scholarly journals Primed CRISPR-Cas Adaptation and Impaired Phage Adsorption in Streptococcus mutans

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Phage Genome ◽  
Multiplicity Of Infection ◽  
Type Ii ◽  
Content Type ◽  
Virulent Phage ◽  
Phage Adsorption ◽  
Protospacer Adjacent Motif

ABSTRACT Streptococcus mutans strain P42S possesses a type II-A CRISPR-Cas system that protects against phage infection and plasmid transformation. The analysis of 293 bacteriophage-insensitive mutants (BIMs) obtained upon exposure to the virulent phage M102AD revealed the acquisition of 399 unique spacers, including several ectopic spacer acquisitions and a few cases of native spacer deletions. The acquisition of multiple spacers was also observed and appears to be mostly due to priming, which has been rarely reported for type II-A systems. Analyses of the acquired spacers indicated that 88% of them are identical to a region of the phage M102AD genome. The remaining 12% of spacers had mismatches with the phage genome, primarily at the 5′ end of the spacer, leaving the seed sequence at the 3′ end largely intact. When a high multiplicity of infection (MOI) was used in the phage challenge assays, we also observed the emergence of CRISPR BIMs that, in addition to the acquisition of new spacers, displayed a reduced phage adsorption phenotype. While CRISPR-Cas and adsorption resistance work in tandem to protect S. mutans P42S against phage M102AD, nonidentified antiviral mechanisms are also likely at play in this strain. IMPORTANCE Bacteria are under the constant threat of viral predation and have therefore developed several defense mechanisms, including CRISPR-Cas systems. While studies on the mode of action of CRISPR-Cas systems have already provided great insights into phage-bacterium interactions, still more information is needed on the biology of these systems. The additional characterization of the type II-A CRISPR-Cas system of Streptococcus mutans P42S in this study provides novel information on the spacer acquisition step, especially regarding protospacer-adjacent motif (PAM) recognition, multiple-spacer acquisition, and priming.

scholarly journals Characterization of a Type II-A CRISPR-Cas System in Streptococcus mutans

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Cas Mosterd ◽  
Sylvain Moineau
Keyword(s):  
Streptococcus Mutans ◽  
Defense Mechanisms ◽  
Bacterial Species ◽  
Oral Microbiota ◽  
Type Ii ◽  
Content Type ◽  
C Terminus ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein

ABSTRACT Streptococcus mutans and its virulent phages are important members of the human oral microbiota. S. mutans is also the primary causal agent of dental caries. To survive in this ecological niche, S. mutans must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of S. mutans strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5′ end of the CRISPR locus and ectopically. In comparisons of the cas genes of P42S to those of other strains of S. mutans, cas1, cas2, and csn2 appear to be highly conserved within the species. However, more diversity was observed with cas9. While the nuclease domains of S. mutans Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain S. mutans UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species. IMPORTANCE CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, Streptococcus mutans strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.

scholarly journals ClpP is required for proteolytic regulation of type II toxin–antitoxin systems and persister cell formation in Streptococcus mutans

2019 ◽  
Vol 1 (8) ◽  
Author(s):  
Xiao-Lin Tian ◽  
Miao Li ◽  
Zachariah Scinocca ◽  
Heather Rutherford ◽  
Yung-Hua Li
Keyword(s):  
Streptococcus Mutans ◽  
Type Species ◽  
Critical Role ◽  
Cell Formation ◽  
Type Ii ◽  
Wild Type ◽  
Content Type ◽  
Link Type ◽  
Viability Assay ◽  
Persister Cell

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

scholarly journals Contribution of the Interaction of Streptococcus mutans SerotypekStrains with Fibrinogen to the Pathogenicity of Infective Endocarditis

2014 ◽  
Vol 82 (12) ◽  
pp. 5223-5234 ◽  
Author(s):  
Ryota Nomura ◽  
Masatoshi Otsugu ◽  
Shuhei Naka ◽  
Noboru Teramoto ◽  
Ayuchi Kojima ◽  
...  
Keyword(s):  
Infective Endocarditis ◽  
Streptococcus Mutans ◽  
Heart Valve ◽  
Heart Valves ◽  
Binding Assays ◽  
Bacterial Dna ◽  
Content Type ◽  
Fibrinogen Binding ◽  
Level Of Aggregation

ABSTRACTStreptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotypek-specific bacterial DNA is frequently detected inS. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE fromS. mutansstrains, focusing on the characterization of serotypekstrains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm+/PA−group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm+/PA−strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm+/PA−strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotypekDNA inS. mutans-positive heart valve clinical specimens.

