Recombinant Rv3261 protein of Mycobacterium tuberculosis induces apoptosis through a mitochondrion-dependent pathway in macrophages and inhibits intracellular bacterial growth

Highlights

• Rv3261 induces macrophage apoptosis via caspase-3/-9 through interaction with TLR4.

• Rv3261 induced loss of mitochondrial transmembrane potential (ΔΨ_m).

• ROS-dependent JNK activation pathway was critical in Rv3261-mediated apoptosis.

• Rv3261-mediated apoptosis may play a role as host defense response.

• Rv3261 might be involved in apoptotic modulation of Mtb-infected macrophages.

Abstract

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.

Introduction

Tuberculosis (TB) is a chronic inflammatory disease of global importance that remains a major global health treat. A recent survey by the World Health Organization (WHO), published in 2019, estimated about 10 million new cases of TB per year globally, with around 1.6 million deaths [1]. While one-third of the world’s population is believed to be infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB, <10% suffer from symptomatic TB [2]. However, in recent years, the appearance of drug-resistant TB has increased, escalating into a public health crisis.

The pathogenesis of TB infection starts when a person inhales droplets of tubercle bacilli found in the air, which eventually reach the alveoli of the lungs. Alveolar macrophages are the first effector immune cells to encounter the bacilli and the first line of host defense against Mtb infection, destroying or inhibiting the majority of the bacterial load [3]. Macrophages function via multiple bactericidal mechanisms, including phago-lysosome fusion, production of reactive oxygen and nitrogen species, and the activation of apoptosis [4], [5]. Although subsequent innate and adaptive immune responses are induced to effectively kill intracellular bacteria, Mtb presents diverse mechanisms to circumvent the protective immune responses of the host. Characterizing the virulent factors and pathogenic mechanisms of Mtb will allow for the development of more effective vaccines and drugs to control this devastating disease.

The innate immune response is initiated by the identification and characterization of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) expressed by the immune cells [6]. TLRs have been shown to play an essential role in recognizing specific mycobacterial antigens and in the activation of macrophages. For example, Mtb components, such as MymA (Rv3083) [7] and PPE26 (Rv1789) [8], bind to TLR2, while other components, such as Rv2882c [9] and Rv3463 [2], bind to TLR4, and then modulate macrophage activation. Recent studies have indicated that reactive oxygen species (ROS) produced by macrophages act as signaling molecules for TLR4-mediated innate immunity [10], resulting in TLR4-mediated ROS generation, followed by ASK1-JNK activation, which in turn leads to enhanced macrophage apoptosis [11]. In addition, TLR2 triggers macrophage apoptosis, leading to a reduction in the viability rate of Mtb [12].

Apoptosis is a programmed cell death where the cells eliminate pathogens at the early stages or present apoptotic bodies containing infected microbes for other phagocytic cells for the subsequent activation of immune defense mechanisms. These cell death pathways do not always benefit the host cells, but can serve as a bridge by which the pathogens escape, spread, and evade immune cells and their microenvironments. A number of studies have showed that virulent factors from mycobacterial species can strategically suppress apoptotic induction in macrophages to facilitate their replication [13], [14] or trigger the apoptotic death of host cells, which can benefit their dissemination [15]. Several mycobacterial proteins have been reported to modulate macrophage apoptosis. Early secreted protein antigen ESAT6 (Rv3763) [16], 19-kDa lipoproteins LpqH (Rv3763) [17], and 38-kDa lipoprotein PstS-1 (Rv0934) [18] induce host cell death via the TLR2 signaling pathway. Other mycobacterial proteins, such as PE_PGRS (Rv0297) [19], PE9 (Rv1088), and PE10 (Rv1089) [20], induce macrophage apoptosis through TLR4.

We have previously identified the immunoreactive proteins through the multidimensional fractionation of Mtb culture filtrates or sonic extracts [2], [9], [21], and have recently identified the Rv3261 protein, which has the potential to induce the death of macrophages. In this study, we produced recombinant Rv3261 in Escherichia coli (E. coli) and its cell death-modulating mechanism was characterized in the murine macrophage cell line RAW264.7. Rv3261 induced macrophage apoptosis by triggering ROS production, release of cytochrome c, and activation of caspase-3 via a TLR4-dependent pathway. We also show that the JNK signaling pathway may be critical in Rv3261-mediated ROS generation.