RESUMEN
The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger cyclic di-GMP (c di-GMP) signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but five of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the four intact DGCs in a S. flexneri strain, where these four DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these four DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN, which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC pseudogenes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.
RESUMEN
The intracellular human pathogen Shigella invades the colonic epithelium to cause disease. Prior to invasion, this bacterium navigates through different environments within the human body, including the stomach and the small intestine. To adapt to changing environments, Shigella uses the bacterial second messenger c-di-GMP signaling system, synthesized by diguanylate cyclases (DGCs) encoding GGDEF domains. Shigella flexneri encodes a total of 9 GGDEF or GGDEF-EAL domain enzymes in its genome, but 5 of these genes have acquired mutations that presumably inactivated the c-di-GMP synthesis activity of these enzymes. In this study, we examined individual S. flexneri DGCs for their role in c-di-GMP synthesis and pathogenesis. We individually expressed each of the 4 intact DGCs in an S. flexneri strain where these 4 DGCs had been deleted (Δ4DGC). We found that the 4 S. flexneri intact DGCs synthesize c-di-GMP at different levels in vitro and during infection of tissue-cultured cells. We also found that dgcF and dgcI expression significantly reduces invasion and plaque formation, and dgcF expression increases acid sensitivity, and that these phenotypes did not correspond with measured c-di-GMP levels. However, deletion of these 4 DGCs did not eliminate S. flexneri c-di-GMP, and we found that dgcE, dgcQ, and dgcN , which all have nonsense mutations prior to the GGDEF domain, still produce c-di-GMP. These S. flexneri degenerate DGC genes are expressed as multiple proteins, consistent with multiple start codons within the gene. We propose that both intact and degenerate DGCs contribute to S. flexneri c-di-GMP signaling.
RESUMEN
Occasionally, obtaining an adequate or acceptable postmortem blood specimen for drug analysis is not possible due to factors such as decomposition, exsanguination, or embalming. Submandibular salivary gland tissue, one of three major types of salivary gland tissue in the oral cavity of humans, has been reported to be a viable alternative postmortem specimen for toxicological testing. In this study, we evaluated the performance of the Randox Evidence Investigator instrument and Randox DOA (Drugs of Abuse) Ultra Whole Blood Array for the semi-quantitative determination of 21 immunoassays in an alternative matrix, submandibular salivary gland tissue. We analyzed 132 submandibular salivary gland tissue specimens and compared the generated results to concomitantly collected postmortem whole blood specimen results. Oxycodone 2, meprobamate, barbiturate, benzodiazepine assay 1, zolpidem, and buprenorphine all showed perfect agreement (Cohen's Kappa Score = 1.00) between the submandibular salivary gland tissue results and the postmortem whole blood results; dextromethorphan, fentanyl, benzoylecgonine, methamphetamine, tricyclic antidepressants, oxycodone 1, and opiate showed an almost perfect agreement (Cohen's Kappa Score = 0.81-0.99); methadone, generic opioids, and amphetamine exhibited substantial agreement (Cohen's Kappa Score = 0.61-0.80). Tramadol demonstrated fair agreement (Cohen's Kappa Score = 0.41-0.60). The lowest measure of agreement was observed with cannabinoids, meeting criteria for slight agreement (Cohen's Kappa Score = 0.01-0.20). An application of the techniques described in this study could be implemented in postmortem toxicology laboratories as well as medical examiners offices to provide preliminary drugs of abuse test results that can be used to direct additional testing. This study highlights the successful integration of a novel specimen matrix and an "off-label" use of an established analytical technique.
RESUMEN
1,4-naphthoquinones (NQs) catalytically oxidize H2S to per- and polysufides and sulfoxides, reduce oxygen to superoxide and hydrogen peroxide, and can form NQ-SH adducts through Michael addition. Here, we measured oxygen consumption and used sulfur-specific fluorophores, liquid chromatography tandem mass spectrometry (LC-MS/MS), and UV-Vis spectrometry to examine H2S oxidation by NQs with various substituent groups. In general, the order of H2S oxidization was DCNQ ~ juglone > 1,4-NQ > plumbagin >DMNQ ~ 2-MNQ > menadione, although this order varied somewhat depending on the experimental conditions. DMNQ does not form adducts with GSH or cysteine (Cys), yet it readily oxidizes H2S to polysulfides and sulfoxides. This suggests that H2S oxidation occurs at the carbonyl moiety and not at the quinoid 2 or 3 carbons, although the latter cannot be ruled out. We found little evidence from oxygen consumption studies or LC-MS/MS that NQs directly oxidize H2S2-4, and we propose that apparent reactions of NQs with inorganic polysulfides are due to H2S impurities in the polysulfides or an equilibrium between H2S and H2Sn. Collectively, NQ oxidation of H2S forms a variety of products that include hydropersulfides, hydropolysulfides, sulfenylpolysulfides, sulfite, and thiosulfate, and some of these reactions may proceed until an insoluble S8 colloid is formed.