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Acinetobacter baumannii is an emerging pathogen that poses a global health threat due to a lack of therapeutic options for treating drug-resistant strains. In addition to acquiring resistance to last-resort antibiotics, the success of A. baumannii is partially due to its ability to effectively compete with the host for essential metals. Iron is fundamental in shaping host-pathogen interactions, where the host restricts availability of this nutrient in an effort to curtail bacterial proliferation. To circumvent restriction, pathogens possess numerous mechanisms to obtain iron, including through the use of iron-scavenging siderophores. A. baumannii elaborates up to ten distinct siderophores, encoded from three different loci: acinetobactin and pre-acinetobactin (collectively, acinetobactin), baumannoferrins A and B, and fimsbactins A-F. The expression of multiple siderophores is common amongst bacterial pathogens and often linked to virulence, yet the collective contribution of these siderophores to A. baumannii survival and pathogenesis has not been investigated. Here we begin dissecting functional redundancy in the siderophore-based iron acquisition pathways of A. baumannii. Excess iron inhibits overall siderophore production by the bacterium, and the siderophore-associated loci are uniformly upregulated during iron restriction in vitro and in vivo. Further, disrupting all of the siderophore biosynthetic pathways is necessary to drastically reduce total siderophore production by A. baumannii, together suggesting a high degree of functional redundancy between the metabolites. By contrast, inactivation of acinetobactin biosynthesis alone impairs growth on human serum, transferrin, and lactoferrin, and severely attenuates survival of A. baumannii in a murine bacteremia model. These results suggest that whilst A. baumannii synthesizes multiple iron chelators, acinetobactin is critical to supporting growth of the pathogen on host iron sources. Given the acinetobactin locus is highly conserved and required for virulence of A. baumannii, designing therapeutics targeting the biosynthesis and/or transport of this siderophore may represent an effective means of combating this pathogen.

Acinetobacter baumannii is an opportunistic pathogen and an emerging global health threat. Within healthcare settings, major presentations of A . baumannii include bloodstream infections and ventilator-associated pneumonia. The increased prevalence of ventilated patients during the COVID-19 pandemic has led to a rise in secondary bacterial pneumonia caused by multidrug resistant (MDR) A . baumannii . Additionally, due to its MDR status and the lack of antimicrobial drugs in the development pipeline, the World Health Organization has designated carbapenem-resistant A . baumannii to be its priority critical pathogen for the development of novel therapeutics. To better inform the design of new treatment options, a comprehensive understanding of how the host contains A . baumannii infection is required. Here, we investigate the innate immune response to A . baumannii by assessing the impact of infection on host gene expression using NanoString technology. The transcriptional profile observed in the A . baumannii infected host is characteristic of Gram-negative bacteremia and reveals expression patterns consistent with the induction of nutritional immunity, a process by which the host exploits the availability of essential nutrient metals to curtail bacterial proliferation. The gene encoding for lipocalin-2 ( Lcn2 ), a siderophore sequestering protein, was the most highly upregulated during A . baumannii bacteremia, of the targets assessed, and corresponds to robust LCN2 expression in tissues. Lcn2 -/- mice exhibited distinct organ-specific gene expression changes including increased transcription of genes involved in metal sequestration, such as S100A8 and S100A9 , suggesting a potential compensatory mechanism to perturbed metal homeostasis. In vitro , LCN2 inhibits the iron-dependent growth of A . baumannii and induces iron-regulated gene expression. To elucidate the role of LCN2 in infection, WT and Lcn2 -/- mice were infected with A . baumannii using both bacteremia and pneumonia models. LCN2 was not required to control bacterial growth during bacteremia but was protective against mortality. In contrast, during pneumonia Lcn2 -/- mice had increased bacterial burdens in all organs evaluated, suggesting that LCN2 plays an important role in inhibiting the survival and dissemination of A . baumannii . The control of A . baumannii infection by LCN2 is likely multifactorial, and our results suggest that impairment of iron acquisition by the pathogen is a contributing factor. Modulation of LCN2 expression or modifying the structure of LCN2 to expand upon its ability to sequester siderophores may thus represent feasible avenues for therapeutic development against this pathogen.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR , enhance the pathogenic potential of S. aureus . Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus . PurR acts as transcriptional repressor of purine biosynthesis genes in some bacterial species. Here, the authors show that purR mutations can arise in Staphylococcus aureus upon exposure to stress, leading to upregulation of fibronectin-binding proteins and increased virulence.

