Dysregulation of the cell cycle underlies many human genetic diseases and cancers, yet numerous organisms, including microbes, also manipulate the cell cycle to generate both morphologic and genetic diversity as a natural mechanism to enhance their chances for survival. The eukaryotic pathogen
Cryptococcus neoformans
generates morphologically distinct polyploid titan cells critical for host adaptation and subsequent disease.
CRISPR-Cas9–based screening with single-guide RNA (sgRNA) libraries has emerged as a revolutionary tool for comprehensive analysis of genetic elements. However, genome-scale sgRNA libraries are currently available only in a few model organisms. The traditional approach is to synthesize thousands to tens of thousands of sgRNAs, which is laborious and expensive. We have developed a simple method, RELATe (restriction/ligation coupled with Agrobacterium-mediated transformation), to generate sgRNA libraries from 10 μg of genomic DNA, targeting over 98% of the protein-coding genes in the human fungal pathogen Cryptococcus neoformans. Functional screens identified 142 potential C. neoformans genes contributing to blood-brain barrier penetration. We selected two cryptococcal genes, SFP1 and WDR1, for a proof-of-concept demonstration that RELATe-identified genes are relevant to C. neoformans central nervous system infection. Our RELATe method can be used in many other fungal species and is powerful and cost-effective for genome-wide high-throughput screening for elucidating functional genomics.
Microbial penetration of the blood–brain barrier, a prerequisite for development of the central nervous system (CNS) infection, involves microbial invasion, intracellular traversal and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of the human brain microvascular endothelial cell (HBMEC) infected with
Escherichia coli
and
Cryptococcus neoformans
, representative bacterial and fungal pathogens common in CNS infection. Among the upregulated targets in response to
E. coli
and
C. neoformans
infection,
PDLIM2
was knocked down by shRNA in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated the microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication and egression. Additionally, the defective exocytosis of internalized
E. coli
from the
PDLIM2
shRNA knockdown cell was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed the proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2-interactors. Among the nine enriched Pdlim2-interactors in response to both
E. coli
and
C. neoformans
infection, we selected
MPRIP
and showed that HBMEC with knockdown of
MPRIP
mimicked the phenotype of
PDLIM2
knockdown cell. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.
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