QsvR integrates into quorum sensing circuit to control Vibrio parahaemolyticus virulence.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, requires the two type-III secretion systems (T3SS1 and T3SS2) and a thermostable direct hemolysin (encoded by tdh1 and tdh2) for full virulence. The tdh genes and the T3SS2 gene cluster constitute an 80 kb pathogenicity island known as Vp-PAI located on the chromosome II. Expression of T3SS1 and Vp-PAI is regulated in a quorum sensing (QS)-dependent manner but its detailed mechanisms remain unknown. Herein, we show that three factors (QS regulators AphA and OpaR and an AraC-type transcriptional regulator QsvR) form a complex regulatory network to control the expression of T3SS1 and Vp-PAI genes. At low cell density (LCD), whereas Vp-PAI expression is repressed, T3SS1 genes are induced by AphA, which directly binds (an operator region of) the exsBAD-vscBCD operon. At high cell density (HCD), the bacterium turns off T3SS1 expression by replacing AphA with OpaR, triggering the induction of Vp-PAI. Furthermore, QsvR binds to the regulatory regions of all the tested T3SS1 and Vp-PAI genes to activate their transcription at HCD. Taken together, our data highlight how multiple QS regulators contribute to the pathogenicity of V. parahaemolyticus by precisely controlling the expression of major virulence determinants during different stages of growth.

Mig-14 may contribute to Salmonella enterica serovar Typhi resistance to polymyxin B by decreasing the permeability of the outer-membrane and promoting the formation of biofilm.

Mig-14 is essential for Salmonella enterica serovar Typhimurium (S. Typhimurium) resistance to antimicrobial peptides, including polymyxin B (PB). However, the molecular mechanism is as yet unknown. In this study, we demonstrated that mig-14 also played a crucial role in Salmonella enterica serovar Typhi (S. Typhi) resistance to PB. A series of genes associated with drug-resistance controlled by Mig-14 were identified in the presence of PB. Among which, ompF and ompC were up-regulated 8 and 6 folds in mig-14 mutant (Δmig-14) strains, respectively. Further, the deletion of ompF or/and ompC in Δmig-14 strains decreased their sensitivity to PB. Besides, the biofilm formation ability was reduced in Δmig-14 strains. Our results indicate that Mig-14 may contribute to PB resistance in S. Typhi by decreasing the permeability of the outer membrane and promoting biofilm formation.

Reciprocal Regulation of OmpR and Hfq and Their Regulatory Actions on the Vi Polysaccharide Capsular Antigen in Salmonella enterica Serovar Typhi.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of human typhoid fever. S. Typhi expresses a major virulence determinant called Vi polysaccharide capsular antigen, which is encoded by the viaB locus containing 10 consecutive genes including tviA and tviB. Expression of Vi antigen is regulated by the two-component regulatory system EnvZ/OmpR and the global RNA-binding factor Hfq. In this study, we show that OmpR coordinates with Hfq to regulate the transcription of Vi antigen genes under osmotic stress conditions. OmpR binds to the promoters of tviA and its own genes to activate their transcription; however, it positively regulates tviB and negatively regulates hfq in an indirect manner. Moreover, Hfq reversely inhibits ompR, tviA and tviB, and positively regulates its own gene expression. Thus, we report of a complex gene regulatory network involving the reciprocal regulation and autoregulation of OmpR and Hfq, and their regulatory actions on Vi polysaccharide capsular antigen genes in S. Typhi.

The malS-5'UTR regulates hisG, a key gene in the histidine biosynthetic pathway in Salmonella enterica serovar Typhi.

