Human Genetic Variation Reveals Novel Regulators of Yersinia pestis Cellular Infection

Abstract

Infectious diseases have been strong agents of natural selection since at least the large-scale use of agriculture. This selection was likely polygenic, acting through multiple additive variants in related genes to confer resistance to infection. Yersinia pestis, the gram-negative bacterium responsible for plague, has infected humans since at least the early Bronze Age, ultimately resulting in three highly deadly and genetically impactful pandemics. These pandemics likely led to changes in the frequency of SNPs that conferred resistance and susceptibility to Y. pestis, as the pathogen likely drove natural selection. Today, Y. pestis is still a threat to human health due to continuing epidemics, global endemicity, and its possible use as a bioterrorist agent. To discover human genetic determinants of Y. pestis infection, we utilized nearly a thousand genetically diverse lymphoblastoid cell lines (LCLs; EBV-immortalized B cells) in a cellular genome-wide association study method called Hi-HOST (High-throughput Human in-vitrO Susceptibility Testing). This bacterium is known to infect phagocytic cells, such as dendritic cells and macrophages, but interactions with non-phagocytic cells of the adaptive immune system are frequently overlooked despite the importance they are likely to hold for human infection. Using a Hi-HOST screen of seven Y. pestis cellular phenotypes in LCLs, we first identified a genome-wide significant hit for intracellular survival of Y. pestis in rs15029, a SNP in the 3’ untranslated region of RTN4 (Reticulon 4). We found that the A allele for this SNP is associated with decreased intracellular survival and decreased expression of RTN4, although modestly, in LCLs of African-ancestry individuals. Previously, RTN4 has been shown to have a role in ER shape and trafficking, the localization of Toll-like Receptor 9 to endosomes, and alteration of the host cellular environment during Legionella infection. I hypothesized that, through one of these mechanisms, RTN4 is an important factor in regulating Y. pestis infection. We also found that RTN4 knockout and knock down LCL assays resulted in increased intracellular survival in LCLs, while it decreased survival in HeLa cells. While more studies need to be completed to tease apart RTN4’s connection to Y. pestis survival in host cells, we found that RTN4 colocalizes with the Yersinia-containing vacuole and may play a role in the cell response to Y. pestis. In a combined Hi-HOST screen utilizing over 1000 genotyped lymphoblastoid cell lines, we discovered the nonsynonymous variant rs2282284 in FCRL3 (Fc receptor-like 3) was associated with the invasion phenotype. FCRL3 belongs to the immunoglobulin superfamily and is primarily expressed in lymphocytes. rs2282284 is within a tyrosine-based signaling motif, causing an asparagine-to-serine mutation (N721S) in the most common FCRL3 isoform. I hypothesized that FCRL3 may be a B cell receptor for Y. pestis. Consistent with this, we find that expressing FCRL3 in HeLa cells results in an increase of bacterial attachment and invasion while an LCL pooled knockout line showed a significant decrease in both phenotypes. FCRL3 also colocalized with extracellularly attached bacteria at sites of invasion. In addition to FCRL3, attachment and invasion were variably conserved across the FCRL family, revealing molecular requirements of attachment and invasion. An Ig-like C2 domain was conserved across all FCRL proteins that facilitated attachment to Y. pestis, while a SYK interaction motif was conserved in the two FCRL proteins that facilitated invasion (FCRL3 and FCRL5). Direct binding of Y. pestis was confirmed with purified FCRL5 extracellular domain. Invasion of Y. pestis was dependent on SYK and decreased with the N721S mutation. Thus, Y. pestis hijacks FCRL proteins, possibly taking advantage of an immune receptor to create a lymphocyte niche during infection. In summary, we have utilized Hi-HOST cellular GWAS to identify human genetic variants that are associated with susceptibility to invasion and intracellular survival of Y. pestis. For infectious diseases such as plague where human infection is no longer common, Hi-HOST provides a means to characterize how human genetic diversity may have been impacted by past pandemic pathogens while further characterizing the molecular interactions that may occur during the disease state. We speculate, and future studies in animals will test, that variation in RTN4 and FCRL3 impacts risk and severity of plague. Such genetic differences likely continue to have an impact on human health. Indeed, the FCRL3 variant is associated with risk of chronic hepatitis C virus infection. Studying these and other candidate genes and variants will result in a better understanding of plague pathogenesis, human genetics, and clinical outcomes of infectious disease.