26 research outputs found
Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences
The global spread of multi- and pan-resistant bacteria has triggered research to identify
novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages.
In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting
Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial
peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can
synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost
of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition
of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected
E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta
strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally,
plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed
the production of satisfactory peptide quantities. When these sequences were integrated into the
T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the
expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable
quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select
phage genome, expression was below detection limits and an effect on the growth of potentially
phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we
were able to show that apidaecins can be integrated into the T7Select phage genome to induce their
expression in host cells, but further research is required to optimize the engineered T7Select phages
for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible
when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures
Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity
Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial
infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae
both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa
heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs,
we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of
PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding
motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a
competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluoresceinlabeled
(Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on
ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression
system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was
investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant
(Kd) of 201 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S
ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other
PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin
and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome.
Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same
time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of
wild-type and DsbmA and DmdtM knockout mutants indicated that substitutions in the ribosomal
binding motif altered also the bacterial uptake, which was generally improved by incorporation of
hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S
ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal
binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502
analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding,
inhibition of translation, and bacterial uptake
Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients
Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the
environment, causes cryptococcal meningitis (CM) mainly in immunocompromised
patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal
protein antigens targeted by the human humoral immune response. Therefore, we used
sera from Colombian CM patients, with or without HIV infection, and from healthy
individuals living in the same region. Serological analysis revealed increased titers of
anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients,
compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected
by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from
healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during
CM was supported by analysis of sera from C. neoformans-infected mice. Stronger
increase in IgG was found in wild type mice with high lung fungal burden compared to
IL-4Ra-deficient mice showing low lung fungal burden. To identify the proteins targeted by
human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome
approach identifying cryptococcal protein spots preferentially recognized by sera from CM
patients or healthy individuals followed by mass spectrometry analysis. Twenty-three
cryptococcal proteins were recombinantly expressed and confirmed to be
immunoreactive with human sera. Fourteen of them were newly described as
immunoreactive proteins. Twelve proteins were classified as disease-associated
antigens, based on significantly stronger immunoreactivity with sera from CM patients
compared to healthy individuals. The proteins identified in our screen significantly expand
the pool of cryptococcal proteins with potential for (i) development of novel anticryptococcal
agents based on implications in cryptococcal virulence or survival, or
(ii) development of an anti-cryptococcal vaccine, as several candidates lack homology
to human proteins and are localized extracellularly. Furthermore, this study defines preexisting
anti-cryptococcal immunoreactivity in healthy individuals at a molecular level,
identifying target antigens recognized by sera from healthy control persons
Identification of Disease-Associated Cryptococcal Proteins Reactive With Serum IgG From Cryptococcal Meningitis Patients
Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons
Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences
The global spread of multi- and pan-resistant bacteria has triggered research to identify
novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages.
In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting
Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial
peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can
synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost
of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition
of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected
E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta
strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally,
plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed
the production of satisfactory peptide quantities. When these sequences were integrated into the
T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the
expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable
quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select
phage genome, expression was below detection limits and an effect on the growth of potentially
phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we
were able to show that apidaecins can be integrated into the T7Select phage genome to induce their
expression in host cells, but further research is required to optimize the engineered T7Select phages
for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible
when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures
Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences
The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures
Construction and Characterization of T7 Bacteriophages Harboring Apidaecin-Derived Sequences
The global spread of multi- and pan-resistant bacteria has triggered research to identify
novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages.
In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting
Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial
peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can
synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost
of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition
of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected
E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta
strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally,
plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed
the production of satisfactory peptide quantities. When these sequences were integrated into the
T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the
expression in infected E. coli Rosetta cells, the GFP–apidaecin analogs were produced in reasonable
quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select
phage genome, expression was below detection limits and an effect on the growth of potentially
phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we
were able to show that apidaecins can be integrated into the T7Select phage genome to induce their
expression in host cells, but further research is required to optimize the engineered T7Select phages
for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible
when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures
Antimicrobial Activity and 70S Ribosome Binding of Apidaecin-Derived Api805 with Increased Bacterial Uptake Rate
In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes
Antimicrobial Activity and 70S Ribosome Binding of Apidaecin-Derived Api805 with Increased Bacterial Uptake Rate
In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes