Arginine Catabolism and Polyamine Biosynthesis Pathway Disparities Within \u3ci\u3eFrancisella tularensis\u3c/i\u3e Subpopulations

Francisella tularensis is a highly infectious zoonotic pathogen with as few as 10 organisms causing tularemia, a disease that is fatal if untreated. Although F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) share over 99.5% average nucleotide identity, notable differences exist in genomic organization and pathogenicity. The type A clade has been further divided into subtypes A.I and A.II, with A.I strains being recognized as some of the most virulent bacterial pathogens known. In this study, we report on major disparities that exist between the F. tularensis subpopulations in arginine catabolism and subsequent polyamine biosynthesis. The genes involved in these pathways include the speHEA and aguAB operons, along with metK. In the hypervirulent F. tularensis A.I clade, such as the A.I prototype strain SCHU S4, these genes were found to be intact and highly transcribed. In contrast, both subtype A.II and type B strains have a truncated speA gene, while the type B clade also has a disrupted aguA and truncated aguB. Ablation of the chromosomal speE gene that encodes a spermidine synthase reduced subtype A.I SCHU S4 growth rate, whereas the growth rate of type B LVS was enhanced. These results demonstrate that spermine synthase SpeE promotes faster replication in the F. tularensis A.I clade, whereas type B strains do not rely on this enzyme for in vitro fitness. Our ongoing studies on amino acid and polyamine flux within hypervirulent A.I strains should provide a better understanding of the factors that contribute to F. tularensis pathogenicity

MS/MS spectrum for the spermidine adduct on Q267 of <i>F</i>. <i>tularensis</i> rUsp/His₆.

The structures of y1 to y9 ions are shown; none of the y-ions include the mass for spermidine. The added mass of 128.1 Da from spermidine is on glutamine. The MH+2 parent ion at m/z 620.8537 supports the presence of spermidine in this peptide. A peak for the parent ion minus water is at 611.8492 in charge state +2.</p

Western blot hybridized with affinity purified polyclonal antibodies to denatured <i>F</i>. <i>tularensis</i> rUsp/His₆.

Lane 1, 0.5 μg F. tularensis lysate. Lane 2, 0.01 μg purified F. tularensis rUsp/His6. Lane 3, protein molecular weight markers with sizes denoted in kilodaltons (kDa). Lanes 4 and 5, combined 0.5 μg F. tularensis lysate plus 0.01 μg F. tularensis rUsp/His6.</p

MS/MS spectrum for acetylated lysine 238 in <i>F</i>. <i>tularensis</i> rUsp/His₆.

The exact location of the 42.01 Da acetyl adduct is defined by the y1 to y10 ion series and the y11 ion whose structures are shown. The mass of the y11 ion at 1279.71 Da is 42.01 Da higher than the mass of the unmodified y11 ion. Masses of the y1 to y10 ions are consistent with the absence of an adduct on residues NDLIVVGSHR. The MH+2 parent ion has a mass of m/z 832.9394.</p

Uncropped, unadjusted Western blot in text as Fig 1.

Uncropped, unadjusted Western blot in text as Fig 1.</p

S1 Graphical abstract -

Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.</div

MS/MS spectrum for putrescine adduct on glutamine 267 of <i>F</i>. <i>tularensis</i> rUsp/His6.

The b5 to b9 ions support putrescine on Q267 because they carry an added mass of 71 Da. The y-ions do not have an added mass of 71, which means putrescine is not on residues VDVLVVR. The MH+3 parent ion mass is m/z 545.3059. The parent ion minus a water molecule, MH-H2O+3, has a peak at m/z 539.2938.</p