Neonatal sepsis continues to be a debilitating toll globally. Among the wide array of pathogens concerned, E. coli has emerged as the commonest cause of early onset sepsis with high case fatality, specifically among pre-term and low birth-weight infants both in developed and developing countries. Increasing antibiotic resistance among the extraintestinal pathogenic E. coli (ExPEC) isolates is a serious hold up in management of the disease. Hence, development of an effective and inexpensive vaccine against neonatal septicemic E. coli (NSEC) should draw serious research attention. SslE or YghJ is a cell associated and secreted lipoprotein having M60 metalloprotease domain of diverse E. coli pathotypes. Importantly, SslE was identified as one of the most potent vaccine candidates for ExPEC isolates by “substractive reverse vaccinology” and was shown to provide almost complete protection from sepsis in a mouse model. We have identified, cloned and purified SslE from a NSEC isolate and showed for the first time that SslE can stimulate the production of different proinflammatory cytokines such as IL-1α, IL-1β and TNF-α in murine macrophages RAW264.7 cells via the activation of TLR2/TLR1 heterodimer. Moreover, SslE induces phosphorylation of ERK1/2, JNK1/2 and p38 and promotes nuclear translocation of both p50 and p65 to trigger the MAP Kinase and NFκB signaling pathways which get impaired in presence of specific inhibitors against each signal molecule and in TLR2 siRNA transfected cells and in cells blocked with mAb against TLR2, suggesting the involvement of TLR2 in NFκB and MAPK activation and subsequent cytokine secretion. Furthermore, our study demonstrates that SslE induces TLR2 dependent production of several other pro-inflammatory hallmarks such as type I chemokines, reactive oxygen (ROS) and reactive nitrogen species (NO) in mouse macrophages. Interestingly, the overexpression of MHC-II and other co-stimulatory molecules such as CD80 and CD86 in SslE-stimulated macrophages essentially suggests macrophage activation and M1 polarization, crucial in framing host’s innate immune response to this protein. To be an ideal vaccine candidate, SslE must unlock the otherwise blunted adaptive immune system through the activation and polarization of CD4+ T cells which interact with the antigens presented by MHC molecules on antigen presenting cells and start to proliferate and differentiate into its two subtypes Th1 and Th2. We found that splenic CD4+ T cells from immunized mice proliferated significantly in response to both SslE pulsed macrophages and SslE pulsed dendritic cells, while the cells from non-immunized mice did not. The Th1 cellular immune response is critical for anti-microbial immunity thus, antigens having potent immunomodulatory effect to induce Th1 polarization are an important tool for vaccination against a particular pathogen.  The CD4+ T cells from SslE immunized BALB/c mice were able to secrete the Th1 cytokines such as IFN-γ, IL-2 and TNF-α in vivo and also when co-cultured with both SslE pulsed macrophages and dendritic cells in vitro, whereas no secretion of IL-4, the Th2 cytokine, could be found. Immune CD4+ T cells also showed overexpression of Th1 chemokines such as RANTES, MIP-1α, MIP-1β and lymphotactin both at RNA and protein levels whereas expression of Th2 chemokines such as MDC, eotaxin-2 and TARC remained unaltered. Among the chemokine receptors, CCR5 got highly expressed in immune T cells while expression of CCR3, CCR4, CCR7 and CXCR3 remained unaltered. Immune T cells also over-expressed mRNA of T-bet, a Th1-specific transcription factor but not GATA-3, a Th2-specific transcription factor. All these clearly depict that SslE activates CD4+ T cells toward type 1 polarization in vitro and in vivo to develop a Th1 skewed adaptive immune response. Moreover, we have found almost complete protection of neonatal mice against a clinical septicemic E. coli isolate by maternal immunization with SslE as compared with the unimmunized mice. Hence, our study delineates that SslE could be a potent immunotherapeutic target against E. coli sepsis in newborns.

Dr. Rima Tapader Ghosh is working as ICMR (Indian Council of Medical Research)-Post Doctoral Researcher in the Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED), Kolkata, India since 2016. She has completed her Ph.D. in Microbiology from University of Calcutta, India in 2016. She is the first to show the significant contribution of secreted proteases such as serine protease autotransporters of Enterobacteriaceae (SPATEs) and SslE (a cell associated and secreted lipoprotein of E. coli) in the pathogenesis of neonatal septicemic Escherichia coli (NSEC). Presently, the major goal of her research work is to develop a vaccine against NSEC using SslE, one of the potent vaccine candidates for extraintestinal pathogenic E. coli. She has shown the role of SslE in activating innate immune response as SslE can trigger TLR2 mediated proinflammation, activation and M1 polarization of macrophages. Furthermore, she is working on SslE mediated activation of adaptive immunity and is trying to establish the protective role of SslE against E. coli causing neonatal septicemia. She has been awarded Best Oral Presentation Award in the 3rd International Conference on Biotechnology & Bioinformatics (ICBB-2016) and Best Poster Presentation Award in the 2nd International Conference on Biotechnology & Bioinformatics (ICBB-2015). She has also been awarded “Bill and Melinda Gates Travel Grant Award for Young Investigators” for presentation of her work at 18th International Congress on Infectious Diseases at Buenos Aires, Argentina in 2018.