Antimycobacterial Activity: A New Pharmacological Target for Conotoxins Found in the First Reported Conotoxin from Conasprella ximenes

Abstract

Mycobacterium tuberculosis is the etiological agent of tuberculosis, an airborne infectious disease that is a leading cause of human morbidity and mortality worldwide. We report here the first conotoxin that is able to inhibit the growth of M. tuberculosis at a concentration similar to that of two other drugs that are currently used in clinics. Furthermore, it is also the first conopeptide that has been isolated from the venom of Conasprella ximenes. The venom gland transcriptome of C. ximenes was sequenced to construct a database with 24,284 non-redundant transcripts. The conopeptide was purified from the venom using reverse phase high performance liquid chromatography (RP-HPLC) and was analyzed using electrospray ionization-mass spectrometry (ESI-MS/MS). No automatic identification above the identity threshold with 1% of the false discovery rate was obtained; however, a 10-amino-acid sequence tag, manually extracted from the MS/MS spectra, allowed for the identification of a conotoxin in the transcriptome database. Electron transfer higher energy collision dissociation (EThcD) fragmentation of the native conotoxin confirmed the N-terminal sequence (1-14), while LC-MS/MS analysis of the tryptic digest of the reduced and S-alkylated conotoxin confirmed the C-terminal region (15-36). The expected and experimental molecular masses corresponded, within sub-ppm mass error. The 37-mer peptide (MW 4109.69 Da), containing eight cysteine residues, was named I1_xm11a, according to the current nomenclature for this type of molecule.

Keywords: Conasprella ximenes; EThcD; antimycobacterial; conotoxins; de novo sequencing; mass spectrometry; transcriptome; tuberculosis.

Figure 1

Fractionation using RP-HPLC of Conasprella ximenes venom on a linear gradient, from 0% to 60% of Solution B. The gray area represents the fraction with activity against Mycobacterium tuberculosis (Mtb) and the arrow indicates the elution time for the conotoxin of interest (I1_xm11a). This fraction was further repurified (inset) with a slower linear gradient, from 22% to 45% of Solution B (only part of the chromatogram is shown). In both RP-HPLC runs, the broken lines indicate the linear gradient of Solution B. This peak was evaluated in further biological assays to determine its minimal inhibitory concentration, and was also characterized using mass spectrometry to reveal its chemical identity (Section 2.4, Section 2.5 and Section 2.6).

Figure 2

(a) Growth-inhibitory effect of I1_xm11a peptide against pathogenic M. tuberculosis (H37Rv strain). Minimal inhibitory concentration (MIC) cut-off values for positive controls, ethambutol (EMB) and isoniazid (INH), were 9.8 and 0.45 µM, respectively (Figure S1). The experimental control (EC) corresponds to sterility media without inoculum; (b) The MIC range at 24–0.75 µM of conotoxin was evaluated. The statistical significance of differences between treatments and growth control were analyzed using a Student’s t-test. ** p < 0.01, *** p < 0.001 vs. Growth Control (GC).

Figure 3

Electrospray ionization-mass spectrometry (ESI-MS) spectra of the native (a) and reduced S-carbamidomethylated; (b) conotoxin, I1_xm11a. In both mass spectra, the insets show the isotopic ion distributions of the corresponding [M + 6H]⁶⁺ ions. The molecular mass difference between the native (4109.69 Da) and S-alkylated peptide (4573.92 Da) allowed the assignment of eight cysteine residues linked by four disulfide bonds.

Figure 4

(a) Manual interpretation of the ESI-MS/MS spectrum of the [M + 6H]⁶⁺ precursor ion (m/z 763.32) of the reduced-S-alkylated conotoxin, I1_xm11a. The cyan rhombus indicates the precursor ion selected for fragmentation by CID. The sequence tag, (333.19, PC*AIVTIV C*T, 1447.74), written in red was extracted by considering the singly-charged y″_n ions, and was used to identify the conotoxin of interest in the transcriptome database; (b) Assignment for the backbone y″_n and b_n fragment ions observed in this MS/MS spectrum. The eight cysteine residues are carbamidomethylated and are represented as C*. Four expanded ranges of this MS/MS spectrum, as well as the assignment of fragment ions, are shown in Figure S2.

Figure 5

Electron transfer higher energy collision dissociation (EThcD) spectrum, recorded on the +6 precursor (m/z 763.32) of peptide I1_xm11a, after reduction and alkylation of cysteine residues, using iodoacetamide. N-terminal fragment; c ions are indicated by ⌉ and C-terminal fragment, y and z·ions are indicated by L. Multiply-charged fragment ions are indicated with the corresponding charge state z·ions resulting from cleavage at cysteine, and loss of the cysteine side chain is indicated with #. Charge-reduced species are labeled in the spectrum with ●, indicating the number of electrons transferred to the precursor ion. z ions with extra protons are indicated by *, where the number of * represents the number of protons added to the z ion.

Figure 6

Alignment for I1_xm11a with similar conopeptides belonging to the I1-superfamily, and with framework XI. Ep11.1 corresponds to a conotoxin from Conus episcopatus; Tx11.3, from Conus textile; Vc11.4, from Conus victoriae; and Mr11.2, from Conus marmoreus. Signal peptides are underlined in blue, while mature peptides are underlined in black. Cys residues are highlighted in yellow and a conservation percentage for each amino acid is also presented in bar graph format at the bottom of the figure. The arrows in red indicate the three possible sites for N-terminal processing, according to the mechanism proposed by Dutertre et al. [6].