Ecological and evolutionary dynamics of CRISPR-Cas systems in Clostridium botulinum: Insights from genome mining and comparative analysis.

Understanding the prevalence and distribution of CRISPR-Cas systems across different strains can illuminate the ecological and evolutionary dynamics of Clostridium botulinum populations. In this study, we conducted genome mining to characterize the CRISPR-Cas systems of C. botulinum strains. Our analysis involved retrieving complete genome sequences of these strains and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Subsequently, we performed an analysis of homology in spacer sequences from identified CRISPR arrays to investigate and characterize the range of targeted phages and plasmids. Additionally, we investigated the evolutionary trajectory of C. botulinum strains under selective pressures from foreign invasive DNA. Our findings revealed that 306 strains possessed complete CRISPR-Cas structures, comprising 58% of the studied C. botulinum strains. Secondary structure prediction of consensus repeats indicated that subtype II-C, with longer stems compared to subtypes ID and IB, tended to form more stable RNA secondary structures. Moreover, protospacer motif analysis demonstrated that strains with subtype IB CRISPR-Cas systems exhibited 5'-CGG-3', 5'-CC-3', and 5'-CAT-3' motifs in the 3' flanking regions of protospacers. The diversity observed in CRISPR-Cas systems indicated their classification into subtypes IB, ID, II-C, III-B, and III-D. Furthermore, our results showed that systems with subtype ID and III-D frequently harbored similar spacer patterns. Moreover, analysis of spacer sequences homology with phage and prophage genomes highlighted the specific activities exhibited by subtype IB and III-B against phages and plasmids, providing valuable insights into the functional specialization within these systems.

Phage Mediated Biocontrol: A Promising Green Solution for Sustainable Agriculture.

In the current scenario of growing world population, limited cultivable land resources, plant diseases, and pandemics are some of the major factors responsible for declining global food security. Along with meeting the food demand, the maintenance of food quality is also required to ensure healthy consumption and marketing. In agricultural fields, pest infestations and bacterial diseases are common causes of crop damage, leading to massive yield losses. Conventionally, antibiotics and several pesticides have been used to manage and control these plant pathogens. However, the overuse of antibiotics and pesticides has led to the emergence of resistant strains of pathogenic bacteria. The bacteriophages are the natural predators of bacteria and are host-specific in their action. Therefore, the use of bacteriophages for the biocontrol of pathogenic bacteria is serving as a sustainable and green solution in crop protection and production. In this review, we have discussed the important plant pathogens and their impact on plant health and yield loss. Further, we have abridged the role of bacteriophages in the protection of crops from bacterial disease by discussing various greenhouse and field trials. Finally, we have discussed the impact of bacteriophages on the plant microbiome, phage resistance, and legal challenges in the registration and commercial production of bacteriophage-based biopesticides. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01204-x.

Phage therapy: an alternative treatment modality for MDR bacterial infections.

The increasing global incidence of multidrug-resistant (MDR) bacterial infections threatens public health and compromises various aspects of modern medicine. Recognising the urgency of this issue, the World Health Organisation has prioritised the development of novel antimicrobials to combat ESKAPEE pathogens. Comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli, such pathogens represent a spectrum of high to critical drug resistance, accounting for a significant proportion of hospital-acquired infections worldwide. In response to the waning efficacy of antibiotics against these resilient pathogens, phage therapy (PT) has emerged as a promising therapeutic strategy. This review provides a comprehensive summary of clinical research on PT and explores the translational journey of phages from laboratory settings to clinical applications. It examines recent advancements in pre-clinical and clinical developments, highlighting the potential of phages and their proteins, alone or in combination with antibiotics. Furthermore, this review underlines the importance of establishing safe and approved routes of phage administration to patients. In conclusion, the evolving landscape of phage therapy offers a beacon of hope in the fight against MDR bacterial infections, emphasising the imperative for continued research, innovation and regulatory diligence to realise its full potential in clinical practice.

Complete genome sequences of Arthrobacter globiformis phages Uzumaki and Argan of cluster AU6.

