Abstract
Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.
Highlights
The genus Yersinia, a member of the enterobacteriaceae family, consists of 11 species, three of which are pathogenic to humans; Yersinia pestis (Yp), Yersinia pseudotuberculosis, and Yersinia enterocolitica
Reverse Phase Protein Microarray (RPMA) ANALYSIS OF HBE CELLS INFECTED WITH YERSINIA PESTIS In order to study host cell signaling pathways that are altered during Yp infection, including pathways that are modulated independently of the TTSS mechanism, we compared host response of 16HBE14o- cells infected with the fully virulent strain CO92 with the response to infection with a derivative avirulent strain, CO92 (Pgm, Pst-)
For the avirulent strain CO92 (Pgm, Pst-), we found that about 5% of the bacteria were taken up by
Summary
The genus Yersinia, a member of the enterobacteriaceae family, consists of 11 species, three of which are pathogenic to humans; Yersinia pestis (Yp), Yersinia pseudotuberculosis, and Yersinia enterocolitica. For a number of pathogenic bacteria, their ability to alter host protein phosphorylation pathways to allow the subversion of host defenses has been reported (Popova et al, 2010; Younossi et al, 2011; Steiner et al, 2014). In the case of Yp, the pCD1 plasmid encodes for bacterial virulence factors known as Yersinia Outer Proteins (YOPs) that are used to evade the host immune response and manipulate the host cell machinery, as well as proteins essential for the assembly of the Type Three Secretion System (TTSS) (Cornelis, 2002b; Pujol and Bliska, 2005; Plano and Schesser, 2013). The following identified Yop effectors are important in regulating host response, including the modulation of host signaling pathways: YopE, YopH, YopJ, YopK, YopM, YopT, and YpkA (Viboud and Bliska, 2005). YopE, H, T, and YpkA have been shown to alter the host cytoskeleton in order to prevent bacterial phagocytosis (Dukuzumuremyi et al, 2000a; Cornelis, 2002b; Sauvonnet et al, 2002; Aepfelbacher et al, 2011; Ke et al, 2013), which has been proposed to be beneficial to Yp because it proliferates much better in the extracellular space
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