Non-pathogenic Escherichia coli acquires virulence by mutating a growth-essential LPS transporter

The molecular mechanisms that allow pathogenic bacteria to infect animals have been intensively studied. On the other hand, the molecular mechanisms by which bacteria acquire virulence functions are not fully understood. In the present study, we experimentally evaluated the evolution of a non-pathogenic strain of Escherichia coli in a silkworm infection model and obtained pathogenic mutant strains. As one cause of the high virulence properties of E. coli mutants, we identified amino acid substitutions in LptD (G580S) and LptE (T95I) constituting the lipopolysaccharide (LPS) transporter, which translocates LPS from the inner to the outer membrane and is essential for E. coli growth. The growth of the LptD and LptE mutants obtained in this study was indistinguishable from that of the parent strain. The LptD and LptE mutants exhibited increased secretion of outer membrane vesicles containing LPS and resistance against various antibiotics, antimicrobial peptides, and host complement. In vivo cross-linking studies revealed that the conformation of the LptD-LptE complex was altered in the LptD and LptE mutants. Furthermore, several clinical isolates of E. coli carried amino acid substitutions of LptD and LptE that conferred resistance against antimicrobial substances. This study demonstrated an experimental evolution of bacterial virulence properties in an animal infection model and identified functional alterations of the growth-essential LPS transporter that led to high bacterial virulence by conferring resistance against antimicrobial substances. These findings suggest that non-pathogenic bacteria can gain virulence traits by changing the functions of essential genes, and provide new insight to bacterial evolution in a host environment. Author summary Pathogenic bacteria developed their virulence properties by changing the functions of various genes after the emergence of the host animals on earth. The types of gene function alterations that confer bacterial virulence properties, however, have remained unclear. We utilized a silkworm infection model to perform an experimental evolution of bacterial virulence activity. From a non-pathogenic strain of Escherichia coli, we obtained a mutant strain that exhibited 500-fold higher virulence than the original strain and identified mutations of the lipopolysaccharide (LPS) transporter, which translocates LPS onto the bacterial surface, as one cause of the high virulence. The mutations changed the structure of the LPS transporter, increased the secretion of outer membrane vesicles, and enabled bacterial survival in the presence of host antimicrobial substances. This mechanism to gain high virulence occurs naturally, as several E. coli clinical isolates carried mutations of the LPS transporter that confer resistance against antimicrobial substances. Our study unveiled a novel mechanism by which bacteria increase their virulence through modifying their gene function.