Guanylate Binding Proteins Regulate Inflammasome Activation in Response to Hyperinjected Yersinia Translocon Components

Abstract

Gram-negative bacterial pathogens utilize virulence-associated secretion systems to inject, or translocate, effector proteins into host cells to manipulate cellular processes and promote bacterial replication. However, translocated bacterial products are sensed by nucleotide binding domain and leucine-rich repeat-containing proteins (NLRs), which trigger the formation of a multiprotein complex called the inflammasome, leading to secretion of interleukin-1 (IL-1) family cytokines, pyroptosis, and control of pathogen replication. Pathogenic Yersinia bacteria inject effector proteins termed Yops, as well as pore-forming proteins that comprise the translocon itself, into target cells. The Yersinia translocation regulatory protein YopK promotes bacterial virulence by limiting hyperinjection of the translocon proteins YopD and YopB into cells, thereby limiting cellular detection of Yersinia virulence activity. How hyperinjection of translocon proteins leads to inflammasome activation is currently unknown. We found that translocated YopB and YopD colocalized with the late endosomal/lysosomal protein LAMP1 and that the frequency of YopD and LAMP1 association correlated with the level of caspase-1 activation in individual cells. We also observed colocalization between YopD and Galectin-3, an indicator of endosomal membrane damage. Intriguingly, YopK limited the colocalization of Galectin-3 with YopD, suggesting that YopK limits the induction or sensing of endosomal membrane damage by components of the type III secretion system (T3SS) translocon. Furthermore, guanylate binding proteins (GBPs) encoded on chromosome 3 (Gbp Chr3 ), which respond to pathogen-induced damage or alteration of host membranes, were necessary for inflammasome activation in response to hyperinjected YopB/-D. Our findings indicate that lysosomal damage by Yersinia translocon proteins promotes inflammasome activation and implicate GBPs as key regulators of this process.

Gain-of-Function Analysis Reveals Important Virulence Roles for the Yersinia pestis Type III Secretion System Effectors YopJ, YopT, and YpkA

“…Some incomplete sets of Yersinia effectors result in increased macrophage death, either via YopE induction of pyroptosis (30,31) or as a result of dysregulated effector and translocon secretion in the absence of YopK (32,41). However, the ΔT3SE::ϩyopHEKM strain expresses both YopM, which prevents YopE-mediated activation of the pyrin inflammasome (30, 31), and YopK, which prevents inflam-masome activation by translocon components (41,42). As expected, therefore, this strain resulted in minimal macrophage death (see Fig.…”

Sensing of invading pathogens by GBPs: At the crossroads between cell-autonomous and innate immunity

“…Most importantly, it is not known what drives GBP recruitment to bacteria or their PCVs. Recent work has suggested that in the case of Yersinia pseudotuberculosis or L. pneumophila , GBP recruitment to the PCVs is dependent on the bacterial type‐3 or ‐4 secretion system, respectively, and the recruitment of galectin‐3, a β‐galactoside‐binding protein (Fig. C).…”

Gain of Function Analysis Reveals Non-Redundant Roles for theYersinia pestisType III Secretion System Effectors YopJ, YopT, and YpkA

“…Some incomplete sets of Yersinia effectors result increased macrophage death, either via YopE induction of pyroptosis (30, 31) or as a result of dysregulated effector and translocon secretion in the absence of YopK (32, 41). However, the ΔT3SE::+yopHEKM strain expresses both YopM, which prevents YopE-mediated activation of the pyrin inflammasome (30, 31), and YopK, which prevents inflammasome activation by translocon components (41, 42). As expected, therefore, this strain resulted in minimal macrophage death (Figure S1).…”