Characterization of the Serralysin-Like Gene of ‘Candidatus Liberibacter solanacearum’ Associated with Potato Zebra Chip Disease

Abstract

The nonculturable bacterium ‘Candidatus Liberibacter solanacearum’ is the causative agent of zebra chip disease in potato. Computational analysis of the ‘Ca. L. solanacearum’ genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to serralysin. Serralysin and other serralysin family metalloprotease are typically characterized as virulence factors and are secreted by the type I secretion system (T1SS). The ‘Ca. L. solanacearum’ serralysin-like gene is located next to and divergently transcribed from genes encoding a T1SS. Based on its relationship to the T1SS and the role of other serralysin family proteases in circumventing host antimicrobial defenses, it was speculated that a functional ‘Ca. L. solanacearum’ serralysin-like protease could be a potent virulence factor. Gene expression analysis showed that, from weeks 2 to 6, the expression of the ‘Ca. L. solanacearum’ serralysin-like gene was at least twofold higher than week 1, indicating that gene expression stays high as the disease progresses. A previously constructed serralysin-deficient mutant of Serratia liquefaciens FK01, an endophyte associated with insects, as well as an Escherichia coli lacking serralysin production were used as surrogates for expression analysis of the ‘Ca. L. solanacearum’ serralysin-like gene. The LsoA and LsoB proteins were expressed as both intact proteins and chimeric S. liquefaciens-‘Ca. L. solanacearum’ serralysin-like proteins to facilitate secretion in the S. liquefaciens surrogate and as intact proteins or as a truncated LsoB protein containing just the putative catalytic domains in the E. coli surrogate. None of the ‘Ca. L. solanacearum’ protein constructs expressed in either surrogate demonstrated proteolytic activity in skim milk or zymogram assays, or in colorimetric assays using purified protein, suggesting that the ‘Ca. L. solanacearum’ serralysin-like gene does not encode a functional protease, or at least not in our surrogate systems.