scholarly journals Computationally-guided technology platform for on-demand production of diversified therapeutic phage cocktails

2020 ◽  
Author(s):  
Catherine M. Mageeney ◽  
Anupama Sinha ◽  
Richard A. Mosesso ◽  
Douglas L. Medlin ◽  
Britney Y. Lau ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Bacterial Pathogens ◽  
Rapid Development ◽  
Opportunistic Pathogen ◽  
Near Neighbor ◽  
Computational Molecular Biology ◽  
Integrase Gene ◽  
Technology Platform ◽  
On Demand

ABSTRACTNew therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements (IGEs) in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five of these were engineered for non-lysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, showed killing of P. aeruginosa PAO1 in vitro as well as in a waxworm (Galleria mellonella) model of infection.SIGNIFICANCE STATEMENTThe antibiotic-resistance crisis in medicine and agriculture has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

scholarly journals Computational Basis for On-Demand Production of Diversified Therapeutic Phage Cocktails

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Catherine M. Mageeney ◽  
Anupama Sinha ◽  
Richard A. Mosesso ◽  
Douglas L. Medlin ◽  
Britney Y. Lau ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Bacterial Pathogens ◽  
Rapid Development ◽  
Opportunistic Pathogen ◽  
Near Neighbor ◽  
Computational Molecular Biology ◽  
Integrase Gene ◽  
Content Type ◽  
Technology Platform ◽  
On Demand

ABSTRACT New therapies are necessary to combat increasingly antibiotic-resistant bacterial pathogens. We have developed a technology platform of computational, molecular biology, and microbiology tools which together enable on-demand production of phages that target virtually any given bacterial isolate. Two complementary computational tools that identify and precisely map prophages and other integrative genetic elements in bacterial genomes are used to identify prophage-laden bacteria that are close relatives of the target strain. Phage genomes are engineered to disable lysogeny, through use of long amplicon PCR and Gibson assembly. Finally, the engineered phage genomes are introduced into host bacteria for phage production. As an initial demonstration, we used this approach to produce a phage cocktail against the opportunistic pathogen Pseudomonas aeruginosa PAO1. Two prophage-laden P. aeruginosa strains closely related to PAO1 were identified, ATCC 39324 and ATCC 27853. Deep sequencing revealed that mitomycin C treatment of these strains induced seven phages that grow on P. aeruginosa PAO1. The most diverse five phages were engineered for nonlysogeny by deleting the integrase gene (int), which is readily identifiable and typically conveniently located at one end of the prophage. The Δint phages, individually and in cocktails, killed P. aeruginosa PAO1 in liquid culture as well as in a waxworm (Galleria mellonella) model of infection. IMPORTANCE The antibiotic resistance crisis has led to renewed interest in phage therapy as an alternative means of treating infection. However, conventional methods for isolating pathogen-specific phage are slow, labor-intensive, and frequently unsuccessful. We have demonstrated that computationally identified prophages carried by near-neighbor bacteria can serve as starting material for production of engineered phages that kill the target pathogen. Our approach and technology platform offer new opportunity for rapid development of phage therapies against most, if not all, bacterial pathogens, a foundational advance for use of phage in treating infectious disease.

scholarly journals ace, Which Encodes an Adhesin in Enterococcus faecalis, Is Regulated by Ers and Is Involved in Virulence

2009 ◽  
Vol 77 (7) ◽  
pp. 2832-2839 ◽  
Author(s):  
Francois Lebreton ◽  
Eliette Riboulet-Bisson ◽  
Pascale Serror ◽  
Maurizio Sanguinetti ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Animal Experiments ◽  
Opportunistic Pathogen ◽  
Transcriptional Analysis ◽  
Binding Assays ◽  
Mobility Shift ◽  
Reverse Transcription Quantitative Pcr

ABSTRACT Enterococcus faecalis is an opportunistic pathogen that causes numerous infectious diseases in humans and is a major agent of nosocomial infections. In this work, we showed that the recently identified transcriptional regulator Ers (PrfA like), known to be involved in the cellular metabolism and the virulence of E. faecalis, acts as a repressor of ace, which encodes a collagen-binding protein. We characterized the promoter region of ace, and transcriptional analysis by reverse transcription-quantitative PCR and mobility shift protein-DNA binding assays revealed that Ers directly regulates the expression of ace. Transcription of ace appeared to be induced by the presence of bile salts, probably via the deregulation of ers. Moreover, with an ace deletion mutant and the complemented strain and by using an insect (Galleria mellonella) virulence model, as well as in vivo-in vitro murine macrophage models, we demonstrated for the first time that Ace can be considered a virulence factor for E. faecalis. Furthermore, animal experiments revealed that Ace is also involved in urinary tract infection by E. faecalis.

