Quantitative Proteomic Analysis of Simian Primary Hepatocytes Reveals Candidate Molecular Markers for Permissiveness to Relapsing Malaria Plasmodium cynomolgi

Abstract

A major obstacle impeding malaria research is the lack of an in vitro system capable of supporting infection through the entire liver stage cycle of the parasite, including that of the dormant forms known as hypnozoites. Primary hepatocytes lose their liver specific functions in long‐term in vitro culture. The malaria parasite Plasmodium initiates infection in hepatocyte. This corresponds to the first step of clinically silent infection and development of malaria parasite Plasmodium in the liver. Thus, the liver stage is an ideal target for development of novel antimalarial interventions and vaccines. However, drug discovery against Plasmodium liver stage is severely hampered by the poor understanding of host–parasite interactions during the liver stage infection and development. In this study, tandem mass tag labeling based quantitative proteomic analysis is performed in simian primary hepatocytes cultured in three different systems of susceptibility to Plasmodium infection. The results display potential candidate molecular markers, including asialoglycoprotein receptor, apolipoproteins, squalene synthase, and scavenger receptor B1 (SR‐BI) that facilitate productive infection and full development in relapsing Plasmodium species. The identification of these candidate proteins required for constructive infection and development of hepatic malaria liver stages paves the way to explore them as therapeutic targets.

Systems biology of malaria explored with nonhuman primates

“…Recently, such in vitro cultures with P. vivax revealed parasite-derived membraneous networks in both schizonts and hypnozoites and possible functions of aquaporin-3 [ 216 ]. This type of research comes with many challenges, given the need to distinguish quiescent infected cells that have low metabolic activity from developing or newly activated forms [ 217 , 218 ]. Today, such culture systems, single-cell -omic technologies [ 219 ], and cellular imaging advances [ 220 , 221 ] are paving the way for using these model systems to understand P. vivax sporozoites [ 222 ] and host–parasite interactions in infected hepatocytes, including dormant and activated hypnozoites [ 223 ].…”

“…These feats have been groundbreaking, but—if P. vivax clinical cases continue to decline, as hoped—logistical challenges will be compounded that include having sufficient availability of patient infected-blood donors, mosquito insectary operations, and experts for establishing, infecting, and analyzing the data coming from hepatocyte cultures. Meanwhile, there is much yet to be learned about the biology of hypnozoites to effectively identify targets for drug intervention, and multi-omic approaches that can distinguish host and parasite targets will be essential [ 218 , 223 ]. With such knowledge and systems biological analyses, vaccination to eliminate hypnozoites, or at a minimum delay or reduce the number of relapses [ 225 ], may one day also become a reality, in addition to targeting the universal parasite developmental life cycle forms or stages known across all species as liver-stage forms (LSFs, or exoerythrocytic stages) and blood-stage forms (BSFs, or erythrocytic stages) (Fig.…”

Elucidating Spatially-Resolved Changes in Host Signaling During Plasmodium Liver-Stage Infection

“…Transcriptomic studies have demonstrated extensive changes in host gene expression that vary over the course of infection, however concordance among these studies has been low, perhaps due to differences in hepatocyte origin and time needed to sort infected cells ( Albuquerque et al., 2009 ; LaMonte et al., 2019 ). We used reverse phase protein array (RPPA) to evaluate changes in host protein and post-translational modifications in an in vitro model of Plasmodium yoelii infection ( Kaushansky et al., 2013a ), and to identify proteins that are expressed proteins that are expressed at different levels between hepatocyte populations of differential susceptibility to LS infection ( Dembele et al., 2019 ; Glennon et al., 2019 ). Several proteins and processes that were identified as altered in infected cells were also found to be important for LS infection [reviewed in ( Glennon et al., 2018 )].…”

Elucidating spatially-resolved changes in host signaling during Plasmodium liver-stage infection

“…Transcriptomic studies have demonstrated extensive changes in host gene expression that vary over the course of infection, however concordance among these studies has been low, perhaps due to differences in hepatocyte origin and time needed to sort infected cells [16, 17]. Protein and post-translational modification level screens have been conducted using reverse phase protein array (RPPA) in an in vitro model of Plasmodium yoelii infection [12], and to identify proteins that are differentially expressed between hepatocyte populations of differential susceptibility to LS infection [11, 18]. Several proteins and processes that were identified as altered in infected cells were also found to be important for LS infection (reviewed in [19]).…”