Modulation of Microtubule Dynamics Affects Brucella abortus Intracellular Survival, Pathogen-Containing Vacuole Maturation, and Pro-inflammatory Cytokine Production in Infected Macrophages

Abstract

The microtubule (MT) cytoskeleton regulates several cellular processes related to the immune system. For instance, an intricate intracellular transport mediated by MTs is responsible for the proper localization of vesicular receptors of innate immunity and its adaptor proteins. In the present study, we used nocodazole to induce MTs depolymerization and paclitaxel or recombinant (r) TIR (Toll/interleukin-1 receptor) domain containing protein (TcpB) to induce MT stabilization in bone marrow-derived macrophages infected with Brucella abortus. Following treatment of the cells, we evaluated their effects on pathogen intracellular replication and survival, and in pro-inflammatory cytokine production. First, we observed that intracellular trafficking and maturation of Brucella-containing vesicles (BCVs) is affected by partial destabilization or stabilization of the MTs network. A typical marker of early BCVs, LAMP-1, is retained in late BCVs even 24 h after infection in the presence of low doses of nocodazole or paclitaxel and in the presence of different amounts of rTcpB. Second, microscopy and colony forming unit analysis revealed that bacterial load was increased in infected macrophages treated with lower doses of nocodazole or paclitaxel and with rTcpB compared to untreated cells. Third, innate immune responses were also affected by disturbing MT dynamics. MT depolymerization by nocodazole reduced IL-12 production in infected macrophages. Conversely, rTcpB-treated cells augmented IL-12 and IL-1β secretion in infected cells. In summary, these findings demonstrate that modulation of MTs affects several crucial steps of B. abortus pathogenesis, including BCV maturation, intracellular survival and IL-12 secretion in infected macrophages.

The TIR-domain containing effectors BtpA and BtpB from Brucella abortus impact NAD metabolism

“…We show here that catalytically dead mutants in BtpA and BtpB TIR domains, still have the ability to self-aggregate and form filaments, proving that both features can be segregated. BtpA has been related to tubulin structures in host cells, specifically by protecting microtubules from depolymerization [19,42]. However, we did not see coincidence of TIR cytoplasmic filaments with tubulin.…”

The TIR-domain containing effectors BtpA and BtpB fromBrucella abortusblock energy metabolism

“…We show here that catalytically dead mutants in BtpA and BtpB TIR domains, still have the ability to self-aggregate and form filaments, proving that both features can be segregated. BtpA has been related to tubulin structures in host cells, specifically by protecting microtubules from depolymerization [18, 40]. However, we did not see coincidence of TIR cytoplasmic filaments with tubulin.…”

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells

“…It has previously been demonstrated that Brucella is able to induce cytoskeleton rearrangements; for instance, BigA, an adhesion protein containing the immunoglobulin-like domain found in B. abortus, induces actin-cytoskeleton rearrangement in Caco-2 and the dog cell line MDCK (Czibener et al, 2016). Alves-Silva et al (2017) demonstrated that BtpA induced microtubule bundling in bone-marrow derived macrophages (BMDM) and increased B. abortus virulence. In addition, BMDM stimulated with BtpA and infected with B. abortus induced IL-12 and IL-1β production (Alves-Silva et al, 2017).…”

“…Alves-Silva et al (2017) demonstrated that BtpA induced microtubule bundling in bone-marrow derived macrophages (BMDM) and increased B. abortus virulence. In addition, BMDM stimulated with BtpA and infected with B. abortus induced IL-12 and IL-1β production (Alves-Silva et al, 2017). Cytoskeleton rearrangement and TNFα production have been linked to cytokine production by way of common signal pathways such as ERK kinases, a component of the mitogen-activated protein kinase (MAPK) signaling pathway (Rosengart et al, 2002).…”