scholarly journals Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity

2013 ◽  
Vol 80 (3) ◽  
pp. 994-1001 ◽  
Author(s):  
Alexandra E. Briner ◽  
Rodolphe Barrangou
Keyword(s):  
Immune System ◽  
Comparative Analysis ◽  
Common Origin ◽  
Type Ii ◽  
Crispr Locus ◽  
Content Type ◽  
Lactobacillus Buchneri ◽  
Protospacer Adjacent Motif ◽  
Associated Sequences ◽  
Short Palindromic Repeat

ABSTRACTClustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping ofLactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems inLactobacillus buchnerigenomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to fourcasgenes, including the universalcas1andcas2genes and the type II signature genecas9. Comparative analysis of CRISPR spacer content in 26L. buchneripickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology toLactobacillusplasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping ofL. buchneri.

scholarly journals Characterization of a Novel Arginine Catabolic Mobile Element (ACME) and Staphylococcal Chromosomal CassettemecComposite Island with Significant Homology to Staphylococcus epidermidis ACME Type II in Methicillin-Resistant Staphylococcus aureus Genotype ST22-MRSA-IV

2011 ◽  
Vol 55 (5) ◽  
pp. 1896-1905 ◽  
Author(s):  
Anna C. Shore ◽  
Angela S. Rossney ◽  
Orla M. Brennan ◽  
Peter M. Kinnevey ◽  
Hilary Humphreys ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Staphylococcus Epidermidis ◽  
Mobile Element ◽  
Type I ◽  
Type Ii ◽  
Coagulase Negative Staphylococci ◽  
Methicillin Resistant ◽  
Content Type ◽  
Arginine Catabolic Mobile Element

ABSTRACTThe arginine catabolic mobile element (ACME) is prevalent among methicillin-resistantStaphylococcus aureus(MRSA) isolates of sequence type 8 (ST8) and staphylococcal chromosomal cassettemec(SCCmec) type IVa (USA300) (ST8-MRSA-IVa isolates), and evidence suggests that ACME enhances the ability of ST8-MRSA-IVa to grow and survive on its host. ACME has been identified in a small number of isolates belonging to other MRSA clones but is widespread among coagulase-negative staphylococci (CoNS). This study reports the first description of ACME in two distinct strains of the pandemic ST22-MRSA-IV clone. A total of 238 MRSA isolates recovered in Ireland between 1971 and 2008 were investigated for ACME using a DNA microarray. Twenty-three isolates (9.7%) were ACME positive, and all were either MRSA genotype ST8-MRSA-IVa (7/23, 30%) or MRSA genotype ST22-MRSA-IV (16/23, 70%). Whole-genome sequencing and comprehensive molecular characterization revealed the presence of a novel 46-kb ACME and staphylococcal chromosomal cassettemec(SCCmec) composite island (ACME/SCCmec-CI) in ST22-MRSA-IVh isolates (n= 15). This ACME/SCCmec-CI consists of a 12-kb DNA region previously identified in ACME type II inS. epidermidisATCC 12228, a truncated copy of the J1 region of SCCmectype I, and a complete SCCmectype IVh element. The composite island has a novel genetic organization, with ACME located withinorfXand SCCmeclocated downstream of ACME. One PVL locus-positive ST22-MRSA-IVa isolate carried ACME located downstream of SCCmectype IVa, as previously described in ST8-MRSA-IVa. These results suggest that ACME has been acquired by ST22-MRSA-IV on two independent occasions. At least one of these instances may have involved horizontal transfer and recombination events between MRSA and CoNS. The presence of ACME may enhance dissemination of ST22-MRSA-IV, an already successful MRSA clone.

scholarly journals Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

2014 ◽  
Vol 197 (4) ◽  
pp. 749-761 ◽  
Author(s):  
M. A. Serbanescu ◽  
M. Cordova ◽  
K. Krastel ◽  
R. Flick ◽  
N. Beloglazova ◽  
...  
Keyword(s):  
Heat Shock ◽  
Streptococcus Mutans ◽  
Antibiotic Resistance Genes ◽  
Transcriptional Analysis ◽  
Cell Physiology ◽  
Type I ◽  
Type Ii ◽  
Content Type ◽  
Bacterial Physiology

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

scholarly journals Disruption of a Novel Iron Transport System Reverses Oxidative Stress Phenotypes of a dpr Mutant Strain of Streptococcus mutans

