The biological functions of protein S-sulfhydration in eukaryotes and the ever-increasing understanding of its effects on bacteria.

As a critical endogenous signaling molecule, hydrogen sulfide may induce reversible post-translational modifications on cysteine residues of proteins, generating a persulfide bond known as S-sulfhydration. A systemic overview of the biofunctions of S-sulfhydration will equip us better to characterize its regulatory roles in antioxidant defense, inflammatory response, and cell fate, as well as its pathological mechanisms related to cardiovascular, neurological, and multiple organ diseases, etc. Nevertheless, the understanding of S-sulfhydration is mostly built on mammalian cells and animal models. We subsequently summarized the mediation effects of this specific post-transcriptional modification on physiological processes and virulence in bacteria. The high-sensitivity and high-throughput detection technologies are required for studying the signal transduction mechanism of H2S and protein S-sulfhydration modification. Herein, we reviewed the establishment and development of different approaches to assess S-sulfhydration, including the biotin-switch method, modified biotin-switch method, alkylation-based cysteine-labelled assay, and Tag-switch method. Finally, we discussed the limitations of the impacts of S-sulfhydration in pathogens-host interactions and envisaged the challenges to design drugs and antibiotics targeting the S-sulfhydrated proteins in the host or pathogens.

Fitness Costs of Tigecycline Resistance in Acinetobacter baumannii and the Resistance Mechanism Revealed by a Transposon Mutation Library.

Acinetobacter baumannii is one of the main pathogens causing nosocomial and community-acquired infections. Tigecycline is an important antibiotic for the treatment of multidrug-resistant A. baumannii infections, but strains resistant to tigecycline have also emerged. There are still many unclear questions concerning the mechanism of tigecycline resistance in A. baumannii. In this study, tigecycline-susceptible and tigecycline-intermediate strains were gradually cultured with sub-minimum inhibitory concentrations of tigecycline to select for tigecycline-resistant mutants, and a tigecycline-resistant strain was cultured under 42 °C to select for tigecycline-susceptible mutants. We found that the acquisition of tigecycline resistance affected the susceptibility of the strains to other antibiotics. Resistance to ampicillin-sulbactam is negatively correlated with tigecycline resistance. The strains will experience fitness costs along with the acquisition of tigecycline resistance. Tigecycline resistance in the strains was not related to 16S rRNA target variation or outer membrane integrity alteration. By constructing a transposon mutation library, we found that transposon insertion of the adeL gene reduced the sensitivity of A. baumannii to tigecycline. This study provides important clues for understanding the mechanism of tigecycline resistance in A. baumannii.

Salmonella Regulator STM0347 Mediates Flagellar Phase Variation via Hin Invertase.

Salmonella enterica is one of the most important food-borne pathogens, whose motility and virulence are highly related to flagella. Flagella alternatively express two kinds of surface antigen flagellin, FliC and FljB, in a phenomenon known as flagellar phase variation. The molecular mechanisms by which the switching orientation of the Hin-composed DNA segment mediates the expression of the fljBA promoter have been thoroughly illustrated. However, the precise regulators that control DNA strand exchange are barely understood. In this study, we found that a putative response regulator, STM0347, contributed to the phase variation of flagellin in S. Typhimurium. With quantitative proteomics and secretome profiling, a lack of STM0347 was confirmed to induce the transformation of flagellin from FliC to FljB. Real-time PCR and in vitro incubation of SMT0347 with the hin DNA segment suggested that STM0347 disturbed Hin-catalyzed DNA reversion via hin degradation, and the overexpression of Hin was sufficient to elicit flagellin variation. Subsequently, the Δstm0347 strain was outcompeted by its parental strain in HeLa cell invasion. Collectively, our results reveal the crucial role of STM0347 in Salmonella virulence and flagellar phase variation and highlight the complexity of the regulatory network of Hin-modulated flagellum phase variation in Salmonella.

Legionella pneumophila regulates host cell motility by targeting Phldb2 with a 14-3-3ζ-dependent protease effector.

The cytoskeleton network of eukaryotic cells is essential for diverse cellular processes, including vesicle trafficking, cell motility, and immunity, thus is a common target for bacterial virulence factors. A number of effectors from the bacterial pathogen Legionella pneumophila have been shown to modulate the function of host actin cytoskeleton to construct the Legionella-containing vacuole (LCV) permissive for its intracellular replication. In this study, we found that the Dot/Icm effector Lem8 (Lpg1290) is a protease whose activity is catalyzed by a Cys-His-Asp motif known to be associated with diverse biochemical activities. Intriguingly, we found that Lem8 interacts with the host regulatory protein 14-3-3ζ, which activates its protease activity. Furthermore, Lem8 undergoes self-cleavage in a process that requires 14-3-3ζ. We identified the Pleckstrin homology-like domain-containing protein Phldb2 involved in cytoskeleton organization as a target of Lem8 and demonstrated that Lem8 plays a role in the inhibition of host cell migration by attacking Phldb2.

