Hooven & Aronoff
CRISPRi repression of group B Streptococcus externalized proteins to identify key mediators of macrophage responses during neonatal infection
Streptococcus agalactiae (group B Streptococcus; GBS) is a major global contributor to infant mortality. In the United States, GBS is the most common cause of neonatal sepsis, affecting approximately 2,500 patients annually with a fatality rate of 6%. GBS infection and resultant inflammation result from molecular interactions at the host-pathogen interface. Some of these interactions involve bacterial surface-associated or secreted proteins, which can be identified based on the presence of an N-terminus signal peptide sequence. Several GBS surface-associated and secreted proteins are known to be important participants in pathogenesis, promoting host epithelial binding, intracellular invasion, and survival within host phagocytes. However, systematic study of GBS proteins with signal peptide sequences has not been feasible because making targeted genetic knockouts in GBS is technically challenging and prone to failure. There are 76 signal peptide encoding genes in the commonly used laboratory strain CTNTC 10/84. Knocking out each gene individually would be prohibitively time-consuming. The Hooven laboratory has recently developed a rapid, reliable, and cost-effective method of downregulating expression of specific GBS genes with CRISPR interference (CRISPRi) technology. Briefly, using a GBS strain in which the programmable endonuclease Cas9 is catalytically inactivated (dCas9), it is possible to direct dCas9 to specific sites on the GBS chromosome by introducing easily designed and inexpensive targeting DNA sequences. If dCas9 is targeted to a site within a protein coding gene, steric hinderance with RNA polymerase reduces gene transcription and protein expression. Our CRISPRi system therefore opens the door to systematic evaluation of the roles of all surface-associated and secreted proteins in the GBS genome, which has never been possible before.
In this study, we propose combining this new CRISPRi technology with high-throughput, in vitro assays of GBS-human macrophage interactions, which have been characterized by the laboratory of David Aronoff at Vanderbilt University. Tissue-resident macrophages are among the first components of the innate immune system encountered by GBS during its transition from a commensal lifestyle to an invasive one but we lack understanding of how GBS evades macrophage defense mechanisms or alters their paracrine signaling responses, such as the release of immunomodulatory cytokines. We hypothesize that GBS surface-associated and/or secreted proteins are likely to modulate macrophage antibacterial responses systems such as internalization (phagocytosis), intracellular killing, and the expression of immunomodulatory cytokines. Proteins with a significant effect on these functions would be logical candidates for future studies of targeting antibodies or pharmaceuticals.
Thomas Hooven, MD
Department of Pediatrics
Division of Neonatology / Newborn Medicine
UPMC / University of Pittsburgh
David Aronoff, MD
Addison B. Scoville, Jr. Professor
Department of Medicine
Division of Infectious Diseases