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.

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)-carboxyfluorescein-labeled (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 (K_d) 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 ΔsbmA and ΔmdtM 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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI− cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI− cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.

Because of the global spread of multi- and pan-resistant bacteria, there is a need to identify, research, and develop new strategies to combat these pathogens. In a previous proof-of-concept study, we presented an innovative strategy by genetically modifying lytic T7 bacteriophages. We integrated DNA fragments encoding for derivatives of the antimicrobial peptide (AMP) apidaecin into the phage genome to induce the production and release of apidaecin within the T7 infection cycle, thereby also targeting phage-resistant Escherichia coli bacteria. In this follow-up study, we optimized the apidaecin encoding insert to improve the expression of the apidaecin derivative Api805 by adding the secretion signal peptide of the OmpA protein. This prevented the detrimental effects of the peptide on the producing bacterial cell after its production. The integration of two copies of the OmpA-Api805 insert into the phage genome resulted in T7Select-2xOmpA-Api805 phages, which had a partially improved activity in inhibiting phage-resistant E. coli compared to the T7Select phages without insert and with only one copy of the OmpA-Api805 insert. Additionally, we showed that the combinatorial use of the lytic bacteriophage T7Select with the highly active and lytic AMPs CRAMP (cathelicidin-related AMP) and melittin against E. coli made the lysis process of the phage and the peptides more effective and prevented the growth of potentially AMP- and phage-resistant E. coli strains. The integration of DNA sequences derived from CRAMP and melittin into the phage genome resulted in the created T7Select-(M)CRAMP and T7Select-(M)melittin phages, which showed a lysis behavior like the phage without insert and partially inhibited the growth of potentially phage-resistant E. coli strains after the phage-mediated lysis.

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.

This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity.