Involvement of RNA-binding protein Hfq in the osmotic-response regulation of invE gene expression in Shigella sonnei

<p>Abstract</p> <p>Background</p> <p>The expression of Type III secretion system (TTSS) in <it>Shigella </it>is regulated in response to changes in environmental osmolarity and temperature. Temperature-dependent regulation of <it>virF</it>, the master regulator of TTSS synthesis, is believed to occur at the transcriptional level. We recently demonstrated, however, that TTSS synthesis also involves post-transcriptional regulation of the synthesis of InvE, a target of <it>virF </it>and key regulator of TTSS synthesis. The mRNA levels of <it>invE </it>(<it>virB</it>) are stable at 37°C, but mRNA stability markedly decreases at low temperatures where the TTSS synthesis is tightly repressed. Deletion of <it>hfq</it>, which encodes an RNA chaperone in Gram-negative bacteria, results in the restoration of expression of <it>invE </it>and other TTSS genes at low temperature due to an increase in the stability of <it>invE </it>mRNA. To date, the molecular details of the regulation of TTSS expression in response to osmotic pressure are not known. In the current study, we investigated the mechanism of regulation of TTSS by osmotic pressure.</p> <p>Results</p> <p>Transcription of <it>virF</it>, which encodes the master regulator of TTSS expression, was partially repressed under low osmotic conditions. Several lines of evidence indicated that osmolarity-dependent changes in TTSS synthesis are controlled at the post-transcriptional level, through the regulation of InvE synthesis. First, the expression InvE protein was tightly repressed under low osmotic growth conditions, even though <it>invE </it>mRNA transcripts were readily detectable. Second, under low osmotic conditions, <it>invE </it>mRNA was rapidly degraded, whereas deletion of <it>hfq</it>, which encodes an RNA chaperone, resulted in increased <it>invE </it>mRNA stability and the production of InvE protein. Third, the binding of purified Hfq <it>in vitro </it>to <it>invE </it>RNA was stronger in low-salt buffer, as assessed by gel-shift analysis and surface plasmon resonance (Biacore analysis).</p> <p>Conclusion</p> <p>Osmolarity-dependent changes in TTSS synthesis in <it>Shigella </it>involve the post-transcriptional regulation of InvE expression, in addition to partial transcriptional activation by <it>virF</it>. The stability of <it>invE </it>mRNA is reduced under low osmotic conditions, similar to the effect of temperature. Deletion of an RNA chaperone gene (<it>hfq</it>) abolished the repression of TTSS synthesis at low osmolarity through a mechanism that involved increased stability of <it>invE </it>mRNA. We propose that the expression of <it>Shigella </it>virulence genes in response to both osmolarity and temperature involves the post-transcriptional regulation of expression of InvE, a critical regulator of TTSS synthesis.</p

A Sensor of the Two-Component System CpxA Affects Expression of the Type III Secretion System through Posttranscriptional Processing of InvE

The chief function of the Cpx two-component system is perceiving various cell envelope stresses, but CpxR is also known to regulate the expression of the type III secretion system (TTSS) of Shigella sonnei through transcription of the primary regulator virF. Here, we have isolated novel cpxA mutants that exhibited decreased TTSS expression from Escherichia coli HW1273, which carries the virulence plasmid of S. sonnei. The cpxA deletion strain of HW1273 expressed β-galactosidase activity levels from the virF-lacZ fusion similar to those of HW1273. However, the second regulator InvE (VirB) and the TTSS component IpaB proteins were apparently expressed at a low level. In the cpxA strain, β-galactosidase activity levels from the invE-lacZ transcriptional fusion remained similar to those of HW1273, whereas the β-galactosidase activity level from the translational fusion of invE-lacZ was reduced to 21% of that of HW1273. Therefore, the deletion of the cpxA gene influenced TTSS expression chiefly at the posttranscriptional processing of InvE. In addition, the cpxA deletion strain of S. sonnei showed the same phenotype. These results indicate that the Cpx two-component system is involved in virulence expression through posttranscriptional processing of the regulatory protein InvE, a novel feature of the Cpx two-component system in posttranscriptional processing and virulence expression of Shigella

