The Effector Protein BPE005 from Brucella abortus Induces Collagen Deposition and Matrix Metalloproteinase 9 Downmodulation via Transforming Growth Factor β1 in Hepatic Stellate Cells

Abstract

The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway. Brucellosis is a worldwide zoonosis characterized by hepatomegaly, splenomegaly, and peripheral lymphadenopathy. It is a chronic and debilitating infection caused by Gram-negative facultative intracellular bacteria that infect domestic and wild animals and that can be transmitted to humans (1, 2). The frequency of liver involvement in active brucellosis ranges from 5% to 52% or more (1). However, although numerous studies have focused on brucellar liver histopathology (1), the pathogenic mechanisms of liver disease caused by Brucella have not been completely investigated at the molecular and cellular levels.Liver fibrosis is a wound-healing response to chronic hepatic injury, which may be caused by alcohol abuse, hepatitis virus infection, or nonalcoholic steatohepatitis, and it is characterized by an excessive accumulation of extracellular matrix proteins in the liver (3, 4). An early event in the development of liver fibrosis is the activation of hepatic stellate cells (HSCs), the major cell type responsible for increased synthesis of extracellular matrix proteins (5). An elevated level of transforming growth factor ␤1 (TGF-␤1) is also observed in the damaged liver, and it has a close correlation with fibrogenic changes in HSCs and liver tissue (6-8). In addition, decreased matrix metalloproteinase 9 (MMP-9) expression was observed in alcoholic liver fibrosis (9). This fibrogenic phenotype involves alterations in the balance of MMPs and their natural inhibitors-tissue inhibitors of metalloproteinases (TIMPs). In particular, MMP-2 and MMP-9 (gelatinase A and B, respectively) are important in regulating fibrogenesis and scar degradation. They can degrade a variety of collagens, including basement membrane (type IV collagen), denatured fibrillar...

Brucella abortus Infection Elicited Hepatic Stellate Cell-Mediated Fibrosis Through Inflammasome-Dependent IL-1β Production

“…Finally, to verify the in vivo significance of our hypothesis, Casp-1, ASC, NLRP3, and AIM2 KO mice and WT mice, as control, were infected with B. abortus, and 4 weeks later, animals were sacrificed to determine the role of inflammasome in the liver fibrosis. Accordingly with our previous results, Masson's trichrome staining revealed the presence of fibrotic patch in livers from B. abortus-infected mice with respect to uninfected control (9,15). In contrast, Casp-1, ASC, NLRP3, and AIM2 KO animals presented a significant reduction in the fibrotic patch (Figure 7).…”

“…Previously, we have demonstrated that upon infection of HSCs, B. abortus triggers a profibrotic response characterized by inhibition of MMP-9 secretion inducing concomitant collagen deposition and transforming growth factor (TGF)-β1 secretion in a way that involves a functional T4SS and its effectors protein BPE005 (9). Taking into account that inflammasome has been documented to be necessary to induce activation to a fibrotic phenotype of HSCs, we hypothesized that Brucella infection might create a microenvironment that would promote inflammasome activation and concomitant profibrogenic phenotype in HSCs.…”

Proteomics of Brucella

“…Surprisingly, the functional aspects of proteases in Brucellae have not been investigated with proteomics so far. This is in sharp contrast to the proven importance of proteases for a great many functions such as host infection and persistence [89][90][91][92], stress response [92][93][94][95][96], morphology [97], communication and signaling [98][99][100], to name the most prominent.…”

Brucella abortus Promotes a Fibrotic Phenotype in Hepatic Stellate Cells, with Concomitant Activation of the Autophagy Pathway

“…It has been demonstrated that autophagy participates in HSC activation (17). We have previously demonstrated that B. abortus infection induces HSC activation, leading to a profibrogenic phenotype (18,19). To determine if B. abortus infection induces the activation of the autophagic pathway in HSCs, we evaluated by Western blotting at 24 h postinfection the expression of LC3 II, the lipidated form of LC3 I, and the only known protein that specifically associates with autophagosomes (20), the autophagy regulator Beclin-1 (21), as well as p62, which participates in the autophagic clearance of ubiquitinated proteins (22).…”