AUTHOR=Deblais Loïc , Drozd Mary , Kumar Anand , Antwi Janet , Fuchs James , Khupse Rahul , Helmy Yosra A. , Rajashekara Gireesh 

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

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1342573

DOI=10.3389/fmicb.2024.1342573

ISSN=1664-302X

ABSTRACT=<sec><title>Introduction</title><p>Control of <italic>Campylobacter</italic> from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of <italic>Campylobacter</italic>. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows <italic>Campylobacter</italic> 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 (CuSO<sub>4</sub>).</p></sec><sec><title>Methods</title><p>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 <italic>Campylobacter</italic> in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (<italic>n</italic> = 50,917 SM) for increased susceptibility to CuSO<sub>4</sub> (1 mM) in <italic>C. jejuni</italic> 81–176, a human isolate which is widely studied.</p></sec><sec><title>Results</title><p>Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO<sub>4</sub> 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 <italic>Campylobacter</italic> strains (<italic>n</italic> = 12) at &gt;10 ng/mL in the presence of 0.5 mM CuSO<sub>4</sub><italic>in vitro</italic>. 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 <italic>C. jejuni</italic> cells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO<sub>4</sub>. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; <italic>p</italic> &lt; 0.01) with minimal disruption of microbiota. <italic>In silico</italic> 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.</p></sec><sec><title>Discussion</title><p>Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to control <italic>C. jejuni</italic> spread on farms.</p></sec>