up to 1 wt.% manganese (II) within, with a further 0.6 wt.% adsorbed onto the surface. At room temperature, Bacillus spores possess average magnetic susceptibilities in the range of 10-6 to 10-5 . Three spore-related biotechnological applications-magnetic sensing, magnetic separation and metal ion adsorption-were assessed subsequently, with the latter two considered as having the most potential for development.

Identification and initial characterisation of a protein involved in Campylobacter jejuni cell shape.

Campylobacter jejuni is the leading cause of bacterial food borne illness. While helical cell shape is considered important for C. jejuni pathogenesis, this bacterium is capable of adopting other morphologies. To better understand how helical-shaped C. jejuni maintain their shape and thus any associated colonisation, pathogenicity or other advantage, it is first important to identify the genes and proteins involved. So far, two peptidoglycan modifying enzymes Pgp1 and Pgp2 have been shown to be required for C. jejuni helical cell shape. We performed a visual screen of ∼2000 transposon mutants of C. jejuni for cell shape mutants. Whole genome sequence data of the mutants with altered cell shape, directed mutants, wild type stocks and isolated helical and rod-shaped 'wild type' C. jejuni, identified a number of different mutations in pgp1 and pgp2, which result in a change in helical to rod bacterial cell shape. We also identified an isolate with a loss of curvature. In this study, we have identified the genomic change in this isolate, and found that targeted deletion of the gene with the change resulted in bacteria with loss of curvature. Helical cell shape was restored by supplying the gene in trans. We examined the effect of loss of the gene on bacterial motility, adhesion and invasion of tissue culture cells and chicken colonisation, as well as the effect on the muropeptide profile of the peptidoglycan sacculus. Our work identifies another factor involved in helical cell shape.

The crystal structure of Clostridium perfringens SleM, a muramidase involved in cortical hydrolysis during spore germination.

Clostridium perfringens spores employ two peptidoglycan lysins to degrade the spore cortex during germination. SleC initiates cortex hydrolysis to generate cortical fragments that are degraded further by the muramidase SleM. Here, we present the crystal structure of the C. perfringens S40 SleM protein at 1.8 Å. SleM comprises an N-terminal catalytic domain that adopts an irregular α/ß-barrel fold that is common to GH25 family lysozymes, plus a C-terminal fibronectin type III domain. The latter is involved in forming the SleM dimer that is evident in both the crystal structure and in solution. A truncated form of SleM that lacks the FnIII domain shows reduced activity against spore sacculi indicating that this domain may have a role in facilitating the position of substrate with respect to the enzyme's active site. Proteins 2016; 84:1681-1689. © 2016 Wiley Periodicals, Inc.

Ellipsoid Localization Microscopy Infers the Size and Order of Protein Layers in Bacillus Spore Coats.

Multilayered protein coats are crucial to the dormancy, robustness, and germination of bacterial spores. In Bacillus subtilis spores, the coat contains over 70 distinct proteins. Identifying which proteins reside in each layer may provide insight into their distinct functions. We present image analysis methods that determine the order and geometry of concentric protein layers by fitting a model description for a spheroidal fluorescent shell image to optical micrographs of spores incorporating fluorescent fusion proteins. The radius of a spherical protein shell can be determined with <10 nm error by fitting an equation to widefield fluorescence micrographs. Ellipsoidal shell axes can be fitted with comparable precision. The layer orders inferred for B. subtilis and B. megaterium are consistent with measurements in the literature. The aspect ratio of elongated spores and the tendency of some proteins to localize near their poles can be quantified, enabling measurement of structural anisotropy.

Structural and functional analysis of SleL, a peptidoglycan lysin involved in germination of Bacillus spores.

A major event in the germination of Bacillus spores concerns hydrolysis of the cortical peptidoglycan that surrounds the spore protoplast, the integrity of which is essential for maintenance of dormancy. Cortex degradation is initiated in all species of Bacillus spores by the combined activity of two semi-redundant cortex-lytic enzymes, SleB and CwlJ. A third enzyme, SleL, which has N-acetylglucosaminidase activity, cleaves peptidoglycan fragments generated by SleB and CwlJ. Here we present crystal structures of B. cereus and B. megaterium SleL at 1.6 angstroms and 1.7 angstroms, respectively. The structures were determined with a view to identifying the structural basis of differences in catalytic efficiency between the respective enzymes. The catalytic (α/ß)8 -barrel cores of both enzymes are highly conserved from a structural perspective, including the spatial distribution of the catalytic residues. Both enzymes are equipped with two N-terminal peptidoglycan-binding LysM domains, which are also structurally highly conserved. However, the topological arrangement of the respective enzymes second LysM domain is markedly different, and this may account for differences in catalytic rates by impacting upon the position of the active sites with respect to their substrates. A chimeric enzyme comprising the B. megaterium SleL catalytic domain plus B. cereus SleL LysM domains displayed enzymatic activity comparable to the native B. cereus protein, exemplifying the importance of the LysM domains to SleL function. Similarly, the reciprocal construct, comprising the B. cereus SleL catalytic domain with B. megaterium SleL LysM domains, showed reduced activity compared with native B. cereus SleL.

Crystal structure of the PepSY-containing domain of the YpeB protein involved in germination of bacillus spores.

The crystal structure of the C-terminal domain of the Bacillus megaterium YpeB protein has been solved by X-ray crystallography to 1.80-Å resolution. The full-length protein is essential in stabilising the SleB cortex lytic enzyme in Bacillus spores, and may have a role in regulating SleB activity during spore germination. The YpeB-C crystal structure comprises three tandemly repeated PepSY domains, which are aligned to form an extended laterally compressed molecule. A predominantly positively charged region located in the second PepSY domain may provide a site for protein interactions that are important in stabilising SleB and YpeB within the spore.

Spore germination mediated by Bacillus megaterium QM B1551 SleL and YpeB.

Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB(N)). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro.