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

Abstract

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.

Type III-Dependent Translocation of HrpB2 by a Nonpathogenic hpaABC Mutant of the Plant-Pathogenic Bacterium Xanthomonas campestris pv. vesicatoria

“…The N-terminal region of HrpB2 contains 10% leucine and 5% serine residues (compared to 6.7% leucine and 8.9% serine residues, respectively, in the rest of the protein); therefore, it does not match the criteria predicted for T3S signals, e.g., a depletion of leucine and an enrichment of serine residues (8,9,(66)(67)(68). Notably, however, in the effector protein AvrPto from Pseudomonas syringae, the functional importance of these characteristic amino acid patterns could not be confirmed by mutational approaches, suggesting a high variability of the T3S signal and the presence of additional targeting patterns (12). The finding that amino acids 10 to 40 of HrpB2 are sufficient for secretion and translocation of HrpB2 in the absence of HpaC suggests that HrpB2 contains one joint secretion and translocation signal which also determines the secretion specificity, i.e., the HpaC-mediated suppression of HrpB2 secretion and translocation.…”

“…We are grateful to U. Bonas for comments on the manuscript and for providing the AvrBs3-specific antibody, to T. Schreiber for discussing unpublished data on AvrBs3, to C. Lorenz and K. Schlien for generating constructs pBhrpB2 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] …”

“…Secretion and translocation of T3S substrates depend on an export signal, which is not conserved on the amino acid level and is often located in the N-terminal 20 to 30 amino acids (2). Despite the lack of amino acid sequence conservation, many T3S signals consist of specific amino acid compositions or patterns and are often structurally disordered (7)(8)(9)(10)(11)(12). In some T3S substrates, export signals have also been identified in the C-terminal protein region or the 5= region of the mRNA (13)(14)(15)(16)(17).…”

Structure–function discrepancy inClostridium botulinumC3 toxin for its rational prioritization as a subunit vaccine

“…Any mutational change in these residues may bring a drastic change in their structure-function integrity by evolutionary consequence. A single mutation in mutable residues rarely can bring major functional changes in a protein (Schechter et al, 2012). The virulence of influenza B virus was broadly neutralized by mutable 190-helix region near the receptor binding site in the hemagglutinin protein (Yasugi et al, 2013).…”

Bacterial Type III Secretion System Effector Proteins are Distinct between Plant Symbiotic, Plant Pathogenic and Animal Pathogenic Bacteria

“…Thus, understanding the characteristic feature of T3SS effector protein is important. Actually, numerous researches tried to discriminate T3SS effector proteins from other proteins [2], [3], [4], [5], [6].…”