Bacterial noncoding RNAs (ncRNA) regulate diverse cellular processes, including virulence and environmental fitness. The malS 5' untranslated region (named malS-5'UTR) was identified as a regulatory ncRNA that increases the invasive capacity of Salmonella enterica serovar Typhi. An IntaRNA search suggested base pairing between malS-5'UTR and hisG mRNA, a key gene in the histidine biosynthetic pathway. Overexpression of malS-5'UTR markedly reduced bacterial growth in minimal medium without histidine. Overexpression of malS-5'UTR increased mRNA from his operon genes, independently of the bax gene, and decreased HisG protein in Salmonella Typhi. RNA structure analysis showed base pairing of the malS-5'UTR RNA with the hisG mRNA across the ribosome binding site. Thus, we propose that malS-5'UTR inhibited hisG translation, probably by base pairing to the Shine-Dalgarno sequence.

OmpR may regulate the putative YehU/YehT two-component system in Salmonella enterica serovar Typhi under hypotonic growth condition.

Decreased expression (twofold) of a putative yehUTS operon of which yehUT encodes a putative YehU/YehT two-component system in the ompR mutant from Salmonella enterica serovar Typhi (S. Typhi) GIFU10007 under hypotonic growth condition was observed by qRT-PCR. Purified recombinant protein OmpR(His6) of GIFU10007 was shown to bind the upstream region of the yehU gene by the gel-shift assay. In addition, the yehT deletion mutant (ΔyehT) displayed differential expression (twofold or higher) of 26 genes under the condition by the DNA microarray analysis. Altogether, OmpR might regulate the YehUT system in S. Typhi under hypotonic growth condition.

RpoE may promote flagellar gene expression in Salmonella enterica serovar typhi under hyperosmotic stress.

Salmonella enterica serovar Typhi z66 positive strain contains a fljBA-like operon on a linear plasmid. The operon contains the gene fljB:z66 which encodes the z66 antigen. RpoE is a sigma factor σ(E) that initiates transcription of a series of genes in Escherichia and Salmonella under environmental stresses. To investigate whether the gene fljB:z66 is regulated by RpoE (σ(E)), a rpoE deletion mutant of S. enterica serovar Typhi (ΔrpoE) was prepared in this study. The defective motility of the ΔrpoE was confirmed firstly. Transcriptional expression of flagellar genes was screened using a genomic DNA microarray. Some class-2 and most class-3 flagellar genes were downregulated in the ΔrpoE after 30 min of hyperosmotic stress. The expression of fliA and fljB:z66, a class-2 flagellar gene and a class-3 flagellar gene, obviously decreased; however, expression of the class-1 flagellar genes flhDC did not change obviously in the ΔrpoE compared to the wild-type strain in the same conditions. Results of quantitative real-time PCR (qRT-PCR) showed that the expression levels of fliA and fljB:z66 in the ΔrpoE after 30 min of hyperosmotic stress decreased about five and eightfold, respectively, compared to the wild-type strain. Similar results were observed at 120 min of hyperosmotic stress. Western blotting and qRT-PCR analysis showed that expression of fliA and fljB:z66 was significantly increased after supplemental expression of rpoE with a recombinant plasmid pBADrpoE in the ΔrpoE strain. These results demonstrated that RpoE promoted the expression of class-3 flagellar genes and it might be performed by initiating the expression of fliA in S. enterica serovar Typhi under hyperosmotic stress.

Coregulation of gene expression by sigma factors RpoE and RpoS in Salmonella enterica serovar Typhi during hyperosmotic stress.

