Dual functions: A coumarin-chalcone conjugate inhibits cyclic-di-GMP and quorum-sensing signaling to reduce biofilm formation and virulence of pathogens.

Antibiotic resistance or tolerance of pathogens is one of the most serious global public health threats. Bacteria in biofilms show extreme tolerance to almost all antibiotic classes. Thus, use of antibiofilm drugs without bacterial-killing effects is one of the strategies to combat antibiotic tolerance. In this study, we discovered a coumarin-chalcone conjugate C9, which can inhibit the biofilm formation of three common pathogens that cause nosocomial infections, namely, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli, with the best antibiofilm activity against P. aeruginosa. Further investigations indicate that C9 decreases the synthesis of the key biofilm matrix exopolysaccharide Psl and bacterial second messenger cyclic-di-GMP. Meanwhile, C9 can interfere with the regulation of the quorum sensing (QS) system to reduce the virulence of P. aeruginosa. C9 treatment enhances the sensitivity of biofilm to several antibiotics and reduces the survival rate of P. aeruginosa under starvation or oxidative stress conditions, indicating its excellent potential for use as an antibiofilm-forming and anti-QS drug.

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system.

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections.

Mycobacterium tuberculosis Rv1473 is a novel macrolides ABC Efflux Pump regulated by WhiB7.

AIM: To characterize a novel macrolide ATP binding cassette efflux pump encoding gene Rv1473 which might be involved in antibiotic resistance. METHODS: Mycobacterium smegmatis was used as a surrogate model for pathogenic mycobacteria, drug susceptibility assays and ethidium bromide accumulation assay were harnessed to verify drug resistance. The real-time quantitative PCR was used to evaluate the transcription levels of WhiB7 and Ms3140 upon exposure to macrolides. RESULTS: Rv1473 contributes to macrolides resistance via efflux mechanisms, and was positively regulated by the transcription factor WhiB7 upon macrolides exposure. CONCLUSION: Rv1473 is a novel ATP binding cassette efflux pump involved in mycobacterium intrinsic antibiotics resistance via efflux mechanism. This finding will facilitate novel antibiotic discovery and the treatment of pathogen, especially for nontuberculous mycobacteria.

The effect of high intensity focused ultrasound on the treatment of liver cancer and patients' immunity.

OBJECTIVE: To investigate the effects of high intensity focused ultrasound on liver function, tumor markers and survival rate of hepatocellular carcinoma patients. METHODS: Ninety six cases with primary liver cancer patients, consisting of 66 males and 30 females, were enrolled in this study and treated with high intensity focused ultrasound combined with stereotactic segmentation dose radiation, low frequency for 10 times, followed by analysis of KPS score of liver cancer, Child-Pugh, grading and staging of liver cancer, 3 months, 6 months, 1 year of clinical symptom remission rate, tumor markers, liver function, survival rate, as well as the change of immune related cytokines. RESULTS: Three months after high intensity focused ultrasound treatment, abdominal distension abdominal pain, jaundice symptoms, anorexia and ascites were significantly relieved compared with before treatment (P< 0.05). At 3 months after treatment, levels of AFP and CA199 were significantly reduced than before treatment (P< 0.05). Meanwhile, Child-Pugh classification score was significantly decreased at 3 months after treatment compared with before treatment, which was further decreased at 6 months after treatment than 3 months after treatment (P< 0.05). In addition, ALT, AST, AKP, propagated and TBIL level at 3 months after treatment displayed no differences to those before treatment but was significantly decreased at 6 months treatment (P< 0.05). Moreover, the late stages of liver cancer, the lower survival rate after treatment. Furthermore, the levels of NK, CD3, CD4, CD8 and CD4/CD8 cytokines were significantly increased at 3 months after treatment (P< 0.05), together with significantly increased levels of IFN-r and IL-2 and decreased levels of IL-4 and IL-10 (P< 0.05). CONCLUSION: High intensity focused ultrasound can effectively improve liver function, increase the survival rate and enhance immune function of patients with liver cancer.

