Lytic Activity Research Articles

Isolation, Characterization of Enterococcus phages and their application in control of E. faecalis in milk.

Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6h at both 30 and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48h at 4°C. The strong bactericidal and biofilm removal activities of the phage cocktail suggests the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk. Enterococcus faecalis is an opportunistic pathogen being transmitted from food to humans and causes disease. This study demonstrated the effectiveness of the phage cocktail consisted of new isolated phages in controlling E. faecalis in food.

Chidamide Induces Epstein-Barr Virus (EBV) Lytic Infection and Acts Synergistically with Tenofovir to Eliminate EBV-Positive Burkitt Lymphoma.

Epstein-Barr virus (EBV) is a type of human γ-herpesvirus, and its reactivation plays an important role in the development of EBV-driven Burkitt lymphoma (BL). Despite intensive chemotherapy, the prognosis of relapsed/refractory BL patients remains unfavorable, and a definitive method to completely eliminate latent EBV infection is lacking. Previous studies have demonstrated that histone deacetylase (HDAC) inhibitors can induce the transition of EBV from latency to the lytic phase. The lytic activation of EBV can be inhibited by tenofovir, a potent inhibitor of DNA replication. Herein, we explored the antitumor effect and EBV clearance potential of a novel HDAC inhibitor called chidamide, combined with tenofovir, in the treatment of EBV-positive BL. In the study, chidamide exhibited inhibitory activity against HDAC. Moreover, chidamide inhibited BL cell proliferation, arrested cell cycle progression, and induced BL cell apoptosis primarily by regulating the MAPK pathways. Additionally, chidamide promoted the transcription of lytic genes, including BZLF1, BMRF1, and BMLF1 Compared with chidamide alone, the addition of tenofovir further induced growth arrest and apoptosis in EBV-positive BL cells and inhibited the transcriptions of EBV lytic genes induced by chidamide alone. Furthermore, our in vivo data demonstrated that the combination of chidamide and tenofovir had superior tumor-suppressive effects in a mouse model of BL cell tumors. The aforementioned findings confirm the synergistic effect of chidamide combined with tenofovir in inducing growth inhibition and apoptosis in EBV-positive BL cells and provide an effective strategy for eliminating EBV and EBV-associated malignancies. SIGNIFICANCE STATEMENT: High levels of Epstein-Barr virus (EBV)-DNA have consistently been associated with unfavorable progression-free survival and overall survival in EBV-associated lymphomas. Therefore, identifying novel strategies to effectively eradicate tumor cells and eliminate EBV is crucial for lymphoma patients. This study confirmed, for the first time, the synergistic effect of chidamide combined with tenofovir in the treatment of Burkitt lymphoma and the eradication of EBV virus.

Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis-causing pathogens.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis.

Rapid bacteriolysis of Staphylococcus aureus by lysin exebacase.

Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among pathogenic bacteria. Exebacase is an antistaphylococcal lysin and the first member of the lysin class to have entered clinical trials in the United States. In this study, the bacteriolytic activity of exebacase was characterized with time-kill assays, turbidity reduction assays, and microscopy. Three methicillin-susceptible Staphylococcus aureus and three methicillin-resistant S. aureus isolates were tested in time-kill assays over a range of concentrations from 0.25 to 8 × MIC. Exebacase demonstrated a concentration-dependent killing and showed bactericidal activity (≥3 log10 kill achieved relative to the starting inoculum) within 3 h at 1 × MIC against all strains tested. Dose-dependent lysis by exebacase was, furthermore, observed in the turbidity reduction assay, wherein decreases in initial OD600 of 50% were observed within ~15 min at concentrations as low as 4 µg/mL. Membrane dissolution, loss of cytoplasmic material, and lysis were confirmed by video and electron microscopy. The demonstrated rapid bacteriolytic effect of exebacase is an important distinguishing feature of this novel modality. IMPORTANCE To guide the development of an investigational new antibacterial entity, microbiological data are required to evaluate the killing kinetics against target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of Staphylococcus aureus. Killing by exebacase was determined in multiple assay formats including time-kill assays, wherein reductions of viability of ≥3 log10 colony-forming units/mL were observed within 3 h for multiple different isolates tested, consistent with very rapid bactericidal activity. Rapid reductions in optical density were likewise observed in exebacase-treated cultures, which were visually consistent with microscopic observations of rapid lysis. Overall, exebacase provides a novel antimicrobial modality against S. aureus, characterized by a rapid cidal and lytic activity.