Salmonella enterica serovar Typhi (S. Typhi) is the cause of typhoid fever, a food-borne disease that is prevalent worldwide, most particularly in developing countries. RNA polymerase sigma factors RpoE (σ(E)) and RpoS (σ(S)) govern transcription initiation of two sets of genes in Escherichia and Salmonella. It was previously suggested that some genes might be coregulated by RpoE and RpoS in Salmonella under conditions of environmental stress, but experimental evidence has been lacking. We therefore constructed rpoS deletion (ΔrpoS) and double rpoE/rpoS deletion (ΔrpoE/ΔrpoS) mutants of S. Typhi and compared their growth properties with an rpoE mutant (ΔrpoE) and wild-type strains under conditions of hyperosmotic stress. We report that the ΔrpoE, ΔrpoS, and ΔrpoE/ΔrpoS strains grew more slowly under hyperosmotic stress conditions than the wild-type strain, and the ΔrpoE/ΔrpoS strain grew most slowly. The global transcriptional profiles of ΔrpoE, ΔrpoS, ΔrpoE/ΔrpoS after 30 min of hyperosmotic stress were investigated using a Salmonella genomic DNA microarray. The results of microarray indicated that the expression levels of 38 genes were markedly reduced during hyperosmotic stress in the double mutant ΔrpoE/ΔrpoS strain, but expression levels were not significantly affected by single ΔrpoE or ΔrpoS mutations. This was confirmed for several key genes by qRT-PCR. This study therefore indicated crosstalk between sigma factors RpoE and RpoS in S. Typhi under hyperosmotic conditions and provides new insights into the regulatory networks of S. Typhi.

Molecular characterization of a functional type VI secretion system in Salmonella enterica serovar Typhi.

The type VI secretion system (T6SS) of Salmonella enterica serovar Typhi (S. typhi) is associated with Salmonella pathogenicity island 6 (SPI-6). Though the T6SS gene cluster is intact in S. typhi, the protein complex is believed to be non-functional due to the presence of a pseudogene form of SciI (VipB homolog), a key component. We detected the SciK-his6 in the supernatant of the wild type strain of S. typhi containing the plasmid over-expressing SciK (hcp homolog) with a his6 epitope at the C-terminus, which suggested that the T6SS in S. typhi is functional. We also identified four genes that were essential to T6SS function: sciC (vasA homolog), sciS (vasK homolog), sciG (clpV homolog), and vrgS (vgrG homolog). Further analysis revealed that S. typhi T6SS is cytotoxic to human epithelial cells, but does not influence bacterial growth and mobility. RcsB, PmrA, and Hfq were identified as regulators of S. typhi T6SS gene expression; however, PhoP appears to not be involved. Taken together, the data demonstrate the functionality of S. typhi T6SS and confirm the important role of T6SS for S. typhi's ability to invade and infect epithelial cells.

Expression of tviA is transiently repressed by Hfq in Salmonella enterica serovar Typhi at hyperosmotic stress.

The putative global post-transcriptional regulator gene hfq was deleted in Salmonella enterica serovar Typhi (Salmonella typhi). Genomic DNA microarray assay and quantitative real time PCR were used to estimate the level of gene expression. The expression of tviA, the gene required for expression of the Vi capsular antigen, was increased in the hfq mutant at 30 min of an up-shift osmotic stress but was not at sustained high or low osmolarity, compared to the wild type strain. In addition, the level of expression of tviA in the ompR mutant of S. typhi was greatly decreased, similar to what is found in the hfq-ompR double mutant. The results indicate that Hfq negatively regulates the expression of tviA in S. typhi transiently at early stage of hyperosmotic stress.

Expression of FLJB:Z66 on a Linear Plasmid of Salmonella Enterica Serovar Typhi is Dependent on FLIA and FLHDC and Regulated By OMPR.

Salmonella enterica serovar Typhi z66-positive strains have two different flagellin genes, fliC:d/j and fljB:z66, located on the chromosome and on a linear plasmid, respectively. To investigate the mechanism underlying the expressional regulation of fljB:z66, gene deletion mutants of the regulators FliA, FlhDC, and OmpR were constructed in this study. The expression levels of fliC and fljB:z66 were analyzed by qRT-PCR in the wild-type strain and mutants at high and low osmolarity. The results show that the expression levels of both fljB:z66 and fliC were greatly reduced in fliA and flhDC mutants under both high and low osmotic conditions. In the ompR mutant, the expression levels of fljB:z66, fliC, fliA, and flhD were increased at low osmotic conditions. SDS-PAGE and western blotting analysis of the secreted proteins revealed that the FljB:z66 was almost absent in the fliA and flhDC mutants at both high and low osmolarity. In the wild-type strain, the fljB:z66 was more highly expressed under high-osmolarity conditions than under low-osmolarity conditions. However, this difference in expression disappeared in the ompR mutant. Translational expression assay of FljB:z66 showed that the FljB:z66 expression was decreased in ompR mutant at both low and high osmolarity. These results suggest that the expression of fljB:z66 in S. enterica serovar Typhi is dependent on FliA and FlihDC, and OmpR can regulate the expression and secretion of FljB:z66 in different osmolarity.

