Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens

Abstract

Introduction: Control of Campylobacter from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of Campylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows Campylobacter to colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO₄).

Methods: Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigate Campylobacter in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO₄ (1 mM) in C. jejuni 81-176, a human isolate which is widely studied.

Results: Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO₄ for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1-T8) were selected for further studies. These selected eight Tat inhibitors cleared all tested Campylobacter strains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO₄in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellular C. jejuni cells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO₄. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; p < 0.01) with minimal disruption of microbiota. In silico analyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein.

Discussion: Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to control C. jejuni spread on farms.

Keywords: Campylobacter jejuni; microbiome; poultry production system; small molecule inhibitor; twin arginine translocase.

Figure 1

Identification of Tat dependent inhibitors in C. jejuni 81–176. (A) Copper sulfate sensitivity assay. C. jejuni 81–176 was challenged for 24 h with 6.25 μg/mL of SM plus 0.5 mM CuSO₄ in microaerophilic condition. The growth inhibition was determined by measuring the optical density and being compared to the DMSO control. (B) Formate Dehydrogenase (FDH) inhibition activity assay. C. jejuni 81–176 was challenged for 24 h with 6.25 μg/mL of SM in microaerophilic condition. The FDH activity was determined by measuring the optical density and being compared to the DMSO control. N = 3 replicates per group.

Figure 2

Copper sulfate sensitivity dose–response assay in vitro. C. jejuni 81–176 was challenged for 24 h with SM concentration ranging between 0.006 and 6.25 μg/mL in presence of 0.5 mM CuSO₄. A total of 19 compounds were tested. N = four replicates per group.

Figure 3

Chemical structure diversity of the eight most potent Tat-dependent inhibitors. The constellation tree was built based on the structure similarity score generated based on 3D Tanimoto scoring method in PubChem (https://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?p=clustering). The circled node represents the root of the tree. Each SM is associated with its chemical structure and its PubChem ID.

Figure 4

Dose-dependent cytotoxicity assay of the eight Tat-dependent inhibitors using colon epithelial (Caco-2) cells. Bar: standard deviation; Cm, chloramphenicol; Kn, kanamycin; LDH, lactate dehydrogenase; N = four replicates per group.

Figure 5

Effect of the four most potent Tat-dependent inhibitors on the persistence of C. jejuni in three- and five-week-old chicken ceca and its microbiota. (A) C. jejuni abundance in five-week-old chicken ceca after SM treatment (n = 5–4 chickens per group). The C. jejuni population in ceca was determined after 5 days of treatment with 0.127 mg/mL of SM. Each dot represents a chicken. Red bar represents the mean. *: significant reduction of the C. jejuni population in ceca compared to the DMSO control group (p < 0.01). (B) C. jejuni abundance in three-week-old chicken ceca after SM treatment. The C. jejuni population in ceca was determined after 5 days of treatment with 0.255 mg/mL of SM (n = six to five chickens per group). (C) Relative abundance at the phylum level in five-week-old chicken ceca after treatment. (D) Relative abundance at the phylum level in three-week-old chicken ceca after treatment. NC: not inoculated not treated chickens; PC: colonized not treated chickens; DMSO, T1, T2, T7, T8: colonized chickens treated with DMSO or one of the selected SM.

Figure 6

In silico docking model between the Tat inhibitors and the TatC system in C. jejuni. Binding interactions of the most active small molecule inhibitors with a homology model of TatC from Aquifex aeolicus, Compound T2 (A), Compound T7 (B). The compounds bind in the same pocket and the interaction with key residue Glu165 and Trp85 is responsible for Tat C inhibition. The ionizability model for T2 (C) and T7 (D) of residues in the binding pocket indicates the Pi-anion interactions with Glu165 and Pi-Pi interaction with Trp85. These docking models indicate that TatC inhibition may be responsible for the anti-C. jejuni activity of T2 and T7.

Figure 7

Role of Tat system in copper (Cu) homeostasis in C Jejuni. C jejuni employs a Tat complex (Tat-A, B, C) for the transport of proteins from cytoplasm to periplasm. TatC is the core transmembrane component of this complex located in inner membrane (IM), responsible for translocation of folded proteins such as multi-copper oxidase (CueO) and formate dehydrogenase (Fdh) from cytoplasm to periplasm. CueO is critical for oxidation of Cu⁺ which are highly toxic as compared to relatively non-toxic Cu²⁺ form. Thus, in the presence of TatC inhibitors the transportation of important cytoplasmic proteins such as CueO and Fdh is hampered. This results in increased sensitivity of C jejuni to copper. OM and IM, outer and inner membrane, respectively; CueO, copper oxidase; Cu, copper.