Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

Abstract

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage. The O1 serogroup of Vibrio cholerae is the most common cause of the potentially fatal diarrheal disease cholera, which remains a significant global health burden worldwide. The O1 antigen is a constituent of the lipopolysaccharide portion of the outer membrane, and its location on the bacterial surface makes it a major target of both the immune system and bacteriophages. We used an O1-specific bacteriophage as a tool to understand if, and how, V. cholerae can alter O1 antigen expression. We discovered that two genes, which are critical for O1 antigen biosynthesis, are subject to phase variation. Additionally, one of the phase variable genes we identified was not previously known to play a role in O1 antigen biosynthesis in V. cholerae. Phase variation is a well-recognized mechanism many other bacterial pathogens use to generate variable expression of surface components, and this is generally thought to help these organisms evade the immune system. Phase variation has not previously been described as a widespread mechanism used by V. cholerae, furthermore, this is the first report that V. cholerae O1 is capable of generating diverse populations with variable and unique O1 antigen expression.