Abstract
Numerous bacterial pathogens express an ortholog of the enzyme TlyA, which is an rRNA 2′-O-methyltransferase associated with resistance to cyclic peptide antibiotics such as capreomycin. Several other virulence traits have also been attributed to TlyA, and these appear to be unrelated to its methyltransferase activity. The bacterial pathogen Campylobacter jejuni possesses the TlyA homolog Cj0588, which has been shown to contribute to virulence. Here, we investigate the mechanism of Cj0588 action and demonstrate that it is a type I homolog of TlyA that 2′-O-methylates 23S rRNA nucleotide C1920. This same specific function is retained by Cj0588 both in vitro and also when expressed in Escherichia coli. Deletion of the cj0588 gene in C. jejuni or substitution with alanine of K80, D162, or K188 in the catalytic center of the enzyme cause complete loss of 2′-O-methylation activity. Cofactor interactions remain unchanged and binding affinity to the ribosomal substrate is only slightly reduced, indicating that the inactivated proteins are folded correctly. The substitution mutations thus dissociate the 2′-O-methylation function of Cj0588/TlyA from any other putative roles that the protein might play. C. jejuni strains expressing catalytically inactive versions of Cj0588 have the same phenotype as cj0588-null mutants, and show altered tolerance to capreomycin due to perturbed ribosomal subunit association, reduced motility and impaired ability to form biofilms. These functions are reestablished when methyltransferase activity is restored and we conclude that the contribution of Cj0588 to virulence in C. jejuni is a consequence of the enzyme's ability to methylate its rRNA.
Highlights
Proteins encoded by orthologs of tlyA genes are present in a diverse range of bacterial pathogens including Mycobacterium sp., Campylobacter jejuni and Brachyspira (Serpulina) hyodysenteriae, and have been linked to various roles in virulence including hemolysis (Wren et al, 1998) and antibiotic resistance (Maus et al, 2005)
MALDIToF mass spectrometric analysis of 16S rRNA from wild-type strains showed that there was no methylation at nucleotide C1409 (Figure S1), whereas a clear top was seen corresponding to the fragment containing 2′-O-methylation at 23S rRNA nucleotide C1920 (Figure 2A)
There was no methylation at these nucleotides in the cj0588-null mutants, and methylation capacity was restored by complementation of null-mutants with an intact copy of the C. jejuni cj0588 gene (Figure 3)
Summary
Proteins encoded by orthologs of tlyA genes are present in a diverse range of bacterial pathogens including Mycobacterium sp., Campylobacter jejuni and Brachyspira (Serpulina) hyodysenteriae, and have been linked to various roles in virulence including hemolysis (Wren et al, 1998) and antibiotic resistance (Maus et al, 2005). Alignments of TlyA sequences from various organisms show that they possess a conserved K-D-K-E amino acid tetrad previously identified at the active core of other 2′-Omethyltransferases such as RrmJ/FtsJ (Bugl et al, 2000; Hager et al, 2002; Feder et al, 2003; Punekar et al, 2012). These structural features are evident in the TlyA ortholog Cj0588 of C. jejuni. Nucleotides C1409 and C1920 are respectively located on the interface of the 30 and 50S ribosomal subunits and come into close proximity at the capreomycin/viomycin binding site upon subunit association (Johansen et al, 2006; Stanley et al, 2010)
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