T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage

Abstract

Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which—in contrast to the direct cytoplasmic injection of T3SS effector proteins—are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida (Phdp). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1β. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

Interleukin-22 level is negatively correlated with neutrophil recruitment in the lungs in a Pseudomonas aeruginosa pneumonia model

“…Tuning appropriate host response, especially PMN recruitment in response to pathogen aggression, is critical for host survival. PMN recruitment and activation during bacterial infection is a double-edged sword 30 . Neutropenic mice display exacerbated susceptibility of PA pneumonia 31 and neutrophil depletion (by i.v.…”

“…administration of Ly6-G neutralisation antibody) which led to a fatal lung infection in our model within 12 hours (data not shown). Failure to properly control PMN accumulation following pulmonary infection will contribute to tissue damage and ARDS 26 , 30 , 32 . In the context of chronic obstructive pulmonary disease (COPD), Guillon et al .…”

Nuclear trafficking of bacterial effector proteins

“…The presence of YopM in the cytoplasm (24% of total protein) 1 hr after incubation with HeLa cells, suggests that this protein can avoid lysosomal degradation by escaping to the cytoplasm before entering the nucleus (18.7% of total protein) (Scharnert et al, 2013) (Figure 1a). rNleC from EPEC also traffics to early and late endosomes, however, it is partially degraded in lysosomes (Stolle et al, 2017) (Figure 1b).…”

Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization

“…Notably, by comparing the ratio of fluorescence from internalized rhodamine-labeled molecules to that of internalized naphthofluorescein-labeled molecules, the relative efficiencies of endosomal escape for different molecules-of-interest could be estimated. 35,163,[165][166][167] While these assays were designed to report exclusively on cytosolic localization, they required additional independent information, and they lacked absolute quantitation. Recently, a new fluorescence-based method was reported that provided absolute quantitation of fluorophore concentration in the cytosol (Figure 3c).…”