bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 4 MrvR, a Group B Streptococcus Transcription Factor that Controls Multiple Virulence Traits 5 6 Allison N. Dammann1, Anna B. Chamby2, Andrew J. Catomeris3, Kyle M. Davidson4, Hervé 7 Tettelin5,6, Jan-Peter van Pijkeren7, Kathyayini P. Gopalakrishna4, Mary F. Keith4, Jordan L. 8 Elder4, Adam J. Ratner1,8, Thomas A. Hooven4,9* 9 10 1 Department of Pediatrics, New York University School of Medicine, New York, NY, USA 11 12 2 University of Vermont Larner College of Medicine, Burlington, VT, USA 13 14 3 Georgetown University School of Medicine, Washington, DC, USA 15 16 4 Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 17 18 5 Department of Microbiology and Immunology, University of Maryland School of Medicine, 19 Baltimore, MD, USA 20 21 6 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 22 USA 23 24 7 Department of Food Science, University of Wisconsin, Madison, WI, USA 25 26 8 Department of Microbiology, New York University, New York, NY, USA 27 28 9 Richard King Mellon Institute for Pediatric Research, UPMC Children’s Hospital of Pittsburgh, 29 Pittsburgh, PA, USA 30 31 * Corresponding author 32 E-mail: [email protected] 33 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 34 35 Abstract 36 37 Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious 38 neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal 39 period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which 40 is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We 41 used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS 42 genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq 43 findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This 44 analysis showed that one gene, which encodes a GntR-class transcription factor that we named 45 MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic 46 targeted knockout of the mrvR gene, which we found to have a growth defect in amniotic fluid 47 relative to the wild type parent strain. In addition to growing poorly in amniotic fluid, the 48 knockout also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed 49 that, while the knockout was able to cause persistent murine vaginal colonization, pregnant 50 mice colonized with the knockout strain did not develop preterm labor despite consistent GBS 51 invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with 52 wild type GBS consistently deliver prematurely. Similarly, in a sepsis model in which 87% of 53 mice infected with wild type GBS died within three days, none of the mice infected with the 54 knockout strain died. In order to better understand the mechanism by which this newly 55 identified transcription factor controls GBS virulence, we performed electrophoresis mobility bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 56 shift assays with recombinant MrvR and whole-genome transcriptomic analysis on the 57 knockout and wild type strains. We show that MrvR binds to its own promoter region, 58 suggesting likely self-regulation. RNA-seq revealed that the transcription factor affects 59 expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and 60 salvage pathways were highly represented among the set of differentially expressed genes, 61 suggesting a linkage between purine or pyrimidine availability and activity of MrvR in multiple 62 GBS virulence traits. 63 64 Introduction 65 66 Streptococcus agalactiae (group B Streptococcus; GBS) is a cause of chorioamnionitis, stillbirth, 67 and neonatal infections including bacteremia, pneumonia, and meningitis (1–10). GBS is a 68 common commensal of the intestinal and reproductive tracts in healthy adults, among whom 69 invasive disease is rare (11, 12). In the pregnant or neonatal host, however, GBS can be highly 70 invasive, breaching anatomic and immunologic barriers with potentially severe consequences 71 (13, 14). 72 73 GBS is known to express a number of virulence factors such as adhesins (15–17), IgA binding 74 proteins (18–20), and the cytotoxic ornithine-rhamnopolyene b-hemolysin/cytolysin (13, 21– 75 30). Many of these are regulated by transcription factors, some of which have been studied and 76 described in detail (31–40), yet many of the predicted transcription factors encoded by GBS 77 remain minimally characterized. bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 78 79 We have previously described development of a GBS saturated transposon mutant library and 80 its use in Tn-seq experiments to identify essential and conditionally essential genes (41, 42). In 81 this study, we performed Tn-seq on GBS grown in human amniotic fluid in order to identify 82 bacterial genes that promote survival in the nutrient-poor, unhospitable growth conditions of 83 the amniotic sac. 84 85 Among the candidate genes was a transcription factor with domain features marking it as a 86 member of the GntR protein superfamily. We named this GntR-class transcription factor MrvR 87 (multiple regulator of virulence). Here we describe the role of MrvR in several GBS virulence- 88 related phenotypes, including survival in human amniotic fluid, in vitro biofilm formation, and 89 invasive bacteremia and preterm labor in murine models. We also use transcriptomic data and 90 electrophoresis mobility shift assay results to demonstrate autoregulation of MrvR expression 91 and its significant role in modifying expression of genes integral to nucleotide metabolism. 92 93 Results 94 95 Five GBS genes are conditionally essential for GBS survival in human amniotic fluid 96 97 Using a previously described transposon mutant library in an A909 (serotype Ia) background 98 (41), we performed Tn-seq after growth challenge in eight human amniotic fluid samples from 99 amniocentesis (two to three technical replicates in three separate biological samples). Prior to bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 100 use for Tn-seq, the amniotic fluid was filter sterilized to remove any contaminating 101 microorganisms and host cells that may have been present. The same mutant library was grown 102 in rich media as an input control. 103 104 We used ESSENTIALS, a publicly available Tn-seq bioinformatics package, to analyze sequencing 105 data from the Tn-seq experiment. This analysis revealed five candidate conditionally essential 106 genes (Figure 1 and Table 1). 107 108 One of the candidate genes, SAK_RS10120, which we termed mrvR, had conserved functional 109 domains suggesting its membership in the GntR superfamily of transcriptional regulators. The 110 other candidate genes encoded an expected DeoR family transcriptional regulator 111 (SAK_RS09545), a predicted membrane-spanning protein of unknown function (SAK_RS05160), 112 an ABC-class transporter (SAK_RS09890), and one protein (SAK_RS02930) with a single domain 113 of unknown function whose cellular localization and role are also unknown. 114 115 CRISPRi knockdown and targeted knockout competition assays validate Tn-seq findings 116 117 To further validate the five candidate genes conditionally essential for GBS survival in amniotic 118 fluid, we developed a CRISPRi gene expression knockdown system, which uses natively 119 expressed, catalytically inactive Cas9 (dCas9) encoded on the GBS chromosome along with a 120 single guide RNA (sgRNA) expression plasmid to generate efficient and flexible gene expression 121 inhibition in GBS. We competed knockdowns of the five candidate conditionally essential genes bioRxiv preprint doi: https://doi.org/10.1101/2020.11.17.386367; this version posted November 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 122 against a sham targeting control dCas9 strain in human amniotic fluid and assayed survival 123 using quantitative PCR analysis of knockdown and control strain persistence. 124 125 Development of a GBS CRISPRi system was aided by the fact that the GBS cas9 gene shows high 126 sequence similarity to the canonical cas9 endonuclease first described in Streptococcus 127 pyogenes (43–46) (Supplemental Figure 1).