MODIFICATION OF THE PHAGE TITER INCREASE REACTION PROCEDURE FOR AEROMONAD BACTERIOPHAGES

The article presents the results of studies on modification of the phage titer increase reaction technique for aeromonad bacteriophages. Using bacteriophages Av-4 of the UlSAU series and Ah-4 of the Ul-SAU series, which are characterized by high titers of lytic activity and a wide spectrum of specific lyt-ic action, the optimal parameters for setting up the phage titer increase reaction (a quantitative indicator of the reaction that has diagnostic value and the optimal time that ensures full interaction of the phage with bacteria) were developed. It was empirically established that the following parameters are the most effective for setting up the RNF for the purpose of indicating A. veronii bv.veronii and A.hydrophila bacteria in sanitary inspection facilities: the concentration of the indicator culture de-tected during the reaction is 103 - 104 m.c./ml/g, the working dilution of the bacteriophage is 105 PFU/ml. Experiments to optimize the methodology for setting up the RNF have shown that the most optimal method for detecting A. hydrophila and A. veronii bv. veronii bacteria in non-sterile water and fish samples in laboratory conditions is preliminary cultivation of the test material for 2 hours at (29±1) 0C, followed by infection with phages Av-4 of the UlSAU series and Ah-4 of the UlSAU series and incubation of the mixture for 4 hours at (29±1) 0C. The study was carried out in accordance with the thematic plan-task of the Ministry of Agriculture of the Russian Federation, registration number EGISU NIOKTR 123031600041-9.

Identification, activity and delivery of new LysFA67 endolysin to target cheese spoilage Clostridium tyrobutyricum

Bacteriophages and their endolysins are potential biocontrol agents for the anaerobic spoilage organism Clostridium tyrobutyricum, which causes cheese late blowing defect. This study sequenced and compared the genomes of eight bacteriophages from Spanish dairy farms that were active against C. tyrobutyricum, to identify novel species and phage proteins. Phages vB_CtyS-FA67 and vB_CtyS-FA70 shared >94% intergenomic similarity to each other but neither phage had significant similarity to ΦCTP1, the unique C. tyrobutyricum phage sequenced to date. Taxonomic analysis indicated that both phages belong to the class Caudoviricetes and are related to dsDNA viruses with long non-contractile tails. vB_CtyS-FA67 had no other close relatives and encoded a novel endolysin, LysFA67, predicted to belong to the glycoside hydrolase GH24 family. LysFA67 lysed 93% of C. tyrobutyricum cells after 4 min in turbidity reduction assays, retaining lytic activity at pHs 4.2–8.1 and at 30–45 °C. The endolysin remained stable after 30 d storage at 4, 12 and 25 °C, while its activity decreased at −20 °C. LysFA67 lysed several clostridia species, while common dairy bacteria were not affected. Lactococcus lactis INIA 437, used as a cheese starter, was engineered to deliver LysFA67 and red fluorescent LysFA67-mCherry to dairy products. We demonstrated that these engineered strains were able to maintain lytic activity and fluorescence without affecting their technological properties in milk.

Algicidal Activity and Microcystin-LR Destruction by a Novel Strain Penicillium sp. GF3 Isolated from the Gulf of Finland (Baltic Sea).

