Abstract
A type III secretion system (T3SS) in pathogenic Yersinia species functions to translocate Yop effectors, which modulate cytokine production and regulate cell death in macrophages. Distinct pathways of T3SS-dependent cell death and caspase-1 activation occur in Yersinia-infected macrophages. One pathway of cell death and caspase-1 activation in macrophages requires the effector YopJ. YopJ is an acetyltransferase that inactivates MAPK kinases and IKKβ to cause TLR4-dependent apoptosis in naïve macrophages. A YopJ isoform in Y. pestis KIM (YopJKIM) has two amino acid substitutions, F177L and K206E, not present in YopJ proteins of Y. pseudotuberculosis and Y. pestis CO92. As compared to other YopJ isoforms, YopJKIM causes increased apoptosis, caspase-1 activation, and secretion of IL-1β in Yersinia-infected macrophages. The molecular basis for increased apoptosis and activation of caspase-1 by YopJKIM in Yersinia-infected macrophages was studied. Site directed mutagenesis showed that the F177L and K206E substitutions in YopJKIM were important for enhanced apoptosis, caspase-1 activation, and IL-1β secretion. As compared to YopJCO92, YopJKIM displayed an enhanced capacity to inhibit phosphorylation of IκB-α in macrophages and to bind IKKβ in vitro. YopJKIM also showed a moderately increased ability to inhibit phosphorylation of MAPKs. Increased caspase-1 cleavage and IL-1β secretion occurred in IKKβ-deficient macrophages infected with Y. pestis expressing YopJCO92, confirming that the NF-κB pathway can negatively regulate inflammasome activation. K+ efflux, NLRP3 and ASC were important for secretion of IL-1β in response to Y. pestis KIM infection as shown using macrophages lacking inflammasome components or by the addition of exogenous KCl. These data show that caspase-1 is activated in naïve macrophages in response to infection with a pathogen that inhibits IKKβ and MAPK kinases and induces TLR4-dependent apoptosis. This pro-inflammatory form of apoptosis may represent an early innate immune response to highly virulent pathogens such as Y. pestis KIM that have evolved an enhanced ability to inhibit host signaling pathways.
Highlights
Microbial pathogens encode numerous types of virulence factors that are used to circumvent or usurp immune responses within cells of their hosts
YopJ inhibits the production of survival factors in naıve macrophages, causing them to die by apoptosis, which is generally considered to be immunologically silent
We have identified a distinct isoform of YopJ in Y. pestis (YopJKIM) that induces high levels of apoptosis and caspase-1 activation in infected macrophages
Summary
Microbial pathogens encode numerous types of virulence factors that are used to circumvent or usurp immune responses within cells of their hosts. A protein export pathway known as the type III secretion system (T3SS) allows Gram-negative bacterial pathogens to deliver effector proteins into or across the plasma membrane of host cells, with the goal of co-opting or disrupting eukaryotic signaling pathways [1,2]. Infection of macrophages with T3SS-expressing bacterial pathogens commonly causes cytotoxicity in the host cell, but the mechanisms of cellular demise and the morphological and immunological characteristics of cell death can be unique for each microbe [3]. T3SS-expressing pathogens and distinguished morphologically and immunologically are apoptosis and pyroptosis [4]. Apoptosis and pyroptosis can be distinguished mechanistically by the fact that only the latter mechanism of cell death is dependent upon the activity of caspase-1, a pro-inflammatory caspase [4,5]. It has been determined that caspase-1 can be activated in macrophages dying of apoptosis [6,7,8], indicating that pathogen-inflicted apoptosis may not be immunologically silent
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