Cas9 Contributes to Group B Streptococcal Colonization and Disease

Abstract

Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20–30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10–15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δ cas9 mutants. Compared to parental strains, Δ cas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δ cas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δ cas9 mutant strain. RNA-Seq analysis of the WT and Δ ciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.

Identification of a novel cationic glycolipid inStreptococcus agalactiaethat contributes to brain entry and meningitis

“…To mimic the human blood-brain barrier (BBB), the human cerebral microvascular endothelial cell line hCMEC/D3 was utilized. The in vitro assays for adhesion and invasion were performed using pre-established protocols (37,38). To control for the presence of the plasmid backbone in the complement strain, COH1(pABG5), COH1Δ mprF (pABG5), and COH1Δ mprF (pGBSMprF) strains were used.…”

“…Assays to determine the total number of bacteria adhered to host cells or intracellular bacteria were performed as described previously (37,38). Briefly, bacteria were grown to mid log phase (OD 600nm 0.4-0.5) and normalized to 1 × 10 8 to infect cell monolayers at a multiplicity of infection (MOI) of 1 (1 × 10 5 CFU per well).…”

The CRISPR-Cas Mechanism for Adaptive Immunity and Alternate Bacterial Functions Fuels Diverse Biotechnologies

“…CRISPR-Cas systems modulate bacterial-host interactions, the mechanisms of which are currently evolving. Relative to the wild-type, Neisseria , Streptococcus , and Campylobacter △ cas9 strains adhere poorly to mammalian host cells ( Ma et al., 2018 ; Shabbir et al., 2018 ; Heidrich et al., 2019 ; Spencer et al., 2019 ; Gao et al., 2019 ). The S. pyogenes mechanism involves Cas9-mediated down-regulation of the FasA/FasB TCS that downregulates adhesion factors ( Gao et al., 2019 ).…”

“…These studies suggest complex interdependence of CRISPR and DNA repair systems, the mechanisms of which are still elusive. Transcriptomic and proteomic studies of △ cas9 S. pyogenes (type II-A) ( Gao et al., 2019 ), Group B Streptococcus (type II-A) ( Spencer et al., 2019 ), and Riemerella anatipestifer (type II-C) ( Wang et al., 2019 ) have indicated both up- and down- regulation of several endogenous genes, the implications of which are not clear. Several studies in Myxococcus xanthus have shown that CRISPR systems are involved in cell-stress dependent sporulation (type I-C) and fruiting body development (type III-B) ( Viswanathan et al., 2007 ; Wallace et al., 2014 ).…”

Global gene expression analysis ofStreptococcus agalactiaeat exponential growth phase

“…Together with improved computational tools for data analysis, innovations in RNA-seq technologies are contributing to a better understanding of RNA biology and intermolecular interactions that govern RNA function, as well as transcriptional dynamics changes driving bacterial response and adaptation, to distinct growth conditions and external stimuli [9]. Recently, RNA-seq technology has been successfully applied to clarify some molecular mechanisms of pathogenesis in S. agalactiae [8,[10][11][12][13][14][15]. The first S. agalactiae comparative transcriptomic study evidenced several genetic factors (in particular related to lactose metabolism) likely important in adaptation to the bovine environment [10].…”