Oral antimicrobial therapy for cellulitis versus outpatient parenteral antimicrobial therapy: a single-centre audit of cellulitis outcomes.

BACKGROUND: Cellulitis is a common acute skin and soft tissue infection that causes substantial morbidity and healthcare costs. AIMS: To audit the impact on cellulitis management, regimen tolerability and outcomes of switching from outpatient parenteral antimicrobial therapy (OPAT) using intravenous (i.v.) cefazolin once daily plus probenecid to oral beta-lactam therapy (OBLT) using oral flucloxacillin plus probenecid. METHODS: We undertook a retrospective audit on cellulitis management, regimen tolerability and outcomes at the Dunedin Public Hospital Emergency Department (ED) before and after a change of the local outpatient cellulitis treatment pathway from OPAT using i.v. cefazolin once daily plus probenecid to OBLT using oral flucloxacillin plus probenecid. RESULTS: OPAT was used in 97/123 (78.9%) patients with cellulitis before compared to 1/70 (1.4%) after the pathway change (odds ratio (OR), 0.04, P < 0.01). OBLT was used in 26/123 (21.1%) patients with cellulitis before and 69/70 (98.6%) after (OR, 218.8, P < 0.01). Antimicrobial change due to intolerance occurred in 4/123 (3.2%) patients with cellulitis before and 4/70 (5.7%) after (OR, 1.8, P, not significant (NS)) the pathway change. Inpatient admission within 28 days occurred in 15/123 (12.2%) cellulitis patients before and 9/70 (12.9%) after (OR, 1.1, P, NS) the pathway change. CONCLUSIONS: Implementation of a change in outpatient cellulitis treatment pathway resulted in a significant change in prescribing practice. Our findings suggest that OBLT was both tolerable and had similar outcomes to OPAT.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism.

Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes.

Mining and visualizing high-order directional drug interaction effects using the FAERS database.

BACKGROUND: Adverse drug events (ADEs) often occur as a result of drug-drug interactions (DDIs). The use of data mining for detecting effects of drug combinations on ADE has attracted growing attention and interest, however, most studies focused on analyzing pairwise DDIs. Recent efforts have been made to explore the directional relationships among high-dimensional drug combinations and have shown effectiveness on prediction of ADE risk. However, the existing approaches become inefficient from both computational and illustrative perspectives when considering more than three drugs. METHODS: We proposed an efficient approach to estimate the directional effects of high-order DDIs through frequent itemset mining, and further developed a novel visualization method to organize and present the high-order directional DDI effects involving more than three drugs in an interactive, concise and comprehensive manner. We demonstrated its performance by mining the directional DDIs associated with myopathy using a publicly available FAERS dataset. RESULTS: Directional effects of DDIs involving up to seven drugs were reported. Our analysis confirmed previously reported myopathy associated DDIs including interactions between fusidic acid with simvastatin and atorvastatin. Furthermore, we uncovered a number of novel DDIs leading to increased risk for myopathy, such as the co-administration of zoledronate with different types of drugs including antibiotics (ciprofloxacin, levofloxacin) and analgesics (acetaminophen, fentanyl, gabapentin, oxycodone). Finally, we visualized directional DDI findings via the proposed tool, which allows one to interactively select any drug combination as the baseline and zoom in/out to obtain both detailed and overall picture of interested drugs. CONCLUSIONS: We developed a more efficient data mining strategy to identify high-order directional DDIs, and designed a scalable tool to visualize high-order DDI findings. The proposed method and tool have the potential to contribute to the drug interaction research and ultimately impact patient health care. AVAILABILITY AND IMPLEMENTATION: http://lishenlab.com/d3i/explorer.html.

Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery.

Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded ß-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis.

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.

HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development.

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Tumour-induced osteomalacia due to residual benign glomangioma.

Tumour-induced osteomalacia (TIO) is a rare paraneoplastic syndrome. The constellation of findings of unprovoked fractures, hypophosphataemia, urinary phosphate wasting and a negative genetic evaluation suggest a TIO diagnosis. Tumours leading to TIO are often small and difficult to localise using standard imaging studies. The 68Ga-DOTATATE CT/positron emission tomography, a somatostatin receptor imaging modality, is the radiographical study of choice for localisation. It is highly sensitive and specific since tumours that cause oncogenic osteomalacia have been shown to express somatostatin receptors. Complete surgical resection is the treatment of choice; however, it may not always be feasible. Burosumab, a human anti-fibroblast growth factor-23 monoclonal antibody, is a therapeutic option in cases of unresectable TIO to normalise phosphorus levels and improve fracture healing. Our patient was initiated on burosumab, which led to healing of his fractures and profound symptomatic improvement of his pain. TIO is often undiagnosed for many years, leading to significant patient morbidity.

BRAFV600E-Driven Lung Adenocarcinoma Requires Copper to Sustain Autophagic Signaling and Processing.

