Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities.

C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities-one for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.

The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a.

Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al. (2020) described the discovery and characterization of 7 Acr families (AcrVIA1-7) that inhibit type VI-A CRISPR systems. We detail several inconsistencies that question the results reported in the Lin et al. (2020) study. These include inaccurate bioinformatics analyses and bacterial strains that are impossible to construct. Published strains were provided by the authors, but MS2 bacteriophage plaque assays did not support the published results. We also independently tested the Acr sequences described in the original report, in E. coli and mammalian cells, but did not observe anti-Cas13a activity. Taken together, our data and analyses prompt us to question the claim that AcrVIA1-7 reported in Lin et al. are type VI anti-CRISPR proteins.

Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.

Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13acrRNA in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.

CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

Leptotrichia Bacteremia: 10-Year Retrospective Clinical Analysis and Antimicrobial Susceptibility Profiles.

Leptotrichia species are anaerobic, Gram-negative bacilli increasingly recognized as pathogens capable of causing invasive infections such as bloodstream infection (BSI), particularly among immunocompromised patients. However, there is a paucity of data regarding epidemiology, antimicrobial susceptibility, optimal treatment, and clinical outcomes among patients with Leptotrichia bacteremia. Patient risk factors, treatment approaches, and outcomes of a retrospective cohort of adult patients with Leptotrichia BSI at a tertiary medical center (Mayo Clinic Rochester [MCR]) were evaluated. Concurrently, species, temporal trends, and antimicrobial susceptibility testing (AST) results of Leptotrichia isolates submitted to a reference laboratory (Mayo Clinic Laboratories) over the past 10 years were examined. We identified 224 blood culture isolates of Leptotrichia species, with 26 isolates from patients treated at MCR. The most frequent species included L. trevisanii (49%), L. buccalis (24%), and L. wadei (16%). Leptotrichia species demonstrated >90% susceptibility to penicillin, metronidazole, ertapenem, and piperacillin-tazobactam. However, 96% (74/77) of isolates were resistant to moxifloxacin. For patients treated at MCR, the mean patient age was 55 years (standard deviation [SD], 17), with 9 females (35%), and all were neutropenic at the time of BSI. The primary sources of infection were gastrointestinal (58%), intravascular catheter (35%), and odontogenic (15%). Patients were treated with metronidazole (42%), piperacillin-tazobactam (27%), or carbapenems (19%). The mean duration of treatment was 11 days (SD, 4.5), with a 60-day all-cause mortality of 19% and no microbiologic relapse. Leptotrichia species are rare but important causes of BSI in neutropenic patients. Due to evolving antimicrobial susceptibility profiles, a review of AST results is necessary when selecting optimal antimicrobial therapy.

RNA targeting with CRISPR-Cas13.

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.

Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

SPRINT: a Cas13a-based platform for detection of small molecules.

Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.

High-throughput sequencing provides insights into oral microbiota dysbiosis in association with inflammatory bowel disease.

Although the prevalence of inflammatory bowel disease (IBD) has been increasing worldwide, the etiology remains elusive. Investigating oral microbiota dysbiosis is essential to understanding IBD pathogenesis. Our study evaluated variations in salivary microbiota and identified potential associations with IBD. The saliva microbiota of 22 IBD patients and 8 healthy controls (HCs) was determined using 16S ribosomal RNA (rRNA) gene sequencing and analyzed using QIIME2. A distinct saliva microbiota dysbiosis in IBD, characterized by alterations in microbiota biodiversity and composition, was identified. Saccharibacteria (TM7), Absconditabacteria (SR1), Leptotrichia, Prevotella, Bulleidia, and Atopobium, some of which are oral biofilm-forming bacteria, were significantly increased. Moreover, levels of inflammatory cytokines associated with IBD were elevated and positively correlated with TM7 and SR1. Functional variations include down-regulation of genetic information processing, while up-regulation of carbohydrate metabolism and protein processing in the endoplasmic reticulum in IBD. Our data implicate salivary microbiota dysbiosis involving in IBD pathogenesis.

In vitro activities and spectrum of lascufloxacin（KRP-AM1977）against anaerobes.

