Mutagenesis Analysis of ABCB8 Gene Promoter of Danio rerio

The ABCB8 is one of the members under the ABCB subfamily of ATP-Binding Cassette (ABC) transporter which possess the ability in regulating the intracellular iron and heme transport. The loss of function mutation of ABCB8 gene leads to iron and heme accumulation in the cell which is highly toxic to human. However, the information regarding the expression regulation of this gene remains scarce. Hence, the objectives of this project are to determine the transcription factors binding site (TFBS) of ABCB8 and to identify the transcriptional roles of the cis-elements through mutagenesis analysis. To examine this, total genomic DNA was extracted from Danio rerio and the promoter sequence was isolated by using specific pair of primers through polymerase chain reaction (PCR). The sample was sent for DNA sequencing and the result showed 98% similarities to the zebrafish DNA sequence from clone DKEYP-87A6 in linkage group 24. Besides, the TFBS was studied in aspect of TFBS abundance, TFBS composition and TFBS distribution. The two most abundant TFBSs based on liver-specific profile were HNF-3β and C/EBPβ, with 38 and 39 binding sites, respectively. The sequence of ABCB8 promoter gene was mutated through substitution of the AP-1 binding site at location 535 with other nucleotides by using a pair of mutagenic primers (forward primer: 5’-TGGGGGTTTAGATATTGAAAC-3’; reverse primer: 5’-AACTCGC ATACATTTCAGTCATC-3’). This result may benefit the development of new diagnostics and therapeutics for iron-associated disorder.

A new model for Trypanosoma cruzi heme homeostasis depends on modulation of TcHTE protein expression

Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life cycle stages of the parasite to gain insight into its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the heme response gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.

Heme Uptake in Lactobacillus sakei Evidenced by a New Energy Coupling Factor (ECF)-Like Transport System

ABSTRACT Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [lsa1836-1840]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δlsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57Fe heme. We showed that in the WT L. sakei, heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems. IMPORTANCE Lactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei. In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system.

Contributions of the heme coordinating ligands of the Pseudomonas aeruginosa outer membrane receptor HasR to extracellular heme sensing and transport

Pseudomonas aeruginosa exhibits a high requirement for iron, which it can acquire via several mechanisms, including the acquisition and utilization of heme. The P. aeruginosa genome encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonas heme utilization (Phu) system. Extracellular heme is sensed via the Has system, which encodes an extracytoplasmic function (ECF) σ factor system. Previous studies have shown that the transfer of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI and upregulation of has operon expression. Here, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting, and 13C-heme uptake experiments, we delineated the differential contributions of the extracellular FRAP/PNPNL loop residue His-624 in HasR and of His-221 in its N-terminal plug domain required for heme capture to heme transport and signaling, respectively. Specifically, we show that substitution of the N-terminal plug His-221 disrupts both signaling and transport, leading to dysregulation of both the Has and Phu uptake systems. Our results are consistent with a model wherein heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His-624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Our results provide critical insight into heme release, signaling, and transport in P. aeruginosa and suggest a functional link between the ECF σ factor and Phu heme uptake system.

Progress towards the Development of a NEAT Vaccine for Anthrax II: Immunogen Specificity and Alum Effectiveness in an Inhalational Model

ABSTRACT Bacillus anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA-approved vaccine to prevent anthrax in humans is anthrax vaccine adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against protective antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five-course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. The cocktail of five NEAT domains was protective against a lethal challenge of inhaled bacillus spores at 3 and 28 weeks after vaccination. The reduction of the formulation to three NEATs (IsdX1, IsdX2, and Bslk) was as effective as a five-NEAT domain cocktail. The adjuvant alum, approved for use in humans, was as protective as Freund’s Adjuvant, and protective vaccination correlated with increased anti-NEAT antibody reactivity and reduced bacterial levels in organs. Finally, the passive transfer of anti-NEAT antisera reduced mortality and disease severity, suggesting the protective component is comprised of antibodies. Collectively, these results provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next-generation anthrax vaccine.

Contributions of the Heme Coordinating Ligands of the Pseudomonas aeruginosa Outer Membrane Receptor HasR to Extracellular Heme Sensing and Transport

ABSTRACTPseudomonas aeruginosa exhibits a high requirement for iron which it can acquire via several mechanisms including the acquisition and utilization of heme. P. aeruginosa encodes two heme uptake systems, the heme assimilation system (Has) and the Pseudomonasheme utilization (Phu) system. Extracellular heme is sensed via the Has system that encodes an extra cytoplasmic function (ECF) σ factor system. Previous studies have shown release of heme from the extracellular hemophore HasAp to the outer membrane receptor HasR is required for activation of the σ factor HasI. Herein, employing site-directed mutagenesis, allelic exchange, quantitative PCR analyses, immunoblotting and 13C-heme uptake studies, we characterize the differential contributions of the outer membrane receptor HasR extracellular FRAP/PNPNL loop residue His-624 and the N-terminal plug residue His-221 to heme transport and signaling, respectively. Specifically, we show mutation of the N-terminal plug His-221 disrupts both signaling and transport. The data is consistent with a model where heme release from HasAp to the N-terminal plug of HasR is required to initiate signaling, whereas His624 is required for simultaneously closing off the heme transport channel from the extracellular medium and triggering heme transport. Furthermore, mutation of His-221 leads to dysregulation of both the Has and Phu uptake systems suggesting a possible functional link that is coordinated through the ECF σ factor system.

Artificial Biosynthetic Pathway for an Unnatural Terpenoid with an Iridiumcontaining P450

<div>Synthetic biology enables microbial hosts to produce complex molecules that are</div><div>otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these</div><div>molecules are limited to chemical reactions catalyzed by natural enzymes. The integration of</div><div>artificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks could</div><div>broaden the cache of molecules produced biosynthetically by microorgansms. We report the</div><div>assembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpene</div><div>producing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArM</div><div>into a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows that</div><div>synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce</div><div>molecules previously inaccessible to nature.</div>

Artificial Biosynthetic Pathway for an Unnatural Terpenoid with an Iridiumcontaining P450

<div>Synthetic biology enables microbial hosts to produce complex molecules that are</div><div>otherwise produced by organisms that are rare or difficult to cultivate, but the structures of these</div><div>molecules are limited to chemical reactions catalyzed by natural enzymes. The integration of</div><div>artificial metalloenzymes (ArMs) that catalyze abiotic reactions into metabolic networks could</div><div>broaden the cache of molecules produced biosynthetically by microorgansms. We report the</div><div>assembly of an ArM containing an iridium-porphyrin complex in the cytoplasm of a terpene</div><div>producing Escherichia coli by a heterologous heme transport machinery, and insertion of this ArM</div><div>into a natural biosynthetic pathway to produce an unnatural terpenoid. This work shows that</div><div>synthetic biology and synthetic chemistry, incorporated together in whole cells, can produce</div><div>molecules previously inaccessible to nature.</div>