Probing the Role of the Mycobacterium tuberculosis ESX-3 Secretion System in Iron Acquisition and Host Macrophage Manipulation in the Nigerian Clinical Environment

The ESX-3 type VII secretion system of Mycobacterium tuberculosis is essential for iron and zinc acquisition and plays a poorly characterized but critical role in manipulating host macrophage biology during infection. This study investigated the functional role of ESX-3 in iron acquisition and macrophage immune evasion using Nigerian clinical M. tuberculosis strains collected at Aminu Kano Teaching Hospital, Kano State, and Federal Medical Centre Abuja, in the North West and North Central geopolitical zones respectively. Clinical isolates were characterized by whole-genome sequencing for ESX-3 gene cluster polymorphisms. A conditional ESX-3 knockdown system was constructed using CRISPRi in high-burden clinical isolates. Macrophage infection assays using primary human monocyte-derived macrophages from Nigerian blood donors tested ESX-3 knockdown effects on intracellular survival, phagosome maturation, cytokine induction, and iron acquisition measured by ferritin immunofluorescence. ESX-3 suppression resulted in significantly reduced intracellular iron acquisition and an 8.5-fold reduction in intracellular survival compared to wild-type strains. ESX-3-deficient strains triggered higher TNF-alpha and IL-1beta production by macrophages. Polymorphisms in the EsxH-EsxG interface region were associated with altered macrophage cytokine profiles among clinical isolate variants. These findings establish ESX-3 as a virulence determinant and therapeutic target in Nigerian M. tuberculosis and provide mechanistic evidence for ESX-3 inhibitor development as a host-directed therapeutic approach. Keywords: Mycobacterium tuberculosis, ESX-3 secretion system, iron acquisition, macrophage, host-directed therapy.