Understanding the contribution of metabolism to Mycobacterium tuberculosis drug tolerance

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Functional and Computational Analysis of Amino Acid Patterns Predictive of Type III Secretion System Substrates in Pseudomonas syringae

Bacterial type III secretion systems (T3SSs) deliver proteins called effectors into eukaryotic cells. Although N-terminal amino acid sequences are required for translocation, the mechanism of substrate recognition by the T3SS is unknown. Almost all actively deployed T3SS substrates in the plant pathogen Pseudomonas syringae pathovar tomato strain DC3000 possess characteristic patterns, including (i) greater than 10% serine within the first 50 amino acids, (ii) an aliphatic residue or proline at position 3 or 4, and (iii) a lack of acidic amino acids within the first 12 residues. Here, the functional significance of the P. syringae T3SS substrate compositional patterns was tested. A mutant AvrPto effector protein lacking all three patterns was secreted into culture and translocated into plant cells, suggesting that the compositional characteristics are not absolutely required for T3SS targeting and that other recognition mechanisms exist. To further analyze the unique properties of T3SS targeting signals, we developed a computational algorithm called TEREE (Type III Effector Relative Entropy Evaluation) that distinguishes DC3000 T3SS substrates from other proteins with a high sensitivity and specificity. Although TEREE did not efficiently identify T3SS substrates in Salmonella enterica, it was effective in another P. syringae strain and Ralstonia solanacearum. Thus, the TEREE algorithm may be a useful tool for identifying new effector genes in plant pathogens. The nature of T3SS targeting signals was additionally investigated by analyzing the N-terminus of FtsX, a putative membrane protein that was classified as a T3SS substrate by TEREE. Although the first 50 amino acids of FtsX were unable to target a reporter protein to the T3SS, an AvrPto protein substituted with the first 12 amino acids of FtsX was translocated into plant cells. These results show that the T3SS targeting signals are highly mutable and that secretion may be directed by multiple features of substrates

Schematic diagram of AvrPto mutants examined in this study.

<p>Plasmids were constructed that express wild-type or mutant versions of the <i>avrPto</i> gene fused in frame to either <i>FLAG</i> epitope tag sequences or <i>cya</i> (calmodulin-dependent adenylate cyclase). Each gene was expressed from an upstream <i>lac</i> promoter (P<i>_lac</i>). The sequences of the first 50 amino acids of each protein are shown above the <i>avrPto</i> gene. Amino acids in the mutant proteins that differ from the wild-type AvrPto sequence are underlined. Dashes within the AvrPto_Δ2–12 sequence indicate deleted residues.</p

Comparison of TEREE to other computational T3SS substrate prediction models.

a<p>Values were calculated by dividing the number of validated effectors, or true positives, by the sum of the true positives and false negatives. The second columns of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036038#pone.0036038.s003" target="_blank">Tables S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036038#pone.0036038.s004" target="_blank">S4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036038#pone.0036038.s004" target="_blank">S5</a> list the validated effectors for <i>P. s.</i> phaseolicola 1448a, <i>S. e.</i> Typhimurium LT2, and <i>R. solanacearum</i> GMI1000, respectively.</p>b<p>Values were calculated by dividing the number of true negatives (non-substrates of the T3SS) by the sum of the false positives and true negatives.</p>c<p>The sensitivity and specificity values for SIEVE and BPBAc were calculated based on published data sets <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036038#pone.0036038-Wang1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036038#pone.0036038-Samudrala1" target="_blank">[43]</a>.</p

Translocation of AvrPto-Cya hybrid proteins into <i>N. benthamiana</i> by <i>P. s.</i> tomato DC3000.

<p>Translocation of AvrPto-Cya hybrid proteins into <i>N. benthamiana</i> by <i>P. s.</i> tomato DC3000.</p

Computational T3SS substrate predictions for proteins experimentally tested in this study.

a<p>This model could be run at more stringent (selective) or less stringent (sensitive) settings. Symbols in this column indicate that the protein was classified as a T3SS substrate at: (+) the selective level, (+/−) the sensitive level, or (−) neither level.</p>b<p>This model could be run using either an ANN or SVM classifer. Symbols in this column indicate that the protein was classified as a T3SS substrate using: (+/−) ANN, (−/+) SVM, (+/+) both, or (−/−) neither classifiers.</p

Bacterial strains and plasmids used in this study.

a<p>Rf^r, Cm^r, Ap^r, Gm^r, Sp^r, and Km^r indicate resistance to rifampicin, chloramphenicol, ampicillin, gentamicin, spectinomycin, and kanamycin, respectively.</p

Expression of AvrPto-Cya hybrid proteins in <i>P. s.</i> tomato DC3000.

<p>DC3000 strains containing plasmids that express Cya fusion proteins were grown in culture and protein samples were separated in a 12.5% SDS–PAGE gel. An immunoblot analysis was performed using primary antibodies against Cya. The protein in each lane and its estimated molecular weight is: Lane 1, empty vector; lane 2, AvrPto_Δ2–12-Cya (60.9 kDa); lane 3, AvrPto_WT(1–164)-Cya (62.0 kDa); lane 4, AvrPto_WT(1–50)-Cya (48.9 kDa); lane 5, AvrPto_SSM(1–164)-Cya (61.9 kDa); lane 6, AvrPto_SSM(1–50)-Cya (48.9 kDa); lane 7, AvrPto_FtsX(1–12)-Cya (62.1 kDa); lane 8, AvrPto_TccB(1–12)-Cya (62.2 kDa); lane 9, FtsX_1–50-Cya (50.8 kDa). The positions of protein standards on the gel are indicated to the left of the blot.</p

DC3000 T3SS substrates and their scores after analysis by the TEREE algorithm.

a<p>Experimentally validated T3SS substrates that were not included in the positive training set are denoted by asterisks.</p

Entropy estimates for the N-terminal regions of DC3000 T3SS substrates and nonsecreted proteins.

<p>The dashed line represents the negative (background) training set, whereas the dotted line represents the T3SS substrate set. The estimates were calculated for residues 2–47 using a sliding window size of 3.</p