Bacteriophages Uzumaki and Argan infect Arthrobacter globiformis B-2880 isolated from soil samples in Long Island, New York. These bacteriophages have lambda-like morphology with prolate capsid and share 97% gene content similarity. These traits place them in cluster AU6 with other related Arthrobacter phages.

A new standardization for the use of chicken embryo: selection of target from the phage display library and infection.

The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: • SP-infected chicken embryo can be a helpful model of systemic infection for different tests. • Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. • Phage M13 from the PD library can be used in chicken embryo model tests.

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

Characterization and genomic analysis of a lytic Stenotrophomonas maltophilia short-tailed phage A1432 revealed a new genus of the family Mesyanzhinovviridae.

Stenotrophomonas maltophilia (S. maltophilia) is an emerging opportunistic pathogen that exhibits resistant to a majority of commonly used antibiotics. Phages have the potential to serve as an alternative treatment for S. maltophilia infections. In this study, a lytic phage, A1432, infecting S. maltophilia YCR3A-1, was isolated and characterized from a karst cave. Transmission electron microscopy revealed that phage A1432 possesses an icosahedral head and a shorter tail. Phage A1432 demonstrated a narrow host range, with an optimal multiplicity of infection of 0.1. The one-step growth curve indicated a latent time of 10 min, a lysis period of 90 min, a burst size of 43.2 plaque-forming units per cell. In vitro bacteriolytic activity test showed that phage A1432 was capable to inhibit the growth of S. maltophilia YCR3A-1 in an MOI-dependent manner after 2 h of co-culture. BLASTn analysis showed that phage A1432 genome shares the highest similarity (81.46%) with Xanthomonas phage Xoo-sp2 in the NCBI database, while the query coverage was only 37%. The phage contains double-stranded DNA with a genome length of 61,660 bp and a GC content of 61.92%. It is predicted to have 79 open reading frames and one tRNA, with no virulence or antibiotic resistance genes. Phylogenetic analysis using terminase large subunit and DNA polymerase indicated that phage A1432 clustered with members of the Bradleyvirinae subfamily but diverged into a distinct branch. Further phylogenetic comparison analysis using Average Nucleotide Identity, proteomic phylogenetic analysis, genomic network analysis confirmed that phage A1432 belongs to a novel genus within the Bradleyvirinae subfamily, Mesyanzhinovviridae family. Additionally, phylogenetic analysis of the so far isolated S. maltophilia phages revealed significant genetic diversity among these phages. The results of this research will contribute valuable information for further studies on their morphological and genetic diversity, will aid in elucidating the evolutionary mechanisms that give rise to them.

DdmABC-dependent death triggered by viral palindromic DNA sequences.

Defense systems that recognize viruses provide important insights into both prokaryotic and eukaryotic innate immunity mechanisms. Such systems that restrict foreign DNA or trigger cell death have recently been recognized, but the molecular signals that activate many of these remain largely unknown. Here, we characterize one such system in pandemic Vibrio cholerae responsible for triggering cell density-dependent death (CDD) of cells in response to the presence of certain genetic elements. We show that the key component is the Lamassu DdmABC anti-phage/plasmid defense system. We demonstrate that signals that trigger CDD were palindromic DNA sequences in phages and plasmids that are predicted to form stem-loop hairpins from single-stranded DNA. Our results suggest that agents that damage DNA also trigger DdmABC activation and inhibit cell growth. Thus, any infectious process that results in damaged DNA, particularly during DNA replication, can in theory trigger DNA restriction and death through the DdmABC abortive infection system.

Evaluation of Reproductive Histology Response of Adult Fasciola hepatica in Goats Vaccinated with Cathepsin L Phage-Exposed Mimotopes.