Functional analysis of the RNA polymerase || Rpd3 subunit of the fission yeast Schizosaccharomyces pombe

Three types of the nuclear RNA polymerase in eukaryotes are all multi-subunit enzymes, each consisting of more than 10 subunits. The RNA polymerase II is involved in the synthesis of mRNA and plays a key role in transcription of protein-coding genes. The RNA polymerase II from the fission yeast Schizosaccharomyces pombe is composed of 12 putative subunits. Sequence analysis indicated that, as in the case of other eukaryotic RNA polymerases, the largest subunit Rpb1 and the second largest subunit Rpb2 of S. pombe RNA polymerase II have notable homology with the β\u27 and β subunits, respectively, of the prokaryotic RNA polymerases. The third largest subunit Rpb3 also has homology with the amino (N)-terminal domain of prokaryotic RNA polymerase α subunit (αNTD), which plays a key role in subunit assembly of this complex enzyme by providing the contact surfaces for both β and β\u27 subunits. The Schizosaccharomyces pombe Rpb3 protein forms a core subassembly together with Rpb2 (the β homologue) and Rpb11 (the second αhomologue) subunits as in the case of the prokaryotic α2β complex. Several lines of evidence indicate that in addition to these core subunits, Rpb1, Rpb5, Rpb7 and Rpb8 also interact with Rpb3.Sequence comparison between prokaryotes and eukaryotes also indicated Rpb3 has four conserved regions A to D. Among which two (A and D) are conserved among Rpb3 homologues from both prokaryotes (α subunits) and eukaryotes and two (B and C) are conserved only within eukaryotes. The regions A and D correspond to the N-terminal proximal and the C-terminal proximal regions of the αNTD, each playing critical roles in the contact with α and β subunits (motif-1) or the contact withα and β\u27 subunits (motif- 2), respectively. On the other hands, two regions in the middle part of Rpb3 protein (Region B and C) do not exist in the prokaryotic RNA polymerases, Region B is specifically conserved in eukaryotic RNA polymerase II, and Region C is conserved among all three types of eukaryotic RNA polymerases. Therefore these two regions have been considered to be involved in eukaryote specific function(s). In order to get insight into in vivo roles of Rpb3 in the assembly and function of RNA polymerase II, we have performed mutant studies for the S. pombe rpb3 gene of RNA polymerase II. First, we carried out a systematic search for temperature-sensitive (Ts-) or cold-sensitive(Cs-)S. pombe mutants with mutations throughout the rpb3 gene. After PCR mutagenesis of the entire rpb3 sequence, we isolated 9 Ts- and 3 Cs- mutants. After the sequence analysis of mutant rpb3 gene, each mutant was found to carry a single (or double in a few cases) mutation in one of the four regions (A to D) conserved among the eukaryotic subunit 3 homologues. The 3 Cs- mutations were all located in the region A, in agreement with its most important role in the assembly of prokaryotic RNA polymerase, while the Ts- mutations were scattered in all four regions. Since the metabolic stability of most Ts- mutant Rpb3 proteins was markedly reduced at a non-permissive temperature, we predict that these mutant Rpb3 proteins are defective in the assembly or the mutant RNA polymerases containing the mutant Rpb3 are thermolabile. The assembly state of mutant RNA polymerase II was tested by treating purified mutant RNA ploymerases with low concentrations of urea. One representative Ts- mutant complex was indeed dissociated more easily than the wild-type RNA polymerase. Moreover, the Ts- phenotype of all the mutants were suppressed to various extents by overexpression of Rpb11, the pairing subunit in the initial stage of RNA polymerase II assembly. We conclude that the majority of rpb3 mutations affect the subunit assembly of Rpb3, even though the extent of influence on the subunit assembly is different depending on the location of mutations.Since the conserved regions B and C are unique for eukaryotic Rpb3 homologues, we thought that eukaryotic-specific transcription factors interact with one or both of these regions. We started the analysis of factor-dependent in vitro transcription activity for the mutant RNA polymerases. For this purpose, we constructed activator-dependent in vitro transcription system of S. pombe using S.cerevisiae GAL4-VP16. Results of the preliminary experiments indicate loss of the factor dependent transcription activity after heat treatment of the cell extracts from rpb3 mutants carrying mutations in the B or C region

