Functional Characterization of the Putative POT from Clostridium perfringens

Abstract

Proton-coupled oligopeptide transporters (POTs) are a fundamental part of the cellular transport machinery that provides plants, bacteria, and mammals with nutrition in the form of short peptides. However, POTs are not restricted to peptide transport; mammalian POTs have especially been in focus due to their ability to transport several peptidomimetics in the small intestine. Herein, we studied a POT from Clostridium perfringens (CPEPOT), which unexpectedly exhibited atypical characteristics. First, very little uptake of a fluorescently labelled peptide β-Ala-Lys-AMCA, an otherwise good substrate of several other bacterial POTs, was observed. Secondly, in the presence of a competitor peptide, enhanced uptake of β-Ala-Lys-AMCA was observed due to trans-stimulation. This effect was also observed even in the absence of a proton electrochemical gradient, suggesting that β-Ala-Lys-AMCA uptake mediated by CPEPOT is likely through the substrate-concentration-driving exchange mechanism, unlike any other functionally characterized bacterial POTs.

Keywords: Clostridium perfringens; POTs; major facilitator superfamily; peptide transport.

Figure 1

YdgR- and CPEPOT-mediated β-Ala-Lys-AMCA uptake inhibition in the presence of A, AA, AAA, AAAA, and compared to background uptake by empty vector (pTTQ18). Fluorescence normalized to the uptake of CPEPOT. Insert is a Western blot showing the expression level of each transporter protein. The blot is from the same gel but assembled for illustrational purposes. Lane 1, 2, 3, 4: CPEPOT, YdgR, empty vector, marker. NF = normalized fluorescence intensity. YdgR shows a significant decrease while CPEPOT shows a significant increase in β-Ala-Lys-AMCA uptake inhibition in the presence of AA and AAA, respectively. Significance level indicated by * for p < 0.05, ** for p < 0.02, and *** for p < 0.01. All experiments performed in triplicate (n = 3).

Figure 2

(A) CPEPOT-mediated uptake of β-Ala-Lys-AMCA in the presence of AA, KA, AD, DA, AF, FA, and AK. Fluorescence normalized according to control. (B) CPEPOT-mediated uptake of β-Ala-Lys-AMCA at pH 5.5, 6.5, and 7.5 in the absence and presence of AA. Fluorescence normalized according to the intensity at pH 6.5 in each experiment. Significance level indicated by * for p < 0.05, ** for p < 0.02, and *** for p < 0.01. All experiments performed in triplicate (n = 3).

Figure 3

(A) Time-dependent CPEPOT-mediated uptake of β-Ala-Lys-AMCA with and without subsequent addition of AA at time = 15 min. (B) Uptake of β-Ala-Lys-AMCA in the absence and presence of CCCP (abolishing proton electrochemical gradient) by CPEPOT, YjdL, and YdgR. Fluorescence normalized according to −CCCP in each experiment. Significance level indicated by ** for p < 0.02. All experiments performed in triplicate (n = 3).

Figure 4

(A) Multiple sequence alignment of CPEPOT, YdgR, YjdL, YhiP, PepTso, PepTst, and GKPOT. (B) Schematic representation of the four different types of POTs: with hairpin and periplasmic salt bridge, without salt bridge but containing hairpin, with neither salt bridge nor hairpin, and without hairpin but containing salt bridge (C) Left: YdgR (PDB:6GS4), a canonical bacterial POT structure showing two additional helices. Middle: AlphaFold model of CPEPOT with missing additional helices. Right: Enlarged view showing induced periplasmic mutations forming salt bridge in CPEPOT.

Figure 5

Uptake inhibition assay of mutants V63E/N283R and E383A in the presence and absence of trans-stimulator AA. Fluorescence normalized to CPEPOT in the absence of AA. Western blot of mutant transporter proteins E383A, V63E/N283R, and WT CPEPOT as an insert. Each band is representative of three independent biological replicates n = 3. The blot is from the same gel but assembled for illustrational purposes. In the presence of AA, WT CPEPOT and V63E/N283R show a significant increase in the uptake of β-Ala-Lys-AMCA. Significance level indicated by *** for p < 0.01. All experiments performed in triplicate (n = 3).