Prediction of miRNA targets by learning from interaction sequences.

MicroRNAs (miRNAs) are involved in a diverse variety of biological processes through regulating the expression of target genes in the post-transcriptional level. So, it is of great importance to discover the targets of miRNAs in biological research. But, due to the short length of miRNAs and limited sequence complementarity to their gene targets in animals, it is challenging to develop algorithms to predict the targets of miRNA accurately. Here we developed a new miRNA target prediction algorithm using a multilayer convolutional neural network. Our model learned automatically the interaction patterns of the experiment-validated miRNA:target-site chimeras from the raw sequence, avoiding hand-craft selection of features by domain experts. The performance on test dataset is inspiring, indicating great generalization ability of our model. Moreover, considering the stability of miRNA:target-site duplexes, our method also showed good performance to predict the target transcripts of miRNAs.

Nucleotide-level Convolutional Neural Networks for Pre-miRNA Classification.

Due to the biogenesis difference, miRNAs can be divided into canonical microRNAs and mirtrons. Compared to canonical microRNAs, mirtrons are less conserved and hard to be identified. Except stringent annotations based on experiments, many in silico computational methods have be developed to classify miRNAs. Although several machine learning classifiers delivered high classification performance, all the predictors depended heavily on the selection of calculated features. Here, we introduced nucleotide-level convolutional neural networks (CNNs) for pre-miRNAs classification. By using "one-hot" encoding and padding, pre-miRNAs were converted into matrixes with the same shape. The convolution and max-pooling operations can automatically extract features from pre-miRNAs sequences. Evaluation on test dataset showed that our models had a satisfactory performance. Our investigation showed that it was feasible to apply CNNs to extract features from biological sequences. Since there are many hyperparameters can be tuned in CNNs, we believe that the performance of nucleotide-level convolutional neural networks can be greatly improved in the future.

Microsatellites that violate Chargaff's second parity rule have base order-dependent asymmetries in the folding energies of complementary DNA strands and may not drive speciation.

Models for meiotic recombination based on Crick's "unpairing postulate" require symmetrical extrusion of stem-loop structures from homologous DNA duplexes. The potential for such extrusion is abundant in many species and, for a given single-strand segment, can be quantitated as the "folding of natural sequence" (FONS) energy value. This, in turn, can be decomposed into base order-dependent and base composition-dependent components. The FONS values of top and bottom strands in most Caenorhabditis elegans segments are close, as are the corresponding base order-dependent and base composition-dependent components; any discrepancies are in the base composition-dependent component. This suggests that the strands would extrude with similar kinetics. However, interspersed among these segments and at the ends of chromosomes (telomeres) are segments containing short tandem repeats (microsatellites) which, by virtue of their high variability, have been postulated to inhibit the pairing of homologous chromosomes and hence drive speciation. In these segments, there are usually wide discrepancies between the FONS values of top and bottom strands, mainly attributable to differences in base order-dependent components. Analyses of artificial microsatellites of different unit sizes and base compositions show that this asymmetrical distribution of folding potential is greatest for microsatellites when the units are short and violate Chargaff's second parity rule. It is proposed that when there is folding asymmetry, recombination proceeds by special, strand-biased, somatic mechanisms analogous to those operating with Chi sequences in Escherichia coli. If meiotic recombination in the germ-line requires extrusion symmetry, then a general inhibitory influence of microsatellite-containing segments could mask the antirecombinational influence of their variability. Thus, microsatellites may not have driven speciation.

