The Chromosomal Gene rfaL Plays a Role in Type III Secretion and Virulence in Yersinia pestis

Yersinia pestis is the causative agent of bubonic, pneumonic and septicemic plague. Y. pestis along with the two other pathogenic Yersinia require the use of a Type III Secretion System (TTSS) in order to cause disease within a mammalian host. The bacterium uses the TTSS to inject cytotoxic proteins, known as Yops, directly into the cytosol of host cells upon contact with the host cell. The TTSS can also be activated by growing the bacterium in vitro at 37°C in the absence of calcium. Under these conditions the bacterium undergoes growth cessation and secretes its Yop proteins into the culture medium, a process known as the low calcium response (LCR). The genes encoding the TTSS machinery, Yop proteins, and regulators are all encoded on a ∼70 kb virulence plasmid, called pCD1. We sought to identify genes on the Y. pestis chromosome that are also required for proper function of the TTSS. We identified five chromosomal genes that were able to overcome the growth cessation phenotype of the LCR. These mutants were also unable to secrete Yops under LCR inducing conditions, but were all able to inject Yops into host cells at various levels upon contact with host cells. One of the mutants contained an insertion in a gene, rfaL, and had three distinct but conflicting phenotypes 1. It was unable to secrete Yops under LCR inducing conditions, 2. but the mutant injected Yops into host cells at wild type levels, 3. however the rfaL mutant was less virulent than wild type. We chose to investigate the cause behind the rfaL mutant's in vitro secretion defect and virulence defect. Through this research, we were able to determine that the rfaL mutant's secretion defect could be overcome by overexpressing the TTSS genes and by adding back the divalent cations Mg2+ and Sr2+ to the medium. The virulence defect of the rfaL mutant is likely the result of a decrease in targeting of innate immune cells along with an increased sensitivity to cationic antimicrobial peptides. RfaL is hypothesized to function as an O-antigen ligase in Y. pestis. Y. pestis does not make O-antigen, however the O-antigen ligases of other Gram negative bacteria have been shown to link enterobacterial common antigen (ECA) to their lipopolysaccharide molecules in place of O-antigen. We therefore investigated the contribution of ECA to the function of the TTSS and virulence in Y. pestis..