An ABC transporter containing a forkhead-associated domain interacts with a serine-threonine protein kinase and is required for growth of Mycobacterium tuberculosis in mice

Abstract

Forkhead-associated (FHA) domains are modular phosphopeptide recognition motifs with a striking preference for phosphothreonine-containing epitopes. FHA domains have been best characterized in eukaryotic signaling pathways but have been identified in six proteins in Mycobacterium tuberculosis, the causative organism of tuberculosis. One of these, coded by gene Rv1747, is an ABC transporter and the only one to contain two such modules. A deletion mutant of Rv1747 is attenuated in a mouse intravenous injection model of tuberculosis where the bacterial load of the mutant is 10-fold lower than that of the wild type in both lungs and spleen. In addition, growth of the mutant in mouse bone marrow-derived macrophages and dendritic cells is significantly impaired. In contrast, growth of this mutant in vitro was indistinguishable from that of the wild type. The mutant phenotype was lost when the mutation was complemented by the wild-type allele, confirming that it was due to mutation of Rv1747. Using yeast two-hybrid analysis, we have shown that the Rv1747 protein interacts with the serine-threonine protein kinase PknF. This interaction appears to be phospho-dependent since it is abrogated in a kinase-dead mutant and by mutations in the presumed activation loop of PknF and in the first FHA domain of Rv1747. These results demonstrate that the protein coded by Rv1747 is required for normal virulent infection by M. tuberculosis in mice and, since it interacts with a serine-threonine protein kinase in a kinase-dependent manner, indicate that it forms part of an important phospho-dependent signaling pathway.

FIG. 1.

Diagram of the genomic region of M. tuberculosis containing the pknF and Rv1747 operon and adjacent genes, showing the extent of the deletion and the complementing plasmid pRv1747⁺. The marks on the chromosome are at 1,000-bp intervals.

FIG. 2.

Growth of the Rv1747 knockout mutant is unimpaired in vitro. The H37Rv wild-type, Rv1747 knockout and the complemented strain were grown in Dubos medium in rolling bottles as described in Materials and Methods, and OD₆₀₀ measurements were made on 1-ml aliquots taken at 24-h intervals. There was no statistical difference between the OD measurements from the three strains.

FIG. 3.

Growth of the Rv1747 knockout mutant is impaired in a mouse intravenous infection. BALB/c mice were inoculated intravenously with ca. 5 × 10⁵ CFU of each strain. The survival and multiplication of the M. tuberculosis strains in the lungs and the spleen were determined by CFU counts and are shown for the H37Rv wild-type (•), Rv1747 knockout mutant (○), and Rv1747 complemented mutant (▾) strains. The results for each time point are the means of CFU determinations performed on organs from three to five infected mice, and error bars indicate the standard deviations. The asterisks indicate that the result is statistically significantly different from that of the wild type as determined by the two-tailed Student t test for groups of unequal variance (P < 0.01), as well as by single-factor analysis of variance (P < 0.01).

FIG. 4.

Growth of the Rv1747 knockout mutant is impaired in in vitro infections into mouse bone marrow-derived macrophages and dendritic cells. Macrophages were isolated from BALB/c mice as described in the text and infected at a multiplicity of infection of one bacterium to two macrophages with each strain. The survival and multiplication of the M. tuberculosis strains were determined by CFU counts and are shown for the H37Rv wild-type (•), Rv1747 knockout mutant (○), and Rv1747 complemented mutant (▾) strains. The experiment was performed twice with similar results; the results of a representative experiment are shown. The results for each time point are the means of CFU determinations performed on triplicate infections, and error bars indicate the standard deviations. The asterisks indicate that the result is statistically significantly different from that of the wild type as determined by the two-tailed Student t test for groups of unequal variance (P < 0.02), as well as by single-factor analysis of variance (P < 0.02).

FIG. 5.

β-Galactosidase assay of yeast two-hybrid constructs showing the effect of the PknF K41A, T173A, T175A, and T178A mutations and the Rv1747 S47A and S248A mutations on the interaction between PknF and Rv1747. S. cerevisiae Y187 were cotransformed with plasmids expressing PknF in the Gal4-binding domain and Rv1747 in the Gal4 activation domain. Transformants were selected on minimal Difco Leu⁻ Trp⁻ plates and further tested for lacZ expression by β-galactosidase assay using ONPG as a substrate. Bars: 1, S. cerevisiae(pGAD/pGBD); 2, S. cerevisiae (pGAD Rv1747/pGBD); 3, S. cerevisiae (pGAD/pGBD PknF); 4, S. cerevisiae (pGAD Rv1747/pGBD Rv1747); 5, S. cerevisiae (pGAD Rv1747/pGBD PknF); 6, S. cerevisiae (pGAD Rv1747 S47A/pGBD PknF); 7, S. cerevisiae (pGAD Rv1747 S248A/pGBD PknF); 8, S. cerevisiae (pGAD Rv1747/pGBD PknF K41A); 9, S. cerevisiae (pGAD Rv1747/pGBD PknF T173A); 10, S. cerevisiae (pGAD Rv1747/pGBD PknF T175A); 11, S. cerevisiae (pGAD Rv1747/pGBD PknF T178A).

FIG. 6.

Structure of the pThr binding site in FHA domains. The top panel shows the interactions of Ser85 and Arg70 from the N-terminal FHA domain (FHA1) of S. cerevisiae Rad53p checkpoint kinase (11). The lower panel shows a sequence overlap within the core FHA homology region between Rad53p FHA1 and the two FHA domains of M. tuberculosis Rv1747. Six of the most highly conserved residues are highlighted (Arg70 and Ser85 in red and Gly69, His88, Asn107, and Asn112 in blue [Rad53 numbering]).