The present article focuses on a strain of ascomycete GF3 isolated from a water sample taken in the Gulf of Finland. Based on phylogenetic analysis data, the isolate was identified as Penicillium sp. GF3. The fungus GF3 demonstrates algicidal activity towards cyanobacteria (98-100%). The algicidal effect on green algae did not exceed 50%. The isolate GF3 exhibits an indirect attack mode by releasing metabolites with algicidal and/or lytic activity into the environment. Moreover, the strain Penicillium sp. GF3 is able to degrade MC-LR. After 72 h of GF3 cultivation, the MC-LR content was reduced by 34.1% and 26.7% at initial 0.1 μg/mL and 0.45 μg/mL concentrations, respectively. The high stress resistance of the GF3 to toxic MC-LR is provided by a 1.5-fold activation of catalase activity and a change in the reduced glutathione content. Additionally, during the MC-LR biotransformation, a MC-LR-GSH conjugate and linearized MC-LR were identified. The linearized MC-LR in the presence of fungi capable of degrading MCs was revealed for the first time. Using Daphnia magna as a bioindicator, it was shown that the MC-LR biotransformation led to the formation of less toxic intermediates. The toxicity of the fungal filtrate is reduced by five times compared to the abiotic control. Our findings enhance the understanding of the role that ascomycete fungi have as potential bioagents for cyanoHABs to control and detoxify water bodies.

Mycobacteriophage D29 Lysin B exhibits promising anti-mycobacterial activity against drug-resistant Mycobacterium tuberculosis.

The emergence of antibiotic resistance in Mycobacterium tuberculosis has been the culprit behind the inefficient treatment of tuberculosis (TB). Mycobacteriophages being rapid and specific in their activity against mycobacteria including M. tuberculosis are an emerging alternative solution to the antibiotic resistance problem. D29 mycobacteriophage is known as an efficient lytic phage. Its lytic cassette encodes for two lytic proteins, Lysin A (LysA) and Lysin B (LysB), and a holin protein responsible for the translocation of the former two. LysB is a mycolyl-arabinogalactan esterase, which has shown a promising lytic effect on M. tuberculosis. In this study, we explored the lytic efficiency of D29 mycobacteriophage LysB protein on drug-resistant strains of M. tuberculosis. We demonstrate that LysB effectively lyses multi-drug-resistant M. tuberculosis both alone and along with anti-TB drugs. We also report that the surfactant Tween-80, although not vital, potentiates LysB activity. We are able to demonstrate that LysB can eliminate M. tuberculosis residing in mouse macrophages. Thus, by combining our in vitro and ex vivo data, we are able to claim that LysB is a potential phage-derived therapeutic which should undergo pre-clinical and clinical trials. IMPORTANCE To combat the rapidly emerging drug-resistant M. tuberculosis, it is now essential to look for alternative therapeutics. Mycobacteriophages can be considered as efficient therapeutics due to their natural ability to infect and kill mycobacteria including M. tuberculosis. Here, we have exploited the mycolyl-arabinogalactan esterase property of LysB encoded from mycobacteriophage D29. This study is novel in terms of targeting a multi-drug-resistant pathogenic strain of M. tuberculosis with LysB and also examining the combination of anti-TB drugs and LysB. All the experiments include external administration of LysB. Therefore, the remarkable lytic activity of LysB overcomes the difficulty to enter the complex cell envelope of mycobacteria. Targeting the intracellularly located M. tuberculosis by LysB and non-toxicity to macrophages take the process of the development of LysB as a drug one step ahead, and also, the interaction studies with rifampicin and isoniazid will help to form a new treatment regimen against tuberculosis.