CBS-derived H2S facilitates host colonization of Vibrio cholerae by promoting the iron-dependent catalase activity of KatB.

Sensing and resisting oxidative stress is critical for Vibrio cholerae to survive in either the aquatic environment or the gastrointestinal tract. Previous studies mainly focused on the mechanisms of oxidative stress response regulation that rely on enzymatic antioxidant systems, while functions of non-enzymatic antioxidants are rarely discussed in V. cholerae. For the first time, we investigated the role of hydrogen sulfide (H2S), the simplest thiol compound, in protecting V. cholerae against oxidative stress. We found that degradation of L-cysteine by putative cystathionine ß-synthase (CBS) is the major source of endogenous H2S in V. cholerae. Our results indicate that intracellular H2S level has a positive correlation with cbs expression, while the enhanced H2S production can render V. cholerae cells less susceptible to H2O2 in vitro. Using proteome analysis and real-time qPCR assay, we found that cbs expression could stimulate the expression of several enzymatic antioxidants, including reactive oxygen species (ROS) detoxifying enzymes SodB, KatG and AhpC, the DNA protective protein DPS and the protein redox regulator Trx1. Assays of ROS detoxification capacities revealed that CBS-derived H2S could promote catalase activity at the post-translational level, especially for KatB, which serves as an important way that endogenous H2S participates in H2O2 detoxification. The enhancement of catalase activity by H2S is achieved through facilitating the uptake of iron. Adult mice experiments showed that cbs mutant has colonization defect, while either complementation of cbs or exogenous supplement of N-Acetyl-L-Cysteine restores its fitness in the host environment. Herein, we proposed that V. cholerae regulates CBS-dependent H2S production for better survival and proliferation under ROS stress.

A Type I-F Anti-CRISPR Protein Inhibits the CRISPR-Cas Surveillance Complex by ADP-Ribosylation.

CRISPR-Cas systems are bacterial anti-viral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here, we report a novel mechanism by which AcrIF11 inhibits the type I-F CRISPR system. Our structural and biochemical studies demonstrate that AcrIF11 functions as a novel mono-ADP-ribosyltransferase (mART) to modify N250 of the Cas8f subunit, a residue required for recognition of the protospacer-adjacent motif, within the crRNA-guided surveillance (Csy) complex from Pseudomonas aeruginosa. The AcrIF11-mediated ADP-ribosylation of the Csy complex results in complete loss of its double-stranded DNA (dsDNA) binding activity. Biochemical studies show that AcrIF11 requires, besides Cas8f, the Cas7.6f subunit for binding to and modifying the Csy complex. Our study not only reveals an unprecedented mechanism of type I CRISPR-Cas inhibition and the evolutionary arms race between phages and bacteria but also suggests an approach for designing highly potent regulatory tools in the future applications of type I CRISPR-Cas systems.

Bisphenol-A induces neurodegeneration through disturbance of intracellular calcium homeostasis in human embryonic stem cells-derived cortical neurons.

Bisphenol-A (BPA) is a representative exogenous endocrine disruptor, which is extensively composed in plastic products. Due to the capability of passing through the blood-brain barrier, evidence has linked BPA exposure with multiple neuropsychological dysfunctions, neurobehavioral disorders and neurodegenerative diseases. However, the underlying mechanism by which BPA induces neurodegeneration still remains unclear. Our study used human embryonic stem cells-derived human cortical neurons (hCNs) as a cellular model to investigate the adverse neurotoxic effects of BPA. hCNs were treated with 0, 0.1, 1 and 10 µM BPA for 14 days. Impacts of BPA exposure on cell morphology, cell viability and neural marker (MAP2) were measured for evaluating the neurodegeneration. The intracellular calcium homeostasis, reactive oxygen species (ROS) generation and organelle functions were also taken into consideration. Results revealed that chronic exposure of BPA damaged the neural morphology, induced neuronal apoptosis and decreased MAP2 expression at the level of both transcription and translation. The intracellular calcium levels were elevated in hCNs after BPA exposure through NMDARs-nNOS-PSD-95 mediating. Meanwhile, BPA led to oxidative stress by raising the ROS generation and attenuating the antioxidant defense in hCNs. Furthermore, BPA triggered ER stress and increased cytochrome c release by impairing the mitochondrial function. Ultimately, BPA triggered the cell apoptosis by regulating Bcl-2 family and caspase-dependent signaling pathway. Taken together, BPA exerted neurotoxic effects on hCNs by eliciting apoptosis, which might due to the intracellular calcium homeostasis perturbation and cell organellar dysfunction.

