Mlp24 (McpX) of Vibrio cholerae implicated in pathogenicity functions as a chemoreceptor for multiple amino acids

Abstract

The chemotaxis of Vibrio cholerae, the causative agent of cholera, has been implicated in pathogenicity. The bacterium has more than 40 genes for methyl-accepting chemotaxis protein (MCP)-like proteins (MLPs). In this study, we found that glycine and at least 18 L-amino acids, including serine, arginine, asparagine, and proline, serve as attractants to the classical biotype strain O395N1. Based on the sequence comparison with Vibrio parahaemolyticus, we speculated that at least 17 MLPs of V. cholerae may mediate chemotactic responses. Among them, Mlp24 (previously named McpX) is required for the production of cholera toxin upon mouse infection. mlp24 deletion strains of both classical and El Tor biotypes showed defects in taxis toward several amino acids, which were complemented by the expression of Mlp24. These amino acids enhanced methylation of Mlp24. Serine, arginine, asparagine, and proline were shown to bind directly to the periplasmic fragment of Mlp24. The structural information of its closest homolog, Mlp37, predicts that Mlp24 has two potential ligand-binding pockets per subunit, the membrane distal of which was suggested, by mutational analyses, to be involved in sensing of amino acids. These results suggest that Mlp24 is a chemoreceptor for multiple amino acids, including serine, arginine, and asparagine, which were previously shown to stimulate the expression of several virulence factors, implying that taxis toward a set of amino acids plays critical roles in pathogenicity of V. cholerae.

Fig 1

Chemotactic responses of the classical V. cholerae strain to amino acids. (A) Capillary assay of responses of strain O395N1 to serine (attractant) and glutamate (nonattractant). Closed circles, O395N1 (Che⁺); open circles, VcheA2 (ΔcheA2). (B) Summary of the results of competition assays. Preincubation with proline failed to inhibit serine taxis, while that with serine inhibited proline taxis. (C) Competition between different attractants in chemotaxis of V. cholerae. O395N1 (closed circles) and VcheA2 (open circles) were preincubated with TMN buffer (+ None), 10 mM cysteine, and 10 mM proline and were examined for responses to serine.

Fig 2

Alignment of the periplasmic domains of MCPs from various bacterial species. Amino acid sequences of MLPs were aligned by using the ClustalW program, version 1.4. The Cache domain (3), consisting of the N- and C-terminal parts, and the pre-Cache motif identified in this study are indicated by separate lines above the sequences. White letters on a black background indicate residues identical to those of Mlp24. The names of the proteins are shown with the abbreviations of the species: Agrc, Agrobacterium tumefaciens; Bs, Bacillus subtilis; Cv, Chromobacterium violaceum; Vp, Vibrio parahaemolyticus; Vv, Vibrio vulnificus; Pa, Pseudononas aeruginosa; Pbpra, Photobacterium profundum; Smc, Sinorhizobium; Yp, Yersinia pestis.

Fig 3

Chemotactic responses of the classical strain of V. cholerae and its Δmlp24 mutant toward amino acids indicated in each panel. Closed circles, O395N1; open circles, Δmlp24. Relative chemotaxis index is defined as a ratio of the number of bacteria in the capillary of interest to that in the control capillary filled with buffer in each set of experiments. Standard errors of triplicate data for each concentration are shown.

Fig 4

Methylation and ligand-binding assays of Mlp24. (A) Methylation patterns of Mlp24 in the presence and absence of various amino acids. O395N1 cells (classical) expressing Mlp24-FLAG were incubated with (+) or without (−) serine (Ser), arginine (Arg), asparagine (Asn), proline (Pro), and glutamate (Glu), and Mlp24-FLAG was detected by immunoblotting with anti-FLAG antibody. (B and C) Binding of serine to the periplasmic fragment of Mlp24 (Mlp24p). ITC measurements of 10 μM Mlp24p were carried out with 10 mM serine (attractant [B]) and 30 mM glutamic acid (nonattractant [C]). Enthalpy changes per mole were plotted as a function of the molar ratio of serine to Mlp24p. The binding parameters calculated from the data are described in the text.

Fig 5

Chemotactic responses of an El Tor biotype strain of V. cholerae and its Δmlp24 mutant toward serine (A) and arginine (B). Closed circles, AC-V66 and O395N1; open circles, its mlp24-deletion derivative AC-V1400 (Δmlp24). Standard errors of triplicate data for each concentration are shown.

Fig 6

Mutations introduced into the potential ligand-binding pockets of Mlp24. (A and B) Side chains of the residues constituting the potential binding pockets I (A) and II (B) of Mlp37 (PDB ID, 3C8C). The alanine molecule (shown with a space filling model) in pocket I (A) exists in the crystal. Residues conserved in Mlp24 are circled. Those corresponding to the Mlp24 mutations constructed in this study are marked with asterisks: *1, Y120A; *2, R125A; *3, Y192A; and *4, H205A. (C) Capillary assays of Vmlp201 (Δmlp24 Δmlp37) cells (derived from O395N1) carrying the vector (pAH901, open bars) or the plasmid encoding wild-type or mutant Mlp24 (wild type, hatched bars; Y120A, dotted bars; R125A, checked bars; Y192A, cross-hatched bars; H205A, closed bars). Capillaries were filled with TMN buffers containing each amino acid (1, 10, or 100 mM) shown at the bottom.