Bacteriophage entrapped chitosan microgel for the treatment of biofilm-mediated polybacterial infection in burn wounds

Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria are most commonly present in burn wound infections. Multidrug resistance (MDR) and biofilm formation make it difficult to treat these infections. Bacteriophages (BPs) are proven as an effective therapy against MDR as well as biofilm-associated wound infections. In the present work, a naturally inspired bacteriophage cocktail loaded chitosan microparticles-laden topical gel has been developed for the effective treatment of these infections. Bacteriophages against MDR S. aureus (BPSAФ1) and P. aeruginosa (BPPAФ1) were isolated and loaded separately and in combination into the chitosan microparticles (BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs), which were later incorporated into the SEPINEO™ P 600 gel (BPSAФ1-CHMPs-gel, BPPAФ1-CHMPs-gel, and MBP-CHMPs-gel). BPs were characterized for their morphology, lytic activity, burst size, and hemocompatibility, and BPs belongs to Caudoviricetes class. Furthermore, BPSAФ1-CHMPs, BPPAФ1-CHMPs, and MBP-CHMPs had an average particle size of 1.19 ± 0.11, 1.42 ± 0.21, and 2.84 ± 0.28 μm, respectively, and expressed promising in vitro antibiofilm eradication potency. The ultrasound and photoacoustic imaging in infected burn wounds demonstrated improved wound healing reduced inflammation and increased oxygen saturation following treatment with BPs formulations. The obtained results suggested that the incorporation of the BPs in the MP-gel protected the BPs, sustained the BPs release, and improved the antibacterial activity.

Actin cytoskeleton remodeling at the cancer cell side of the immunological synapse: good, bad, or both?

Cytotoxic lymphocytes (CLs), specifically cytotoxic T lymphocytes and natural killer cells, are indispensable guardians of the immune system and orchestrate the recognition and elimination of cancer cells. Upon encountering a cancer cell, CLs establish a specialized cellular junction, known as the immunological synapse that stands as a pivotal determinant for effective cell killing. Extensive research has focused on the presynaptic side of the immunological synapse and elucidated the multiple functions of the CL actin cytoskeleton in synapse formation, organization, regulatory signaling, and lytic activity. In contrast, the postsynaptic (cancer cell) counterpart has remained relatively unexplored. Nevertheless, both indirect and direct evidence has begun to illuminate the significant and profound consequences of cytoskeletal changes within cancer cells on the outcome of the lytic immunological synapse. Here, we explore the understudied role of the cancer cell actin cytoskeleton in modulating the immune response within the immunological synapse. We shed light on the intricate interplay between actin dynamics and the evasion mechanisms employed by cancer cells, thus providing potential routes for future research and envisioning therapeutic interventions targeting the postsynaptic side of the immunological synapse in the realm of cancer immunotherapy. This review article highlights the importance of actin dynamics within the immunological synapse between cytotoxic lymphocytes and cancer cells focusing on the less-explored postsynaptic side of the synapse. It presents emerging evidence that actin dynamics in cancer cells can critically influence the outcome of cytotoxic lymphocyte interactions with cancer cells.

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization

Staphylococcus aureus (S.aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S.aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

Broad-Spectrum Antimicrobial Activity of Synthetic Peptides GV185 and GV187.

