Although genetic polymorphisms in the LRRK2 gene are associated with a variety of diseases, the physiological function of LRRK2 remains poorly understood. In this study, we report a crucial role for LRRK2 in the activation of the NLRC4 inflammasome during host defense against Salmonella enteric serovar Typhimurium infection. LRRK2 deficiency reduced caspase-1 activation and IL-1β secretion in response to NLRC4 inflammasome activators in macrophages. Lrrk2−/− mice exhibited impaired clearance of pathogens after acute S. Typhimurium infection. Mechanistically, LRRK2 formed a complex with NLRC4 in the macrophages, and the formation of the LRRK2–NLRC4 complex led to the phosphorylation of NLRC4 at Ser533. Importantly, the kinase activity of LRRK2 is required for optimal NLRC4 inflammasome activation. Collectively, our study reveals an important role for LRRK2 in the host defense by promoting NLRC4 inflammasome activation.

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis, we found that a cluster of E scherichia coli v irulence f actors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli -HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

Enterohemorrhagic Escherichia coli (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of katN is promoted by RpoS and OxyR and repressed by H-NS, and katN contributes to bacterial growth under oxidative stress in vitro. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of katN had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells. The type VI secretion system (T6SS) is a specific macromolecular protein export apparatus, and widely distributed in Gram-negative bacteria. Generally, T6SS has been shown to play an important role in anti-bacterial competition and virulence to eukaryotic hosts. Enterohemorrhagic Escherichia coli (EHEC) can cause severe foodborne disease, including abdominal cramps and diarrhea that may progress to bloody diarrhea and hemolytic uremic syndrome. In the current study, we show that the T6SS of EHEC is involved in its intracellular survival and virulence in mice. Specifically, the novel effector KatN, a Mn-catalase identified in this work updates the general role of the T6SS in the pathogenesis of EHEC, in the context of oxidative stress in host cytoplasm. Combined with the biased distribution of katN in the T6SS-containing bacteria, our data suggest that KatN, as a new T6SS effector, is a key virulence factor in the pathogenesis of T6SS⁺ bacteria.

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms. Salmonella Typhimurium is a facultative intracellular pathogen capable of survival within host phagocytic cells. The crucial two-component system regulator PhoP-PhoQ is involved in response to different stresses and essential for the virulence of S. Typhimurium. In this study, we show that PhoP can be acetylated by the protein acetyltransferase Pat, or deacetylated by the NAD-dependent deacetylase CobB. Acetylation level of the conserved lysine residue 201 (K201) on PhoP varies with environmental factors including low magnesium, acid stress and phagocytosis, which consequently modulates the DNA-binding ability of PhoP. Furthermore, acetylation of PhoP K201 is critical for host inflammation response and bacterial survival in the host. Taken together, these results highlight the fact that Salmonella may utilize reversible lysine acetylation to quickly adapt themselves to the hostile environments.