The transition metal copper (Cu) is an essential micronutrient required for development and proliferation, but the molecular mechanisms by which Cu contributes to these processes is not fully understood. Although traditionally studied as a static cofactor critical for the function of Cu-dependent enzymes, an expanding role for Cu is emerging to include its novel function as a dynamic mediator of signaling processes through the direct control of protein kinase activity. We now appreciate that Cu directly binds to and influences MEK1/2 and ULK1/2 kinase activity, and show here that reductions in MAPK and autophagic signaling are associated with dampened growth and survival of oncogenic BRAF-driven lung adenocarcinoma cells upon loss of Ctr1. Efficient autophagy, clonogenic survival, and tumorigenesis of BRAF-mutant cells required ULK1 Cu-binding. Although treatment with canonical MAPK inhibitors resulted in the upregulation of protective autophagy, mechanistically, the Cu chelator tetrathiomolybdate (TTM) was sufficient to target both autophagic and MAPK signaling as a means to blunt BRAF-driven tumorigenic properties. These findings support leveraging Cu chelation with TTM as an alternative therapeutic strategy to impair autophagy and MAPK signaling. As traditional MAPK monotherapies initiate autophagy signaling and promote cancer cell survival. IMPLICATIONS: We establish that copper chelation therapy inhibits both autophagy and MAPK signaling in BRAFV600E-driven lung adenocarcinoma, thus overcoming the upregulation of protective autophagy elicited by canonical MAPK pathway inhibitors.

Copper Modulates the Catalytic Activity of Protein Kinase CK2.

Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.

Upregulation of Receptor Tyrosine Kinase Activity and Stemness as Resistance Mechanisms to Akt Inhibitors in Breast Cancer.

The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial-mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer.

Loss of p19Arf promotes fibroblast survival during leucine deprivation.

Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

One Health security lessons from a year-long webinar series on international COVID-19 response.

Following the principles outlined by the Global Outbreak Alert and Response Network, the Federal Bureau of Investigation's International Biosecurity and Prevention Forum, the European Commission's Joint Research Centre, and the Middlebury Institute of International Studies' James Martin Center for Nonproliferation Studies cohosted a webinar series from April 2020 to January 2021 on COVID-19 management across Africa, Europe, and North America. We provide here an overview of the webinar series and discuss how lessons learned during the COVID-19 pandemic and debated during the webinars can be used to bridge One Health with biological threat-driven health security. This report can be used to inform recommendations for future One Health security approaches to strengthen global capacity and multidisciplinary cooperation.

Effect of cytokine interplay on macrophage polarization during chronic pulmonary infection with Cryptococcus neoformans.

The immune response to Cryptococcus neoformans following pulmonary infection of C57BL/6 wild-type (WT) mice results in the development of persistent infection with characteristics of allergic bronchopulmonary mycosis (ABPM). To further clarify the role of Th1/Th2 polarizing cytokines in this model, we performed kinetic analysis of cytokine responses and compared cytokine profiles, pathologies, and macrophage (Mac) polarization status in C. neoformans-infected WT, interleukin-4-deficient (IL-4(-/-)), and gamma interferon-deficient (IFN-γ(-/-)) C57BL/6 mice. Results show that cytokine expression in the infected WT mice is not permanently Th2 biased but changes dynamically over time. Using multiple Mac activation markers, we further demonstrate that IL-4 and IFN-γ regulate the polarization state of Macs in this model. A higher IL-4/IFN-γ ratio leads to the development of alternatively activated Macs (aaMacs), whereas a higher IFN-γ/IL-4 ratio leads to the generation of classically activated Macs (caMacs). WT mice that coexpress IL-4 and IFN-γ during fungal infection concurrently display both types of Mac polarization markers. Concurrent stimulation of Macs with IFN-γ and IL-4 results in an upregulation of both sets of markers within the same cells, i.e., formation of an intermediate aaMac/caMac phenotype. These cells express both inducible nitric oxide synthase (important for clearance) and arginase (associated with chronic/progressive infection). Together, our data demonstrate that the interplay between Th1 and Th2 cytokines supports chronic infection, chronic inflammation, and the development of ABPM pathology in C. neoformans-infected lungs. This cytokine interplay modulates Mac differentiation, including generation of an intermediate caMac/aaMac phenotype, which in turn may support chronic "steady-state" fungal infection and the resultant ABPM pathology.

Copper biology.

Metals are vital for life as they are necessary for essential biological processes. Traditionally, metals are categorized as either dynamic signals or static cofactors. Redox-inactive metals such as calcium (Ca), potassium (K), sodium (Na), and zinc (Zn) signal through large fluctuations in their metal-ion pools. In contrast, redox-active transition metals such as copper (Cu) and iron (Fe) drive catalysis and are largely characterized as static cofactors that must be buried and protected within the active sites of proteins, due to their ability to generate damaging reactive-oxygen species through Fenton chemistry. Cu has largely been studied as a static cofactor in fundamental processes from cellular respiration to pigmentation, working through cytochrome c oxidase and tyrosinase, respectively. However, within the last decade, a new paradigm in nutrient sensing and protein regulation - termed 'metalloallostery' - has emerged, expanding the repertoire of Cu beyond the catalytic proteins to dynamic signaling molecules essential for cellular processes that impact normal physiology and disease states. In this Primer we introduce both the 'traditional' and emerging roles for Cu in biology and the many ways in which Cu intersects with human health.

Glutamine deprivation triggers NAGK-dependent hexosamine salvage.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.

Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

Tiny Earth: A Big Idea for STEM Education and Antibiotic Discovery.

The world faces two seemingly unrelated challenges-a shortfall in the STEM workforce and increasing antibiotic resistance among bacterial pathogens. We address these two challenges with Tiny Earth, an undergraduate research course that excites students about science and creates a pipeline for antibiotic discovery.

Three Yersinia pestis adhesins facilitate Yop delivery to eukaryotic cells and contribute to plague virulence.

To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.

Ail binding to fibronectin facilitates Yersinia pestis binding to host cells and Yop delivery.

Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Delta ail mutant had decreased binding to host cells, while a KIM5 Delta pla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Delta pla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.