Optimizing synthetic antimicrobial peptides for safe and enhanced activity against fungal and bacterial pathogens is useful for genetic engineering of plants for resistance to plant pathogens and their associated mycotoxins. Nine synthetic peptides modeled after lytic peptides tachyplesin 1, D4E1 from cecropin A, and protegrin 1 were added to germinated spores of fungal species Aspergillus flavus, Rhizopus stolonifer, Fusarium oxysporum f. sp. vasinfectum, F. verticillioides, F. graminearum, Claviceps purpurea, Verticillium dahliae, and Thielaviopsis basicola and bacterial cultures of Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. campestris at different doses and inhibitory dose response curves, and were modeled to assess antimicrobial activity. Peptides GV185 and GV187, modified from tachyplesin 1, had superior abilities to inhibit fungal and bacterial growth (50% inhibitory concentrations [IC50] ranging from 0.1 to 8.7 µM). R. stolonifer (IC50 = 8.1 µM), A. flavus (IC50 = 3.1 µM), and F. graminearum (IC50 = 2.2 µM) were less inhibited by GV185 and GV187 than all the remaining fungi (IC50 = 1.4 µM) and bacteria (IC50 = 0.1 µM). Of the remaining peptides, GV193, GV195, and GV196 (IC50 range of 0.9 to 6.6 µM) inhibited fungal growth of A. flavus, F. verticillioides, and F. graminearum less than GV185 and GV187 (IC50 range of 0.8 to 3.9 µM), followed by GV197 (IC50 range of 0.8 to 9.1 µM), whereas GV190 and GV192 inhibited poorly (IC50 range of 28.2 to 36.6 µM and 15.5 to 19.4 µM, respectively) and GV198 stimulated growth. GV185 and GV187 had slightly weaker hydrophobic and cationic residues than other tachyplesin 1 modified peptides but still had unexpectedly high lytic activity. Germinated fungal spores of R. stolonifer and F. graminearum exposed to these two peptides and D4E1 and AGM182 appeared wrinkled, with perforations near potential cytoplasmic leakage, which provided evidence of plasma membrane and cell wall lysis. We conclude that peptides GV185 and GV187 are promising candidates for genetic engineering of crops for resistance to plant-pathogenic bacteria and fungi, including A. flavus and aflatoxin contamination.

Expression of a recombinant endolysin from bacteriophage CAP 10-3 with lytic activity against Cutibacterium acnes

The bacteriophage CAP 10-3 forming plaques against Cutibacterium acnes which causes skin acne was previously isolated from human skin acne lesion. Incomplete whole genome sequence (WGS) of the bacteriophage CAP 10-3 was obtained and it had 29,643 bp long nucleotide with 53.86% GC content. The sequence was similar to C. acnes phage PAP 1-1 with a nucleotide sequence identity of 89.63% and the bacteriophage belonged to Pahexavirus. Bioinformatic analysis of the WGS predicted 147 ORFs and functions of 40 CDSs were identified. The predicted endolysin gene of bacteriophage CAP 10-3 was 858 bp long which was deduced as 285 amino acids (~ 31 kDa). The protein had the highest similarity with amino acid sequence of the endolysin from Propionibacterium phage PHL071N05 with 97.20% identity. The CAP 10-3 endolysin gene was amplified by PCR with primer pairs based on the gene sequence, cloned into an expression vector pET-15b and transformed into Escherichia coli BL21(DE3) strain. The predicted protein band (~ 33 kDa) for the recombinant endolysin was detected in an SDS-PAGE gel and western blot assay. The concentrated supernatant of cell lysate from E. coli BL21(DE3) (pET-15b_CAP10-3 end) and a partially purified recombinant CAP 10-3 endolysin showed antibacterial activity against C. acnes KCTC 3314 in a dose-dependent manner. In conclusion, the recombinant CAP 10-3 endolysin was successfully produced in E. coli strain and it can be considered as a therapeutic agent candidate for treatment of human skin acne.

Genomic characterization of the novel bacteriophage IME183, infecting Klebsiella pneumoniae of capsular type K2.

Klebsiella pneumoniae causes a wide range of serious and life-threatening infections. Klebsiella phage IME183, isolated from hospital sewage, exhibited lytic activity against K. pneumoniae of capsular type K2. Transmission electron microscopy revealed that phage IME183 has a head with a diameter of 50 nm and a short tail. Its genome is 41,384 bp in length with a GC content of 52.92%. It is predicted to contain 50 open reading frames (ORFs). The results of evolutionary analysis suggest that phage IME183 should be considered a member of a new species in the genus Przondovirus.