scholarly journals Characterisation of the antimicrobial mode of action of gallium maltolate

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Magdalena Piatek ◽  
Darren Griffith ◽  
Kevin Kavanagh
Keyword(s):  
Mode Of Action ◽  
Galleria Mellonella ◽  
Iron Acquisition ◽  
Structural Similarity ◽  
Opportunistic Pathogen ◽  
Inhibitory Effect ◽  
Label Free ◽  
Iron Storage

To human health worldwide. Existing treatments are becoming inefficacious and therefore there is an urgent need for the development of treatments with alternative modes of action. The use of gallium as an antimicrobial agent has been of interest due to its unconventional mode of action involving the inhibition of iron acquisition and metabolism. The structural similarity and inability to reduce from a trivalent to divalent form under normal physiological conditions allows gallium to act as an iron mimetic and inhibit many iron-dependent biological pathways, respectively. The antimicrobial potential of gallium maltolate (GaM), Ga(III) coordination complex of maltol, was investigated on the opportunistic pathogen Pseudomonas aeruginosa. In vitro and in vivo analyses using Galleria mellonella (greater wax moth) larvae demonstrated the potent bacteriostatic and non-toxic effect of the complex. Subsequent analysis of GaM treated P. aeruginosa via label-free quantitative proteomics provided an insight into the intrinsic mechanisms of action of GaM. Increased expression of iron-storage protein Bacterioferritin B, the HemO component of iron-sulfur clusters and several stress response proteins (Chaperone Proteins ClpB, HtpG and DnaJ) indicate cell stress in response to inhibited iron uptake. Decreased expression of LasA Protease and LasB Elastase quorum-sensing proteins and flagellar motility proteins FlgM and FlgG further demonstrate the growth inhibitory effect of GaM. These findings provide a basis for a better understanding of the mode of action of GaM, a requirement for the improvement of synthesis and efficacy of the treatment.

scholarly journals c-di-AMP is essential for the virulence of Enterococcus faecalis

2021 ◽  
Author(s):  
Shivani Kundra ◽  
Ling Ning Lam ◽  
Jessica K. Kajfasz ◽  
Leila Casella ◽  
Marissa J Andersen ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Biological Significance ◽  
Opportunistic Pathogen ◽  
Bacterial Fitness ◽  
Key Aspects

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP and ΔdhhPΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of ΔcdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results reveal that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

scholarly journals c-di-AMP is essential for the virulence of Enterococcus faecalis

2021 ◽  
Author(s):  
Shivani Kundra ◽  
Ling Ning Lam ◽  
Jessica K. Kajfasz ◽  
Leila G. Casella ◽  
Marissa J. Andersen ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Biofilm Formation ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Biological Significance ◽  
Opportunistic Pathogen ◽  
Bacterial Fitness ◽  
Key Aspects

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis ( cdaA ) and degradation ( dhhP and gdpP ). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Δ cdaA strain) or c-di-AMP accumulation (Δ dhhP , Δ gdpP and Δ dhhP Δ gdpP strains) drastically impaired general cell fitness and virulence of E. faecalis . In particular, the Δ cdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo , and was virtually avirulent in an invertebrate ( Galleria mellonella ) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of Δ cdaA could be also attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

The In Vitro Antibiofilm Activity of Water Leaf Extract of Papaya (Carica papaya L.) against Pseudomonas aeruginosa

2017 ◽  
Vol 2 (3) ◽  
pp. 150-163
Author(s):  
Ekajayanti Kining ◽  
Syamsul Falah ◽  
Novik Nurhidayat
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Contact Time ◽  
Cell Attachment ◽  
Water Extract ◽  
Opportunistic Pathogen ◽  
Bacterial Biofilm ◽  
Antibiofilm Activity ◽  
Bacterial Survival ◽  
Optimum Conditions

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.

scholarly journals A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo

2020 ◽  
Vol 8 (10) ◽  
pp. 1627
Author(s):  
Tecla Ciociola ◽  
Pier Paolo Zanello ◽  
Tiziana D’Adda ◽  
Serena Galati ◽  
Stefania Conti ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Human Serum ◽  
Fungicidal Activity ◽  
Galleria Mellonella ◽  
Serum Proteins ◽  
Morphological Alterations ◽  
C Terminus ◽  
Different Sources

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

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