Host factors underlying susceptibility to enteric infection in humans

Host factors are indispensable for infectious disease processes and influence disease susceptibility and severity. Many pathogens usurp host factors by direct binding to or use of specialized secretion systems to exploit the downstream signaling processes facilitating infection. Thus, the host-pathogen interactions implicated in development of disease are key components of infection and transmission. In disease control, the host elicits a myriad of defense mechanisms entailing interplay between the innate and adaptive immune response to clear the infectious agent. Hence, communication between non-immune and immune cells is integral in the host response. Understanding how host factors are involved with and effect disease outcome offers an opportunity for new insight into pathogenesis and therapy.

The apicomplexan parasite Cryptosporidium is the causative agent of cryptosporidiosis, a disease that primarily consists of but is not limited to watery diarrhea. Cryptosporidiosis is a leading cause of diarrhea-associated morbidity and mortality in young children, globally. Notably, clinical presentation of Cryptosporidium infection is variable in severity; some patients experience severe diarrheal illness while others remain asymptomatic. Though the cause for this wide spectrum of disease is poorly understood, several studies have demonstrated long-term clinical implications of both asymptomatic and symptomatic infection. Importantly, nitazoxanide, currently the only FDA-approved drug for Cryptosporidium infection has poor efficacy in patients most at-risk, immunocompromised persons. Thereby, the identification of novel host factors as potential therapeutic targets to combat cryptosporidiosis is warranted.

The highly diverse, classical human leukocyte antigen (HLA) genes are traditional genetic determinants of immunity. We systematically evaluated the impact of HLA alleles and haplotypes on susceptibility to 12 common enteric infections during the first year of life. A birth cohort of 601 Bangladeshi infants was prospectively monitored for diarrheal disease. We compared the frequency of HLA class I (A and B) alleles, class II (DRB1, DQA1, and DQB1) alleles, and haplotypes between infected and uninfected infants. We identified six individual allele associations and one five-locus haplotype association. Two alleles B*38:02 and DQA1*01:01 were associated with increased risk of Cryptosporidium infection, however no protective HLA-Cryptosporidium associations were identified. We discovered an additional three alleles associated with increased risk to other enteric infections: A*24:17—typical EPEC, B*15:01—astrovirus, B*38:02—astrovirus. One allele, A*24:02, was associated with protection from EAEC infection. A single five-locus haplotype was associated with protection: A*11:01~B*15:02~DRB1*12:02~DQA1*06:01~DQB1*03:01—adenovirus 40/41. Our findings validate the role for HLA in susceptibility to five pathogens. Understanding the genetic contribution of HLA in susceptibility has important implications for vaccine design and understanding regional differences in incidence of enteric infection.

To discover novel genetic links to cryptosporidiosis alternate to those with traditional roles in immunity (e.g., HLA), our lab performed a genome-wide association study (GWAS) of a birth cohort in an endemic area of Dhaka, Bangladesh during the first year of life. Our lab discovered single nucleotide polymorphisms (SNP) in an intron region of the human protein kinase C alpha (PRKCA) gene associated with increased susceptibility to Cryptosporidium. Protein Kinase C-α (PKCα) is a host serine/threonine kinase that has been shown to modulate susceptibility to several enteric infections through regulation of the actin cytoskeleton. Cryptosporidium infection has been linked to changes in PRKCA expression and PKCα activity in vitro, independently. Previous work has found Cryptosporidium requires host cell actin polymerization at the site of invasion while in-parallel identifying critical actin-associated mediators. However, a role for host PKCα during Cryptosporidium invasion remains unexplored. Since PKCα is a primary regulator of the actin cytoskeleton, and during intracellular invasion Cryptosporidium sporozoites induce host cell actin remodeling we examined the role of PKCα during this process.

Here, we used an established Cryptosporidium in vitro model of infection: HCT-8 intestinal epithelial cells and infection with Cryptosporidium parvum sporozoites from excysted oocysts. To delineate the role of PKCα in infection, we developed a fluorescence-based imaging assay to differentiate adherent from invaded parasite. We tested pharmacologic agonists and antagonists of PKCα activity and measured the effect on C. parvum sporozoite adherence and invasion. We demonstrated both PKCα agonists and antagonists significantly alter parasite adherence and invasion of HCT-8 cells. We additionally found HCT-8 cell PKCα is activated by C. parvum infection. Altogether, our findings suggest intestinal epithelial cell PKCα as a host therapeutic target for cryptosporidiosis and implicate PKCα activity as a mediator of parasite adherence and invasion.

The results from these studies present a primary role for the host in response to enteric infection and demonstrate that differences in activity of host factors directly influence susceptibility. The work presented in this thesis will focus on host-pathogen interactions in generating a protective or deleterious response to various infectious agents. Overall, this thesis furthers our understanding of the host immune response in enteric infection and explicitly implicates a host-derived kinase, PKCα, in Cryptosporidium pathogenesis.