Lytic polysaccharide monooxygenase activity increases productive binding capacity of cellobiohydrolases on cellulose

Cellobiohydrolases are crucial for cellulose breakdown, but their efficiency on crystalline cellulose is hampered by limited access to single chain ends to initiate hydrolysis. As a result, they depend on enzymes like lytic polysaccharide monooxygenases (LPMOs), which directly target the crystalline cellulose surface. This study investigated how LPMO pretreatment affected the productive binding capacity of a Trichoderma longibrachiatum cellobiohydrolase, TlCBHI, on crystalline cellulose by applying an amperometric cellobiose dehydrogenase biosensor. After the 24-hour of LPMO pretreatment, the productive binding capacity of TlCBHI significantly increased in all reactions. However, with a shorter 5-hour LPMO pretreatment, minimal to no effect on productive binding capacity was observed. Of note, all LPMO reactions were inactivated around this time point. This delayed LPMO effect suggests that the improved binding capacity for cellulases does not directly result from cellulose chain cleavage by LPMOs but rather from the cellulose decrystallization following the oxidative cleavage.

Characterization of Lactobacilli Phage Endolysins and Their Functional Domains-Potential Live Biotherapeutic Testing Reagents.

Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and ΦJB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and ΦJB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and ΦJB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The ΦJB-EAD exhibited stronger lytic activity than ΦJB-lys, possibly due to ΦJB-CBD-mediated sequestration of ΦJB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and ΦJB-lys and their EADs are good candidates for LBP purity reagent development.

Cholesterol catalyzes unfolding in membrane-inserted motifs of the pore forming protein cytolysin A

Plasma membrane-induced protein folding and conformational transitions play a central role in cellular homeostasis. Several transmembrane proteins are folded in the complex lipid milieu to acquire a specific structure and function. Bacterial pore forming toxins (PFTs) are proteins expressed by a large class of pathogenic bacteria that exploit the plasma membrane environment to efficiently undergo secondary structure changes, oligomerize, and form transmembrane pores. Unregulated pore formation causes ion imbalance, leading to cell death and infection. Determining the free energy landscape of these membrane-driven-driven transitions remains a challenging problem. Although cholesterol recognition is required for lytic activity of several proteins in the PFT family of toxins, the regulatory role of cholesterol for the α-PFT, cytolysin A expressed by Escherichia coli remains unexplained. In a recent free energy computation, we showed that the β tongue, a critical membrane-inserted motif of the ClyA toxin, has an on-pathway partially unfolded intermediate that refolds into the helix-turn-helix motif of the pore state. To understand the molecular role played by cholesterol, we carry out string-method-based computations in membranes devoid of cholesterol, which reveals an increase of ∼30 times in the free energy barrier for the loss of β sheet secondary structure when compared with membranes containing cholesterol. Specifically, the tyrosine-cholesterol interaction was found to be critical to creating the unfolded intermediate. Cholesterol also increases the packing and hydrophobicity of the bilayer, resulting in enhanced interactions of the bound protein before complete membrane insertion. Our study illustrates that cholesterol is critical to catalyzing and stabilizing the membrane-inserted unfolded state of the β tongue motif of ClyA, opening up fresh insights into cholesterol-assisted unfolding of membrane proteins.

Development and characterization of a bacteriophage cocktail with high lytic efficacy against field-isolated Salmonella enterica

Salmonella spp. is a prevalent pathogen that causes great public health concern worldwide. Bacteriophage-based cocktails have arisen as an alternative to antibiotics to inhibit the growth of Salmonella. However, the bactericidal effect of bacteriophage cocktails in vivo largely differs from their observed effect in vitro. This is partly because in vitro developments of cocktails do not always consider the bacterial diversity nor the environmental conditions where bacteriophages will have to replicate. Here, we isolated and sequenced 47 bacteriophages that showed variable degrees of lytic activity against 258 Salmonella isolates from a commercial broiler company in Brazil. Three of these bacteriophages were characterized and selected to assemble a cocktail. In vitro quantitative assays determined the cocktail to be highly effective against multiple serovars of Salmonella, including Minnesota and Heidelberg. Remarkably, the in vitro lytic activity of the cocktail was retained or improved in conditions that more closely resembled the chicken gut, such as anaerobiosis, 42°C, and Salmonella mono-strain biofilms. Analysis of bacterial cross-resistance between the 3 bacteriophages composing the cocktail revealed limited or no generation of cross-resistance. Our results highlight the relevance of an optimized flux of work to develop bacteriophage cocktails against Salmonella with high lytic efficacy and strong potential to be applied in vivo in commercial broiler farms.

