Meta-analytic approach to the accurate prediction of secreted virulence effectors in gram-negative bacteria

Background: Many pathogens use a type III secretion system to translocate virulence proteins (called effectors) in order to adapt to the host environment. To date, many prediction tools for effector identification have been developed. However, these tools are insufficiently accurate for producing a list of putative effectors that can be applied directly for labor-intensive experimental verification. This also suggests that important features of effectors have yet to be fully characterized. Results: In this study, we have constructed an accurate approach to predicting secreted virulence effectors from Gram-negative bacteria. This consists of a support vector machine-based discriminant analysis followed by a simple criteria-based filtering. The accuracy was assessed by estimating the average number of true positives in the top-20 ranking in the genome-wide screening. In the validation, 10 sets of 20 training and 20 testing examples were randomly selected from 40 known effectors of Salmonella enterica serovar Typhimurium LT2. On average, the SVM portion of our system predicted 9.7 true positives from 20 testing examples in the top-20 of the prediction. Removal of the N-terminal instability, codon adaptation index and ProtParam indices decreased the score to 7.6, 8.9 and 7.9, respectively. These discrimination features suggested that the following characteristics of effectors had been uncovered: unstable N-terminus, non-optimal codon usage, hydrophilic, and less aliphathic. The secondary filtering process represented by coexpression analysis and domain distribution analysis further refined the average true positive counts to 12.3. We further confirmed that our system can correctly predict known effectors of P. syringae DC3000, strongly indicating its feasibility. Conclusions: We have successfully developed an accurate prediction system for screening effectors on a genome-wide scale. We confirmed the accuracy of our system by external validation using known effectors of Salmonella and obtained the accurate list of putative effectors of the organism. The level of accuracy was sufficient to yield candidates for gene-directed experimental verification. Furthermore, new features of effectors were revealed: non-optimal codon usage and instability of the N-terminal region. From these findings, a new working hypothesis is proposed regarding mechanisms controlling the translocation of virulence effectors and determining the substrate specificity encoded in the secretion system.