Brucella secretory protein VceA promotes FOXO1 entry into the nucleus to shift host cell metabolism toward glycolysis

Abstract

Increased glycolytic metabolism is a key step in the reproduction of Brucella and the induction of brucellosis, however, little is known about how this process is regulated during infection. Forkhead box protein O1 (FOXO1) is a transcription factor that regulates energy metabolism. In this study, we employ the yeast two-hybrid system (Y2H) and immunoprecipitation (Co-IP) to reverse screen for the FOXO1 for the first time and identify interactions between FOXO1 and the Brucella secretory protein VceA. Our findings reveal that the Brucella secretory protein VceA colocalizes with FOXO1 in the cytoplasm. Additionally, we observe that infection of macrophages with Brucella abortus 2308 (S2308) promotes FOXO1 entry into the nucleus, leading to a significant upregulation of glycolysis level in macrophage. Conversely, in a VceA mutant strain (S2308-ΔVceA), we note a significant reduction in the ability of FOXO1 to enter the nucleus, accompanied by a decrease in glycolysis level. Furthermore, Brucella interacts with FOXO1 through the secreted protein VceA, promoting the entry of FOXO1 into the nucleus and thereby altering host metabolic patterns. This study provides insights into the mechanisms by which Brucella invades host macrophages and induces unique metabolic changes. These insights may offer a novel rationale for developing metabolic therapeutic strategies for the treatment and prevention of related diseases.

1. Introduction

Brucella is a gram-negative bacterium capable of infecting humans and livestock, leading to brucellosis. It commonly presents with undulant fever, weakness, weight loss, and chronic debilitating symptoms. Abortion and infertility associated with infection can result in significant economic losses [1,2]. The remarkable capacity of Brucella to persist and replicate within host macrophages underlies the establishment of chronic infection [3]. Brucella enters the host cell and undergoes a continuous cycle, progressing from endosomal Brucella-containing vacuoles (eBCVs) to replicative Brucella-containing vacuoles (rBCVs) and then to autophagic Brucella-containing vacuoles (aBCVs) [4,5]. One of the principal functions of the type IV secretion system (T4SS) in Brucella is to regulate the intracellular trafficking of Brucella vacuoles within host macrophages, thereby preventing their degradation within phagolysosomes [6]. For example, BSPB interacts with the conserved oligomeric Golgi (COG) complex in host cells to facilitate the orientation of Golgi-derived vesicles toward rBCVs [7]. The T4SS effector SepA is able to eliminate the lysosomal marker LAMP-1 during the conversion of eBCV to rBCV [8]. The T4SS of Brucella can also modulate the immune response. For example, the T4SS effector VceC interacts with the host cell endoplasmic reticulum (ER) chaperone Bip, resulting in ER stress. This interaction stimulates the production of inflammatory cytokines, which leads to granuloma formation and, consequently, promotes chronic infections [9].

Intracellular Brucella may also increase glucose accessibility by regulating the activity of host cell enzymes involved in carbohydrate metabolism via the T4SS secretion system. For example, the T4SS effector BPE123 interacts with and stimulates the activity of the host cell glycolytic enzyme α-enolase. This interaction is necessary for the replication of Brucella abortus 2308 (S2308) [10]. Specific modulation of glycolytic energy flux determines macrophage polarity. The metabolic divergence of different macrophage phenotypes is of particularly relevance to the study of intracellular infections because it can directly affect pathogen survival and is likely to be a key factor in determining disease control and progression [11]. Studies have demonstrated that Brucella abortus (B. abortus) depends on glucose metabolites, such as lactate, for survival within the host organism [12]. Glucose is the preferred carbon source for Brucella during the chronic phase of infection in mice, when it resides in alternatively activated macrophages [11]. The wild-type virulence of Brucella strains in mice depends on pyruvate kinase (PykM), whereas gluconeogenesis depends on PpdK, which is critical for glucose catabolism [13]. Glycolysis plays a pivotal role in the pathogenesis of Brucella brucellosis. Nevertheless, the precise mechanisms by which Brucella induces alterations in glycolytic metabolism in host immune cells during infection remain largely unexplored.