Genomic Analyses Of Rickettsia Heilongjiangensis And Transcriptomic Analyses Of R. Heilongjiangensis Infected Mice

Rickettsia heilongjiangensis, the causative agent of far eastern spotted fever, is anobligate intracellular Gram-negative bacterium that belongs to the spotted fever grouprickettsiae. Here we present the whole genome sequence of R. heilongjiangensis andcompare it to other published rickettsial genomes. The genome is a1,278,471bpcircular chromosome that contains1,333genes, including1,297protein coding genes,33tRNA coding genes and3rRNAs (5S rRNA16S rRNA23S rRNA) genes.Further analyses showed that the genome contains121pseudo genes,54insertionsequences,39tandem repeats and318simple direct and inverted repeats. Sixteengenes encode the major type IV secretion system proteins. Thirty seven-barrel outermembrane proteins were predicted and8of which have been proved to be outermembrane proteins. Two hundred and sixty six potential virulence factors werepredicted through blasting against related database. Seven partially deleted antibioticresistance genes were found in the genome. One genomic island, which encodes aVirB1-like protein, a transposase-like protein and five hypothetical proteins, wasfound in the genome.Comparative genomic analyses showed that R. heilongjiangensis and R. japonicashare one unique sequence that can be a target sequence of diagnostic assay. R.heilongjiangensis has37genes present in R. rickettsii str. Sheila Smith but deficient inR. rickettsii str. Iowa, which may explain why R. heilongjiangensis is pathogenic.Pan-genome analyses showed R. heilongjiangensis and42sequenced rickettsiaeshare693core genes with a pan-genome size of4,837genes. The pan-genome basedphylogenies provide new insight into the evolution of Rickettsia spp. This study systematically analyzed the expression levels of mRNAs, lncRNAsand miRNAs of spleens form C3H/HeN mice with R. heilongjiangensis infection, inorder to understand the infection process and pathogenesis of R. heilongjiangensis inthe acute phase of far eastern spotted fever. Function enrichment analyses ofdifferentially expressed mRNAs showed that the function of il6, mcp-1was upregulated3hours post-infection.For the mRNAs,39,37and41differentially expressed genes were found to beassociated with innate immunity, adaptive immunity, and inflammation, respectively.After infection, the expression level of the innate immune related genes ccl2, gbp10,irg1, il12b and ubd were up-regulated. On the contrary, il12b, isg20, trim10, rsad2,trim59, cr2and snca were down-regulated. Gbp gene family, ccl2, and irg1were up-regulated sharply3hours post infection, suggesting that host cells were stimulated torespond to interferon after infection. Because rickettsial infections can activate NF-Band NF-B thus promotes the expression of early immune response genes andinflammation process. Up-regulation of cfb coupled with down-regulation of fcnasuggested that the complement system were activated by an alternative pathway. Wesupposed that LPS of R. heilongjiangensis strongly stimulated the proliferation ofCD14+immune cells, triggering a series of immune response against the endotoxin.Three days after infection, the up-regulation of il6and il12b implemented theactivation of TLR related pathways and up-regulation of pro-inflammatory cytokine,suggesting Th1and Th17immune response was induced.Acquired immune response related gene, gzmb, was up-regulated after infection,indicating enhanced NK cell activity. NK cells produce GZMB to kill rickettsiae.C4b, c1rb and some other genes related to complement activation were up-regulatedat3days post infection, indicating that the complement system was activated andparticipated in the clearance of rickettsial infection. The expression level of genemyd88was increased about3times after infection. The expression levels of sema4a,gadd45g, relb, il18bp, relb, bcl3were up-regulated after infection, which implemented that R. heilongjiangensis infection mainly activates Th1immuneresponse. The up-regulation of the expression level of batf suggested that Th17immune response was also activated.The expression level analyses of inflammation-related genes showed that cxcl, ccland their receptors were originally up-regulated and then down regulated, while ccl8was gradually up-regulated. The expression levels of il1a, il1b, il10, il27were similarto chemokine. The increased expression level of IL-27may promote Th1response butsuppress Th17response. The up-regulation of apoptosis-related genes, bcl-2-associated gene3and fas, implemented that the rickettsial infection induced anti-apoptotic mechanism.There were1,915lncRNAs predicted in the present study. The expression levelsof all lncRNA transcripts were calculated. The expression level of lncRNAs wasfound to be lower than that of mRNAs, but the specificity was higher than mRNAs.We filtered eight high expressed and differentially expressed miRNAs, includingmmu-miR-378c, mmu-miR-222-3p, mmu-miR-210-3p, mmu-miR-146b-5p, mmu-miR-21a-3p, mmu-miR-21a-5p, mmu-miR-122-5p, and mmu-miR-378a-5p.Moreover, we predicted72potential miRNAs and predicted target genes. We alsofiltered eight potential miRNAs that highly differentially expressed. These miRNAsmay play important roles in the regulation of immune response against rickettsialinfection and we will focus on them in further studies.