Aerobactin, a citryl-hydroxamate siderophore, is produced by a number of pathogenic Gram-negative bacteria to aid in iron assimilation. Interest in this well-known siderophore was reignited by recent investigations suggesting that it plays a key role in mediating the enhanced virulence of a hypervirulent pathotype of (hvKP). In contrast to classical opportunistic strains of, hvKP causes serious life-threatening infections in previously healthy individuals in the community. Multiple contemporary reports have confirmed fears that the convergence of multidrug-resistant and hvKP pathotypes has led to the evolution of a highly transmissible, drug-resistant, and virulent "super bug." Despite hvKP harboring four distinct siderophore operons, knocking out production of aerobactin led to a significant attenuation of virulence. Herein, we continue our structural and functional studies on the biosynthesis of this crucial virulence factor. heterologous production and reconstitution of aerobactin biosynthesis from hvKP was carried out, demonstrating the specificity, stereoselectivity, and kinetic throughput of the complete pathway. Additionally, we present a steady-state kinetic analysis and the X-ray crystal structure of the second aerobactin synthetase IucC, as well as describe a surface entropy reduction strategy that was employed for structure determination. Finally, we show solution X-ray scattering data that support a unique dimeric quaternary structure for IucC. These new insights into aerobactin assembly will help inform potential antivirulence strategies and advance our understanding of siderophore biosynthesis.

Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N 6 -acetyl-N 6hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with sitedirected mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

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Toll-like receptors (TLRs) represent a subset of pathogen-recognition receptors (PRRs) employed by the innate immune system to detect pathogen-associated molecular patterns (PAMPs) and initiate the response to invading microbes. The transcription factor interferon regulatory factor 5 (IRF5) functions as an important mediator of the inflammatory response downstream of myeloid differentiation factor 88 (MyD88)-dependent TLR activation. While the dysregulation of IRF5 activity has been implicated in the development of several autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, the factors that modulate TLR-induced IRF5 post-translational modifications (PTMs) are poorly understood. Therefore, the focus of this study is to identify protein kinases involved in the regulation of TLR7/8 signaling. We performed a kinome-based siRNA screen in human THP-1 monocytic cells and human primary myeloid cells to identify important mediators of TLR7/8-induced TNF-α and IL-6 production. We identified serine/threonine protein kinase 25 (STK25) as a positive regulator of proinflammatory cytokine production in response to TLR7/8 activation in human myeloid cells. We determined that STK25 phosphorylates IRF5 in vitro via multiple biochemical assays. Phosphopeptide mapping by mass spectrometry revealed that STK25 phosphorylates IRF5 at a highly conserved threonine residue. Thus, our data implicate STK25 as a regulator of TLR7/8 signaling through the modulation of IRF5 activity.

The recruitment of leukocytes to sites of infection is an essential component of the innate immune response to pathogens. Defects in leukocyte migration can render the host susceptible to recurrent infections. In our efforts to identify new mediators of innate immunity, we found that serine/threonine protein kinase 25 (STK25) promotes TLR-induced proinflammatory cytokine production. While STK25 has previously been implicated in the regulation of cell migration, its role in leukocyte trafficking has not been defined. To investigate the involvement of STK25 in leukocyte migration, we evaluated baseline hematological parameters from Stk25-deficient (Stk25−/−) mice via a Hemavet. We found that Stk25−/− mice exhibit reduced circulating lymphocytes, neutrophils, and monocytes at steady-state compared to age- and gender-matched wild-type littermate (WT) mice. Immunophenotyping by flow cytometry confirmed these findings and revealed a significant reduction in the frequency of Ly6Chi inflammatory monocytes in Stk25−/− peripheral blood (PB) relative to WT. In contrast, we observed no apparent change in the frequencies of neutrophils and Ly6Chi inflammatory monocytes in Stk25−/− bone marrow, suggesting a defect in leukocyte migration. Finally, we examined the role of STK25 in neutrophil trafficking via an in vitro chemotaxis assay. We found that PB neutrophils from Stk25−/− mice displayed a defect in migration toward WT serum, suggesting a cell-intrinsic role for STK25 in the control of cell trafficking. Additionally, PB neutrophils from WT mice exhibited reduced migration toward Stk25−/− serum, supporting a cell-extrinsic function for STK25. Thus, our data support a new role for STK25 as a mediator of leukocyte trafficking.

Despite the progress made in identifying cellular factors and mechanisms that predict progression and metastasis, breast cancer remains the second leading cause of death for women in the US. Using The Cancer Genome Atlas and mouse models of spontaneous and invasive mammary tumorigenesis, we identified that loss of function of interferon regulatory factor 5 (IRF5) is a predictor of metastasis and survival. Histologic analysis of Irf5-/- mammary glands revealed expansion of luminal and myoepithelial cells, loss of organized glandular structure, and altered terminal end budding and migration. RNA-seq and ChIP-seq analyses of primary mammary epithelial cells from Irf5+/+ and Irf5-/- littermate mice revealed IRF5-mediated transcriptional regulation of proteins involved in ribosomal biogenesis. Using an invasive model of breast cancer lacking Irf5, we demonstrate that IRF5 re-expression inhibits tumor growth and metastasis via increased trafficking of tumor infiltrating lymphocytes and altered tumor cell protein synthesis. These findings uncover a new function for IRF5 in the regulation of mammary tumorigenesis and metastasis.

Generation of antibody secreting cells (ASCs) requires multiple B cell activation pathways, including B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptors (TLRs). In human naïve B cells, we reported that knockdown of interferon regulatory factor 5 (IRF5) resulted in IgD retention, reduced proliferation and plasma cell differentiation, and reduced IgG secretion. Defects were due to early impairments in B cell activation and clonal expansion. Conversely, murine Irf5 was reported to regulate antibody production through direct control of class switch recombination (CSR) at the γ2a locus. To further elucidate distinct and overlapping roles between human and murine IRF5 in the regulation of plasma cell differentiation and antibody production, we performed in vivo analysis of B and T cell differentiation and function in wild-type (wt) and Irf5 knockout (ko) littermate mice after immunization. B and T cell subsets were analyzed by multi-color flow cytometry. Naïve B cells were in vitro differentiated to ASCs with CpG-B, anti-IgM, anti-CD40 and IL-21. Kinetics of B cell activation, AID expression and IgG production were determined. BCR and TCR signaling were examined by phospho-flow. A combination of T-dependent/independent and TLR-dependent/independent immunizations were used to study ASC differentiation. RAG−/− mice were used to determine cell type-specific effects. Results indicate distinct roles for human and murine IRF5 at the early stages of BCR signaling and redundant roles at the later stages of CSR, plasma cell differentiation and antibody secretion via regulation of AID.