Iron is a versatile redox-active catalyst and a required cofactor within a diverse array of biological processes. To almost all organisms, iron is both essential and potentially toxic, where homeostatic concentrations must be stringently maintained. Within the iron-restricted host, the survival and proliferation of microbial invaders is contingent upon exploiting the host iron pool. Bacteria express a multitude of complex, and often redundant means of acquiring iron, including surface-associated heme-uptake pathways, high affinity iron-scavenging siderophores and transporters of free inorganic iron. Within the last decade, our understanding of iron acquisition by Gram-positive pathogens has expanded substantively, from the discovery of the iron-regulated surface-determinant pathway and numerous unique siderophores through to the detailed elucidation of heme–iron extraction, and heme and siderophore coordination and transfer. This review provides a comprehensive summary of the iron acquisition strategies of notorious Gram-positive pathogens and highlights how both conserved and distinct tactics for acquiring iron contribute to the pathophysiology of these bacteria. Further, a focus on recent structural and mechanistic studies details how these iron acquisition systems may be exploited in the development of novel therapeutics. In this review, we discuss both the conserved and distinct strategies that Gram-positive pathogens use to acquire iron within the context of the iron-restricted host.

Lipoproteins fulfill diverse roles in antibiotic resistance, adhesion, protein secretion, signaling and sensing, and many also serve as the substrate binding protein (SBP) partner to ABC transporters for the acquisition of a diverse array of nutrients including peptides, sugars, and scarcely abundant metals. In the staphylococci, the iron-regulated SBPs are significantly upregulated during iron starvation and function to sequester and deliver iron into the bacterial cell, enabling staphylococci to circumvent iron restriction imposed by the host environment. Accordingly, this subset of lipoproteins has been implicated in staphylococcal pathogenesis and virulence. Lipoproteins also activate the host innate immune response, triggered through Toll-like receptor-2 (TLR2) and, notably, the iron-regulated subset of lipoproteins are particularly immunogenic. In this review, we discuss the iron-regulated staphylococcal lipoproteins with regard to their biogenesis, substrate specificity, and impact on the host innate immune response.

Serine hydrolases play diverse roles in regulating host–pathogen interactions in a number of organisms, yet few have been characterized in the human pathogen Staphylococcus aureus. Here we describe a chemical proteomic screen that identified ten previously uncharacterized S. aureus serine hydrolases that mostly lack human homologs. We termed these enzymes fluorophosphonate-binding hydrolases (FphA–J). One hydrolase, FphB, can process short fatty acid esters, exhibits increased activity in response to host cell factors, is located predominantly on the bacterial cell surface in a subset of cells, and is concentrated in the division septum. Genetic disruption of fphB confirmed that the enzyme is dispensable for bacterial growth in culture but crucial for establishing infection in distinct sites in vivo. A selective small molecule inhibitor of FphB effectively reduced infectivity in vivo, suggesting that it may be a viable therapeutic target for the treatment or management of Staphylococcus infections.

Siderophores, iron-scavenging small molecules, are fundamental to bacterial nutrient metal acquisition and enable pathogens to overcome challenges imposed by nutritional immunity. Multimodal imaging mass spectrometry allows visualization of host−pathogen iron competition, by mapping siderophores within infected tissue. We have observed heterogeneous distributions of Staphylococcus aureus siderophores across infectious foci, challenging the paradigm that the vertebrate host is a uniformly iron-depleted environment to invading microbes.

Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.

Staphylococcus lugdunensis is both a commensal of humans and an opportunistic pathogen. Little is currently known about the molecular mechanisms underpinning the virulence of this bacterium. Here, we demonstrate that in contrast to S. aureus, S. lugdunensis makes neither staphyloferrin A (SA) nor staphyloferrin B (SB) in response to iron deprivation, owing to the absence of the SB gene cluster, and a large deletion in the SA biosynthetic gene cluster. As a result, the species grows poorly in serum‐containing media, and this defect was complemented by introduction of the S. aureus SA gene cluster into S. lugdunensis. S. lugdunensis expresses the HtsABC and SirABC transporters for SA and SB, respectively; the latter gene set is found within the isd (heme acquisition) gene cluster. An isd deletion strain was significantly debilitated for iron acquisition from both heme and hemoglobin, and was also incapable of utilizing ferric‐SB as an iron source, while an hts mutant could not grow on ferric‐SA as an iron source. In iron‐restricted coculture experiments, S. aureus significantly enhanced the growth of S. lugdunensis, in a manner dependent on staphyloferrin production by S. aureus, and the expression of the cognate transporters by S. lugdunensis. We demonstrate that S. lugdunensis, an opportunistic human pathogen, grows poorly in iron‐restricted media lacking heme or hemoglobin. We demonstrate that this phenotype is due to the combination of having a functional iron‐regulated surface determinant system, for acquisition of heme, and nonfunctional or missing genes for synthesis of siderophores. The growth of S. lugdunensis can be significantly enhanced if the species is provided with functional S. aureus genes for SA synthesis or with exogenous supplied siderophores. Moreover, S. lugdunensis growth is also significantly enhanced when grown in coculture with staphyloferrin‐producing S. aureus, in a transporter‐dependent manner.