Kathryn P. Haley

A battle for iron: host sequestration and Staphylococcus aureus acquisition

The use of iron as an enzymatic cofactor is pervasive in biological systems. Consequently most living organisms, including pathogenic bacteria, require iron to survive and replicate. To combat infection vertebrates have evolved sophisticated iron sequestration systems against which, pathogenic bacteria have concomitantly evolved equally elaborate iron acquisition mechanisms.

Staphylococcus lugdunensis IsdG Liberates Iron from Host Heme

Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization.

The Human Antimicrobial Protein Calgranulin C Participates in Control of Helicobacter pylori Growth and Regulation of Virulence

ABSTRACT During infectious processes, antimicrobial proteins are produced by both epithelial cells and innate immune cells. Some of these antimicrobial molecules function by targeting transition metals and sequestering these metals in a process referred to as “nutritional immunity.” This chelation strategy ultimately starves invading pathogens, limiting their growth within the vertebrate host. Recent evidence suggests that these metal-binding antimicrobial molecules have the capacity to affect bacterial virulence, including toxin secretion systems. Our previous work showed that the S100A8/S100A9 heterodimer (calprotectin, or calgranulin A/B) binds zinc and represses the elaboration of the H. pylori cag type IV secretion system (T4SS). However, there are several other S100 proteins that are produced in response to infection. We hypothesized that the zinc-binding protein S100A12 (calgranulin C) is induced in response to H. pylori infection and also plays a role in controlling H. pylori growth and virulence. To test this, we analyzed gastric biopsy specimens from H. pylori-positive and -negative patients for S100A12 expression. These assays showed that S100A12 is induced in response to H. pylori infection and inhibits bacterial growth and viability in vitro by binding nutrient zinc. Furthermore, the data establish that the zinc-binding activity of the S100A12 protein represses the activity of the cag T4SS, as evidenced by the gastric cell “hummingbird” phenotype, interleukin 8 (IL-8) secretion, and CagA translocation assays. In addition, high-resolution field emission gun scanning electron microscopy (FEG-SEM) was used to demonstrate that S100A12 represses biogenesis of the cag T4SS. Together with our previous work, these data reveal that multiple S100 proteins can repress the elaboration of an oncogenic bacterial surface organelle.

The flexible loop of Staphylococcus aureus IsdG is required for its degradation in the absence of heme.

Degradation of specific native proteins allows bacteria to rapidly adapt to changing environments when the activity of those proteins is no longer required. Although these processes are vital to bacterial survival, relatively little is known regarding how bacterial proteins are recognized and targeted for degradation. Staphylococcus aureus is an important human pathogen that requires iron for growth and pathogenesis. In the vertebrate host, S. aureus fulfills its iron requirement by obtaining heme iron from host hemoproteins via IsdG- and IsdI-mediated heme degradation. IsdG and IsdI are structurally and mechanistically analogous but are differentially regulated by iron and heme availability. Specifically, IsdG is targeted for degradation in the absence of heme. Therefore, we utilized the differential regulation of IsdG and IsdI to investigate the mechanism of regulated proteolysis. In contrast to canonical protease recognition sequences, we show that IsdG is targeted for degradation by internally coded sequences. Specifically, a flexible loop near the heme-binding pocket is required for IsdG degradation in the absence of heme.

Characterization of Key Helicobacter pylori Regulators Identifies a Role for ArsRS in Biofilm Formation

UNLABELLED Helicobacter pylori must be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation, H. pylori encodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ΔnikR ΔarsS, and Δfur ΔnikR ΔarsS mutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus, H. pylori ArsRS has a previously unrecognized role in biofilm formation. IMPORTANCE Despite a paucity of regulatory proteins, adaptation is key to the survival of H. pylori within the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

Helicobacter pylori: Genomic Insight into the Host-Pathogen Interaction

The advent of genomic analyses has revolutionized the study of human health. Infectious disease research in particular has experienced an explosion of bacterial genomic, transcriptomic, and proteomic data complementing the phenotypic methods employed in traditional bacteriology. Together, these techniques have revealed novel virulence determinants in numerous pathogens and have provided information for potential chemotherapeutics. The bacterial pathogen, Helicobacter pylori, has been recognized as a class 1 carcinogen and contributes to chronic inflammation within the gastric niche. Genomic analyses have uncovered remarkable coevolution between the human host and H. pylori. Perturbation of this coevolution results in dysregulation of the host-pathogen interaction, leading to oncogenic effects. This review discusses the relationship of H. pylori with the human host and environment and the contribution of each of these factors to disease progression, with an emphasis on features that have been illuminated by genomic tools.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm. This pathway has been dissected through numerous in vitro studies. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Group B Streptococcus Induces Neutrophil Recruitment to Gestational Tissues and Elaboration of Extracellular Traps and Nutritional Immunity

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a gram-positive bacterial pathogen associated with infection during pregnancy and is a major cause of morbidity and mortality in neonates. Infection of the extraplacental membranes surrounding the developing fetus, a condition known as chorioamnionitis, is characterized histopathologically by profound infiltration of polymorphonuclear cells (PMNs, neutrophils) and greatly increases the risk for preterm labor, stillbirth, or neonatal GBS infection. The advent of animal models of chorioamnionitis provides a powerful tool to study host-pathogen relationships in vivo and ex vivo. The purpose of this study was to evaluate the innate immune response elicited by GBS and evaluate how antimicrobial strategies elaborated by these innate immune cells affect bacteria. Our work using a mouse model of GBS ascending vaginal infection during pregnancy reveals that clinically isolated GBS has the capacity to invade reproductive tissues and elicit host immune responses including infiltration of PMNs within the choriodecidua and placenta during infection, mirroring the human condition. Upon interacting with GBS, murine neutrophils elaborate DNA-containing extracellular traps, which immobilize GBS and are studded with antimicrobial molecules including lactoferrin. Exposure of GBS to holo- or apo-forms of lactoferrin reveals that the iron-sequestration activity of lactoferrin represses GBS growth and viability in a dose-dependent manner. Together, these data indicate that the mouse model of ascending infection is a useful tool to recapitulate human models of GBS infection during pregnancy. Furthermore, this work reveals that neutrophil extracellular traps ensnare GBS and repress bacterial growth via deposition of antimicrobial molecules, which drive nutritional immunity via metal sequestration strategies.

An Iron-regulated Autolysin Remodels the Cell Wall to Facilitate Heme Acquisition in Staphylococcus lugdunensis

ABSTRACT Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection.

Nutrition and Helicobacter pylori: Host Diet and Nutritional Immunity Influence Bacterial Virulence and Disease Outcome

Helicobacter pylori colonizes the stomachs of greater than 50% of the worlds human population making it arguably one of the most successful bacterial pathogens. Chronic H. pylori colonization results in gastritis in nearly all patients; however in a subset of people, persistent infection with H. pylori is associated with an increased risk for more severe disease outcomes including B-cell lymphoma of mucosal-associated lymphoid tissue (MALT lymphoma) and invasive adenocarcinoma. Research aimed at elucidating determinants that mediate disease progression has revealed genetic differences in both humans and H. pylori which increase the risk for developing gastric cancer. Furthermore, host diet and nutrition status have been shown to influence H. pylori-associated disease outcomes. In this review we will discuss how H. pylori is able to create a replicative niche within the hostile host environment by subverting and modifying the host-generated immune response as well as successfully competing for limited nutrients such as transition metals by deploying an arsenal of metal acquisition proteins and virulence factors. Lastly, we will discuss how micronutrient availability or alterations in the gastric microbiome may exacerbate negative disease outcomes associated with H. pylori colonization.