ApoE Mimetic Peptide COG1410 Kills Mycobacterium smegmatis via Directly Interfering ClpC's ATPase Activity.

Antimicrobial peptides (AMPs) hold promise as alternatives to combat bacterial infections, addressing the urgent global threat of antibiotic resistance. COG1410, a synthetic peptide derived from apolipoprotein E, has exhibited potent antimicrobial properties against various bacterial strains, including Mycobacterium smegmatis. However, our study reveals a previously unknown resistance mechanism developed by M. smegmatis against COG1410 involving ClpC. Upon subjecting M. smegmatis to serial passages in the presence of sub-MIC COG1410, resistance emerged. The comparative genomic analysis identified a point mutation in ClpC (S437P), situated within its middle domain, which led to high resistance to COG1410 without compromising bacterial fitness. Complementation of ClpC in mutant restored bacterial sensitivity. In-depth analyses, including transcriptomic profiling and in vitro assays, uncovered that COG1410 interferes with ClpC at both transcriptional and functional levels. COG1410 not only stimulated the ATPase activity of ClpC but also enhanced the proteolytic activity of Clp protease. SPR analysis confirmed that COG1410 directly binds with ClpC. Surprisingly, the identified S437P mutation did not impact their binding affinity. This study sheds light on a unique resistance mechanism against AMPs in mycobacteria, highlighting the pivotal role of ClpC in this process. Unraveling the interplay between COG1410 and ClpC enriches our understanding of AMP-bacterial interactions, offering potential insights for developing innovative strategies to combat antibiotic resistance.

[An improved extraction and nonradioactive thin-layer chromatography detection method of mycolic acid].

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, 14C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of 14C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.

Structural insights into RNase J that plays an essential role in Mycobacterium tuberculosis RNA metabolism.

Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the ß-CASP and the ß-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.

Tuning the corona-core ratio of polyplex micelles for selective oligonucleotide delivery to hepatocytes or hepatic immune cells.

Targeted delivery of oligonucleotides or small molecular drugs to hepatocytes, the liver's parenchymal cells, is challenging without targeting moiety due to the highly efficient mononuclear phagocyte system (MPS) of the liver. The MPS comprises Kupffer cells and specialized sinusoidal endothelial cells, efficiently clearing nanocarriers regardless of their size and surface properties. Physiologically, this non-parenchymal shield protects hepatocytes; however, these local barriers must be overcome for drug delivery. Nanocarrier structural properties strongly influence tissue penetration, in vivo pharmacokinetics, and biodistribution profile. Here we demonstrate the in vivo biodistribution of polyplex micelles formed by polyion complexation of short interfering (si)RNA with modified poly(ethylene glycol)-block-poly(allyl glycidyl ether) (PEG-b-PAGE) diblock copolymer that carries amino moieties in the side chain. The ratio between PEG corona and siRNA complexed PAGE core of polyplex micelles was chemically varied by altering the degree of polymerization of PAGE. Applying Raman-spectroscopy and dynamic in silico modeling on the polyplex micelles, we determined the corona-core ratio (CCR) and visualized the possible micellar structure with varying CCR. The results for this model system reveal that polyplex micelles with higher CCR, i.e., better PEG coverage, exclusively accumulate and thus allow passive cell-type-specific targeting towards hepatocytes, overcoming the macrophage-rich reticuloendothelial barrier of the liver.

Acetylation of Cyclic AMP Receptor Protein by Acetyl Phosphate Modulates Mycobacterial Virulence.

