Listeria monocytogenes crosses blood brain barrier through Rho GTPases induced migration of macrophages and inflammatory interleukin expression

Highlights

• Listeria monocytogenes infection increases the expression of Rho GTPases.

• Enhanced expression of RhoA stimulates the migration of macrophages.

• RhoA induces the expression of inflammatory cytokines.

• Inflammatory cytokines help the Listeria infected macrophages to cross that blood-brain barrier.

Abstract

Listeria monocytogenes crossing the blood-brain barrier in the form of “Trojan Horse” is of great significance for the establishment of bacterial encephalitis and meningitis. Induction of cell migration and crossing the blood-brain barrier is very important to understand the Listeria pathogenesis. The Rho GTPases family is considered a key factor in regulating cell migration. This study was designed to investigate the expression of Rho GTPases and their effect on the behavior of cell migration and the stimulation of immune factors. Selective Rho GTPases were investigated by real-time PCR and Western blot. Among these, the expression of RhoA was significantly increased following the infection of Listeria monocytogenes in macrophages. Further, we found that RhoA improves the migration of macrophages and expression of IL-1β, IL-6, and TNF-α. The expression of IL-1β, IL-6 and TNF-α possibly facilitates the migration and adhesion of macrophages to cross the blood-brain barrier. This study provides preliminary ground to investigate the detailed mechanism of Listeria monocytogenes crossing the blood-brain barrier.

Introduction

Listeria monocytogenes is a short Gram-positive flagellar and ubiquitous intracellular bacterium that causes food-borne illnesses with highly pathogenic outcomes such as meningitis, encephalitis, septicemia, abortion and even death [1,2]. The majority of Listeria monocytogenes cases are food-borne or of zoonotic origin and pathogenesis is known as listeriosis. Being an intracellular pathogen, Listeria monocytogenes has the ability to induce its uptake into numerous types of cells, phagocytic and non-phagocytic cells (endothelial cells, macrophages, and hepatocytes, etc). In the early stages, Listeria monocytogenes is entrapped in the endosome temporarily. However, the virulence genes of Listeria pathogenicity island help the bacterium to evade this intrinsic trap and entry into the cell cytoplasm [3]. It replicates inside the cytoplasm of the infected cells and spreads to the neighboring cells while remaining in the cytoplasm [4,5]. While residing in the cytoplasm, bacteria get nutrition and protection from the extracellular immune surveillance. With recent advances in the field of microbiology, Listeria monocytogenes has become the model pathogen to study cellular and infection microbiology due to its notorious interference with microbial recognition and immune signaling pathways [6]. Tilney and Portnoy first time reported the detailed uptake of Listeria monocytogenes by cells, hemolysin based phagosomal escape, and the use of actin cytoskeleton for the movement and spread of bacterium [7]. In brief, Listeria monocytogenes gains entry through the plasma membrane and mediates the host actin cytoskeleton. Factors helping the ingestion and replication of bacterium vary in different cells. As professional phagocytes, for instance, macrophages are naturally able to engulf the pathogens and initiate antibody and complement response which increases the ingestion of bacterium without involving the microbial virulence factors [4]. However, in comparison to phagocytic cells, Listeria monocytogenes uses specific bacterial surface proteins, called internalins to gain entry into non-phagocytic cells such as epithelial cells, hepatocytes, endothelial cells [8,9]. Studies have shown that the virulence factors InlA and InlB on the surface of Listeria monocytogenes bind with the receptors E-cadherin and c-Met respectively to cross the intestinal barrier [10]. However, it was recently found that host GTPases Arf1 and its effectors AP1 and PICK1 play their role in actin polymerization and exocytosis during InlB-dependent internalization [11]. The Listeria monocytogenes also breaches the blood-brain barrier either by direct infection of brain microvascular endothelial cells or through circulating bacteria in the blood, free or associated with leukocytes [12]. Literature has shown that InlB induces polymerization of actin filaments, which facilitates infection of hepatocytes and cell diffusion [8]. It also helps in the colonization of the intestine, trophoblast tissue in the placenta and other barriers in body [13].