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Tridib Ganguly ◽  
Jessica K. Kajfasz ◽  
James H. Miller ◽  
Eric Rabinowitz ◽  
Lívia C. C. Galvão ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Streptococcus Mutans ◽  
Hydroxyl Radicals ◽  
Transport System ◽  
Iron Uptake ◽  
Iron Transport ◽  
Transport Systems ◽  
Phenotypic Characterization ◽  
Content Type

ABSTRACT The Dps-like peroxide resistance protein (Dpr) is essential for H 2 O 2 stress tolerance and aerobic growth of the oral pathogen Streptococcus mutans . Dpr accumulates during oxidative stress, protecting the cell by sequestering iron ions and thereby preventing the generation of toxic hydroxyl radicals that result from the interaction of iron with H 2 O 2 . Previously, we reported that the SpxA1 and SpxA2 regulators positively regulate expression of dpr in S. mutans . Using an antibody raised against S. mutans Dpr, we confirmed at the protein level the central and cooperative nature of SpxA1 and SpxA2 regulation in Dpr production. During phenotypic characterization of the S. mutans Δ dpr strain, we observed the appearance of distinct colony variants, which sometimes lost the oxidative stress sensitivity typical of Δ dpr strains. Whole-genome sequencing of these phenotypically distinct Δ dpr isolates revealed that a putative iron transporter operon, smu995-smu998 , was a genomic hot spot with multiple single nucleotide polymorphisms identified within the different isolates. Deletion of smu995 or the entire smu995-smu998 operon in the Δ dpr background strain completely reversed the oxidative stress-sensitive phenotypes associated with dpr inactivation. Conversely, inactivation of genes encoding the ferrous iron transport system FeoABC did not alleviate phenotypes of the Δ dpr strain. Preliminary characterization of strains lacking smu995-smu998 , feoABC , and the iron/manganese transporter gene sloABC revealed the interactive nature of these three systems in iron transport but also indicated that there may be additional iron uptake systems in S. mutans . IMPORTANCE The dental caries-associated pathogen Streptococcus mutans routinely encounters oxidative stress within the human plaque biofilm. Previous studies revealed that the iron-binding protein Dpr confers protection toward oxidative stress by limiting free iron availability, which is associated with the generation of toxic hydroxyl radicals. Here, we report the identification of spontaneously occurring mutations within Δ dpr strains. Several of those mutations were mapped to the operon smu995-smu998 , revealing a previously uncharacterized system that appears to be important in iron acquisition. Disruption of the smu995-smu998 operon resulted in reversion of the stress-sensitive phenotype typical of a Δ dpr strain. Our data suggest that the Smu995-Smu998 system works along with other known metal transport systems of S. mutans , i.e., FeoABC and SloABC, to coordinate iron uptake.

scholarly journals A catalogue of biochemically diverse CRISPR-Cas9 orthologs

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Giedrius Gasiunas ◽  
Joshua K. Young ◽  
Tautvydas Karvelis ◽  
Darius Kazlauskas ◽  
Tomas Urbaitis ◽  
...  
Keyword(s):  
Temperature Dependence ◽  
Genome Editing ◽  
Type Ii ◽  
Single Nucleotide ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Biochemical Analyses ◽  
Sequence Requirements ◽  
Biochemical Diversity

Abstract Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Parasite defense mechanisms in bees: behavior, immunity, antimicrobials, and symbionts

2019 ◽  
Vol 4 (1) ◽  
pp. 59-76 ◽  
Author(s):  
Alison E. Fowler ◽  
Rebecca E. Irwin ◽  
Lynn S. Adler
Keyword(s):  
Defense Mechanisms ◽  
Poor Quality ◽  
Future Research ◽  
Immune Priming ◽  
Social Bees ◽  
Bee Health ◽  
Landscape Scales ◽  
And Function ◽  
Solitary Species

Parasites are linked to the decline of some bee populations; thus, understanding defense mechanisms has important implications for bee health. Recent advances have improved our understanding of factors mediating bee health ranging from molecular to landscape scales, but often as disparate literatures. Here, we bring together these fields and summarize our current understanding of bee defense mechanisms including immunity, immunization, and transgenerational immune priming in social and solitary species. Additionally, the characterization of microbial diversity and function in some bee taxa has shed light on the importance of microbes for bee health, but we lack information that links microbial communities to parasite infection in most bee species. Studies are beginning to identify how bee defense mechanisms are affected by stressors such as poor-quality diets and pesticides, but further research on this topic is needed. We discuss how integrating research on host traits, microbial partners, and nutrition, as well as improving our knowledge base on wild and semi-social bees, will help inform future research, conservation efforts, and management.

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