Strain dependent variations in iron metabolism of Mycobacterium avium subsp. paratuberculosis

Abstract

Johne’s disease is a major animal health problem of ruminant species worldwide and imposes significant economic losses to the industry. Our ability to culture the causative agent--Mycobacterium avium subsp. paratuberculosis (MAP)--and therefore its rapid diagnosis and our understanding of its virulence is limited. MAP is difficult to culture because of its unusually strict iron requirements. For optimal growth in laboratory media, MAP requires a siderophore (mycobactin) supplementation that makes MAP fastidious, often requiring eight to sixteen weeks to produce colonies in culture – a major hurdle in timely diagnosis and therefore implementation of optimal control measures. Understanding iron regulatory networks in the pathogen in vitro is therefore of great importance. Several microbiological and genotyping studies and clinical observations suggest that Johne’s in certain hosts such as sheep, goats, deer, and bison is caused by a distinct set of strains that show a relatively high degree of host preference. At least two microbiologically distinct types of MAP have been recognized. A less readily cultivable type is the common, but not invariable, cause of paratuberculosis in sheep (type I), while another readily cultivable type is the most common cause of the disease in cattle (type II). In addition, since the MAP genome sequence was published in 2005, very little research has focused on iron physiology and its contribution to metabolic networks of this fastidious organism. Based on these observations, I hypothesize that iron dependent gene regulation is different between type I and type II MAP strains. Iron dependent Regulator (IdeR), a transcription factor, is an essential gene in MAP and differentially controls the expression of genes involved in iron physiology in the two strain types of MAP. We identified polymorphisms in the IdeR open reading frame (ORF) and the promoters of putative IdeR regulated genes between the type I and type II strains of MAP. Structure-function association studies revealed repression of an iron storage gene, bfrA in the presence of iron by type I MAP strain alone. In contrast, bfrA was upregulated in the presence of iron in type II MAP strain. This leads us to propose that type I MAP strains may experience iron toxicity when excess iron is provided in the medium. The rationale is that excess free iron is detrimental to the cells and must be stored in bacterioferritins, a feature that type I strains lack. Transcriptional and proteomic profiling of these MAP strains under iron-replete or –deplete conditions revealed that iron-sparing response to iron limitation was unique to the type II strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Under iron-replete conditions, type II MAP alone increased expression of BfrA (bacterioferritin) and MhuD (mycobacterial heme utilization, degrader) protein, which is intricately involved in iron recycling. These findings further supported the contention that type I MAP strains are metabolically inept under iron-replete conditions. The intracellular lifestyle of MAP in the intestines and lymph nodes of natural infection revealed that MAP deployed genes involved in maintaining iron homeostasis under iron stress in the tissues of infected animals. There was a clear dichotomy in in vitro infected macrophages and natural infection in the expression profiles of both iron acquisition genes and other virulence factors involved in MAP survival inside the host. In summary, our studies revealed that IdeR of type II strain regulates mycobactin synthesis and iron storage genes, similar to the function of IdeR in M. tuberculosis (MTB), while the type I strain is deficient in iron storage function. Given our inability to delete ideR, it appears that this is an essential gene (as in MTB) for MAP survival. MAP IdeR regulon studies led us to define a novel operon carrying genes encoding a potential secretory apparatus (ESX-3/type VII secretory system). Functional analysis of the iron-induced proteome also identified novel ESAT-6 (early secreted antigenic target) family of proteins belonging to ESX-5, which have been identified as major virulence factors in MTB. We also established that, type I MAP strains are more sensitive to fluctuations of environmental iron due to defective regulation of bfrA and may grow better under lower iron levels in the culture media. Taken together, our studies suggest that MAP employs a sophisticated repertoire of proteins that are inter-connected and function in response to environmental stress.