Macrophages are key players of the innate immune system and professional phagocytic cells to ingest bacteria, cell debris and other foreign particles. Macrophages are decorated with specific pathogen recognition receptors (PRRs) [14], externally on the cell membrane and internally in the cytosol. These receptors allow them to recognize the external and internal presence of pathogenic bacteria. Once the invading bacteria are recognized, PRRs, initiate the signaling pathways for the polymerization of actin to form a phagocytic cup which subsequently engulfs the bacterium in the form of a phagosome. Afterward, the phagosome undergoes some gradual changes and produces antibacterial nitric oxide (NO) and reactive oxygen species (ROS) to eliminate bacterial infection [15]. However, Listeria monocytogenes escapes from the phagolysosome smartly by expressing the virulence factors hemolysin, pore-forming toxin listeriolysin O (LLO), PlcA and PlcB phospholipases and subsequently Listeria monocytogenes grow rapidly in the cytosol and captures the cellular actin tails to carry out its inter and intra cellular movements [16]. Infected phagocytes potentially can transfer bacteria to the brain through blood circulation and cause infection same as Trojan Horse [17]. Another virulence factor of Listeria monocytogenes is pore forming hemolysin O, which can activate the NF-κB pathway of endothelial cells [18], and induce cell surface adhesion molecules P, E selectin and ICAM-1, VCAM-1. Activation of NF-κB subsequently initiates the transcription of inflammatory factors such as IL-1β, IL-6, TNF-α, and IL-10 [19], which further destroys the integrity of the blood-brain barrier by accumulating lymphocytes, endothelial and inflammatory cells in nerve tissues [20]. The expression of inflammatory cytokines such as IL-1β and TNF-α enhances the induction of ICAM-1 which mediates the cell adhesion and promotes leukocytes to enter the central nervous system [21]. In patients with bacterial meningitis, significantly elevated levels of TNF-α and IL-1β can be detected, and TNF-α is directly proportional to the severity of the disease [22]. The expression levels of TNF-α and IL-1β have important reference cytokines for monitoring the occurrence and development of bacterial meningitis.

GTPases play a key role in the regulation of biochemical strategies to control complex cellular signaling processes. Among the GTPases super family, Rho GTPases regulate the cellular signal transduction during bacterial infections. Rho GTPases principally play their crucial role to regulate the actin cytoskeleton but also influence the microtubule dynamics, membrane transport channels and transcription factor activity [23]. However, GTPases, of the Rab, Arf, and Dynamin families also play an integral role in the regulation of cytoskeletal assembly, vesicular trafficking, and plasma membrane structure remodeling. Whereas, the Listeria monocytogenes has evolved the strategies to exploits or manipulate the GTPases molecular switching and promote its internalization, survival in the phagosome, actin-based motility, or exocytosis. Listeria monocytogenes, redistributes the Dynamine 2 GTPases to facilitate its intracellular vesicle fusion [24]. The intracellular replication of Listeria directs the polymerization of actin cytoskeleton and remodels the plasma membrane that pushes the plasma membrane around the adherent bacterium [25]. However, this kind of bacterial manipulation is sensed by NLR proteins NOD1 and NOD2. NOD1 and NOD2 can recognize the presence of peptidoglycan and ultimately activate NF-κB. Interestingly, peptidoglycan recognition by NOD1 depends upon the manipulation of small Rho GTPases by pathogens [26,27]. Several studies indicate that GTPases promote actin polymerization during bacterial infection to uptake the bacterium.

However, the exact role of GTPases in terms of inflammatory cytokines is limited. In this study, we have investigated the role of GTPases on the expression of inflammatory cytokines. We found that the RhoA is expressed significantly in the cells infected with Listeria monocytogenes and expression of Interleukins such as IL-1β, IL-6 and TNF-α was also increased. Collectively, we suggest that GTPases play role in the uptake of Listeria monocytogenes and interleukins promote the adhesion of leukocytes to endothelial cells and increase the permeability of the endothelial barrier to cross the blood-brain barrier.