Transcriptional expression of fljB:z66, a flagellin gene located on a novel linear plasmid of Salmonella enterica serovar Typhi under environmental stresses.

A previous study identified that z66+ strain of Salmonella enterica serovar Typhi contains two different flagellin genes, the fliC encoding d or j antigen in chromosome and the fljB-like gene encoding z66 antigen in a novel linear plasmid, respectively. The promoter of fljB:z66 is different from that of fliC:d/j and z66+ strain alters flagellin expression in only one orientation, from z66 to d orj antigen, raising the suspicion that z66+ strain is a special biphasic strain. To clarify the expressional characteristics of flagellin genes of z66+ strain, expression patterns of fljB:z66 and fliC were investigated by RT-PCR under a series of environmental stresses during infection, such as acidic stress, osmotic stress, bile acid stress and oxidative stress. Results showed that the expression level of fljB:z66 is over 10-fold higher than the level of fliC in low and middle osmotic conditions before stresses. Only the expressional regulatory tendency of fljB:z66 in response to bile acid stress is similar to that of fliC. Differential expressional patterns between fljB:z66 and fliC of S. enterica serovar Typhi were seen under osmotic stress, bile acid stress and oxidative stress. These results support the hypothesis that the z66+ strain is a special biphasic strain of S. enterica serovar Typhi.

Identification and Characterization of a Cis Antisense RNA of the rpoH Gene of Salmonella enterica Serovar Typhi.

Antisense RNAs from complementary strands of protein coding genes regulate the expression of genes involved in many cellular processes. Using deep sequencing analysis of the Salmonella enterica serovar Typhi (S. Typhi) transcriptome, a novel antisense RNA encoded on the strand complementary to the rpoH gene was revealed. In this study, the molecular features of this antisense RNA were assessed using northern blotting and rapid amplification of cDNA ends. The 3,508 nt sequence of RNA was identified as the antisense RNA of the rpoH gene and was named ArpH. ArpH was found to attenuate the invasion of HeLa cells by S. Typhi by regulating the expression of SPI-1 genes. In an rpoH mutant strain, the invasive capacity of S. Typhi was increased, whereas overexpression of ArpH positively regulates rpoH mRNA levels. Results of this study suggest that the cis-encoded antisense RNA ArpH is likely to affect the invasive capacity of S. Typhi by regulating the expression of rpoH.

A 3' UTR-derived non-coding RNA RibS increases expression of cfa and promotes biofilm formation of Salmonella enterica serovar Typhi.

Bacterial non-coding RNAs (ncRNAs) are widely studied and found to play important roles in regulating various cellular processes. Recently, many ncRNAs have been discovered to be transcribed or processed from 3' untranslated regions (3' UTRs). Here we reported a novel 3' UTR-derived ncRNA, RibS, which could influence biofilm formation of Salmonella enterica serovar Typhi (S. Typhi). RibS was confirmed to be a ∼700 nt processed product produced by RNase III-catalyzed cleavage from the 3' UTR of riboflavin synthase subunit alpha mRNA, RibE. Overexpression of RibS increased the expression of the cyclopropane fatty acid synthase gene, cfa, which was located at the antisense strand. Biofilm formation of S. Typhi was enhanced by overexpressing RibS both in the wild type strain and cfa deletion mutant. Deletion of cfa attenuated biofilm formation of S. Typhi, while complementation of cfa partly restored the phenotype. Moreover, overexpressing cfa enhanced the biofilm formation of S. Typhi. In summary, RibS has been identified as a novel ncRNA derived from the 3' UTR of RibE that promotes biofilm formation of S. Typhi, and it appears to do so, at least in part, by increasing the expression of cfa.

