Ferritin from Mycobacterium abscessus is involved in resistance to antibiotics and oxidative stress

Abstract

Mycobacterium abscessus subsp. massiliense (Mycma) is a rapidly growing Mycobacterium belonging to the M. abscessus complex that is often associated with lung and soft tissue infection outbreaks. Mycma is resistant to many antimicrobials, including those used for treating tuberculosis. Therefore, Mycma infections are difficult to treat and may lead to high infectious complication rates. Iron is essential for bacterial growth and establishment of infection. During infection, the host reduces iron concentrations as a defense mechanism. To counteract the host-induced iron deficiency, Mycma produces siderophores to capture iron. Mycma has two ferritins (encoded by mycma_0076 and mycma_0077) modulated by different iron concentrations, which allow the survival of this pathogen during iron scarcity. In this study, we constructed knockout (Mycma 0076KO) and complemented (Mycma 0076KOc) gene strains for mycma_0076 to understand the function of 0076 ferritin. Deletion of mycma_0076 in Mycma led to the transition in colony morphology from smooth to rough, alteration of the glycopeptidolipids spectra, increased permeability of the envelope, reduction in biofilm formation, increased susceptibility to antimicrobials and hydrogen peroxide-induced oxidative stress, and decreased internalization by macrophages. This study shows that Mycma_0076 ferritin in Mycma is involved in resistance to oxidative stress and antimicrobials, and alteration of cell envelope architecture. KEY POINTS: • Deletion of the mycma_0076 gene altered colony morphology to rough; • Mycma 0076KO changed GPL profile; • Absence of Mycma_0076 ferritin results in increased susceptibility to antimicrobials and oxidative stress in Mycma. Legend: a In wild-type M. abscessus subsp. massiliense strain, iron is captured from the environment by carboxymycobactins and mycobactins (1). Iron-dependent regulator (IdeR) proteins bind to ferrous iron (Fe⁺²) in the bacterial cytoplasm leading to the activation of the IdeR-Fe⁺² complex (2). The activated complex binds to the promoter regions of iron-dependent genes, called iron box, which in turn help in the recruitment of RNA polymerase to promote transcription of genes such as mycma_0076 and mycma_0077 ferritin genes (3). Mycma_0076 and Mycma_0077 ferritins bind to excess iron in the medium and promote Fe²⁺ oxidation into ferric iron (Fe³⁺) and store iron molecules to be released under iron scarcity conditions. (4) Genes related to biosynthesis and transport of glycopeptidolipids (GPL) are expressed normally and the cell envelope is composed of different GPL species (colored squares represented on the cell surface (GPLs). Consequently, WT Mycma present smooth colony phenotype (5). b In Mycma 0076KO strain, the lack of ferritin 0076 causes overexpression of mycma_0077 (6), but does not restore wild-type iron homeostasis and thus may result in free intracellular iron, even in the presence of miniferritins (MaDps). The excess iron potentiates oxidative stress (7) by generating hydroxyl radicals through Fenton Reaction. During this process, through an unknown mechanism, that could involve Lsr2 (8), the expression of GPL synthesis locus is regulated positively and/or negatively, resulting in alteration of GPL composition in the membrane (as represented by different colors of squares on the cell surface), resulting in a rough colony phenotype (9). The changes of GPL can increase cell wall permeability, contributing to antimicrobial susceptibility (10).

Keywords: Antimicrobial susceptibility; Biofilm; Ferritin; GPL; Mycobacterium abscessus subsp. massiliense; Phenotypic change.