The in vitro antibacterial spectra and activities of five antimicrobial agents, including lascufloxacin (LSFX) and two quinolones, were investigated against 69 species of anaerobes in 31 genera and 188 strains in 9 genera, respectively. In this study, minimum inhibitory concentrations (MICs) of lascufloxacin against the reference strains associated with respiratory and head and neck infections. LSFX inhibited the growth of 33 gram-positive and gram-negative reference strains at ≤0.015-2 µg/mL, except for Leptotrichia buccalis. MICs ranges of LSFX against the clinical isolates of 44 Porphyromonas spp., 45 Prevotella spp., 25 Fusobacterium spp., 7 Leptotrichia spp., 25 Parvimonas micra, 25 other gram-positive anaerobic cocci, and 17 Veillonella spp., were ≤0.015-4, 0.125-4, 0.06-0.5, 2, 0.25-16, ≤0.015-2, ≤0.015-16 µg/mL, respectively. LSFX demonstrated potent antibacterial efficacy against a wide range of species isolated from specimens involved in respiratory as well as head and neck infections.

Bacterial levels and amount of endotoxins in carious dentin within reversible pulpitis scenarios.

OBJECTIVES: the objective of the present exploratory study was to determine bacterial diversity and endotoxin levels in deep carious lesions of teeth presenting symptoms of reversible pulpitis. MATERIALS AND METHODS: Twenty patients with deep carious lesions, reporting clinical symptomatology compatible with reversible pulpitis (n = 10) or not reporting clinical symptomatology (n = 10), were selected. Carious dentin samples were obtained with the aid of sterile and pyrogen-free spoon excavators and harvested in two steps: before and after infected dentin removal. Samples were collected for checkerboard and for kinetic chromogenic LAL assay for determination of microbial profile and quantitation of endotoxin, respectively. Data were analyzed by Mann Whitney for bacteria and two-way ANOVA for endotoxins (5%). RESULTS: No difference on the studied bacteria was detected between the superficial and deep dentin layers. Symptomatic teeth showed greater presence of Lactobacillus species, Capnocytophaga sputigena, and Leptotrichia buccalis. For the endotoxins, symptomatic teeth resulted in greater quantity of endotoxins (p = 0.047), being 4.13 log10 EU/mL/µg dentin and 3.45 log10 EU/mL/µg dentin, for symptomatic and asymptomatic teeth, respectively. Dentin collected in different areas presented similar number of endotoxins (p = 0.139). CONCLUSION: The amount of the studied bacteria does not seem to be related to reported symptomatology of deep carious lesions, while endotoxins quantity is greater in symptomatic scenarios, regardless of the harvesting area. CLINICAL RELEVANCE: The understanding of bacterial amount in reversible pulpitis is important to establish a clinical protocol of treatment.

Proposal to reclassify Leptotrichia goodfellowii into a novel genus as Pseudoleptotrichia goodfellowii gen. nov., comb. nov.

The reclassification of Leptotrichia goodfellowii as Pseudoleptotrichia goodfellowii gen. nov., comb. nov. is proposed because of the separate phylogenetic position on the basis of the results of 16S rRNA gene sequence analysis, the genomic differences from all other Leptotrichia species and phenotypic differences from Leptotrichia species. The species Pseudoleptotrichia goodfellowii is the type species of the genus. The type strain is LB 57T, CCUG 32286 T, DSM 19756T.

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing.

In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference.

Sneathia amnii and Maternal Chorioamnionitis and Stillbirth, Mozambique.

We report a case of Sneathia amnii as the causative agent of maternal chorioamnionitis and congenital pneumonia resulting in a late fetal death in Mozambique, with strong supportive postmortem molecular and histopathologic confirmation. This rare, fastidious gram-negative coccobacillus has been reported to infrequently cause abortions, stillbirths, and neonatal infections.

High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity.

MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. It has been proved that the aberrant expression of miRNAs is related to disease and miRNAs can serve as potential biomarkers for early tumor diagnosis. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a is a recently discovered CRISPR-RNA (crRNA) guided RNA manipulation tool. The recognition of target RNA can morphologically activate the robust nonspecific trans ribonuclease activity of Cas13a. This unique property makes Cas13a ideal for nucleic acid detection. Herein, we first exploited CRISPR/LbuCas13a to directly detect miRNAs with high specificity and simplicity. A limit of detection (LOD) as low as 4.5 amol was achieved by this one-step assay within 30 min, and the dynamic range spanned 4 orders of magnitude from 10 amol to 100 fmol. More importantly, single nucleotide variation, even at the end of target miRNA, can be discriminated by rationally programmed crRNA. In addition, the practical application ability of this Cas13a/crRNA-based signal amplification strategy was demonstrated by miRNA quantification in complex biological samples (total small RNA). With excellent reliability, sensitivity, and simple to implement features, this method promises a great potential for early diagnosis of miRNA-related disease. Moreover, the systematic analysis of the crRNA design could provide guidance to further develop Cas13a-based molecular diagnoses.