Mycobacterium tuberculosis PPE44 (Rv2770c) is involved in response to multiple stresses and promotes the macrophage expression of IL-12 p40 and IL-6 via the p38, ERK, and NF-κB signaling axis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H2O2, SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage.

Proteomic analysis of lysine succinylation of the human pathogen Histoplasma capsulatum.

Histoplasma capsulatum, the causative agent of histoplasmosis (also called "Darling's disease"), can affect both immunocompetent and immunocompromised hosts. Post-translational protein modification by lysine succinylation (Ksuc) is a frequent occurrence in eukaryote and prokaryote. Recently, the roles of succinylation and its regulatory enzymes in regulating metabolic pathway in bacteria, mammalian and fungus were highlighted. Here, we report the first global profiling of lysine succinylation, with 463 modification sites in 202 proteins from H. capsulatum NAM1 identified, coupling immune-affinity enrichment using an anti-succinyllysine antibody with mass spectrometry. The bioinformatics results including GO functional and enrichment analysis showed that these succinylated proteins are mainly involved in central metabolism and protein synthesis, consistent with previous reports. 13 lysine succinylation sites on histones including H2A, H2B, H3 and H4 in H. capsulatum were firstly reported. The data is a good resource for further functional characterization of lysine succinylation in H. capsulatum. BIOLOGICAL SIGNIFICANCE: H. capsulatum is the causative agent of lung disease histoplasmosis. The ability of H. capsulatum yeasts to infect and proliferate within macrophages as an intracellular pathogen can be contributed to several virulence factors and metabolic regulation. Lysine succinylation was recently shown to play a critical role in the metabolism regulation of Candida albicans. H. capsulatum succinylated proteins were firstly characterized in this work, and bioinformatics results showed that this modification may also be relevant with central metabolism in H. capsulatum. New succinylation sites on histones were reported. This represents an important resource to address the function of H. capsulatum lysine succinylation.

Overexpression of Rv2788 increases mycobacterium stresses survival.

Mycobacterium tuberculosis, the causative agent of tuberculosis-one of the most devastating infectious diseases, is a successful intracellular pathogen capable of surviving diverse stresses. Unveiling the molecular mechanisms governing this superior adaptation will inspire better control measures against tuberculosis. To define the role of Rv2788, a manganese-dependent transcriptional repressor, M.smegmatis was used as the host strain for heterologous expression Rv2788. Rv2788 can significantly change the colony morphology and fatty acids and permeability of cell wall, enhance the growth of the recombinants and resistance to diverse stresses, such as hydrogen peroxide (H2O2), diamide exposure, surface stress, acidic condition, multiple antibiotics treatment including chloramphenicol, vancomycin and amikacin. The dysregulation of the target genes of Rv2788, such as whiB1 and lexA, might underpin such phenotypes. The results implicate important roles of Rv2788 in the survival of Mycobacterium under stresses, and might represent ideal novel antibiotics target candidate.

Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators.

Mycobacterium tuberculosis PE13 (Rv1195) manipulates the host cell fate via p38-ERK-NF-κB axis and apoptosis.

PE/PPE family proteins are mycobacteria unique molecules, named after their N-terminal conserved PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains. Mycobacterium tuberculosis (Mtb) PE family gene encoded cell surface proteins are previously reported to be involved in virulence and interaction with host. To explore the role of a novel PE member (PE13, Rv1195), M. smegmatis was used as surrogate host. The study showed that Rv1195 was a cell wall associated protein. Rv1195 can enhance the survival of recombinants under stress conditions such as H2O2, SDS, low pH. This is largely due to the upregulated transcription of Rv1195, since diverse stresses can increase the promoter activity of Rv1195 gene, consistent with enhanced survival within macrophages. Ms_Rv1195 infection also increased the production of interlukin-6 (IL-6) and IL-1ß from macrophages, while decreased the secretion of suppressor of cytokine signaling 3 (SOCS3) in comparison with the vector-only control. The cell death was also precipitated by the Ms_Rv1195 infection. Inhibitors treatment showed that the p38-ERK-NF-κB axis was involved in the Rv1195 triggered change of IL-6 and IL-1ß expression. In summary, we showed that PE13 (Rv1195) is a new PE family member actively engaged in the interaction between Mycobacterium and host, signaling through p38-ERK-NF-κB axis and apoptosis.

