Signal transduction pathway mediated by the novel regulator LoiA for low oxygen tension induced <i>Salmonella</i> Typhimurium invasion

<div><p><i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium) is a major intestinal pathogen of both humans and animals. <i>Salmonella</i> pathogenicity island 1 (SPI-1)-encoded virulence genes are required for <i>S</i>. Typhimurium invasion. While oxygen (O₂) limitation is an important signal for SPI-1 induction under host conditions, how the signal is received and integrated to the central SPI-1 regulatory system in <i>S</i>. Typhimurium is not clear. Here, we report a signal transduction pathway that activates SPI-1 expression in response to low O₂. A novel regulator encoded within SPI-14 (STM14_1008), named LoiA (<u>l</u>ow <u>o</u>xygen <u>i</u>nduced factor <u>A</u>), directly binds to the promoter and activates transcription of <i>hilD</i>, leading to the activation of <i>hilA</i> (the master activator of SPI-1). Deletion of <i>loiA</i> significantly decreased the transcription of <i>hilA</i>, <i>hilD</i> and other representative SPI-1 genes (<i>sipB</i>, <i>spaO</i>, <i>invH</i>, <i>prgH</i> and <i>invF</i>) under low O₂ conditions. The response of LoiA to the low O₂ signal is mediated by the ArcB/ArcA two-component system. Deletion of either <i>arcA</i> or <i>arcB</i> significantly decreased transcription of <i>loiA</i> under low O₂ conditions. We also confirmed that SPI-14 contributes to <i>S</i>. Typhimurium virulence by affecting invasion, and that <i>loiA</i> is the virulence determinant of SPI-14. Mice infection assays showed that <i>S</i>. Typhimurium virulence was severely attenuated by deletion of either the entire SPI-14 region or the single <i>loiA</i> gene after oral infection, while the virulence was not affected by either deletion after intraperitoneal infection. The signal transduction pathway described represents an important mechanism for <i>S</i>. Typhimurium to sense and respond to low O₂ conditions of the host intestinal tract for invasion. SPI-14-encoded <i>loiA</i> is an essential element of this pathway that integrates the low O₂ signal into the SPI-1 regulatory system. Acquisition of SPI-14 is therefore crucial for the evolution of <i>S</i>. Typhimurium as an intestinal pathogen.</p></div

Model of the LoiA regulatory network in <i>S</i>. Typhimurium invasion.

<p>Under low O₂ concentrations, the phosphorylated ArcA after sensing of low O₂ signal by ArcB can indirectly activate <i>loiA</i> gene transcription, LoiA can then activate SPI-1 T3SS through directly activating HilD, thus promoting epithelial cell invasion.</p

The activation of <i>LoiA</i> gene function by low O₂ conditions is mediated by ArcAB.

<p>(A, B) qRT-PCR analysis of <i>loiA</i> gene expression in wild-type, <i>arcA</i> mutant, <i>arcB</i> mutant, and complemented strains for ArcA and ArcB. Bacteria were grown in LB medium (0.17 M NaCl) either with low O₂ (A) or high O₂ (B). (C, D) qRT-PCR analysis of <i>hilD</i> gene expression in wild-type, <i>arcA</i> mutant, <i>arcB</i> mutant, <i>loiA</i> mutant, <i>arcA</i>/<i>loiA</i> double mutant, <i>arcB</i>/<i>loiA</i> double mutant or complemented strains. Bacteria were grown in LB medium (0.17 M NaCl) either with low O₂ (C) or high O₂ (D). Data from graphs (A) to (D) are representative of at least three independent experiments and are presented as mean ±SD. <i>P</i> values were determined by student’s t test (^*<i>P</i><0.05; ^**<i>P</i><0.01). (E) Bacterial counts recovered from ileum of the BALB/c mice orally infected with 5×10⁶ CFU of wild-type, <i>loiA</i> mutant, <i>arcA</i> mutant or <i>arcA</i>/<i>loiA</i> double mutant at day 5 post-infection. Data are combined from three independent experiments. Bars represent mean CFU of all mice, with significance determined by the Mann-Whitney U test (^*<i>P</i><0.05; ^**<i>P</i><0.01; ns, not significant).</p

The gene <i>loiA</i> (<i>STM14_1008</i>) is the virulence determinant in SPI-14 influencing <i>S</i>. Typhimurium invasion.

