Shungao Xu

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.

Predicted co-receptor tropism and sequence characteristics of China HIV-1 V3 loops: implications for the future usage of CCR5 antagonists and AIDS vaccine development

BACKGROUND The co-receptor tropism of any given HIV-1 isolate is closely associated with the progression of AIDS. Understanding the co-receptor tropism and genetic diversity of circulating HIV-1 strains is critical for AIDS treatment and vaccine development. METHODS All available China HIV-1 V3 sequences with known subtypes/circulating recombinant forms (CRFs) and transmission routes were retrieved from the Los Alamos HIV Sequence Database. HIV-1 co-receptor tropism was predicted using online tool HIV-1 PhenoPred. RESULTS All C/CRF07_BC/CRF08_BC strains appeared to use CCR5 for cell entry (R5 strains), while 61.1% of subtype B and 38.7% of CRF01_AE were also R5, indicating a higher prevalence of R5 (76.9%) than X4. The prevalence of R5 remained relatively stable over the different sample years regardless of C/CRF07_BC/CRF08_BC, B, or CRF01_AE subtypes. The co-receptor usage of HIV-1 appeared to be associated with the different subtypes, rather than transmission route. Furthermore, the V3 sequences of C/CRF07_BC/CRF08_BC were more genetically homogeneous relative to both subtypes B and CRF01_AE. CONCLUSIONS The higher prevalence of R5 and higher level of homogeneity of V3 sequences in C/CRF07_BC/CRF08_BC suggest that CCR5 antagonists will be promising drugs for future AIDS treatment in China, and that circulating R5 strains are valuable candidates for AIDS vaccine development.

Global transcriptional response of Salmonella enterica serovar Typhi to anti‐z66 antiserum

Recent studies have shown that flagella may modulate physiological processes by sensing environmental changes in temperature and moisture. When the z66(+) strain of Salmonella enterica serovar Typhi (S. Typhi) was exposed to an antiserum against the z66 flagellar antigen, the fljBA operon was deleted from a linear plasmid, leading to the unidirectional flagellar phase variation from FljB to FliC. We hypothesized that flagella may serve as a sensor that responded to the antiserum by altering gene expression and triggering the unidirectional flagellar phase variation. To test this hypothesis, Salmonella genomic DNA microarrays were used to determine the gene expression profile of the z66(+) wild-type strain of S. Typhi treated with the anti-z66 antiserum for 30 min. The results showed that expression levels of 187 genes were altered by more than threefold compared with the same strain treated with control serum. The microarray expression patterns of representative genes were validated by reverse transcriptase-PCR. Importantly, no significant changes in gene expression were observed in the fljB:z66 deletion mutant that was similarly treated with the anti-z66 antiserum. To the best of our knowledge, this is the first study to show the global transcriptional response of Salmonella to antiflagellin antiserum.

Identification of a fljA gene on a linear plasmid as the repressor gene of fliC in Salmonella enterica serovar Typhi

Salmonella enterica serovar Typhi z66-positive strain contains an fljBA-like operon on a linear plasmid and the fliC gene on the chromosome encoding d or j antigen. The fljB-like gene has been identified as the gene encoding z66 antigen. To investigate the function of the fljA-like gene in S. enterica serovar Typhi, a z66-positive wild-type strain GIFU10007 and its phase variation j positive strain 007j+ were used in the present study. After deletion of the fljA-like gene, the wild-type strain that only expressed FljB:z66 could express both FliC:j and FljB:z66 at the same time. A recombinant plasmid, pBADfljA, containing the fljA-like gene was prepared. SDS-PAGE and western blot analysis of secreted proteins showed that when the strain 007j+ was transformed by the recombinant plasmid, pBADfljA, the expression of FliC:j was inhibited. The fliC::lacZ fusion of the strain 007j+ was constructed. Beta-Galactosidase activity was decreased approximately 100-fold after harboring the plasmid pBADfljA in the fliC::lacZ mutant strain. However, the fliC mRNA level investigated by RT-PCR was decreased only approximately seven-fold in the strain 007j+ after harboring the plasmid pBADfljA. These results demonstrated that thefljA-like gene on a linear plasmid of S. enterica serovar Typhi is a fljA gene, the repressor of fliC gene, and the inhibition by FljA is likely at the post-transcriptional level.

Identification and Characterization of a Cis-Encoded Antisense RNA Associated with the Replication Process of Salmonella enterica Serovar Typhi

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise whiles others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cells transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

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.

Identification and characterization of a cis antisense RNA of the parC gene encoding DNA topoisomerase IV of Salmonella enterica serovar Typhi.

Bacterial cis-encoded antisense RNAs are transcribed from the opposite strand of protein coding genes, and their regulatory roles adapt cells to changing environmental conditions. By deep sequencing of the transcriptome of Salmonella enterica serovar Typhi, an antisense RNA that is encoded in cis to the parC gene was found. parC encodes the subunit A component of topoisomerase IV, a class of enzymes that relax both positively and negatively supercoiled DNA and are also required for segregation of daughter chromosomes in bacteria. Transcription of the 871 nucleotide antisense RNA was confirmed by northern blot and RACE analysis to be expressed mostly in the stationary phase of bacterial growth and also upregulated in iron limitation and osmotic stress conditions. Overexpression of the antisense RNA resulted in a significant increase in parC mRNA levels. Further analysis revealed that expression of the antisense RNA stabilizes the target mRNA, probably by protecting it from endoribonucleases. Our findings confirm and add to the ever increasing knowledge of the important role that regulatory antisense RNAs play in bacteria.

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.

The novel cis-encoded antisense RNA AsrC positively regulates the expression of rpoE-rseABC operon and thus enhances the motility of Salmonella enterica serovar typhi

Bacterial non-coding RNAs are essential in many cellular processes, including response to environmental stress, and virulence. Deep sequencing analysis of the Salmonella enterica serovar typhi (S. typhi) transcriptome revealed a novel antisense RNA transcribed in cis on the strand complementary to rseC, an activator gene of sigma factor RpoE. In this study, expression of this antisense RNA was confirmed in S. typhi by Northern hybridization. Rapid amplification of cDNA ends and sequence analysis identified an 893 bp sequence from the antisense RNA coding region that covered all of the rseC coding region in the reverse direction of transcription. This sequence of RNA was named as AsrC. After overexpression of AsrC with recombinantant plasmid in S. typhi, the bacterial motility was increased obviously. To explore the mechanism of AsrC function, regulation of rseC and rpoE expression by AsrC was investigated. We found that AsrC increased the levels of rseC mRNA and protein. The expression of rpoE was also increased in S. typhi after overexpression of AsrC, which was dependent on rseC. Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. typhi.

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 OmpRHis6 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.