Over the last few decades, T. pyogenes has been demonstrated as a cause of different clinical manifestations in domestic animals (Amos et al., 2014, Ribeiro et al., 2015), and thus our research is of particular importance for domestic animal husbandry. Next-generation sequencing technology has proven to be a potent approach to generate large-scale genomic resources for most organisms and provide useful information on the underlying pathogenic mechanisms and interactions following the microorganism infection (Livny et al., 2014, Schulze et al., 2015, Villarino et al., 2017). Given that organ or tissue specific transcriptomes research has great advantages in the understanding of general biological processes, we used T. pyogenes infected blood and liver samples of mice to perform a genome-wide investigation by transcriptional sequencing and gene expression profile analysis. A total of 272 DEGs were identified in the blood and liver infected samples compared to normal samples, which demonstrated a similar functional distribution of GO categories and indicated the sequence diversity of the transcriptome study. Hierarchical clustering to group proteins according to the level of expression provided the comprehensive landscape of the host transcriptome. Taken together, these analyses suggested that T. pyogenes infection might be associated with many DEGs, which are more likely to play a role in cell invasion and regulation of the host immune system.
To elucidate the molecular pathways and DEGs involved in immune defenses of mice infected with T. pyogenes, the KEGG pathway-based and PPI networks analysis was performed to further understand the DEGs functions and interactions. Among the 26 KEGG pathways that obtained DEGs between infected and control samples in blood and liver were assigned, “endocytosis”, “pathways in cancer”, and “HTLV-I infection” represented the three largest category in blood transcriptome. It is possible that T. pyogenes infection might impair cellular immunity by regulating the expression of immune-related genes, which is consistent with our previous study that the Th1 type immune response was considered to be the major host response against T. pyogenes infection (Huang et al., 2016). In addition, we found that “metabolic pathways” represented the largest category in liver transcriptome, followed by “PI3K-Akt signaling pathway” (Xie et al., 2014), “pathways in cancer,” and “Jak-STAT signaling pathway” (Villarino et al., 2015, Villarino et al., 2017), implying that T. pyogenes might also affect the host by altering the host cell microenvironment for their own benefit. Although a slightly greater number of transcripts were associated with metabolic processes than the immune system, most of the pathways in signal transduction are involved in and play important roles in the immune system, such as PI3K-Akt, Jak-STAT, and mTOR signaling pathways (Saleiro et al., 2015, Saxton et al., 2017). In addition, we found that Ddx18, Rps8, Jak3, and Mtor had higher degrees and betweenness values in the PPI network constructed by STRING database, suggesting that these proteins may interact with each other in their own PPI network.
In the present study, the mRNA level of mTOR were significantly increased in the infected mice compared to the control group. A possible explanation is that T. pyogenes invasion might inhibit host autophagy to survive in the host cells by up-regulating mTOR expression. Given the previous reports that T. pyogenes was able to survive within host phagocytes such as macrophages (Jost et al., 2005) and mTOR was a key regulator of autophagy (CH et al., 2010), we thus focused on mTOR and further investigated its roles throughout this study. As a hub gene in the liver transcriptome, mtor coded for an evolutionarily conserved protein in all eukaryotic species and functions as a critical regulator in protein synthesis, autophagy, and manipulates the progress of cancer and diabetes (Saleiro et al., 2015, Saxton et al., 2017). To investigate whether autophagy induction could reduce T. pyogenes burdens in macrophages, RAW264.7 murine macrophages were infected with T. pyogenes and then treated with rapamycin. We found that treatment with rapamycin induced autophagy and increased bacterial elimination in cells. However, blocking autophagy with ATG7 siRNA or autophagy inhibitor 3-MA partially reversed these effects with increased CFU in cells following T. pyogenes infection. In agreement with our findings, other reports have indicated that autophagy enhanced bacterial elimination and played a key role in immune defense against pathogen invasion (K et al., 2012, R et al., 2015). Mechanistically, the inhibition of mTOR regulated oxidation and cytokines expression such as IL-1? against T. pyogenes in murine macrophages by using mTOR siRNA or rapamycin. Indeed, the activation of autophagy by rapamycin could decrease bacterial toxin-mediated ROS production and protect cells against the toxin-caused impairment (Yuan et al., 2009). These interactions might determine the defense mechanisms of the host that are necessary to defeat T. pyogenes infections. More importantly, to inhibit mTOR expression significantly protected mice from T. pyogenes challenge and increased mice survival rate, which suggested mTOR might be a novel target to control T. pyogenes related diseases. These data also imply that T. pyogenes might alter the defense responses of the host at the transcriptomic level by increasing expression levels of immune-related and antibacterial defense-related genes and might subsequently impair the immune functions of the host. Taken together, these findings provide a robust overview of the pathways and DEGs involved in the immune defense responses of mice during T. pyogenes infection.
Surprisingly, the key genes identified in blood in this study did not overlap with those identified in the liver, suggesting that liver and peripheral blood transcriptome process of T. pyogenes infection were different. Although the liver contains numerous innate and adaptive immune cells that specialize in detection and capture of pathogens from the blood, there are still considerable differences in the response of liver and blood to infectious organisms (Knolle et al., 2000, Jenne et al., 2013). More studies can be undertaken to build on the work we have done, such as dual transcriptome analysis testing in mammals or further investigate the functions of the key DEGs in blood transcriptome. This future work and our current study will contribute to determining the immune responses based on host-pathogen interaction during T. pyogenes infection.
In summary, we identified numerous immune-associated DEGs and signal pathways by gene expression profiling in mice transcriptome following infection with T. pyogenes. We found that mTOR is a potent functional regulator involved in immune defense responses with T. pyogenes infection. More importantly, our findings are the first to pinpoint the role of autophagy that could protect mice from T. pyogenes challenge by inhibiting the expression of mTOR. Overall, this study provides the first informative reference dataset for future studies on global and specific response to T. pyogenes infection at the molecular level and will facilitate gene discovery and biomarker identification for prevention and control of T. pyogenes-related diseases.