<p>(A) Invasion assays of wild-type, <i>SPI-14</i> mutant, <i>STM14_1001-STM14_1004</i> mutant, <i>STM14_1005-STM14_1008</i> mutant, <i>STM14_1005</i> mutant, <i>STM14_1006</i> mutant, <i>STM14_1007</i> mutant and <i>loiA</i> (<i>STM14_1008</i>) mutant. (B) Invasion assays of wild-type, <i>SPI-14</i> mutant, <i>loiA</i> (<i>STM14_1008</i>) mutant and complemented strains. For (A) and (B), Caco-2 cells were infected with bacteria at an MOI of 10. The invasion ability of mutants is reported as percentages relative to the wild-type strain. Data are representative of at least three independent experiments and are presented as mean ±SD. <i>P</i> values were determined by student’s t test (^***<i>P</i><0.001). (C) Survival plots of BALB/c mice over a 30-day period after orally infected with ~5×10⁶ CFU of indicated bacterial strains. Data presented are the combination of three independent experiments, ^***<i>P</i><0.001 by log-rank curve comparison test. (D) Bacterial counts recovered from ileum, liver and spleen of the orally infected mice. At day 5 post-infection, mice organs were harvested and homogenized for colony enumeration. Data are combined from three independent experiments. Bars represent mean CFU of all mice, with <i>P</i> value determined by the Mann-Whitney U test (^**<i>P</i><0.01; ns, not significant).</p

LoiA contributes to invasion through activating SPI-1.

<p>(A) Wild-type, <i>loiA</i> mutant and complemented strain were grown under SPI-1-inducing conditions (high salt, low O₂) to late-exponential phase. Transcript levels of the SPI-1 genes in different strains were assessed by qRT-PCR. <i>16S rRNA</i> gene was used as the internal control. Data are representative of three independent experiments and are presented as mean ±SD. <i>P</i> values were determined by student’s t test (^**<i>P</i><0.01; ^*** <i>P</i><0.001). (B) Bacterial counts recovered from ileum, liver and spleen of the BALB/c mice orally infected with 5×10⁶ CFU of wild-type, <i>loiA</i> mutant, SPI-1 mutant or SPI-1/<i>loiA</i> double mutant at day 5 post-infection. Data are combined from three independent experiments. Bars represent mean CFU of all mice, with <i>P</i> values determined by the Mann-Whitney U test (^**<i>P</i><0.01; ns, not significant).</p

Lack of SPI-14 decreased <i>S</i>. Typhimurium virulence in mice by affecting intestinal invasion.

<p>(A) Survival plots of BALB/c mice for a 30-day period after orally infected with ~5×10⁶ CFU of wild-type or SPI-14 mutant. Data presented are the combination of three independent experiments, ^***<i>P</i><0.001 by log-rank curve comparison test. (B) Bacterial counts recovered from ileum, liver and spleen of the orally infected mice. At day 5 post-infection, mice organs were harvested and homogenized for colony enumeration. Data are combined from three independent experiments. Bars represent mean CFU of all mice, with <i>P</i> value determined by the Mann-Whitney U test (^**<i>P</i><0.01). (C) Survival plots of BALB/c mice after inoculation intraperitoneally (i.p.) with 1×10⁴ CFU of wild-type or SPI-14 mutant. Data presented are the combination of two independent experiments, with <i>P</i> value determined by log-rank curve comparison test (ns, not significant). (D) Bacterial counts recovered from liver and spleen of the i.p. infected mice. At day 3 post-infection, mice organs were harvested and homogenized for colony enumeration. Data are combined from two independent experiments. Bars represent mean CFU of all mice, with <i>P</i> value determined by the Mann-Whitney U test (ns, not significant).</p

Expression of LoiA was induced by low O₂ concentration.

