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. 2021 Jan 13;11(1):972.
doi: 10.1038/s41598-020-79611-5.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

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Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

Jan Felix et al. Sci Rep. .

Abstract

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Phylogenetic position of LdcF sequences within the wing-domain containing LAOdc family. (a) Tree showing the relationships of 553 WING-containing AAT-fold decarboxylase sequences. The tree is a cladogram, meaning that the length of the branches has no evolutionary significance. The cladogram is rooted according to Carriel et al.. The colour of leaves corresponds to the LdcI, LdcC, AdcI, OdcC, OdcI, and LdcA subfamilies. The group corresponding to Francisellaceae sequences (referred as to LdcF) is indicated in pink. F. tularensis, E. coli, and P. aeruginosa sequences are indicated by grey arrows. Grey circles at branches correspond to ultrafast bootstrap values > 95%. The taxonomy of species (Class) is represented by a coloured strip. (b) Phylogram corresponding to the LdcI, LdcC, and LdcF subtree (122 sequences). The scale bar corresponds to the average number of substitutions per site. The length of branches is proportional to genetic divergence.
Figure 2
Figure 2
Genomic context of LdcI, LdcC, and LdcF coding genes (black arrows) in a subsample of representative species. Other conserved neighbour genes are highlighted with colour. The taxonomy of species (Class) is indicated in brackets.
Figure 3
Figure 3
Crystal structure of the F. novicida lysine decarboxylase LdcF. (a) Front (upper panel) and side view (lower panel) of decameric LdcF, with one highlighted dimer coloured blue and purple, while other dimers are coloured light and dark grey. The covalently bound pyridoxal phosphate (PLP) cofactor is shown as yellow spheres. (b) Front (upper panel) and side view (lower panel) of an LdcF dimer extracted from the decamer shown in (a). In one monomer, different domains are coloured according to a rainbow scheme (WING domain: blue, linker: green, PLP-binding domain: yellow, AAT-like domain: orange, C-terminal domain: red), with accompanying annotated amino acid residue ranges. (c) Comparison between the AAT-like domains (termed ppGpp binding domain in E. coli LdcI) of F. novicida LdcF (left) and E. coli LdcI (right). Residues of E. coli LdcI involved in ppGpp binding, and the corresponding residues in the AAT-like domain of F. novicida LdcF are annotated and shown as sticks. Domains are coloured as in (b), but using lighter tints for E. coli LdcI. (d) Comparison between the RavA-binding site in E. coli LdcI, and the corresponding region in F. novicida LdcF. Residues of E. coli LdcI involved in RavA binding, and the corresponding residues of F. novicida LdcF are annotated and shown as sticks.
Figure 4
Figure 4
Alignment of F. novicida LdcF, E. coli LdcI and LdcC, and P. aeruginosa LdcA using Clustal Omega. Partially and fully conserved residues are annotated with ‘:’ and ‘*’ respectively. Domains are coloured according to a rainbow scheme (WING domain: blue, linker: green, PLP-binding domain: yellow, AAT-like domain: orange, C-terminal domain: red), and secondary structure elements are annotated. ppGpp and RavA-interaction sites are highlighted using red and blue transparent boxes respectively.
Figure 5
Figure 5
Growth and biofilm formation of F. novicida. (a) F. novicida WT (solid lines) and ΔldcF (dotted lines) were grown under shaking at 37 °C in MMH adjusted at pH 2.5 (green), pH 4 (blue), pH 6.6 (red), pH 8 (purple) or pH 10 (black) and the bacterial growth was monitored by OD600nm measurement. Results are representative of three independent trials. (b) F. novicida WT (grey columns) and ΔldcF (dotted columns) were grown for 24 h under static conditions at 37 °C in a 96-wells plates. The bacterial growth was evaluated by measurement of OD600nm and the biofilm biomass was further determined by OD595nm after Crystal violet staining. This graph corresponds to mean ± s.e.m. of three independent experiments, with at least 4 technical replicates each.
Figure 6
Figure 6
Sensitivity of F. novicida to oxidative stress. Exponential growth phase bacteria diluted in MMH were exposed to increasing concentration of (a) H2O2 (b) methyl viologen or (c) menadione for 1 h at 37 °C and 3 µl of the cell suspensions were spotted on PVX-CHA plates. These pictures are representative of at least 3 distinct experiments performed in duplicate each. The antibacterial activity of oxidative compounds was also quantified by (d) cfu counting from a cell suspension containing 108 bacteria incubated for 1 h under shaking in presence of H2O2 or by disk diffusion assays with (e) methyl viologen or menadione (f) as detailled in materials and methods section. Histograms correspond to the mean ± s.e.m. of at least 3 distinct experiments performed in duplicate (*P < 0.05, **P < 0.01).
Figure 7
Figure 7
Replication of F. novicida strains within the J774 macrophage-like cell line. (a) F. novicida WT (black circles, solid line), ΔldcF (white circles, dotted line) and ΔldcF::ldcF (black triangles, solid line) were inoculated at a MOI of 100:1 and intracellular bacteria were enumerated by cfu counting at different times post infection (b) Production of ROS evaluated at 24 h after infection of macrophages with a MOI of 1,000:1 using the redox-sensitive dye DCFA detected by fluorescence spectroscopy. Data correspond to mean ± s.e.m. of 4 distinct experiments and after subtraction of background values obtained with uninfected macrophages. *P < 0.05.

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