Actin-Dependent Regulation of Borrelia burgdorferi Phagocytosis by Macrophages.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, nervous system, and joints. If an infection with Borrelia proceeds unchecked, the disease can also enter a chronic stage, leading to the development of neuroborreliosis or cardiac arrhythmia. Successful elimination of B. burgdorferi by the host immune system is thus decisive for the positive outcome of a respective infection. Accordingly, host immune cells such as macrophages and dendritic cells have to be able to efficiently internalize and degrade infecting spirochetes. These processes are based on closely controlled rearrangements of the actin cytoskeleton, which enables the spatiotemporally fine-tuned formation of cellular protrusions and compartments that assist in the capturing, immobilization, and uptake of borreliae, as well as their further intracellular processing. Here, we discuss actin-based structures, in particular filopodia and coiling pseudopods that are involved in phagocytosis of B. burgdorferi by macrophages, their regulation by actin-associated proteins such as formins and Arp2/3 complex, as well as the subsequent intracellular processing of borreliae.

Daam1 is a regulator of filopodia formation and phagocytic uptake of Borrelia burgdorferi by primary human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, an infectious disease that primarily affects the skin, nervous system, and joints. Uptake of borreliae by immune cells is decisive for the course of the infection, and remodelling of the host actin cytoskeleton is crucial in this process. In this study, we showed that the actin-regulatory formin Daam1 is important in Borrelia phagocytosis by primary human macrophages. Uptake of borreliae proceeds preferentially through capture by filopodia and formation of coiling pseudopods that enwrap the spirochetes. Using immunofluorescence, we localized endogenous and overexpressed Daam1 to filopodia and to F-actin-rich uptake structures. Live-cell imaging further showed that Daam1 is enriched at coiling pseudopods that arise from the macrophage surface. This filopodia-independent step was corroborated by control experiments of phagocytic cup formation with latex beads. Moreover, siRNA-mediated knockdown of Daam1 led to a 65% reduction of borreliae-induced filopodia, and, as shown by the outside-inside staining technique, to a 50% decrease in phagocytic uptake of borreliae, as well as a 37% reduction in coiling pseudopod formation. Collectively, we showed that Daam1 plays a dual role in the phagocytic uptake of borreliae: first, as a regulator of filopodia, which are used for capturing spirochetes, and second, in the formation of the coiling pseudopod that enwraps the bacterial cell. These data identify Daam1 as a novel regulator of B. burgdorferi phagocytosis. At the same time, this is the first demonstration of a role for Daam1 in phagocytic processes in general.-Hoffmann, A.-K., Naj, X., Linder, S. Daam1 is a regulator of filopodia formation and phagocytic uptake of Borrelia burgdorferi by primary human macrophages.

The formins FMNL1 and mDia1 regulate coiling phagocytosis of Borrelia burgdorferi by primary human macrophages.

Spirochetes of the Borrelia burgdorferi sensu lato complex are the causative agent of Lyme borreliosis, a tick-borne infectious disease primarily affecting the skin, nervous system, and joints. During infection, macrophages and dendritic cells are the first immune cells to encounter invading borreliae. Phagocytosis and intracellular processing of Borrelia by these cells is thus decisive for the eventual outcome of infection. Phagocytic uptake of Borrelia by macrophages proceeds preferentially through coiling phagocytosis, which is characterized by actin-rich unilateral pseudopods that capture and enwrap spirochetes. Actin-dependent growth of these pseudopods necessitates de novo nucleation of actin filaments, which is regulated by actin-nucleating factors such as Arp2/3 complex. Here, we demonstrate that, in addition, also actin-regulatory proteins of the formin family are important for uptake of borreliae by primary human macrophages. Using immunofluorescence, live-cell imaging, and ratiometric analysis, we find specific enrichment of the formins FMNL1 and mDia1 at macrophage pseudopods that are in contact with borreliae. Consistently, small interfering RNA (siRNA)-mediated knockdown of FMNL1 or mDia1 leads to decreased formation of Borrelia-induced pseudopods and to decreased internalization of borreliae by macrophages. Our results suggest that macrophage coiling phagocytosis is a complex process involving several actin nucleation/regulatory factors. They also point specifically to the formins mDia1 and FMNL1 as novel regulators of spirochete uptake by human immune cells.

Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation.

Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], as well as mTOR-raptor.

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes.

Candida albicans hyphae can reach enormous lengths, precluding their internalization by phagocytes. Nevertheless, macrophages engulf a portion of the hypha, generating incompletely sealed tubular phagosomes. These frustrated phagosomes are stabilized by a thick cuff of F-actin that polymerizes in response to non-canonical activation of integrins by fungal glycan. Despite their continuity, the surface and invaginating phagosomal membranes retain a strikingly distinct lipid composition. PtdIns(4,5)P2 is present at the plasmalemma but is not detectable in the phagosomal membrane, while PtdIns(3)P and PtdIns(3,4,5)P3 co-exist in the phagosomes yet are absent from the surface membrane. Moreover, endo-lysosomal proteins are present only in the phagosomal membrane. Fluorescence recovery after photobleaching revealed the presence of a diffusion barrier that maintains the identity of the open tubular phagosome separate from the plasmalemma. Formation of this barrier depends on Syk, Pyk2/Fak and formin-dependent actin assembly. Antimicrobial mechanisms can thereby be deployed, limiting the growth of the hyphae.

ER-Coordinated Activities of Rab22a and Rab5a Drive Phagosomal Compaction and Intracellular Processing of Borrelia burgdorferi by Macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting the skin, joints, and nervous system. Macrophages and dendritic cells counteract Borrelia dissemination through internalization and degradation of spirochetes. We now show that Borrelia internalization by primary human macrophages involves uptake and compaction into Rab22a-positive phagosomes that are in close contact with Rab5a-positive vesicles. Compaction of borreliae involves membrane extrusion from phagosomes, is driven by Rab22a and Rab5a activity, and is coordinated by ER tubules forming contact sites of Rab22a phagosomes with Rab5a vesicles. Importantly, Rab22a and Rab5a depletion leads to reduced localization to lysosomes and to increased intracellular survival of spirochetes. These data show that Rab22a- and Rab5a-driven phagosomal uptake is a crucial step in the vesicular cascade that leads to elimination of spirochetes by macrophages. Rab22a and Rab5a thus present as potential molecular targets for the modulation of intracellular processing of borreliae in human immune cells.