Discovery of a Biological Mechanism of Active Transport through the Tympanic Membrane to the Middle Ear

Abstract

Otitis media (OM) is a common pediatric disease for which systemic antibiotics are often prescribed. While local treatment would avoid the systemic treatment side-effects, the tympanic membrane (TM) represents an impenetrable barrier unless surgically breached. We hypothesized that the TM might harbor innate biological mechanisms that could mediate trans-TM transport. We used two M13-bacteriophage display biopanning strategies to search for mediators of trans-TM transport. First, aliquots of linear phage library displaying 10(10th) 12mer peptides were applied on the TM of rats with active bacterial OM. The middle ear (ME) contents were then harvested, amplified and the preparation re-applied for additional rounds. Second, the same naïve library was sequentially screened for phage exhibiting TM binding, internalization and then transit. Results revealed a novel set of peptides that transit across the TM to the ME in a time and temperature dependent manner. The peptides with highest transport capacities shared sequence similarities. Historically, the TM was viewed as an impermeable barrier. However, our studies reveal that it is possible to translocate peptide-linked small particles across the TM. This is the first comprehensive biopanning for the isolation of TM transiting peptidic ligands. The identified mechanism offers a new drug delivery platform into the ME.

Figure 1. Candidate TM-targeting peptide sequences identified during direct in vivo ME phage screening.

(A) Peptide sequences expressed by bacteriophage recovered from the ME, 1-hr after exposure of the TM. Different colors represent amino acids (aa) with similar characteristics; Blue are basic aa, red are acidic aa, orange are aromatic aa, yellow are sulfur containing aa, polar hydroxylic aa are light red, polar amidic aa are light blue, and green is proline. (B) Sequence alignment of the different recovered phage peptides showing a phylogenetic family division. The peptides fall into two phylogenetic families based on amino acid characteristics. (C) Frequency of occurrence (Logo plot) of the different amino acids in the phage display selected ligands from Strategy 1.

Figure 2. Candidate TM-binding and transiting peptide sequences identified during successive in vivo phage screening.

(A) Peptide sequences expressed by bacteriophage recovered from the ME, 1-hr after exposure of the TM to successively enriched phage display libraries. The color schematics are same as in Fig. 1. (B) Phylogenetic tree analysis shows the different family divisions.

Figure 3. Quantification of amount of translocated phage particles present in the ME, after 1-hr incubation of 10¹⁰ PFUs of individual phage clones over the TM.

The top clones (present at 3 or more copies) identified in the screening from strategies 1 and 2 were compared versus WT phage particles. (A) Quantification of transport rate of the five phage peptides recovered in Strategy 1. Phage particles bearing TMT-3 peptide were recovered the greatest compared to WT phage or phage bearing the other peptides. (B) Comparison of 1-hr transport of selected phage recovered in Strategy 2 with that of TMT-3 identifies two additional peptides that mediate comparable transport rates. Values represent the mean and SD from six animals per condition.

Figure 4. Sequence analysis of TM translocation peptide candidates from the top positive phage clones.

(A) Phylogenetic tree analysis grouping the different peptide sequences by similarity. Comparison of these six peptides primary sequence reveals the structural similarities in the side chains. The color schematics are same as in Fig. 1. (B,C) Consensus Logo plot contour of the two families of peptides that target TM showing the core motifs identified for group 1: “ST(K/R)T” and group 2: “PxxP”.

Figure 5. Kinetics of TMT-3 phage translocation and concentration dependence.

(A) Quantification of TMT-3 phage recovery with respect to increased time. (B) A linear X-axis illustrates the superiority of TM-3 phage over WT phage in TM translocation. (C) Quantification of TMT-3 phage recovery as increasing amounts of phage were applied to the TM. TMT-3 translocation increases after 1-hr proportionally to concentration and time, indicating that the transport mechanism is not saturated at these copy numbers. Values are the mean and SD number of phage recovered from six animals for each WT and TMT-3 group.

Figure 6. Competitive binding of the synthetic TMT-3 peptide to the TM.

The transport of TMT-3 phage was effectively completed by the corresponding synthetic TMT-3 peptide after 1-hr incubation indicating that they could compete for the same binding site. A scrambled synthetic peptide had no effect on the transport of the positive TMT-3 phage clone. Values are the mean and SD number of phages recovered from ME fluids of six rats.

Figure 7. Temperature and metabolic requirement of TM transport.

(A) TM transport declines after death, suggesting a metabolic requirement. (B) Recovery of TMT-3 peptide phage from the ME after incubation of 10¹⁰ phage particles on the exterior surface of the ex vivo for 1-hr at 37 °C versus 4 °C. The temperature dependence of TMT-3 peptide recovery may also suggest active transport across the TM.