Fasciolosis, a globally re-emerging zoonotic disease, is mostly caused by the parasitic infection with Fasciola hepatica, often known as the liver fluke. This disease has a considerable impact on livestock productivity. This study aimed to evaluate the fluke burdens and faecal egg counts in goats that were administered phage clones of cathepsin L mimotopes and then infected with F. hepatica metacercariae. Additionally, the impact of vaccination on the histology of the reproductive system, specifically related to egg generation in adult parasites, was examined. A total of twenty-four goats, which were raised in sheds, were divided into four groups consisting of six animals each. These groups were randomly assigned. The goats were then subjected to two rounds of vaccination. Each vaccination involved the administration of 1 × 1013 phage particles containing specific mimotopes for cathepsin L2 (group 1: PPIRNGK), cathepsin L1 (group 2: DPWWLKQ), and cathepsin L1 (group 3: SGTFLFS). The immunisations were carried out on weeks 0 and 4, and the Quil A adjuvant was used in combination with the mimotopes. The control group was administered phosphate-buffered saline (PBS) (group 4). At week 6, all groups were orally infected with 200 metacercariae of F. hepatica. At week 22 following the initial immunisation, the subjects were euthanised, and adult F. hepatica specimens were retrieved from the bile ducts and liver tissue, and subsequently quantified. The specimens underwent whole-mount histology for the examination of the reproductive system, including the testis, ovary, vitellaria, Mehlis' gland, and uterus. The mean fluke burdens following the challenge were seen to decrease by 50.4%, 62.2%, and 75.3% (p < 0.05) in goats that received vaccinations containing cathepsin L2 PPIRNGK, cathepsin L1 DPWWLKQ, and cathepsin L1 SGTFLFS, respectively. Animals that received vaccination exhibited a significant reduction in the production of parasite eggs. The levels of IgG1 and IgG2 isotypes in vaccinated goats were significantly higher than in the control group, indicating that protection is associated with the induction of a mixed Th1/Th2 immune response. The administration of cathepsin L to goats exhibits a modest level of efficacy in inducing histological impairment in the reproductive organs of liver flukes, resulting in a reduction in egg output.

Soil Giant Phage: Genome and Biological Characteristics of Sinorhizobium Jumbo Phage.

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.

Phage Display Technology in Biomarker Identification with Emphasis on Non-Cancerous Diseases.

In recent years, phage display technology has become vital in clinical research. It helps create antibodies that can specifically bind to complex antigens, which is crucial for identifying biomarkers and improving diagnostics and treatments. However, existing reviews often overlook its importance in areas outside cancer research. This review aims to fill that gap by explaining the basics of phage display and its applications in detecting and treating various non-cancerous diseases. We focus especially on its role in degenerative diseases, inflammatory and autoimmune diseases, and chronic non-communicable diseases, showing how it is changing the way we diagnose and treat illnesses. By highlighting important discoveries and future possibilities, we hope to emphasize the significance of phage display in modern healthcare.

Investigating Propionibacterium acnes antibiotic susceptibility and response to bacteriophage in vitro and in vivo.

Introduction: A total of 94 Propionibacterium acnes (P. acnes) isolates were obtained from a hospital in Beijing to evaluate their susceptibility to erythromycin, clarithromycin, doxycycline, and minocycline. As well as the determination of the effectiveness of P. acnes phages in vitro and in P. acnes-induced lesions mouse model. Methods: Patients with acne vulgaris (AV) were enrolled from August 2021 to October 2022. Standard methods were employed for specimen collection, culture, and identification of P. acnes. Susceptibility testing was conducted using E-strips for erythromycin, clarithromycin, minocycline, and doxycycline. Phage culture and identification followed standard procedures. A mouse model with P. acnes-induced skin lesions was established, and data was analyzed using χ 2 test. Results: The results showed that all isolates were susceptible to minocycline and doxycycline, while 53 (56.4%) and 52 (55.3%) isolates were susceptible to erythromycin and clarithromycin, respectively. Interestingly, younger patients and those with lower acne severity exhibited reduced resistance. Phage cleavage rates ranged from 88.30 to 93.60%. Multilocus sequence typing (MLST) analysis was conducted on eight randomly selected P. acnes isolates, and the IA-2 subtype was used in experiments to address P. acnes-induced lesions in mice. Phage therapy proved effective in this model. Discussion: This study highlights the high susceptibility of P. acnes to doxycycline and tetracycline, while erythromycin and clarithromycin exhibited elevated resistance. Additionally, P. acnes phages demonstrated high cleavage rates and potential effectiveness in treating P. acnes-induced lesions. These findings suggest promising avenues for further exploration of phage therapy in acne treatment.

