Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands

The Roles of Transition Metals in the Physiology and Pathogenesis of Streptococcus pneumoniae

For bacterial pathogens whose sole environmental reservoir is the human host, the acquisition of essential nutrients, particularly transition metals, is a critical aspect of survival due to tight sequestration and limitation strategies deployed to curtail pathogen outgrowth. As such, these bacteria have developed diverse, specialized acquisition mechanisms to obtain these metals from the niches of the body in which they reside. To oppose the spread of infection, the human host has evolved multiple mechanisms to counter bacterial invasion, including sequestering essential metals away from bacteria and exposing bacteria to lethal concentrations of metals. Hence, to maintain homeostasis within the host, pathogens must be able to acquire necessary metals from host proteins and to export such metals when concentrations become detrimental. Furthermore, this acquisition and efflux equilibrium must occur in a tissue-specific manner because the concentration of metals varies greatly within the various microenvironments of the human body. In this review, we examine the functional roles of the metal import and export systems of the Gram-positive pathogen Streptococcus pneumoniae in both signaling and pathogenesis

Molecular and Evolutionary Analysis of NEAr-Iron Transporter (NEAT) Domains

<div><p>Iron is essential for bacterial survival, being required for numerous biological processes. <u>NEA</u>r-iron <u>T</u>ransporter (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 <i>Eggerthella</i> 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 <i>Paenibacillus polymyxa</i> 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.</p></div

Structure of the <i>B. anthracis</i> IsdX1 NEAT domain.

<p>The ribbon structure of the heme-bound form of IsdX1 is shown. NEAT domains share a conserved β-barrel fold including a 3₁₀-helix (green) that conventionally begins with a serine and ends with a tyrosine, and eight β-strands (teal). The two tyrosine residues (Tyr-109 and Tyr-113) within the heme-binding sequence YXXXY are indicated in purple. Tyr-109 non-covalently binds to the iron atom within the heme molecule at a predicted distance of 2.2 Å. Tyr-113 H-bonds with Tyr-109 as indicated by the dotted black line (2.4 Å). Ser-24 (green) H-bonds with the buried propionate group of the heme porphyrin, increasing binding affinity (2.6 Å). PDB code: 3SIK; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104794#pone.0104794-Ekworomadu1" target="_blank">[36]</a>.</p

Phylogenetic tree demonstrating the relationship of the 343 identified putative NEAT domains.

<p>Each unique NEAT domain is included and each genus is indicated by a different color. Branch lengths are not representative phylogenetic distance; numbers on branches are bootstrap values obtained from 100 replicates. Colored arrowheads point to NEAT domains that have been previously characterized. The black arches indicate NEAT Groups discussed chosen for analysis. The green and magenta asterisks indicate Pp-p4 and Cb-p4, which are described in detail in the text.</p

Amino acid sequence of Pp-IsdC and heme binding analysis.

<p><b><i>A</i></b>- Amino acid sequence of full-length Pp-IsdC. The signal cleavage site is indicated by the arrowhead, and the NEAT domain (Pp-IsdC_N) is highlighted in grey. The 3₁₀-helix is boxed in black, and the two tyrosine residues within the heme-binding signature sequence are bolded. <b><i>B</i></b>- Spectral analysis of purified Pp-IsdC shows the presence of a Soret band at 404 nm, indicating that the protein can bind heme. The red spectra, magnified ×5, indicates Q bands at 500 and 630 nm, demonstrating that the heme bound by Pp-IsdC is oxidized (Fe³⁺) and is in a high-spin coordination <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104794#pone.0104794-Berry1" target="_blank">[67]</a>. <b><i>Inset:</i></b> SDS-PAGE of purified Pp-IsdC_N at 14 kDa.</p

Alignment of Clostridial Group NEAT domains.

<p>The alignment was generated by ClustalX. The canonical 3₁₀-helix sequence, SXXXXY, present in 33 of the 51 NEAT domains in this group, is highlighted in blue. The locations of two highly conserved phenylalanine residues are boxed in green. The methionine residue predicted to interact with heme (<a><b>Figure 6</b></a>) is highlighted in red and boxed in orange. The <i>C. botulinum</i> protein4, which was chosen for structural modeling analysis, is highlighted in magenta text.</p

Alignment of IsdC Group NEAT domains.

<p>ClustalX generated alignment of the 35 IsdC Group NEAT domains. The 3₁₀-helix is boxed in blue, and the arrowheads indicate the tyrosine residues within the heme-binding signature sequence. The two IsdC proteins of known function, from <i>B. anthracis</i> and <i>S. aureus</i>, are highlighted in black and red text, respectively. The putative IsdC-like NEAT from <i>P. polymyxa</i> is highlighted in green.</p

Increased Zinc Availability Enhances Initial Aggregation and Biofilm Formation of Streptococcus pneumoniae

Bacteria growing within biofilms are protected from antibiotics and the immune system. Within these structures, horizontal transfer of genes encoding virulence factors, and promoting antibiotic resistance occurs, making biofilms an extremely important aspect of pneumococcal colonization and persistence. Identifying environmental cues that contribute to the formation of biofilms is critical to understanding pneumococcal colonization and infection. Iron has been shown to be essential for the formation of pneumococcal biofilms; however, the role of other physiologically important metals such as copper, zinc, and manganese has been largely neglected. In this study, we investigated the effect of metals on pneumococcal aggregation and early biofilm formation. Our results show that biofilms increase as zinc concentrations increase. The effect was found to be zinc-specific, as altering copper and manganese concentrations did not affect biofilm formation. Scanning electron microscopy analysis revealed structural differences between biofilms grown in varying concentrations of zinc. Analysis of biofilm formation in a mutant strain lacking the peroxide-generating enzyme pyruvate oxidase, SpxB, revealed that zinc does not protect against pneumococcal H2O2. Further, analysis of a mutant strain lacking the major autolysin, LytA, indicated the role of zinc as a negative regulator of LytA-dependent autolysis, which could affect biofilm formation. Additionally, analysis of cell-cell aggregation via plating and microscopy revealed that high concentrations of zinc contribute to intercellular interaction of pneumococci. The findings from this study demonstrate that metal availability contributes to the ability of pneumococci to form aggregates and subsequently, biofilms