ABSTRACT
The efficient natural transformation of Neisseria meningitidis allows the rapid construction of bacterial mutants in which the genes of interest are interrupted or replaced by antibiotic-resistance cassettes. However, this proved to be a double-edged sword, i.e., although facilitating the genetic characterization of this important human pathogen, it has limited the development of strategies for constructing markerless mutants without antibiotic-resistance markers. In addition, efficient tools for complementation or labeling are also lacking in N. meningitidis. In this study, we significantly expand the meningococcal genetic toolbox by developing new and efficient tools for the construction of markerless mutants (using a dual counterselection strategy), genetic complementation (using integrative vectors), and cell labeling (using a self-labeling protein tag). This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.IMPORTANCENeisseria meningitidis and Neisseria gonorrhoeae are two important human pathogens. Research focusing on these bacteria requires genetic engineering, which is facilitated by their natural ability to undergo transformation. However, the ease of mutant engineering has led the Neisseria community to neglect the development of more sophisticated tools for gene editing, particularly for N. meningitidis. In this study, we have significantly expanded the meningococcal genetic toolbox by developing novel and efficient tools for markerless mutant construction, genetic complementation, and cell tagging. This expanded toolbox paves the way for more in-depth genetic characterization of N. meningitidis and might also be useful in other Neisseria species.
Subject(s)
Aminophenols , Benzodioxoles , Cystic Fibrosis , Cytokines , Indoles , Mucosal-Associated Invariant T Cells , Pyrazoles , Quinolones , Humans , Cystic Fibrosis/drug therapy , Aminophenols/therapeutic use , Child , Benzodioxoles/therapeutic use , Indoles/therapeutic use , Quinolones/therapeutic use , Pyrazoles/therapeutic use , Male , Female , Mucosal-Associated Invariant T Cells/immunology , Mucosal-Associated Invariant T Cells/drug effects , Cytokines/metabolism , Pyridines/therapeutic use , Adolescent , Drug Combinations , Quinolines/therapeutic use , Pyrroles/therapeutic use , Child, Preschool , PyrrolidinesABSTRACT
Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.