More evolvable bacteriophages better suppress their host.

The number of multidrug-resistant strains of bacteria is increasing rapidly, while the number of new antibiotic discoveries has stagnated. This trend has caused a surge in interest in bacteriophages as anti-bacterial therapeutics, in part because there is near limitless diversity of phages to harness. While this diversity provides an opportunity, it also creates the dilemma of having to decide which criteria to use to select phages. Here we test whether a phage's ability to coevolve with its host (evolvability) should be considered and how this property compares to two previously proposed criteria: fast reproduction and thermostability. To do this, we compared the suppressiveness of three phages that vary by a single amino acid yet differ in these traits such that each strain maximized two of three characteristics. Our studies revealed that both evolvability and reproductive rate are independently important. The phage most able to suppress bacterial populations was the strain with high evolvability and reproductive rate, yet this phage was unstable. Phages varied due to differences in the types of resistance evolved against them and their ability to counteract resistance. When conditions were shifted to exaggerate the importance of thermostability, one of the stable phages was most suppressive in the short-term, but not over the long-term. Our results demonstrate the utility of biological therapeutics' capacities to evolve and adjust in action to resolve complications like resistance evolution. Furthermore, evolvability is a property that can be engineered into phage therapeutics to enhance their effectiveness.

Phage display-derived alpaca nanobodies as potential therapeutics for Naja atra snake envenomation.

Naja atra, the Chinese cobra, is a major cause of snake envenomation in Asia, causing hundreds of thousands of clinical incidents annually. The current treatment, horse serum-derived antivenom, has unpredictable side effects and presents manufacturing challenges. This study focused on developing new-generation snake venom antidotes by using microbial phage display technology to derive nanobodies from an alpaca immunized with attenuated N. atra venom. Following confirmation of the immune response in the alpaca, we amplified VHH genes from isolated peripheral blood mononuclear cells and constructed a phage display VHH library of 1.0 × 107 transformants. After four rounds of biopanning, the enriched phages exhibited increased binding activity to N. atra venom. Four nanobody clones with high binding affinities were selected: aNAH1, aNAH6, aNAH7, and aNAH9. Specificity testing against venom from various snake species, including two Southeast Asian cobra species, revealed nanobodies specific to the genus Naja. An in vivo mouse venom neutralization assay demonstrated that all nanobodies prolonged mouse survival and aNAH6 protected 66.6% of the mice from the lethal dosage. These findings highlight the potential of phage display-derived nanobodies as valuable antidotes for N. atra venom, laying the groundwork for future applications in snakebite treatment.IMPORTANCEChinese cobra venom bites present a formidable medical challenge, and current serum treatments face unresolved issues. Our research applied microbial phage display technology to obtain a new, effective, and cost-efficient treatment approach. Despite interest among scientists in utilizing this technology to screen alpaca antibodies against toxins, the available literature is limited. This study makes a significant contribution by introducing neutralizing antibodies that are specifically tailored to Chinese cobra venom. We provide a comprehensive and unbiased account of the antibody construction process, accompanied by thorough testing of various nanobodies and an assessment of cross-reactivity with diverse snake venoms. These nanobodies represent a promising avenue for targeted antivenom development that bridges microbiology and biotechnology to address critical health needs.

Dual phage-incorporated electrospun polyvinyl alcohol-eudragit nanofiber matrix for rapid healing of diabetic wound infected by Pseudomonas aeruginosa and Staphylococcus aureus.