Bisphenol-A exposure induced neurotoxicity in glutamatergic neurons derived from human embryonic stem cells.

Bisphenol-A (BPA) is a lipophilic, organic, synthetic compound that has been used as an additive in polycarbonate plastics manufacturing since 1957. Studies have shown that BPA interferes with the development and functions of the brain, but little is known about the effects of BPA on human glutamatergic neurons (hGNs) at the molecular and cellular levels. We investigated the impact of chronic exposure to BPA to hGNs derived from human embryonic stem cells (hESCs). The results showed that chronic exposure of different concentrations of BPA (0, 0.1, 1.0 and 10 µM) to hGNs for 14 days reduced neurite outgrowth in a concentration-dependent manner. Using presynaptic protein synaptophysin and postsynaptic protein PSD-95 antibodies, immunofluorescence staining and western blotting results indicated that BPA exposure altered the morphology of dendritic spines and increased synaptophysin and PSD-95 expression. Furthermore, BPA exposure at concentrations higher than 1.0 µM resulted in the increase of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression and deterioration of dendritic spines. In addition, our results suggested that these BPA mediated neurotoxicity effects were due to an increased production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) via increased nitric oxide synthase (iNOS), neuronal nitric oxide synthase (nNOS), 3-nitrotyrosine expression and Ca2+ influx. These results imply that hESC-based neuronal differentiation is an excellent cellular model to examine BPA-induced neurotoxicity on human neurons at the cellular and molecular level.

Gut microbiota characterization and lipid metabolism disorder found in PCB77-treated female mice.

Although the production of polychlorinated biphenyl 77 (PCB77) has already been banned globally, PCB77 is still used for a wide range of commercial purposes. Previous evidence has demonstrated that the PCB77 administration should be responsible for the gut microbiota variations and the host health risk. However, the host disorders and bacterial functions involved in PCB77 exposure remain largely unknown. Few studies have been performed to illuminate the correlation between the bacterial functions and disorders. Furthermore, it is urgently needed to find specific strains as potential biomarkers to monitor PCB77 pollution and associated disorders. This study was designed to investigate the effects of PCB77 on gut microbiota and induced disorders in female mice. Obtained results indicated that PCB77 exposure induced gut microbiota dysbiosis, obesity, hyperlipidemia, hepatic lipid accumulation, and liver injury in mice. Functional prediction based on the phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) algorithm showed that exposure to PCB77 weakened the bacterial functions relating to lipid and energy metabolism, and immune system disease. Experimental findings were consistent with the result of the PICRUSt functional prediction. Importantly, three PCB77-associated bacterial taxa were screened out as potential biomarkers for the assessment of PCB77 pollution. This study provides previously unknown knowledge linking PCB77 administration, gut microbiota functional profile and lipid abnormalities, which is of important clinical significance for therapies treating PCB77-associated diseases.

A human embryonic stem cell-based model for benzo[a]pyrene-induced embryotoxicity.

Benzo[a]pyrene (B[a]P) is one of the most common polycyclic aromatic hydrocarbons. In utero B[a]P exposure exerts multiple adverse effects on embryo development, although the underlying molecular mechanisms have still not been clearly elucidated. In the present study, we used human embryonic stem cell derived embryoid body (EB) as an in vitro model to investigate the embryotoxicity effects of B[a]P. EBs were exposed to B[a]P for 14 days, and the morphology, viability and differentiation of the cells were analyzed, in addition to the molecular changes. The results showed that B[a]P exposure repressed cell growth, impaired the morphology, and triggered apoptosis in the EBs. In addition, B[a]P reduced the gene expression levels of the ectoderm, mesoderm and endoderm biomarkers. Finally, B[a]P inhibited the epithelial-mesenchymal transition (EMT) process and the Akt/GSK-3ß signaling pathway. Taken together, B[a]P-induced aberrant EB development and apoptosis were related to EMT process and the Akt/GSK-3ß signaling pathway modulation.

