Transcriptomic Research On Leptospiral Infection And Structural Research On Leptospira Spp.

Leptospira interrogans is the major causative agent of leptospirosis, the most widespread zoonosis in the world. Recent studies show that phagocytosis plays an important role in the innate immune response to Leptospira infection, and L. interrogans can evade the killing of host phagocytes. However, little is known about the host adaptation of L. interrogans in this process.To better understand the molecular mechanisms during the initial interaction of pathogenic Leptospira and host innate immunity, we employed high-density microarray and comparative genomic approaches detecting transcriptomic changes in L. interrogans Serovar Lai Strain Lai 56601 using macrophage-derived cell models. Our results showed that upon interaction with host cells, L. interrogans dramatically altered expression of many genes involved in carbohydrate metabolism, energy production, lipid metabolism, signal transduction, transcription, translation, oxygen tolerance and outer membrane protein profile. Expression of the heme-containing catalase gene(katE) was significantly up-regulated, suggesting catalase may contribute to resisting the oxidative pressure of the macrophage. Furthermore, several major outer membrane protein (OMP) genes (e.g., ompLl, lipL32, lipL41, lipL48 and ompL47) were dramatically down-regulated (10-50 folds) upon interaction with macrophages, consistent with previous observations that OMPs are regulated in vivo. The persistent down-regulations of representative OMPs, including OmpLl, LipL32 and LipL41, were confirmed by immunoblotting. Lastly, to gain initial insight into the molecular mechanisms underlying differential gene expression in L. interrogans, we re-defined the putative transcriptional factors (TF) present in the genome and identified the major OmpR TF gene (LB333) is concurrently regulated with the down-regulated major OMP genes, suggesting a potential role of LB333 in regulation of these OMPs.This is the first report on transcriptional response of pathogenic Leptospira to host innate immunity and systemic analysis of TFs present in the Leptospira genomes. Alterations of the leptospiral OMP profile up interaction with host antigen-presenting cells (APC) provide critical information for selecting candidates as vaccine targets. Global surveys of the transcriptome primarily reveal the immune evasion strategies of L. interrogans in the early stage of infection and the potential TF vital for the reduction of the major OMPs. Leptospirosis is an important tropical disease around the world, particularly in humid tropical and subtropical countries. The pathogenic Leptospira can infect human through mucous membrane or abrasions in the skin, penetrate rapidly into blood stream, and diffuse into liver, lung, kidney and other organs. The clinical symptoms of different pathogenic Leptospira strains are complicated and complex, including hemorrhage, diarrhea, jaundice, severe renal impairment, and aseptic meningitis, etc. The high virulent strain, for example, Leptospira interrogans acutely infect human and cause severe organs failure. The mortality of the severe lung hemorrhage reach up to 15%.Recently, the innate immunity had been verified to be important for the host to response to the acute infection of leptospirosis. Pathogenic Leptospira can resist the phagocytosis and killing of macrophage, induce the apoptosis of macrophage. As one of the major immune cell of innate immunity, mature macrophage can phagocytize and kill pathogens, process and present antigens for adaptive immunity system, and secret a series of cytokines and chemokines to regulate host immune response. Leptospira interrogans was also found to survive and replicate in human macrophage, but was killed in murine macrophage, which may partially contribute the different severity of chronic infection in reservoir hosts and acute infection in human. To data, approaches to studying the capacity of pathogenic Leptospira to provoke macrophage response had been limited to individual genes and limited pathways. It is also expected to find more activated inflammatory genes and signaling pathways in macrophage infected by pathogenic Leptospira.In this study, we employed whole genome gene expression microarray and GO/KEGG gene function database analyzing the different responses of murine and human macrophages to pathogenic Leptospira interrogans serovar Lai strain Lai 56601. The comparative microarray results partially revealed the different innate immune responses between chronic infection in reservoir hosts and acute infection in humans. The gene regulations in antigen process and presentation of human cells are much less than those of murines. The key component of complement pathway, C3, was only significantly up-regulated in murine cells, which may contribute the complement activation process during the early stage of the infection in murine host. The CASP8 and FADD-like apoptosis regulator genes were significantly up-regulated, which strongly supported our previous results that the caspase-8/3 pathway plays an important role in apoptosis.It is very difficult to reveal the infection mechanism of leptospirosis, mainly due to the facts that the clinical symptoms of leptospirosis are complex, as well as the leptospiral components are complicated. This study revealed some transcriptional differences of the murine and human macrophage cell lines infected by L. interrogans, which set the foundation for further study on the innate immune response to leptospiral infection. The gene regulation system of Leptospira biflexa is much more complicated than that of Borrelia burgdoriferi, mainly due to the fact that L. biflexa has a large genome and more than 100 specific transcription factors (TF). There is no alternative Sigma S (RpoS) homolog in the genome of L. biflexa, which suggests that the Sigma N (Sigma54, RpoN) factor may play an important role in the gene regulation. However, the function of the RpoN of L. biflexa may be quite different from that of Borrelia burgdoriferi because L. biflexa has lots of specific TF.The RpoN gene of L. biflexa was inactivated by the homologous recombination gene knock method in this study. Then, the gene regulation profile of RpoN was analyzed by whole genome microarray. The RpoN of L. biflexa mainly regulated the genes of nitrogen metabolism, which is similar with RpoN function of the research model, Escherichia coli. The RpoN mutant utilized much less Tween-80 lipid component than the wild type. The Cryo-ET structural analysis and specific Nile red staining revealed that the RpoN can not synthesize the Poly-3-Hydroxybutyrate (PHB) storage, which may be associated with the microarray data that RpoN regulated the nitrogen utilize genes. In addition, the death rate of RpoN mutant in pure water is always higher than that of the wild type.This is the first molecular leptospiral research that an important alternative TF was inactivated by the target gene knock method. L. biflexa RpoN mutant lost the ability of synthesizing PHB storage and the high survivability in pure water, which indicated that leptospiral RpoN is essential for the resistant ability in low nutriment. Considering that the Sigma TF system of L. biflexa is almost similar with that of pathogenic L. interrogans, it is possible that the RpoN of L. interrogans is also essential for in vitro survivability and transmission. Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. Despite its significance in global public health, the molecular biology and pathogenesis of L. interrogans are poorly understood, largely due to the lack of robust genetic techniques and in-depth structural insight of this pathogen. In this study, cryo-electron tomography was utilized to compare pathogenic and saprophytic species and uncover the unique biological and pathogenic features of Leptospira spp. A major difference between L. interrogans and L. biflexa is located at the lipopolysaccharide (LPS) layer, providing direct evidence that LPS varied considerably among leptospiral serovars. Novel periplasmic filaments might function as "skeleton" of the characteristic helical morphology. Bacterial DNA fingerprints captured in living organisms indicate that intracellular DNA forms tightly packed bundles (with～3.3nm spacing) in the middle of cylindrical body. A novel chemotaxis receptor array, a unique "hat" at cell end, as well as high resolution structure of in situ flagellar motor, provide not only the structural insights into the unique morphology of Leptospira spp. but also the plausible mechanism on attaching and penetrating mammalian cells during the spirochete-host interaction.