The small RNA PrrH aggravates Pseudomonas aeruginosa-induced acute lung injury by regulating the type III secretion system activator ExsA.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. Small regulatory RNAs (sRNAs) regulate multiple bacterial adaptations to environmental changes, especially virulence. Our previous study showed that sRNA PrrH negatively regulates the expression of a number of virulence factors, such as pyocyanin, rhamnolipid, biofilm, and elastase in the P. aeruginosa strain PAO1. However, previous studies have shown that the prrH-deficient mutant attenuates virulence in an acute murine lung infection model. All ΔprrH-infected mice survived the entire 28-day course of the experiment, whereas all mice inoculated with the wild-type or the complemented mutant succumbed to lung infection within 4 days of injection, but the specific mechanism is unclear. Herein, we explored how PrrH mediates severe lung injury by regulating the expression of virulence factors. In vivo mouse and in vitro cellular assays demonstrated that PrrH enhanced the pathogenicity of PAO1, causing severe lung injury. Mechanistically, PrrH binds to the coding sequence region of the mRNA of exsA, which encodes the type III secretion system master regulatory protein. We further demonstrated that PrrH mediates a severe inflammatory response and exacerbates the apoptosis of A549 cells. Overall, our results revealed that PrrH positively regulates ExsA, enhances the pathogenicity of P. aeruginosa, and causes severe lung injury. IMPORTANCE: Pseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.

The small RNA PrrH of Pseudomonas aeruginosa regulates hemolysis and oxidative resistance in bloodstream infection.

Small regulatory RNAs (sRNAs) regulate multiple physiological functions in bacteria, and sRNA PrrH can regulate iron homeostasis and virulence. However, the function of PrrH in Pseudomonas aeruginosa (P. aeruginosa) bloodstream infection (BSI) is largely unknown. The aim of this study was to investigate the role of PrrH in P. aeruginosa BSI model. First, P. aeruginosa PAO1 was co-cultured with peripheral blood cells for 6 h. qRT-PCR results showed a transient up-regulation of PrrH expression at 1 h. Simultaneously, the expression of iron uptake genes fpvA, pvdS and phuR were upregulated. In addition, the use of iron chelator 2,2'-dipyridyl to create low-iron conditions caused up-regulation of PrrH expression, a result similar to the BSI model. Furthermore, the addition of FeCl3 was found to decrease PrrH expression. These results support the hypothesis that the expression of PrrH is regulated by iron in BSI model. Then, to clarify the effect of PrrH on major cells in the blood, we used PrrH mutant, overexpressing and wild-type strains to act separately on erythrocytes and neutrophils. On one hand, the hemolysis assay revealed that PrrH contributes to the hemolytic activity of PAO1, and its effect was dependent on the T3SS system master regulator gene exsA, yet had no association with the hemolytic phospholipase C (plcH), pldA, and lasB elastase genes. On the other hand, PrrH mutant enhanced the oxidative resistance of PAO1 in the neutrophils co-culture assay, H2O2-treated growth curve and conventional plate spotting assays. Furthermore, the katA was predicted to be a target gene of PrrH by bioinformatics software, and then verified by qRT-PCR and GFP reporter system. In summary, dynamic changes in the expression of prrH are iron-regulated during PAO1 bloodstream infection. In addition, PrrH promotes the hemolytic activity of P. aeruginosa in an exsA-dependent manner and negatively regulates katA to reduce the oxidative tolerance of P. aeruginosa.

Olfactory impact of guaiacol, ortho-vanillin, 5-methyl, and 5-formyl-vanillin as byproducts in synthetic vanillin.

To better control the quality of synthetic vanillin obtained by using the guaiacol synthesis method, the olfactory impacts of byproducts on the aroma of the synthetic vanillin samples were evaluated and their optimum concentration ranges were determined. Four byproducts (guaiacol, ortho-vanillin, 5-methyl-vanillin, and 5-formyl-vanillin) were identified by gas chromatography-mass spectrometry (GC-MS) and quantified by gas chromatography-flame ionization detection (GC-FID) in the synthetic vanillin samples with different degrees of purity. The aroma intensities (AIs) of the four byproducts obtained by gas chromatography-olfactometry (GC-O) were: guaiacol (AI: 3.5-4.0, smoke), ortho-vanillin (AI: 1.6-2.5, almond), 5-methyl-vanillin (AI: 2.5-3.3, aldehyde), and 5-formyl-vanillin (AI: 3.2-3.8, green). The aroma perceptual interactions of the four byproducts and the vanillin in the synthetic vanillin samples were determined by S-curve analysis. Guaiacol and 5-methyl-vanillin showed synergistic effects by Feller's additive model. Combined with the results of an addition experiment, when the contents of guaiacol, ortho-vanillin, 5-methyl-vanillin, and 5-formyl-vanillin were within 50, 10, 400, and 1,000 mg/kg respectively, the byproducts had no effects on the aroma quality of the synthetic vanillin samples. PRACTICAL APPLICATION: Synthetic vanillin is one of the most commonly used food additives. Currently, the purity of synthetic vanillin can reach 99.9%, but trace byproducts are still present. Continuing to improve the purity of synthetic vanillin will significantly increase its production costs. Therefore, it is necessary to determine whether the presence of these byproducts affects the aroma quality of the synthetic vanillin samples or not. If they have a negative effect on its aroma, it will be important to reduce their content. If they have no influence or positive role, there is no need to control the content of these byproducts to very low levels. This study determined the content of the byproducts produced during the synthesis of vanillin by guaiacol glyoxylic acid method, judged the perceptual interaction between the byproducts and the vanillin in the synthetic vanillin samples, and determined the optimum range within which the byproducts had no effects on the aroma quality. This study provides a theoretical basis for improving the aroma quality of synthetic vanillin while controlling the production costs.