Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Roberto Rosales-Reyes,Amal O Amer,Daniel F Aubert,Miguel A Valvano,Jennifer S Tolman,Laurel L Lenz

doi:10.1371/journal.pone.0041726

Abstract

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

Highlights

Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc) [1], a group of opportunistic bacteria that cause respiratory tract infections in patients with cystic fibrosis (CF) [2]
Macrophages transfected with the fluorescent chimeric probe Lact-C2-GFP that binds to cytosolic exposed phosphatidylserine showed fluorescence associated with the inner leaflet of the plasma membrane as well as with bacteriacontaining membrane vacuoles (BcCVs) (Figure 1E)
By comparison with uninfected cells (Figure 1F, top panel) B. cenocepacia infection did not alter the normal distribution of the GFP-PLCd-PH probe at the plasma membrane and no GFPPLCd-PH signal was detectable on the BcCV (Figure 1F, bottom panel)

Summary

Introduction

Burkholderia cenocepacia belongs to the Burkholderia cepacia complex (Bcc) [1], a group of opportunistic bacteria that cause respiratory tract infections in patients with cystic fibrosis (CF) [2]. Intracellular bacterial survival may explain the persistence of the infection and contribute to proinflammatory responses that become detrimental to the patient [5]. Initiation of the inflammatory response upon bacterial infection requires participation of cell membrane and cytoplasmic recognition systems. The Toll-like receptors (TLRs) usually recognize pathogen-associated molecular patterns (PAMPs) directly at the plasma or vacuolar membranes, leading to the synthesis and secretion of specific cytokines, including pro-IL-1b and pro-IL-18 [15]. This mechanism operates in macrophages infected by B. cenocepacia whereby the bacterial O antigen lipopolysaccharide contributes to TLR4-mediated IL-1b secretion [16]. PAMP recognition in the cytoplasm depends on a family of nucleotide-binding oligomerization domain-like receptor (NLR) proteins that mediate caspase-1 activation, which in turns regulates the proteolytic processing of inactive precursors pro-IL1b and pro-IL-18 into mature IL-1b and IL-18 respectively and their subsequent release [17]

Methods

Results

Conclusion