ABSTRACT
Single nucleotide polymorphisms (SNPs) are present in the global transcriptional regulator cyclic AMP (cAMP) receptor protein (CRP) of the attenuated vaccine strain Mycobacterium bovis, bacillus Calmette-Guérin (BCG). We have found that these SNPs resulted in small but significant changes in the expression of a number of genes in M. tuberculosis when a deletion of the Rv3676 CRP was complemented by the BCG allele, compared to complementation by the M. tuberculosis allele. We can explain these changes in gene expression by modeling the structure of the mycobacterial protein on the known structure of CRP from Escherichia coli. Thus, the SNP change in the DNA-binding domain, Lys178, is predicted to form a hydrogen bond with the phosphate backbone of the DNA, as does the equivalent residue in E. coli, whereas Glu178 in M. tuberculosis/M. bovis does not, thus explaining the stronger binding reported for CRP of BCG to CRP-binding sites in mycobacterial DNA. In contrast, the SNP change in the nucleotide binding domain (Leu47Pro) is predicted to result in the loss of one hydrogen bond, which is accommodated by the structure, and would not therefore be expected to cause any change in function relating to cAMP binding. The BCG allele fully complemented the growth defect caused by the deletion of the Rv3676 protein in M. tuberculosis, both in vitro and in macrophage and mouse infections, suggesting that these SNPs do not play any role in the attenuation of BCG. However, they may have allowed BCG to grow better under the in vitro-selective conditions used in its derivation from the M. bovis wild type.
Subject(s)
Cyclic AMP Receptor Protein/genetics , Cyclic AMP Receptor Protein/metabolism , Gene Expression Regulation, Bacterial , Mycobacterium bovis/physiology , Mycobacterium bovis/pathogenicity , Polymorphism, Single Nucleotide , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP Receptor Protein/chemistry , DNA, Bacterial/metabolism , Female , Gene Deletion , Gene Expression Profiling , Genetic Complementation Test , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Sequence Data , Mycobacterium Infections/microbiology , Mycobacterium bovis/genetics , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , VirulenceABSTRACT
Physeal fracture of the proximal tibia is a rare injury, comprising less than 2% of all physeal injuries. The literature distinguishes between tibial tubercle avulsions (apophyseal injuries) classified by Ogden, Tross, and Murphy as type I, II, and III and Salter-Harris II fractures. An extensive review of the literature located only 5 cases in which patients sustained a combined fracture of the proximal tibial physis and tibial tubercle. We report 2 such cases, which are not amenable to classification by current systems, and agree with Ryu and Debenham's suggestion to add a fourth type, avulsion hinge fracture of the proximal tibial epiphysis, to the Watson-Jones/Ogden classification.
