Comparisons Of Regulatory And Physiological Characterization Of SRNA RyhB Homologs In Yersinia Pestis

BackgroundYersinia pestis, a nonmotile Gram-negative bacillus, is the causative agent of plague. The maintenance of plague in nature is dependent upon cyclic transmission between flea vectors and mammal reservoirs including humans as occasional alternatives. Its growth and proliferation within the macrophage cell upon early-stage entry into host is considered as a key step for further infection and pathogenesis. During normally cycles between fleas and rodent hosts, Y. pestis encountered most signal stimuli including temperature, ion concentration, osmotic pressure, pH, oxygen, free radical, superoxide. In response to the different signals, Bateria was adjusted to adapt the environmental changes by regulating specific sets of genes. To establish a successful infection, Y. pestis must overcome many physiological obstacles produced by the hosts including iron starvation. Iron haemin uptake and storage system are used to acquire iron from mammalian hosts as important virulence factors.Small non-coding regulator RNAs (sRNA) are short (50-500nt) RNA molecules that typically do not encode proteins and are located in the intergenic region (IGR) and form some special loop-stem structures. The vast majority of sRNAs act as post-transciptional regulators that can either affect translation and/or stability of mRNA targets by base-pairing with mRNA. sRNAs are increasly recognized to play the crucial role in modulating physiological functions and virulence in prokaryotes. In response to different environment signals, sRNA was involved in iron homoeostasis, stress response, sugar ulitization, envelope homoeostasis, virulence. The RNA chaperone Hfq is required for trans-encoded sRNA-mediated regulation. The hexameric Hfq ring, which is homologous to Sm and Sm-like proteins is recognized as a key post-transciptional regulators. It has been characterized as a global post-transcriptional regulator that acts in numerous bacterial pathways and mediates interactions between many regulatory small RNAs (sRNAs) and their mRNA targets.Iron (Fe) is one of the most improtant nutrients for bacterial metabolism. RyhB was identified as a key regulator of bacterial iron metabolism conserved in Escherichia coli and other Enterobacteriae, implicating the downregulation of numerous genes during iron depletion. It is tightly regulated by the ferric uptake regulator (Fur), interacts with the RNA chaperone Hfq and involves in the post-transcriptional regulation of iron homeostasis. RyhB was shown to be involved in the bacterial growth, biofilm formation, chemotaxis and acid resistance. To compare the regulatory and physiological features between in RyhB1and RyhB2in Y, pestis, we will firstly compare the regulatory features between Y. pestis RyhB1and RyhB2in response to iron deficiency, Fur and Hfq. Then, we will analyze their respective regulons by using microarray-based transcriptomics and2D-based differential proteomics experiments combining with computational prediction of direct pairing of RyhB at the5’UTR of mRNA targets. Their roles in Y. pestis physiology and pathogenesis would be also evaluated by comparing phenotypes using in vitro assays and infecting mice with Y. pestis ryhB deletion strain via different inoculating routes.MethodsOn the basis of homologous recombination technology, RyhB1and RyhB2single or double mutant were successfully constructed by using two-step PCR. To eliminate the effect of Fur on regulation,△ryhB1:△ryhB2:△fur mutant was constructed based on△ryhB1:△ryhB2mutant and pBAD、pBAD-RyhB1or pBAD-RyhB2plasmid was separately electroporated into△ryhB1:AryhB2:△fur mutant. There was the phenotype of RyhB1or RyhB2overproduction in△ryhB1:△ryhB2:△fur mutant. Total RNA of mid-log phase of Y. pestis under iron-depletion were extracted and the specific primers of RyhB1and RyhB2were designed, transcriptional start site was determined by primer extension experiment. Under the condition of high-iron or low-iron,△ryhB1,△ryhB2,△ryhB1:ryhB2,△fur and Ahfq were induced and total RNA were extracted, Northern blot detected expression of RyhB1and RyhB2. After WT and hfq mutant were treated about0,2,4,8,16,32min with Rifampicin, half-time of RyhB1and RyhB2can be determined by Northern blot in indicated time.Once Y. pestis produced arabinose-inducible expression of overproduce of RyhB1and RyhB2, total RNA or proteins were extracted. Differential target mRNA and protein regulated by RyhB1and RyhB2was identified by the microarray and proteomics technology from mRNA and protein levels and were classified by biological function.Considering regulon of RyhB and pathogenic mechanism of Y. pestis, the chief content of phenotype study was determined based on growth curve and colony count. By using growth and survival experiments in vitro, comparisons of sensitivity or resistance