<i>cas9</i>
            Enhances Bacterial Virulence by Repressing the
            <i>regR</i>
            Transcriptional Regulator in Streptococcus agalactiae

Ma, Ke; Cao, Qing; Luo, Sisi; Wang, Zhaofei; Liu, Guangjin; Lu, Chengping; Liu, Yongjie

doi:10.1128/iai.00552-17

Cited by 30 publications

(42 citation statements)

References 56 publications

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“…Recently, it has been reported that CRISPR-Cas systems may also play a regulatory role on endogenous genes. Thus, in addition to protecting from phages, these systems are increasingly being recognized as regulators of bacterial virulence ( Louwen et al, 2014 ; Ma et al, 2018 ).…”

Section: Discussionmentioning

confidence: 99%

Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity

et al. 2018

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The marine bacterium Photobacterium damselae subsp. damselae is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the toxR gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that P. damselae subsp. damselae outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.

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Section: Discussionmentioning

confidence: 99%

Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity

et al. 2018

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“…In Type II CRISPR interference, CRISPR-associated protein 9 (Cas9), an RNA-guided DNA endonuclease, complexes with a trans- activating small RNA (tracrRNA) and small guide CRISPR RNAs (crRNA, derived from the CRISPR array), and this ribonucleoprotein complex targets foreign invading DNA for degradation (Marraffini and Sontheimer, 2010). Recently, Cas9 has been associated with bacterial virulence, immune evasion, and interaction of pathogens with host cells via endogenous regulation of gene expression (Sampson and Weiss, 2013a, b; Dugar et al, 2018; Ma et al, 2018). However, the Cas9 regulon and the mechanism by which Cas9 regulates gene expression has only begun to be elucidated and varies between bacteria; therefore, further study into the conservation of the Cas9 regulon and regulatory mechanism across other cas9- encoding pathogens, such as GBS, is needed.…”

Section: Introductionmentioning

confidence: 99%

“…However, the Cas9 regulon and the mechanism by which Cas9 regulates gene expression has only begun to be elucidated and varies between bacteria; therefore, further study into the conservation of the Cas9 regulon and regulatory mechanism across other cas9- encoding pathogens, such as GBS, is needed. A previous study on a GBS strain isolated from tilapia indicated that Cas9 influenced virulence (Ma et al, 2018); however, the role of Cas9 in both GBS colonization and disease in clinically relevant human isolates, such as those of ST-17 lineage, has not been determined.…”

Section: Introductionmentioning

confidence: 99%

Cas9 Contributes to Group B Streptococcal Colonization and Disease

et al. 2019

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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.

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“…Our results show that intrinsic control does not require dedicated transcription factors and may be a more general property of CRISPR-Cas biology. It remains to be seen whether other CRISPR-Cas types have evolved their own RNA-guided intrinsic regulators, for instance using degenerate self-targeting spacers 60 .…”

Section: Discussionmentioning

confidence: 99%

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

Workman

Pammi

Nguyen

et al. 2020

Preprint

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CRISPR-Cas systems provide their prokaryotic hosts with acquired immunity against viruses and other foreign genetic elements, but how these systems are regulated to prevent auto-immunity is poorly understood. In type II CRISPR-Cas systems, a transactivating CRISPR RNA (tracrRNA) scaffold functions together with a CRISPR RNA (crRNA) guide to program Cas9 for the recognition and cleavage of foreign DNA targets. Here, we show that a long-form tracrRNA performs an unexpected second function by folding into a natural single guide that directs Cas9 to transcriptionally repress its own promoter. Further, we demonstrate that Pcas9 serves as a critical regulatory node; de-repression causes a dramatic induction of Cas genes, crRNAs and tracrRNAs resulting in a 3,000-fold increase in immunization rates against unrecognized viruses. Heightened immunity comes at the cost of increased auto-immune toxicity, demonstrating the critical importance of the controller. Using a bioinformatic analysis, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

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cas9 Enhances Bacterial Virulence by Repressing the regR Transcriptional Regulator in Streptococcus agalactiae

Cited by 30 publications

References 56 publications

Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity

Molecular Epidemiology of Photobacterium damselae subsp. damselae Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity

Cas9 Contributes to Group B Streptococcal Colonization and Disease

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

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