Diabetic wound healing remains a healthcare challenge due to co-occurring multidrug-resistant (MDR) bacterial infections and the constraints associated with sustained drug delivery. Here, we integrate two new species of phages designated as PseuPha1 and RuSa1 respectively lysing multiple clinical MDR strains of P. aeruginosa and S. aureus into a novel polyvinyl alcohol-eudragit (PVA-EU†) nanofiber matrix through electrospinning for rapid diabetic wound healing. PVA-EU† evaluated for characteristic changes that occurred due to electrospinning and subjected to elution, stability and antibacterial assays. The biocompatibility and wound healing ability of PVA-EU† were assessed through mouse fibroblast cell line NIH3T3, followed by validation through diabetic mice excision wound co-infected with P. aeruginosa and S. aureus. The electrospinning resulted in the incorporation of ~ 75% active phages at PVA-EU†, which were stable at 25 °C for 30 days and at 4 °C for 90 days. PVA-EU† showed sustained release of phages for 18 h and confirmed to be detrimental to both mono- and mixed-cultures of target pathogens. The antibacterial activity of PVA-EU† remained unaltered in the presence of high amounts of glucose, whereas alkaline pH promoted the activity. The matrix exerted no cytotoxicity on NIH3T3, but showed significant (p < 0.0001) wound healing in vitro and the process was rapid as validated through a diabetic mice model. The sustained release, quick wound closure, declined abundance of target MDR bacteria in situ and histopathological signs of recovery corroborated the therapeutic efficacy of PVA-EU†. Taken together, our data signify the potential application of PVA-EU† in the rapid treatment of diabetic wounds without the aid of antibiotics.

Biological characterization of the phage lysin AVPL and its efficiency against Aerococcus viridans-induced mastitis in a murine model.

Aerococcus viridans (A. viridans) is an important opportunistic zoonotic pathogen that poses a potential threat to the animal husbandry industry, such as cow mastitis, due to the widespread development of multidrug-resistant strains. Phage lysins have emerged as a promising alternative antibiotic treatment strategy. However, no lysins have been reported to treat A. viridans infections. In this study, the critical active domain and key active sites of the first A. viridans phage lysin AVPL were revealed. AVPL consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two conserved LysM. H40, N44, E52, W68, H147, T157, F60, F64, I77, N92, Q97, H159, V160, D161, and S42 were identified as key sites for maintaining the activity of the catalytic domain. The LysM motif plays a crucial role in binding AVPL to bacterial cell wall peptidoglycan. AVPL maintains stable activity in the temperature range of 4-45°C and pH range of 4-10, and its activity is independent of the presence of metal ions. In vitro, the bactericidal effect of AVPL showed efficient bactericidal activity in milk samples, with 2 µg/mL of AVPL reducing A. viridans by approximately 2 Log10 in 1 h. Furthermore, a single dose (25 µg) of lysin AVPL significantly reduces bacterial load (approximately 2 Log10) in the mammary gland of mice, improves mastitis pathology, and reduces the concentration of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in mammary tissue. Overall, this work provides a novel alternative therapeutic drug for mastitis induced by multidrug-resistant A. viridans. IMPORTANCE: A. viridans is a zoonotic pathogen known to cause various diseases, including mastitis in dairy cows. In recent years, there has been an increase in antibiotic-resistant or multidrug-resistant strains of this pathogen. Phage lysins are an effective approach to treating infections caused by multidrug-resistant strains. This study revealed the biological properties and key active sites of the first A. viridans phage lysin named AVPL. AVPL can effectively kill multidrug-resistant A. viridans in pasteurized whole milk. Importantly, 25 µg AVPL significantly alleviates the symptoms of mouse mastitis induced by A. viridans. Overall, our results demonstrate the potential of lysin AVPL as an antimicrobial agent for the treatment of mastitis caused by A. viridans.

Trojan Horse virus delivering CRISPR-AsCas12f1 controls plant bacterial wilt caused by Ralstonia solanacearum.

