627 - Genome-wide Determination of Group B Streptococcus Factors Conditionally Essential for Intestinal Colonization

Publication Number: 627.4930

Michelle J. Vaz, Hassenfeld Children's Hospital at NYU Langone, Tenafly, NJ, United States; Sophia A. Dongas, New York Medical College, Chicago, IL, United States; Sanjana Sankaran, Hassenfeld Children's Hospital at NYU Langone, BROOKLYN, NY, United States; Adam J. Ratner, New York University Grossman School of Medicine, New York, NY, United States

Background: Group B Streptococcus (GBS) intestinal colonization is critical for the pathogenesis of late-onset (LO) disease in infants. Using a murine model of gastrointestinal (GI) colonization, we have investigated specific bacterial and host factors that influence GBS colonization efficiency. Transposon insertion sequencing (Tn-seq) is a powerful method for genome-scale functional genetics in bacteria that can identify conditionally essential (CE) GBS genes. We performed a genome wide analysis of GBS required for intestinal colonization in vivo.

Objective: To identify conditionally essential genes required for GBS GI colonization using Tn-seq.

Design/Methods: Pre-weaning C57BL/6J mice (12-14 days of life) were orally infected with a GBS A909 transposon mutant library consisting of ~150,000 unique TA site mutants. The colon was harvested at 3,7- and 14-days post infection, and tissue homogenates were plated on selective media to recover transposon insertion mutants. Genomic DNA underwent MmeI digestion, barcoded terminal adapter ligation, PCR and sequencing on the Illumina NovaSeq 6000 platform. Comparing insertion sites in the input library to the GBS recovered from colonic tissue, we determined the set of CE genes using TRANSIT. Of these, we performed validation studies with two candidate genes: sodA (Mn-cofactored superoxide dismutase) and ciaRH (two component system sensor histidine kinase) in cocolonization experiments using a wild-type (WT) GBS A909 and respective isogenic mutant GBS strains (A909 sodA::Himar1 and A909 ciaRH::Himar1). Co-cocolonization experiments were performed by orally inoculating preweaning C57BL/6J mice with sets of both strains in 2 separate experimental cohorts. Multiple GI tract sites were harvested on day 7 post infection. Competitive indices were calculated to determine the contribution of each gene to GI colonization.

Results: We identified 117 underrepresented mutants with a log2 fold-change (LFC) cutoff value of -5.0 and adjusted p value cutoff of < 0.05 (Fig. 1). These mutants were involved in morphology, stress response, transcriptional regulation and nutrient import and efflux systems of GBS. WT GBS A909 outcompeted both of the mutants selected for validation, A909 sodA::Himar1 and A909 ciaRH::Himar1, in co-colonization experiments (Fig. 2 A, B).

Conclusion(s): Tn-seq identified CE genes for GBS persistence in the intestinal tract. Candidate CE genes were validated in cocolonization studies. GBS Tn-seq is a valuable tool to understand the role of GBS factors in GI colonization and progression to invasive disease.

Figure 1. Genome-wide fitness analysis of GBS to identify CE genes in intestinal colonization.
Frequency of insertion count versus LFC histogram plot, with LFC threshold between nonessential and CE genes (-5) indicated (red line).


Figure 2. Validation of individual candidate CE genes using GI tract cocolonization.
Wild type (WT) A909 outcompetes sodA (A) and ciaRH (B) transposon insertion strains in a cocolonization model. Anatomic site and sample size are displayed on the x-axis. Data points represent geometric mean competition indices and error bars represent 95% confidence intervals.