Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

Intracellular metalloporphyrin metabolism in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus is responsible for a significant amount of human morbidity and mortality, and the ability of S. aureus to cause disease is absolutely dependent on the acquisition of iron from the host. The most abundant iron source to invading staphylococci is in the form of the porphyrin heme. S. aureus is capable of acquiring nutrient iron from heme and hemoproteins via two heme-acquisition systems, the iron-regulated surface determinant system (Isd) and the heme transport system (Hts). Heme acquisition through these systems is involved in staphylococcal pathogenesis suggesting that the intracellular fate of heme plays a significant role in the infectious process. The valuable heme molecule presents a paradox to invading bacteria because although heme is an abundant source of nutrient iron, the extreme reactivity of heme makes it toxic at high concentrations. Therefore, bacteria must regulate the levels of intracellular heme to avoid toxicity. Although the molecular mechanisms responsible for staphylococcal heme acquisition are beginning to emerge, the mechanisms by which S. aureus regulate intracellular heme homeostasis are largely unknown. In this review we describe three potential fates of host-derived heme acquired by S. aureus during infection: (i) degradation for use as a nutrient iron source, (ii) incorporation into bacterial heme-binding proteins for use as an enzyme cofactor, or (iii) efflux through a dedicated ABC-type transport system. We hypothesize that the ultimate fate of exogenously acquired heme in S. aureus is dependent upon the intracellular and extracellular availability of both iron and heme.     

Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides

Staphylococcus aureus cause many diseases by producing toxins, whose synthesis is regulated by quorum-sensing mechanisms. S. aureus secretes a protein termed RNAIII activating protein (RAP) which autoinduces toxin production via the phosphorylation of is target protein TRAP. Mice vaccinated with RAP were protected from S. aureus infection, suggesting that RAP is an useful target for selecting potential therapeutic molecules to inhibit S. aureus pathogenesis. We show here that RAP (native and recombinant) was used to select RAP-binding peptides (RBPs) from a random 12-mer phage-displayed peptide library. Two RBPs were shown to inhibit RNAIII production in vitro (used a marker for pathogenesis). The peptide WPFAHWPWQYPR, which had the strongest inhibitory activity, was chemically synthesized and also expressed in Escherichia coli as a GST-fusion. Both synthetic peptide and GST-fusion peptide decreased RNAIII levels in a dose-dependent manner. The GST-fusion peptide was also shown to protect mice from a S. aureus infection in vivo (tested in a murine cutaneous S. aureus infection model). Our results suggest the potential use of RAP-binding proteins in treating clinical S. aureus infections.     

Biosynthesis of Staphylococcus aureus autoinducing peptides by using the synechocystis DnaB mini-intein

The Agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). The peptides are seven to nine residues in length and have the C-terminal five residues constrained in a thiolactone ring. We have developed a new method to generate AIP structures using an engineered DnaB mini-intein from Synechocystis sp. strain PCC6803. In the method, an oligonucleotide encoding the AIP is ligated to the intein and the fusion protein is expressed and purified by affinity chromatography. To produce the correct AIP structure, intein splicing is interrupted, allowing the cysteine side chain to catalyze thiolactone ring formation and release AIP from the resin. The technique is simple and robust, and we have successfully produced the three main classes of AIPs using the intein system. The intein-generated AIPs possessed the correct thiolactone ring modification based on biochemical analysis, and, importantly, all the samples were bioactive against S. aureus. The AIP activity was confirmed through Agr interference and activation profiling with developed S. aureus reporter strains. The simplicity of the method, benefits of DNA encoding, and scalable nature enable the production of S. aureus AIPs for many biological applications.     

Survey of taurine uptake and metabolism in Staphylococcus aureus

Taurine has been reported to be a component of the capsular polysaccharide of the encapsulated M strain of Staphylococcus aureus. This led to a study of the uptake and metabolism of [1,2-14C]taurine in a variety of encapsulated and unencapsulated S. aureus strains. Taurine was taken up by all strains studied. A discrepancy between uptake measured as depletion of radioactivity from growth medium and as cell-associated radioactivity suggested that taurine may be catabolized to CO2 in some strains. In most strains, cell-associated radioactivity was located mainly in cold TCA-soluble (pool metabolites) fractions. About 90% of the cell-associated radioactivity was present in the pool metabolites fraction in the M strain, and about 10% in hot TCA-soluble (nucleic acid-teichoic acid-capsular polysaccharide) fraction. Radioactivity in spent medium and the capsular polysaccharide-containing fraction appeared to be present as taurine in this strain. Radioactivity in the pool metabolites fraction of three of the strains examined did not chromatograph as taurine, indicating that taurine was converted into other cell metabolites. One strain incorporated radioactivity from taurine into cellular macromolecules, thus revealing a heterogeneity of staphylococcal taurine metabolism.     

Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria

There are two basic types of bacterial communication systems--those in which the signal is directed solely at other organisms and those in which the signal is sensed by the producing organism as well. The former are involved primarily in conjugation; the latter in adaptation to the environment. Gram-positive bacteria use small peptides for both types of signaling, whereas Gram-negative bacteria use homoserine lactones. Since adaptation signals are autoinducers the response is population-density-dependent and has been referred to as "quorum-sensing". Gram-negative bacteria internalize the signals which act upon an intracellular receptor, whereas Gram-positive bacteria use them as ligands for the extracellular receptor of a two-component signaling module. In both cases, the signal activates a complex adaptation response involving many genes.     

Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.     

The structure of bacilysin and other products of Bacillus subtillis

1. Mass spectra of the trimethylsilyl derivative and the methyl ester of the N-trifluoroacetyl derivative of bacilysin indicated that the antibiotic had a molecular weight of 270. Several peaks in the spectrum of the methyl ester were consistent with the presence of an N-terminal alanine residue in the molecule. 2. The proton-magnetic-resonance spectrum of bacilysin confirmed that the antibiotic contained an epoxide group and the spin-spin splitting of the protons of the epoxide group indicated that the side chain of the epoxycyclohexanone ring was attached at C-4 and was alphabeta to the keto group. 3. The formation of an alphabeta-unsaturated ketone on reduction of bacilysin with chromous chloride also showed that the epoxide was alphabeta to the keto group. 4. The optical-rotatory-dispersion curve of bacilysin showed a positive Cotton effect. On the assumption that the reversed Octant rule for alphabeta-epoxyketones was applicable this revealed the absolute stereochemistry and enabled a definitive structure to be assigned to the molecule. 5. Similar measurements showed that substance AA1, isolated from culture supernatants, was the C-terminal amino acid of bacilysin. 6. Hydrolysis of substance P2 with leucine aminopeptidase and the mass spectrum of the methyl ester of its N-trifluoroacetyl derivative showed that this substance was l-analyl-l-alanine. 7. These results are discussed in relation to the biogenesis of bacilysin.     

The impact of metal sequestration on Staphylococcus aureus metabolism

The Gram-positive pathogen Staphylococcus aureus poses a serious risk to public health due to its prevalence as a commensal organism, its ability to cause a multitude of diseases, and the increasing incidence of antibiotic resistant strains. S. aureus infects diverse niches within vertebrates despite being challenged by a robust immune response. The host-pathogen confrontation occurs in an environment nearly devoid of metals that are essential for bacterial proliferation. S. aureus is able to flourish in these conditions and often causes significant morbidity and mortality. This review highlights current themes pertaining to the process of host-mediated metal sequestration known as 'nutritional immunity', S. aureus metal acquisition strategies, and how proliferating within a metal restricted environment impacts bacterial physiology.