Affiliation:
1. Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, Liaoning, China
2. Department of Cell Engineering, Beijing Institute of Biotechnology, Beijing, China
Abstract
ABSTRACT
Brucella
is a zoonotic intracellular bacterium that poses threats to human health and economic security. Intracellular infection is a hallmark of the agent
Brucella
and a primary cause of distress, through which the bacterium regulates the host intracellular environment to promote its own colonization and replication, evading host immunity and pharmaceutical killing. Current studies of
Brucella
intracellular processes are typically premised on bacterial phenotype such as intracellular bacterial survival, followed by biochemical or molecular biological approaches to reveal detailed mechanisms. While such processes can deepen the understanding of
Brucella
-host interaction, the insights into host alterations in infection would be easily restricted to known pathways. In the current study, we applied CRISPR Cas9 screen to identify host genes that are most affected by
Brucella
infection on cell viability at the genomic level. As a result of CRISPR screening, we firstly identified that knockout of the negatively selected genes
GOLGA6L6
,
DEFB103B
,
OR4F29
, and
ERCC6
attenuate the viability of both the host cells and intracellular
Brucella
, suggesting these genes to be potential therapeutic targets for
Brucella
control. In particular, knockout of
DEFB103B
diminished
Brucella
intracellular survival by altering host cell autophagy. Conversely, knockout of positive screening genes promoted intracellular proliferation of
Brucella
. In summary, we screened host genes at the genomic level throughout
Brucella
infection, identified host genes that are previously not recognized to be involved in
Brucella
infection, and provided targets for intracellular infection control.
IMPORTANCE
Brucella
is a Gram-negative bacterium that infects common mammals causing arthritis, myalgia, neuritis, orchitis, or miscarriage and is difficult to cure with antibiotics due to its intracellular parasitism. Therefore, unraveling the mechanism of
Brucella
-host interactions will help controlling
Brucella
infections. CRISPR-Cas9 is a gene editing technology that directs knockout of individual target genes by guided RNA, from which genome-wide gene-knockout cell libraries can be constructed. Upon infection with
Brucella
, the cell library would show differences in viability as a result of the knockout and specific genes could be revealed by genomic DNA sequencing. As a result, genes affecting cell viability during
Brucella
infection were identified. Further testing of gene function may reveal the mechanisms of
Brucella
-host interactions, thereby contributing to clinical therapy.
Funder
MOST | National Key Research and Development Program of China
MOST | National Natural Science Foundation of China
NSFC International (Regional) Cooperation and Exchange Program
Publisher
American Society for Microbiology