of WT and AryhB mutant to short-term (H2O2) or long-term stimuli (high-salt, lack-nutrients or iron-depletion) was performed. Competition exprinments between WT and RyhB double mutant and LD50exprinments was used to detect the virulence.The statistical analysis was used by SPSS13.0software. Means comparison of two samples were carried out by Independent-samples T Test, however, multiple comparisons of more samples was first tested by homogeneity of variances. If equal variances is assumed, One-Way ANOVA were performed, LSD was used to analyze multiple comparisons. If equal variances is not assumed, Welch test was applied instead of F test, multiple comparisons were performed by Dunnett’s T3. The three-factor factorial analysis was applied to analyzing the effect of iron-depletion or iron-repletion on RyhB1and RyhB2of wt, Ahfq and Afur. but, ANONA for the repeated measures was used to analyze the effect of different DIP treatment time on RyhB, if Mauchly’s test of spherictiy is not assumed, ε of Greenhouse-Geisser was adopted. ResultsThe△ryhB1,△ryhB,△ryhB1:△ryhB2△ryhB1:△ryhB2:△fur mutant were succ-essfully constructed. Y. pestis have two homologs of RyhB, named RyhB1and RyhB2, which have typical promoter sequence, including the-35,-10and Fur binding sites, and also contain the highly conserved core of RyhB(31nucleotides),5’stem-loop and p-independent terminators. Primer extension test is convincing that transcriptional start site of RyhB1and RyhB2is consistent with bioinformatics prediction.The Northern blot results was shown that expression of WT RyhB1and RyhB2are very high under low-iron condition. Expression of RyhB in fur mutant enhanced, The results revealed that RyhB1or RyhB2expression was directly repressed by Fur and Fe, the result are similar in E.coli.Expression of RyhB between WT and hfq mutant can be detected by Northern blot during differnent time. The results indicate that stability of RyhB1and RyhB2of WT under low-iron are very stable during30min, however, RyhB2of hfq mutant are very stable but RyhB1stablility are bad. Half-life of RyhB1is only8min, but RyhB2is more32min. As were shown, RyhB1stability is depend on Hfq, but RyhB2is independent.The microarray-based results indicates that RyhB1and RyhB2have the same or different regulon. There are47the same regulon regulated by RyhB1and RyhB2. RyhB1regulated individually31transcripts. RyhB2regulated respectively58transcripts. According to function,136transcripts are classified into12groups, including iron uptake, respiration, chaperonin, oxidative stress.The proteomics technology-based results indicated that RyhB1and RyhB2have the same or differnernt target proteins. There are18target proteins, which are classified into10groups,6and16proteins regualted by RyhB1and RyhB2respectively. In agreement with microarray, The mass spectrometry analysis reveal that target protein hmsF are upregulated by RyhB2the Northern blot results were shown that RyhB1and RyhB2in infected lung have higher expression than in vitro. This revealed that RyhB plays in important role in physiology, adaptation response and pathopoiesis of Y. pestis. Phenotype and animal experiments are used to study the effect of RyhB1and RyhB2on physiology and virulence Under the different conditions such as rich or lack nutrients, low-iron, high-salt, deletion of RyhB1and RyhB2does not affect growth of the bacteria. Growth survival rate of△ryhB1:△ryhB2slow comparing with WT, the result indicated that RyhB promoted resistance to H2O2. In addtion, RyhB had no effect on biofilm. Equal WT and ryhB double-mutant mixed and were injected into mice, for calculating competition index(CI). CI in lung is3.75(48h),1.14(72h). CI in spleen is0.7(48h),1.7(72h). The above data was presented that RyhB gene deletion did not affect growth of the bacterium in mice’s lungs. BALB/C mice survival experiments revealed that WT and ryhB gene deletion died in during4-8d and RyhB wasn’t contributed to virulence. As are shown that it is possible that RyhB isn’t directly involved in virulence of mice.Conclusion1. We have established three technologies for sRNA analysis, including two-step gene knock-out, nonradioactive-labeling Northern blot, overexpression of sRNA.2. RyhB1or RyhB2expression was repressed by Fur and Fe.3. RyhB1stability is depend on Hfq, but RyhB2is not. The results provide the direct envidence that stability of RyhB homologs have significant difference.4. RyhB1and RyhB2have same or differential regulon, which reveal that RyhB1and RyhB2can regulate iron metabolism, or both are involved in regulating other metabolic pathway respectively.5. RyhB mutant impair resistance to H2O2and play a role in antioxidative stress. In agreement with microarray, The mass spectrometry analysis reveal that target protein hmsF are mediated by RyhB2. The resutlts will be benefical to investigate whether RyhB2can participate in regualtion of biofilm.