Identification of endometrial biofilm-specific proteins for in vivo diagnostics

Bacteria enter the uterus in a free-floating planktonic state, which makes them vulnerable to the host-immune system as well as exposure to effective antimicrobials. If not eliminated in a timely fashion, the bacteria will adhere to the endometrial surface and may form micro-colonies, develop a complex matrix, and switch into a biofilm community. The ability of bacteria to shift from planktonic to biofilm phenotypes has been described for both Gram-negative and Gram-positive bacteria in human mucosal tissue, indwelling catheters, and implants as well as other organs, including the equine endometrium. Unfortunately, there is currently no available assay to diagnose the condition in vivo. Literature in human medicine suggests that biofilm matrices contain both secretory and non-secretory proteins, and while some of the proteins are common among all biofilms, others may be specific for bacterial species. Therefore, the objective of this research was to identify specific proteins from biofilm-producing bacteria from mares with endometritis. Whole-cell extracts from potentially biofilm-forming bacterial isolates of Streptococcus zooepidemicus (SEZ) (n=3), Pseudomonas aeruginosa (n=1), and Escherichia coli (n=4) were analyzed by 2-D PAGE. Differentially expressed proteins were selected by overlapping 2-D PAGE biofilm proteins with planktonic culture proteins.In-gel analysis of differences (with a 3-fold cut-off)identified a total of 96 proteins for SEZ, 83 proteins for P. aeruginosa, and 69 proteins for E. coli as potential biofilm markers. Select proteins were further identified by MALDI-TOF/TOF. In SEZ, glutathione-disulfide reductase (48.8 kDa, PI 5.6), which catalyzes the reduction of glutathione disulfide to glutathione, a major cellular antioxidant, and TIGR00266 (25.3 kDa, PI 5.4) were identified as proteins of interest. For P. aeruginosa, thioredoxin A (11.8 kDa, PI 4.7), which participates in various redox reactions, osmotically inducible protein C (15.6 kDa, PI 5.9), important for cellular defense mechanism against oxidative stress, and bacterial extracellular solute-binding s, 3 family protein (33 kDa, PI 8.3) serve as potential targets. Potential biomarker candidates for E. coli include the serine protease pet autotransporter (139 kDa, PI 6.5) and outer membrane protein X (18 kDa, PI 6.6), a protein involved in numerous physiological processes including invasion, signal transduction, and binding external proteins. In conclusion, we identified biofilm-specific proteins that may serve as in vivo diagnostic markers for biofilms in uterine lavage from mares with biofilm-associated bacterial endometritis. Future research will pursue these proteins of interest for their use as diagnostic tools in the field.