Improved prediction of coreceptor usage and phenotype of HIV-1 based on combined features of V3 loop sequence using random forest.

HIV-1 coreceptor usage and phenotype mainly determined by V3 loop are associated with the disease progression of AIDS. Predicting HIV-1 coreceptor usage and phenotype facilitates the monitoring of R5-to-X4 switch and treatment decision-making. In this study, we employed random forest to predict HIV-1 biological phenotype, based on 37 random features of V3 loop. In comparison with PSSM method, our RF predictor obtained higher prediction accuracy (95.1% for coreceptor usage and 92.1% for phenotype), especially for non-B non-C HIV-1 subtypes (96.6% for coreceptor usage and 95.3% for phenotype). The net charge, polarity of V3 loop and five V3 sites are seven most important features for predicting HIV-1 coreceptor usage or phenotype. Among these features, V3 polarity and four V3 sites (22, 12, 18 and 13) are first reported to have high contribution to HIV-1 biological phenotype prediction.

Identification and Characterization of a Gene stp17 Located on the Linear Plasmid pBSSB1 as an Enhanced Gene of Growth and Motility in Salmonella enterica Serovar Typhi.

The linear plasmid pBSSB1 mediates the flagellar phase variation in H:z66 positive Salmonella enterica serovar Typhi (S. Typhi). The gene named stp17 (S. Typhi plasmid number 17 gene) is located on pBSSB1 and encodes the protein STP17. The expression pattern at the protein-level and function of STP17 remains unknown. In this study, the recombinant protein STP17His6 was expressed, purified and used to prepare the polyclonal anti-STP17 antibody. We detected protein-level expression of stp17 in S. Typhi and further investigated the protein expression characteristics of stp17 in different growth phases by western blot analysis. The effects of STP17 on bacterial growth and motility were analyzed. In addition, the structure of STP17 was predicted and the active site of STP17 was identified by site-directed mutagenesis. The results showed that STP17 was expressed stably in the wild type strain of S. Typhi. STP17 expression at the protein level peaks when cultures reach an OD600 value of 1.2. The growth rate and motility of the Δstp17 strain were significantly decreased compared with the wild type strain (P < 0.05) and this phenotype was restored in the stp17 complementary strain. Moreover, the growth rate and motility of the stp17 over-expression strain was greater than the wild type strain. STP17 contains nine Helix segments, six Stand segments and some Coil segments in the secondary structural level. The top-ranked 3-D structure of STP17 predicted by I-TASSER contains a putative ATPase domain and the amino acid residues of GLY16, GLY19, LYS20, ASN133, LYS157, and LYS158 may be the active site residues of STP17. Finally, STP17 was able to catalyze the ATP to ADP reaction, suggesting that STP17 may be an ATPase. To our knowledge, this is the first report describing the protein expression characteristics of STP17 in S. Typhi, showing that STP17 promotes bacterial growth and motility, which may be associated with its potential ATPase activity.

RpoE promotes invasion and intracellular survival by regulating SPI-1 and SPI-2 in Salmonella enterica serovar Typhi.

AIM: To demonstrate the role of RpoE during the later stage of hyperosmotic stress in Salmonella. MATERIALS & METHODS: Expressions of SPI-1 and SPI-2 under hyperosmotic stress for 120 min were investigated by a microarray, and the invasion and intracellular survival of wild-type and ΔrpoE strains were compared. The global differential expression of bacterial proteins between the wild-type and ΔrpoE strains was examined after 120 min of hyperosmotic stress. RESULTS: SPI-1 and SPI-2 were repressed, and the invasion and intracellular survival were defected in the ΔrpoE strain. Thirteen bacterial-associated proteins and 11 secreted proteins differed significantly between the wild-type and ΔrpoE strains. CONCLUSION: RpoE may promote invasion and intracellular survival by regulating the expression of SPI-1 and SPI-2.