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ABSTRACT
FipB, an essential virulence factor in the highly virulent Schu S4 strain of F. tularensis subsp. tularensis, shares sequence similarity with Disulfide Bond formation (Dsb) proteins, which can have oxidoreductase, isomerase, or chaperone activity. To further explore FipB's role in virulence potential substrates were identified by co-purification and 2D gel electrophoresis, followed by protein sequencing using mass spectrometry. A total of 119 potential substrates were identified. Proteins with predicted enzymatic activity were prevalent, and there were 19 proteins that had been previously identified as impacting virulence. Among the potential substrates were IglC, IglB, and PdpB, three components of the Francisella Type Six Secretion System (T6SS), which is also essential for virulence. T6SS are widespread in Gram-negative pathogens, but have not been reported to be dependent on Dsb-like proteins for assembly or function. The presented results suggest that FipB affects IglB and IglC substrates differently. In a fipB mutant there were differences in free sulfhydryl accessibility of IglC, but not IglB, when compared to wild-type bacteria. However, for both proteins FipB appears to act as a chaperone that facilitates proper folding and conformation. Understanding the role FipB plays the assembly and structure in this T6SS may reveal critical aspects of assembly that are common and novel among this widely distributed class of secretion systems.
KEYWORDS:
Abbreviations
| AMS | = | 4-acetoamido-4′-maleimidylstilbene 2,2′-disulfonate |
| CDM | = | Chamberlain's defined media |
| COG | = | Clusters of Orthologous Genes |
| CXXC | = | Cysteine- any amino acid- any amino acid-Cysteine |
| Dsb | = | disulfide bond |
| DTT | = | 1,4-Dithiothreitol |
| Hcp | = | Hemolysin coregulated protein |
| IPTG | = | Isopropyl β-D-1-thiogalactopyranoside |
| LVS | = | Live Vaccine Strain |
| MAL-PEG | = | Methoxypolyethylene glycol maleimide |
| MW | = | molecular weight |
| nOctylGlu | = | n-Octyl glucoside |
| T6SS | = | Type six secretion system |
| TCEP | = | Tris (2-carboxyethyl) phosphine |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the W.M. Keck Biomedical Mass Spectrometry Laboratory and Nick Sherman for protein sequencing support. We also thank Thomas Kawula for the plasmid encoding His-tagged IglC, Carol Gilchrist and Girija Ramakrishnan for critical reading of the manuscript.
Funding
The Keck lab is funded by a grant from the University of Virginia's School of Medicine. This work was supported by R56 AI091746 to BJM, and T32 AI 055432 to MEC and GBM.