RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

TADB 3.0: an updated database of bacterial toxin-antitoxin loci and associated mobile genetic elements.

TADB 3.0 (https://bioinfo-mml.sjtu.edu.cn/TADB3/) is an updated database that provides comprehensive information on bacterial types I to VIII toxin-antitoxin (TA) loci. Compared with the previous version, three major improvements are introduced: First, with the aid of text mining and manual curation, it records the details of 536 TA loci with experimental support, including 102, 403, 8, 14, 1, 1, 3 and 4 TA loci of types I to VIII, respectively; Second, by leveraging the upgraded TA prediction tool TAfinder 2.0 with a stringent strategy, TADB 3.0 collects 211 697 putative types I to VIII TA loci predicted in 34 789 completely sequenced prokaryotic genomes, providing researchers with a large-scale dataset for further follow-up analysis and characterization; Third, based on their genomic locations, relationships of 69 019 TA loci and 60 898 mobile genetic elements (MGEs) are visualized by interactive networks accessible through the user-friendly web page. With the recent updates, TADB 3.0 may provide improved in silico support for comprehending the biological roles of TA pairs in prokaryotes and their functional associations with MGEs.

DeepSecE: A Deep-Learning-Based Framework for Multiclass Prediction of Secreted Proteins in Gram-Negative Bacteria.

Proteins secreted by Gram-negative bacteria are tightly linked to the virulence and adaptability of these microbes to environmental changes. Accurate identification of such secreted proteins can facilitate the investigations of infections and diseases caused by these bacterial pathogens. However, current bioinformatic methods for predicting bacterial secreted substrate proteins have limited computational efficiency and application scope on a genome-wide scale. Here, we propose a novel deep-learning-based framework-DeepSecE-for the simultaneous inference of multiple distinct groups of secreted proteins produced by Gram-negative bacteria. DeepSecE remarkably improves their classification from nonsecreted proteins using a pretrained protein language model and transformer, achieving a macro-average accuracy of 0.883 on 5-fold cross-validation. Performance benchmarking suggests that DeepSecE achieves competitive performance with the state-of-the-art binary predictors specialized for individual types of secreted substrates. The attention mechanism corroborates salient patterns and motifs at the N or C termini of the protein sequences. Using this pipeline, we further investigate the genome-wide prediction of novel secreted proteins and their taxonomic distribution across ~1,000 Gram-negative bacterial genomes. The present analysis demonstrates that DeepSecE has major potential for the discovery of disease-associated secreted proteins in a diverse range of Gram-negative bacteria. An online web server of DeepSecE is also publicly available to predict and explore various secreted substrate proteins via the input of bacterial genome sequences.

CpxR promotes the carbapenem antibiotic resistance of Klebsiella pneumoniae by directly regulating the expression and the dissemination of blaKPC on the IncFII conjugative plasmid.

Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene blaKPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying blaKPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by blaKPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of blaKPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of blaKPC. Notably, CpxR was also found to activate the transfer of the blaKPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.

SecReT6 update: a comprehensive resource of bacterial Type VI Secretion Systems.

Type VI Secretion System (T6SS) plays significant roles in microbial activities via injecting effectors into adjacent cells or environments. T6SS increasingly gained attention due to its important influence on pathogenesis, microbial competition, etc. T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource. The SecReT6 version 3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors. To assist T6SS researches like microbial competition and regulatory mechanisms, SecReT6 v3 developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk. We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3. 17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected, analyzed and incorporated into the database. The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/ .

Proteomics analysis of lipid droplets in mouse neuroblastoma cells.

Lipid droplets (LDs) are intracellular droplets containing phospholipids and neutral lipids. It is well known that LDs are organelles with a rich proteome. In the nervous system, these droplets may play an important role in maintaining the normal physiological function of nerve cells. Moreover, LDs may relate to the neurodegenerative disorders, such as Alzheimer's disease (AD). However, more information is still needed about the function of LDs. In the study presented here, we identified the protein composition of mouse neuroblastoma (N2a) cell LDs using immunodetection and high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Seventy three LDs proteins were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to investigate the potential functions of these proteins. Subsequently, the relationships among the proteins were analyzed by constructing a protein-protein interaction (PPI) network. N2a cell LDs contain multiple Rab GTPases, chaperones, and proteins involved in ubiquitination and transport. Some of these proteins were known to modulate LD formation and were related to the function of nerve cells. This work presents the proteome of N2a cell LDs and will help to identify the role of LDs in the nervous system.