Proteome-wide Lysine Glutarylation Profiling of the Mycobacterium tuberculosis H37Rv.

Lysine glutarylation, a new protein posttranslational modification (PTM), was recently identified and characterized in both prokaryotic and eukaryotic cells. To explore the distribution of lysine glutarylation in Mycobacterium tuberculsosis, by using a comprehensive method combining the immune affinity peptide enrichment by the glutaryl-lysine antibody with LC-MS, we finally identified 41 glutarylation sites in 24 glutarylated proteins from M. tuberculosis. These glutarylated proteins are involved in various cellular functions such as translation and metabolism and exhibit diverse subcellular localizations. Three common glutarylated proteins including 50S ribosomal protein L7/L12, elongation factor Tu, and dihydrolipoamide succinyltransferase are shared between Escherichia coli and M. tuberculosis. Moreover, comparison with other PTMs characterized in M. tuberculosis, 15 glutarylated proteins, are found to be both acetylated and succinylated. Notably, several stress-response-associated proteins including HspX are glutarylated. Our data provide the first analysis of M. tuberculosis lysine glutarylated proteins. Further studies on the role of the glutarylated proteins will unveil the molecular mechanisms of glutarylation underlying M. tuberculosis physiology and pathogenesis.

Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum.

Histoplasma capsulatum is the causative agent of human histoplasmosis, which can cause respiratory and systemic mycosis in immune-compromised individuals. Lysine acetylation, a protein posttranslational protein modification, is widespread in both eukaryotes and prokaryotes. Although increasing evidence suggests that lysine acetylation may play critical roles in fungus physiology, very little is known about its extent and function in H. capsulatum. To comprehensively profile protein lysine acetylation in H. capsulatum, we performed a global acetylome analysis through peptide prefractionation, antibody enrichment, and LC-MS/MS analysis, identifying 775 acetylation sites on 456 acetylated proteins; and functionally analysis showing their involvement in different biological processes. We defined six types of acetylation site motifs, and the results imply that lysine residue of polypeptide with tyrosine at the -1 and +1 positions, histidine at the +1 position, and phenylalanine (F) at the +1 and +2 position is a preferred substrate of lysine acetyltransferase. Moreover, some virulence factors candidates including calmodulin and DnaK are acetylated. In conclusion, our data set may serve as an important resource for the elucidation of associations between functional protein lysine acetylation and virulence in H. capsulatum.

Roles of Protein N-Myristoylation and Translational Medicine Applications.

Protein N-myristoylation is a ubiquitous cotranslational and posttranslational modification catalyzed by myristoyl CoA:protein N-myristoyltransferase (NMT), which attaches myristate, a rare 14-carbon saturated fatty acid, to an N-terminal glycine of some eukaryotic and virus proteins. This protein modification triggers dynamic protein-protein and protein-membrane interactions implicated in diverse physiological processes. This review summarizes the NMT catalytic mechanism and demyristoylation. Of special interest are the primary roles of N-myristoylated protein in signaling, protein targeting, tumorigenesis, apoptosis, virus assembly, and morphology change, as well as the regulation of N-myristoylation and NMT inhibitors.

[The roles of epigenetics and protein post-translational modifications in bacterial antibiotic resistance].

The increasing antibiotic resistance is now threatening to take us back to a pre-antibiotic era. Bacteria have evolved diverse resistance mechanisms, on which in-depth research could help the development of new strategies to control antibiotic-resistant infections. Epigenetic alterations and protein post-translational modifications (PTMs) play important roles in multiple cellular processes such as metabolism, signal transduction, protein degradation, DNA replication regulation and stress response. Recent studies demonstrated that epigenetics and PTMs also play vital roles in bacterial antibiotic resistance. In this review, we summarize the regulatory roles of epigenetic factors including DNA methylation and regulatory RNAs as well as PTMs such as phosphorylation and succinylation in bacterial antibiotic resistance, which may provide innovative perspectives on selecting antibacterial targets and developing antibiotics.