Increased motility inCampylobacter jejuniand changes in its virulence, fitness and morphology following protein expression on ribosomes with altered RsmA methylation

Abstract

AbstractInfection withCampylobacter jejuniis the major cause of human gastroenteritis in the United States and Europe, leading in many cases to debilitating autoimmune sequelae. While considerable progress has been made in detailing the infectious cycle ofC. jejuni, a full understanding of the molecular mechanisms responsible for virulence remains to be elucidated. Here, we apply a novel approach by modulating protein expression on the pathogen’s ribosomes by inactivating a highly conserved rRNA methyltransferase. Loss of the RsmA methyltransferase results in a more motile strain with greater adhesive and cell invasive properties. These phenotypical effects correlate with enhanced expression of specific proteins related to flagellar formation and function, together with enzymes involved in cell wall/membrane and amino acid synthesis. Despite the enhancement of certain virulent traits, the null-strain grows poorly on minimal media and is rapidly out-competed by the wild-type strain. Complementation with an active copy of thersmAgene rescues most of the traits changed in the mutant. However, the complemented strain overexpressesrsmAand displays new flaws including loss of the spiral cell shape that is distinctive forC. jejuni. Proteins linked with altered virulence and morphology are identified here by mass spectrometry proteomic analyses of the strains.Author summaryInfections withCampylobacter, and particularly with the most prevalent species of this genus,Campylobacter jejuni, are the leading cause of human bacterial gastroenteritis. Sadly, illnesses caused byC. jejunioften do not end with the infection itself, but continue with serious post-infectious autoimmune sequelae including the peripheral neuropathies Guillain–Barré syndrome, Miller Fisher syndrome and irritable bowel syndrome. Despite decades of research into theC. jejuniinfection cycle, the molecular mechanisms by which pathogenicity progresses are not yet fully understood. Here, we described a novel approach whereby we modulate protein synthesis on the pathogen’s ribosomes to interfere with specific virulence traits. This is followed up with proteome analyses of the engineered strains using state-of-the-art mass spectrometry techniques, enabling us to link specific proteins with steps in the infection cycle. Specifically, we disabled the ribosomal RNA modification enzyme RsmA, and this causes unexpectedly large changes inC. jejunimotility and its ability to attach to and invade human epithelial cells. We then identified specific flagellar, transpeptidase and peptidoglycan peptidase proteins involved in these processes. We propose that this approach of modulating protein synthesis via changes in rRNA methylation can be applied to study the infection cycles of other bacterial pathogens.