Plant bacterial wilt caused by Ralstonia solanacearum results in huge losses. Accordingly, developing an effective control method for this disease is urgently required. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential biocontrol solution. A filamentous phage RSCq that infects R. solanacearum was isolated in this study through genome mining. We constructed engineered filamentous phages based on RSCq by employing our proposed approach with wide applicability to non-model phages, enabling the exogenous genes delivery into bacterial cells. CRISPR-AsCas12f1 is a miniature class 2 type V-F CRISPR-Cas system. A CRISPR-AsCas12f1-based gene editing system that targets the key virulence regulator gene hrpB was developed, generating the engineered phage RSCqCRISPR-Cas. Similar to the Greek soldiers in the Trojan Horse, our findings demonstrated that the engineered phage-delivered CRISPR-Cas system could disarm the key "weapon," hrpB, of R. solanacearum, in medium and plants. Remarkably, pretreatment with RSCqCRISPR-Cas significantly controlled tobacco bacterial wilt, highlighting the potential of engineered filamentous phages as promising biocontrol agents against plant bacterial diseases.IMPORTANCEBacterial disease, one of the major plant diseases, causes huge food and economic losses. Phage therapy, an environmentally friendly control strategy, has been frequently reported in plant bacterial disease control. However, host specificity, sensitivity to ultraviolet light and certain conditions, and bacterial resistance to phage impede the widespread application of phage therapy in crop production. Filamentous phages, which do not lyse host bacteria and exert minimal burden, offer a potential solution to overcome the limitations of lytic phage biocontrol. This study developed a genetic engineering approach with wide applicability to non-model filamentous phages and proved the application possibility of engineered phage-based gene delivery in plant bacterial disease biocontrol for the first.

Protease-activated CendR peptides targeting tenascin-C: mitigating off-target tissue accumulation.

To achieve precision and selectivity, anticancer compounds and nanoparticles (NPs) can be targeted with affinity ligands that engage with malignancy-associated molecules in the blood vessels. While tumor-penetrating C-end Rule (CendR) peptides hold promise for precision tumor delivery, C-terminally exposed CendR peptides can accumulate undesirably in non-malignant tissues expressing neuropilin-1 (NRP-1), such as the lungs. One example of such promiscuous peptides is PL3 (sequence: AGRGRLVR), a peptide that engages with NRP-1 through its C-terminal CendR element, RLVR.Here, we report the development of PL3 derivatives that bind to NRP-1 only after proteolytic processing by urokinase-type plasminogen activator (uPA), while maintaining binding to the other receptor of the peptide, the C-domain of tenascin-C (TNC-C). Through a rational design approach and screening of a uPA-treated peptide-phage library (PL3 peptide followed by four random amino acids) on the recombinant NRP-1, derivatives of the PL3 peptide capable of binding to NRP-1 only post-uPA processing were successfully identified. In vitro cleavage, binding, and internalization assays, along with in vivo biodistribution studies in orthotopic glioblastoma-bearing mice, confirmed the efficacy of two novel peptides, PL3uCendR (AGRGRLVR↓SAGGSVA) and SKLG (AGRGRLVR↓SKLG), which exhibit uPA-dependent binding to NRP-1, reducing off-target binding to healthy NRP-1-expressing tissues. Our study not only unveils novel uPA-dependent TNC-C targeting CendR peptides but also introduces a broader paradigm and establishes a technology for screening proteolytically activated tumor-penetrating peptides.

Inhalable Polymeric Microparticles for Phage and Photothermal Synergistic Therapy of Methicillin-Resistant Staphylococcus aureus Pneumonia.

Acute methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is a common and serious lung infection with high morbidity and mortality rates. Due to the increasing antibiotic resistance, toxicity, and pathogenicity of MRSA, there is an urgent need to explore effective antibacterial strategies. In this study, we developed a dry powder inhalable formulation which is composed of porous microspheres prepared from poly(lactic-co-glycolic acid) (PLGA), internally loaded with indocyanine green (ICG)-modified, heat-resistant phages that we screened for their high efficacy against MRSA. This formulation can deliver therapeutic doses of ICG-modified active phages to the deep lung tissue infection sites, avoiding rapid clearance by alveolar macrophages. Combined with the synergistic treatment of phage therapy and photothermal therapy, the formulation demonstrates potent bactericidal effects in acute MRSA pneumonia. With its long-term stability at room temperature and inhalable characteristics, this formulation has the potential to be a promising drug for the clinical treatment of MRSA pneumonia.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.