Metabolomics approach reveals metabolic disorders and potential biomarkers associated with the developmental toxicity of tetrabromobisphenol A and tetrachlorobisphenol A.

Tetrabromobisphenol A and tetrachlorobisphenol A are halogenated bisphenol A (H-BPA), and has raised concerns about their adverse effects on the development of fetuses and infants, however, the molecular mechanisms are unclear, and related metabolomics studies are limited. Accordingly, a metabolomics study based on gas chromatography-mass spectrometry was employed to elucidate the molecular developmental toxicology of H-BPA using the marine medaka (Oryzias melastigmas) embryo model. Here, we revealed decreased synthesis of nucleosides, amino acids and lipids, and disruptions in the TCA (tricarboxylic acid) cycle, glycolysis and lipid metabolism, thus inhibiting the developmental processes of embryos exposed to H-BPA. Unexpectedly, we observed enhanced neural activity accompanied by lactate accumulation and accelerated heart rates due to an increase in dopamine pathway and a decrease in inhibitory neurotransmitters following H-BPA exposure. Notably, disorders of the neural system, and disruptions in glycolysis, the TCA cycle, nucleoside metabolism, lipid metabolism, glutamate and aspartate metabolism induced by H-BPA exposure were heritable. Furthermore, lactate and dopa were identified as potential biomarkers of the developmental toxicity of H-BPA and related genetic effects. This study has demonstrated that the metabolomics approach is a useful tool for obtaining comprehensive and novel insights into the molecular developmental toxicity of environmental pollutants.

Biological impact of environmental polycyclic aromatic hydrocarbons (ePAHs) as endocrine disruptors.

Polycyclic aromatic hydrocarbons (PAHs) are often detected in the environment and are regarded as endocrine disruptors. We here designated mixtures of PAHs in the environment as environmental PAHs (ePAHs) to discuss their effects collectively, which could be different from the sum of the constituent PAHs. We first summarized the biological impact of environmental PAHs (ePAHs) found in the atmosphere, sediments, soils, and water as a result of human activities, accidents, or natural phenomena. ePAHs are characterized by their sources and forms, followed by their biological effects and social impact, and bioassays that are used to investigate their biological effects. The findings of the bioassays have demonstrated that ePAHs have the ability to affect the endocrine systems of humans and animals. The pathways that mediate cell signaling for the endocrine disruptions induced by ePAHs and PAHs have also been summarized in order to obtain a clearer understanding of the mechanisms responsible for these effects without animal tests; they include specific signaling pathways (MAPK and other signaling pathways), regulatory mechanisms (chromatin/epigenetic regulation, cell cycle/DNA damage control, and cytoskeletal/adhesion regulation), and cell functions (apoptosis, autophagy, immune responses/inflammation, neurological responses, and development/differentiation) induced by specific PAHs, such as benz[a]anthracene, benzo[a]pyrene, benz[l]aceanthrylene, cyclopenta[c,d]pyrene, 7,12-dimethylbenz[a]anthracene, fluoranthene, fluorene, 3-methylcholanthrene, perylene, phenanthrene, and pyrene as well as their derivatives. Estrogen signaling is one of the most studied pathways associated with the endocrine-disrupting activities of PAHs, and involves estrogen receptors and aryl hydrocarbon receptors. However, some of the actions of PAHs are contradictory, complex, and unexplainable. Although several possibilities have been suggested, such as direct interactions between PAHs and receptors and the suppression of their activities through other pathways, the mechanisms underlying the activities of PAHs remain unclear. Thus, standardized assay protocols for pathway-based assessments are considered to be important to overcome these issues.

Potential mechanisms of benzyl isothiocyanate suppression of invasion and angiogenesis by the U87MG human glioma cell line.

Glioma is one of the most common tumors in China and chemotherapy is critical for its treatment. Recent studies showed that benzyl isothiocyanate (BITC) could inhibit the growth of glioma cells, but the mechanisms are not fully understood. This study explored the inhibitory effect of BITC on invasion and angiogenesis of U87MG human glioma cells in vitro and in vivo, as well as potential mechanisms. It was found that BITC could inhibit invasion and angiogenesis of human glioma U87MG cells by inducing cell cycle arrest at phase G2/M. It also was demonstrated that BITC decreased expression of cyclin B1, p21, MMP-2/9, VE-cadherin, CD44, CXCR4 and MTH1, the activity of the telomerase and PKCζ pathway. Microarray analysis was thus useful to explore the potential target genes related to tumorigenic processes. BITC may play important roles in the inhibition of invasion and angiogenesis of human glioma cells.