A Carboxyethylchitosan Gel Cross-Linked with Glutaraldehyde as a Candidate Carrier for Biomedical Applications.

To date, few publications describe CEC's properties and possible applications-thus, further evaluation of these properties is a point of interest. The present in vitro model study aimed to evaluate a carboxyethylchitosan (CEC) gel with a degree of substitution of 1, cross-linked with glutaraldehyde at a polymer:aldehyde molar ratio of 10:1, as a potential carrier for delivering bacteriophages to various pH-fixed media (acidic, alkaline), and including gastrointestinal tract (GIT) variable medium. A quantitative analysis of bacteriophages released from the gel was performed using photon correlation spectrophotometry, and phage activity after emission into medium was evaluated using the spot test. The results showed that the CEC gel's maximum swelling ratios were at a nearly neutral alkaline pH. Increasing temperature enhances the swelling ratio of the gel independent from pH, up to 1127% at 37 °C and alkaline pH. The UV and photon correlation spectrophotometry showed equal gel release kinetics in both fixed media with acidic (pH = 2.2) and alkaline (pH = 7.4) pH environments at 37 °C, with the maximum release within two hours. However, phage lytic activity in the spot test during this simulation was absent. At the same time, we obtained an opaque phage lytic activity in the alkaline pH-fixed medium for at least three hours. Phages released from the tested CEC gel in different pHs suggest that this gel could be used for applications that require fast release at the treatment site both in acidic and alkaline pH. Such treatment sites could be a wound or even soil with mild acidic or alkaline pH. However, such CEC gel is not suitable as a delivery system to the GIT because of possible transported acid-sensitive agent (such as phages) release and destruction already in the stomach.

Biopharmaceutical properties of polyvalent bacteriophage capsules

Scientific relevance. Rational phage therapy is a highly effective way to combat bacterial infections, especially in conditions of steadily increasing antibiotic resistance. Most bacteriophage preparations are formulated as oral and topical solutions. However, oral administration of liquid phage preparations results in significant inactivation in the stomach. To shield active ingredients from the acidic environment, Sextaphag® Pyobacteriophage, polyvalent, has been formulated into capsules.Aim. This study evaluated the polyvalent bacteriophage preparation in capsules in terms of its potency and biopharmaceutical properties.Materials and methods. The study compared the polyvalent bacteriophage preparation formulated as capsules with the polyvalent bacteriophage preparation formulated as a solution. The potency was evaluated by the Appelmans method, and phage particles were quantified by the Gratia method. To evaluate the acid-neutralising capacity, the authors placed test samples of the bacteriophage preparation in 0.1 M hydrochloric acid and analysed their potency by the Appelmans method. Chinchilla rabbits were used to analyse anti-phage immune responses, and their antibody levels were measured using an enzyme immunoassay test system developed by the authors. The pharmacokinetic parameters were studied in outbred white mice after oral dosing.Results. The polyvalent bacteriophage preparation exhibited high lytic activity towards Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aegidinosa, Staphylococcus aureus, and Streptococcus pneumoniae, which accounted for a dilution factor of 10-6. Following oral administration of the polyvalent bacteriophage preparation in capsules to mice, the level of absorption was 3.1–3.7 times higher than that observed with the solution. Repeated oral administration of therapeutic doses did not induce anti-phage antibodies in rabbits. The stability study showed that the polyvalent bacteriophage preparation retained high lytic activity for 18 months.Conclusions. According to the study results, the polyvalent bacteriophage preparation in capsules exerts significant antibacterial activity against the studied microorganisms, has a high level of absorption, retains its lytic activity for a long time, and does not induce anti-phage antibodies after oral dosing, which confirms its safety and efficacy.