<p>(A) qRT-PCR analysis of <i>loiA</i> gene expression under high O₂ or low O₂ (LB, low salt), and high salt (0.3M NaCl) or low salt (0.17M NaCl) conditions (LB, high O₂). Data are representative of at least three independent experiments and are presented as mean ±SD. <i>P</i> values were determined by student’s t test (^**<i>P</i><0.01; ns, not significant). (B) Western blot analysis of LoiA protein level under high O₂ or low O₂ (LB, low salt), and high salt or low salt conditions (LB, high O₂). The expression of the tagged LoiA protein was determined using that of DnaK as the internal control.</p

Tellurium/Bovine Serum Albumin Nanocomposites Inducing the Formation of Stress Granules in a Protein Kinase R‑Dependent Manner

The effect of nanoparticles (NPs) on cellular stress responses is important to the understanding of nanotoxicities and developing safe therapies. Although the relationship between NPs and cellular stress responses has been preliminarily investigated, stress responses to NPs remain unclear. Here, tellurium/bovine serum albumin (Te/BSA) nanocomposites were prepared using sodium tellurite, BSA, and glutathione as precursors. The as-prepared Te/BSA nanocomposites, with particle size similar to that of many viruses, are found to induce the formation of stress granules (SGs), a kind of cytoplasmic RNA granule formed under various stresses. The SGs in Te/BSA nanocomposite-treated cells are composed of T-cell internal antigen 1 (TIA1), TIA1-related protein, and eukaryotic initiation factor 3η. Using chemical inhibitors and small interfering RNA-mediated silencing, protein kinase R (PKR) is identified as the α-subunit of eukaryotic initiation factor 2 (eIF2α)-kinase activated upon Te/BSA nanocomposite incubation, which is also the dominant kinase responsible for eIF2α activation under virus infection. Mechanistically, PKR is activated in a heparin-dependent manner. This study reveals a biological effect of Te/BSA nanocomposites on stress responses, providing a preliminary basis for further research on viruslike particles and the application of NPs in biology

Tellurium/Bovine Serum Albumin Nanocomposites Inducing the Formation of Stress Granules in a Protein Kinase R‑Dependent Manner

The effect of nanoparticles (NPs) on cellular stress responses is important to the understanding of nanotoxicities and developing safe therapies. Although the relationship between NPs and cellular stress responses has been preliminarily investigated, stress responses to NPs remain unclear. Here, tellurium/bovine serum albumin (Te/BSA) nanocomposites were prepared using sodium tellurite, BSA, and glutathione as precursors. The as-prepared Te/BSA nanocomposites, with particle size similar to that of many viruses, are found to induce the formation of stress granules (SGs), a kind of cytoplasmic RNA granule formed under various stresses. The SGs in Te/BSA nanocomposite-treated cells are composed of T-cell internal antigen 1 (TIA1), TIA1-related protein, and eukaryotic initiation factor 3η. Using chemical inhibitors and small interfering RNA-mediated silencing, protein kinase R (PKR) is identified as the α-subunit of eukaryotic initiation factor 2 (eIF2α)-kinase activated upon Te/BSA nanocomposite incubation, which is also the dominant kinase responsible for eIF2α activation under virus infection. Mechanistically, PKR is activated in a heparin-dependent manner. This study reveals a biological effect of Te/BSA nanocomposites on stress responses, providing a preliminary basis for further research on viruslike particles and the application of NPs in biology

DataSheet_1_Two vacuolar invertase inhibitors PpINHa and PpINH3 display opposite effects on fruit sugar accumulation in peach.docx

Soluble sugars are an important determinant of fruit taste, but their accumulation mechanisms remain elusive. In this study, we report two vacuolar invertase inhibitor genes involved in sugar accumulation in peach, PpINHa and PpINH3. Transient overexpression of PpINH3 in peach fruits resulted in an increase in sugar content, while the opposite trend was detected for PpINHa. Unexpectedly, PpINH3 and PpINHa both had no physical interaction with vacuolar invertase (VIN). Moreover, the PpVIN genes had no or extremely low expression in fruits at the ripening stage. These results suggested that the regulatory role of PpINHa and PpINH3 in sugar accumulation is unlikely due to their interaction with PpVINs. Additionally, overexpression of PpINHa and PpINH3 had an impact on transcription of genes related to fruit sugar metabolism and transport, which is likely responsible for their regulatory role in fruit sugar accumulation. Altogether, these results indicated an important role of PpINHs in fruit accumulation in peach. Our study provides new insights into molecular mechanisms underlying sugar accumulation, which could be useful for genetic improvement of fruit taste in breeding programs of peach and other fruit crops.</p