Abstract
Iron is essential for bacterial survival, being required for numerous biological processes. NEAr-iron Transporter (NEAT) domains have been studied in pathogenic Gram-positive bacteria to understand how their proteins obtain heme as an iron source during infection. While a 2002 study initially discovered and annotated the NEAT domain encoded by the genomes of several Gram-positive bacteria, there remains a scarcity of information regarding the conservation and distribution of NEAT domains throughout the bacterial kingdom, and whether these domains are restricted to pathogenic bacteria. This study aims to expand upon initial bioinformatics analysis of predicted NEAT domains, by exploring their evolution and conserved function. This information was used to identify new candidate domains in both pathogenic and nonpathogenic organisms. We also searched metagenomic datasets, specifically sequence from the Human Microbiome Project. Here, we report a comprehensive phylogenetic analysis of 343 NEAT domains, encoded by Gram-positive bacteria, mostly within the phylum Firmicutes, with the exception of Eggerthella sp. (Actinobacteria) and an unclassified Mollicutes bacterium (Tenericutes). No new NEAT sequences were identified in the HMP dataset. We detected specific groups of NEAT domains based on phylogeny of protein sequences, including a cluster of novel clostridial NEAT domains. We also identified environmental and soil organisms that encode putative NEAT proteins. Biochemical analysis of heme binding by a NEAT domain from a protein encoded by the soil-dwelling organism Paenibacillus polymyxa demonstrated that the domain is homologous in function to NEAT domains encoded by pathogenic bacteria. Together, this study provides the first global bioinformatics analysis and phylogenetic evidence that NEAT domains have a strong conservation of function, despite group-specific differences at the amino acid level. These findings will provide information useful for future projects concerning the structure and function of NEAT domains, particularly in pathogens where they have yet to be studied.
Highlights
All bacteria must acquire iron from their environment to survive, with mammalian pathogens exploiting host iron reservoirs during an infection [1]
Identification of putative NEAr-iron Transporter (NEAT) domains To illustrate the features of the conserved NEAT domain, a structure of the B. anthracis IsdX1 NEAT protein is shown in Figure 1 (PDB code: 3SIK; [36])
The second functional region, the ‘‘heme-binding signature’’, is comprised of five amino acids within the hemebinding pocket on the eighth b-strand, and the motif generally begins and ends with a tyrosine (Figure 1). The presence of these two tyrosine residues correlates with the proposal that a NEAT domain should bind heme, as the first tyrosine non-covalently binds to the iron atom, and the second tyrosine hydrogen bonds (H-bonds) to the first tyrosine [17,27,41]
Summary
All bacteria must acquire iron from their environment to survive, with mammalian pathogens exploiting host iron reservoirs during an infection [1]. To protect against the toxic effects of free iron, and as a form of ‘‘nutritional immunity’’, the host sequesters approximately 80% of iron within heme, which is further coordinated by proteins such as hemoglobin [2,3]. It should be noted that heme is primarily used by mammals as a cofactor in several proteins, including hemoglobin (oxygen transport), myoglobin (oxygen storage), and peroxidases (e.g., glutathione peroxidase, which protects mammalian cells against oxidative stress [4]). The mechanism of heme-iron acquisition in Gram-negative pathogens has been characterized in detail: secreted bacterial proteins bind free heme spontaneously released from hemoglobin and they interact with bacterial TonB-dependent cell surface receptors, where heme is imported into the periplasm [5,6,7,8,9]. Due to differences in cell envelope architecture, the action of heme-iron capture and import by Gram-positive organisms is most likely mediated by an alternative mechanism
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