Additional file 1 of Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei

Additional file 1: Figure S1. Schematic representation of the gene-targeting vector used to disrupt PBANKA_1019700, PBANKA_1101300 (SBP1) and PBANKA_0519900. Figure S2. Generation of parasites to investigate the localization of PBANKA_1019700.Figure S3. Generation of parasites to investigate the localization of PBANKA_0519900::GFP and PBANKA_1359300::GFP

Determination of the InvE Binding Site Required for Expression of IpaB of the Shigella sonnei Virulence Plasmid: Involvement of a ParB BoxA-Like Sequence

The InvE protein positively regulates the expression of virulence genes ipaBCD in Shigella sonnei. The InvE has significant homology with ParB of plasmid P1, which is known as a plasmid partitioning factor with DNA binding ability. Although the DNA binding activity of InvE has been predicted, it is not known whether the DNA binding activity is necessary for type III secretion system-associated gene expression. In this study, we determined the transcription start site of the icsB-ipaBCD operon (ipa operon) and constructed a series of deletions of the icsB promoter region in the Escherichia coli K-12 background. The deletion study revealed that an 86-bp region upstream of the icsB transcription start site was essential for expression of the ipa operon, where the ParB binding motif (ParB BoxA-like sequence) was observed. Purified glutathione S-transferase-InvE fusion protein bound directly to the −93 to −54 region (designating the icsB transcription start site as nucleotide +1) containing the ParB BoxA-like sequence. These results indicated that InvE bound directly to the promoter region

Differential Expression of the Smb Bacteriocin in Streptococcus mutans Isolates ▿

The two-component lantibiotic Smb is produced by Streptococcus mutans GS5. In the present study, we identified seven strains of S. mutans containing the smb gene cluster. These strains could be classified into high- and low-level Smb producers relative to the levels of Smb production by indicator strains in vitro. This classification was dependent upon the transcription levels of the structural smbA and smbB genes. Sequence analysis upstream of smbA in the high- and low-level Smb-producing strains revealed differences at nucleotide position −46 relative to the smbA start codon. Interestingly, the transcription start site was present upstream of the point mutation, indicating that both groups of strains have the same promoter constructs and that the differential expression of smbA and smbB mRNA occurred subsequent to transcription initiation. In addition, smbA::lacZ fusion expression was higher when it was regulated by the sequences of strains with high-level Smb activity than when it was regulated by the comparable region from strains with low-level Smb activity. Taken together, we conclude that high- or low-level Smb expression is dependent on the presence of a G or a T nucleotide at position −46 relative to the smbA translational start site in S. mutans Smb producers

An attenuated <i>Shigella</i> mutant lacking the RNA-binding protein Hfq provides cross-protection against <i>Shigella</i> strains of broad serotype

<div><p>Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of <i>Shigella flexneri</i> 2a protected guinea pigs against subsequent infection by <i>S</i>. <i>dysenteriae</i> type 1 and <i>S</i>. <i>sonnei</i> strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against <i>Shigella</i> strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with <i>S</i>. <i>dysenteriae</i> type 1 or <i>S</i>. <i>sonnei</i> strains. Antibodies against common virulence proteins and the O-antigen of <i>S</i>. <i>flexneri</i> 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive <i>Escherichia coli</i>, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of <i>S</i>. <i>sonnei</i> and <i>S</i>. <i>flexneri</i> 2a, but also various <i>Shigella</i> strains, including <i>S</i>. <i>dysenteriae</i> type 1, which produces Shiga toxin.</p></div

List of accession numbers.

<p>List of accession numbers.</p