Biofilm Formation and Motility Are Promoted by Cj0588-Directed Methylation of rRNA in Campylobacter jejuni

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.

Virulence properties of Campylobacter jejuni are enhanced by displaying a mycobacterial TlyA methylation pattern in its rRNA

“…The natural version of the TlyA methyltransferase found in C. jejuni is a type I (TlyA I ) enzyme encoded by the cj0588 gene, and stoichiometrically methylates the 2´‐ O ‐ribose of 23S rRNA nucleotide C1920 (Sałamaszyńska‐Guz et al, ). Type II (TlyA II ) variants, found in some Gram‐positive bacteria including the Actinobacteria, methylate not only at nucleotide C1920 but also at 16S rRNA nucleotide C1409 (Johansen et al, ; Monshupanee et al, ).…”

“…In actinobacterial species, which include Mycobacterium tuberculosis , TlyA enzymes belong to the type II group (TlyA II ) and have the function of 2′‐ O ‐methylating nucleotide C1409 in 16S rRNA and nucleotide C1920 in 23S rRNA (Johansen, Maus, Plikaytis, & Douthwaite, ). Slightly shorter versions of TlyA truncated at their N‐ and C‐termini are found in C. jejuni , Helicobacter pylori and Brachyspira hyodysenteriae , and belong to the type I group of enzymes (TlyA I ) that methylate only at 23S rRNA nucleotide C1920 (Monshupanee, Johansen, Dahlberg, & Douthwaite, ; Sałamaszyńska‐Guz et al, ).…”

“…Loss of TlyA in C. jejuni cells results in a wide range of defects including decreased ribosome subunit association, impeded motility and reduced biofilm formation, which collectively reduce virulence (Sałamaszyńska‐Guz et al, ). In addition, sensitivity to the antibiotic capreomycin is altered.…”

“…In addition, sensitivity to the antibiotic capreomycin is altered. Complementation with natively folded variants of TlyA containing point mutations that abolish methyltransferase activity showed that all the physiological defects were caused by loss of rRNA methylation rather than absence of the protein itself (Sałamaszyńska‐Guz et al, ). These findings indicate that TlyA influences the physiology and pathogenicity of C. jejuni solely through its rRNA methylation activity.…”

Bridging the Gap: A Role for Campylobacter jejuni Biofilms

“…Motility is one of the more characterized elements involved in biofilm formation in C. jejuni. There are many examples suggesting that directed motility or chemotaxis and biofilm formation are processes that are correlated in C. jejuni [15][16][17]. The chemotactic pathway of C. jejuni shares many features with that of Escherichia coli.…”