The success of Mycobacterium tuberculosis (Mtb) as a pathogen is partly attributed to its ability to sense and respond to dynamic host microenvironments. The cyclic AMP (cAMP) receptor protein (CRP) is closely related to the pathogenicity of Mtb and plays an important role in this process. However, the molecular mechanisms guiding the autoregulation and downstream target genes of CRP while Mtb responds to its environment are not fully understood. Here, it is demonstrated that the acetylation of conserved lysine 193 (K193) within the C-terminal DNA-binding domain of CRP reduces its DNA-binding ability and inhibits transcriptional activity. The reversible acetylation status of CRP K193 was shown to significantly affect mycobacterial growth phenotype, alter the stress response, and regulate the expression of biologically relevant genes using a CRP K193 site-specific mutation. Notably, the acetylation level of K193 decreases under CRP-activating conditions, including the presence of cAMP, low pH, high temperature, and oxidative stress, suggesting that microenvironmental signals can directly regulate CRP K193 acetylation. Both cell- and murine-based infection assays confirmed that CRP K193 is critical to the regulation of Mtb virulence. Furthermore, the acetylation of CRP K193 was shown to be dependent on the intracellular metabolic intermediate acetyl phosphate (AcP), and deacetylation was mediated by NAD+-dependent deacetylases. These findings indicate that AcP-mediated acetylation of CRP K193 decreases CRP activity and negatively regulates the pathogenicity of Mtb. We believe that the underlying mechanisms of cross talk between transcription, posttranslational modifications, and metabolites are a common regulatory mechanism for pathogenic bacteria. IMPORTANCE Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, and the ability of Mtb to survive harsh host conditions has been the subject of intensive research. As a result, we explored the molecular mechanisms guiding downstream target genes of CRP when Mtb responds to its environment. Our study makes a contribution to the literature because we describe the role of acetylated K193 in regulating its binding affinity to target DNA and influencing the virulence of mycobacteria. We discovered that mycobacteria can regulate their pathogenicity through the reversible acetylation of CRP K193 and that this reversible acetylation is mediated by AcP and a NAD+-dependent deacetylase. The regulation of CRPMtb by posttranslational modifications, at the transcriptional level, and by metabolic intermediates contribute to a better understanding of its role in the survival and pathogenicity of mycobacteria.

Mycobacterium tuberculosis PE_PGRS19 Induces Pyroptosis through a Non-Classical Caspase-11/GSDMD Pathway in Macrophages.

The PE/PPE protein family commonly exists in pathogenic species, such as Mycobacterium tuberculosis, suggesting a role in virulence and its maintenance. However, the exact role of most PE/PPE proteins in host-pathogen interactions remains unknown. Here, we constructed a recombinant Mycobacterium smegmatis expressing M. tuberculosis PE_PGRS19 (Ms_PE_PGRS19) and found that PE_PGRS19 overexpression resulted in accelerated bacterial growth in vitro, increased bacterial survival in macrophages, and enhanced cell damage capacity. Ms_PE_PGRS19 also induced the expression of pro-inflammatory cytokines, such as IL-6, TNF-α, IL-1ß, and IL-18. Furthermore, we demonstrated that Ms_PE_PGRS19 induced cell pyroptosis by cleaving caspase-11 via a non-classical pathway rather than caspase-1 activation and further inducing the cleavage of gasdermin D, which led to the release of IL-1ß and IL-18. To the best of our current knowledge, this is the first report of a PE/PPE family protein activating cell pyroptosis via a non-classical pathway, which expands the knowledge on PE/PPE protein functions, and these pathogenic factors involved in bacterial survival and spread could be potential drug targets for anti-tuberculosis therapy.

[Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis].

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Atomistic Descriptors for Machine Learning Models of Solubility Parameters for Small Molecules and Polymers.

Descriptors derived from atomic structure and quantum chemical calculations for small molecules representing polymer repeat elements were evaluated for machine learning models to predict the Hildebrand solubility parameters of the corresponding polymers. Since reliable cohesive energy density data and solubility parameters for polymers are difficult to obtain, the experimental heat of vaporization ΔHvap of a set of small molecules was used as a proxy property to evaluate the descriptors. Using the atomistic descriptors, the multilinear regression model showed good accuracy in predicting ΔHvap of the small-molecule set, with a mean absolute error of 2.63 kJ/mol for training and 3.61 kJ/mol for cross-validation. Kernel ridge regression showed similar performance for the small-molecule training set but slightly worse accuracy for the prediction of ΔHvap of molecules representing repeating polymer elements. The Hildebrand solubility parameters of the polymers derived from the atomistic descriptors of the repeating polymer elements showed good correlation with values from the CROW polymer database.