Emergent genital infection by Leptotrichia trevisanii.

We report the first case of an association between Leptotrichia trevisanii and an episode of pelvic inflammatory disease (PID) and the second case of the isolation of this infection in the cervical canal. A 45-yr-old woman was admitted to our emergency department with clinical and radiological signs and symptoms compatible with an episode of PID. She was hospitalized for intravenous antibiotic control and treatment and the subsequent surgical drainage of abscesses. Cultures were taken throughout the process, but only cultures from cervical canal exudate were positive, with the growth of L. trevisanii species. It appears important to carry out a complete microbiological screening, not limited to conventional agents, on adequate clinical samples to detect possible infectious agents that may be missed in these cases.

Leptotrichia trevisanii bacteremia in a woman with systemic lupus erythematosus receiving high-dose chemotherapy.

BACKGROUND: Leptotrichia species are aerotolerant, Gram-negative fusiform bacteria. Cases of bacteremia caused by Leptotrichia trevisanii in immunocompromised patients have been rarely reported. CASE PRESENTATION: A 33-year-old female with systemic lupus erythematosus (SLE) was admitted to the department of rheumatology with bleeding from a mucosal ulcer. One month previously, she had visited our hospital and begun to receive methotrexate therapy, but mis-dosed for nearly 1 month at home. Methotrexate toxicity resulted in a severe oral ulcer and bone marrow suppression. On day-7 of hospital admission, she developed a fever, and Gram-negative rods (Leptotrichia trevisanii) were detected in blood cultures. She was diagnosed with methotrexate poisoning followed by L. trevisanii bacteremia. After antibiotic and detoxification therapy, she recovered from bacteremia, and the oral ulcer and bone-marrow suppression improved obviously. CONCLUSIONS: This is the first reported case of Leptotrichia trevisanii bacteremia in a SLE patient who took mis-dosed an immunosuppressant and had an oral mucosal lesion.

Chorioamnionitis due to Leptotrichia trevisanii.

Very long fusiform gram-negative bacilli were observed after Gram staining of amniotic fluid from a 36-year-old multigravida woman. At 24 hours, pure, abundant growth of smooth, gray, only slightly convex catalase-positive and oxidase-negative colonies measuring about 2 mm were observed. Growth was greater in anaerobic than in aerobic conditions. The bacterium was identified as Leptotrichia trevisanii by matrix-assisted laser desorption ionization time of flight mass spectrometry. Ampicillin and gentamicin were prescribed for chorioamnionitis, and vaginal prostaglandins were administered to terminate the pregnancy. The patient remained afebrile throughout 48 hours and was discharged. Microscopic examination of the placenta revealed severe acute chorioamnionitis with a maternal inflammatory response and abundant bacillary-shaped microorganisms. To our knowledge, this isolate constitutes the first reported case of chorioamnionitis caused by L. trevisanii.

Impact of Periodic Presumptive Treatment for Bacterial Vaginosis on the Vaginal Microbiome among Women Participating in the Preventing Vaginal Infections Trial.

Background: Evidence suggests that specific vaginal bacteria associated with bacterial vaginosis (BV) may increase the risk of adverse health outcomes in women. Among women participating in a randomized, double-blinded trial, we assessed the effect of periodic presumptive treatment (PPT) on detection of select vaginal bacteria. Methods: High-risk women from the United States and Kenya with a recent vaginal infection received intravaginal metronidazole 750 mg plus miconazole 200 mg or placebo for 5 consecutive nights each month for 12 months. Vaginal fluid specimens were collected via polyester/polyethylene terephthalate swabs every other month and tested for bacteria, using quantitative polymerase chain reaction (PCR) assays targeting the 16S ribosomal RNA gene. The effect of PPT on bacterium detection was assessed among all participants and stratified by country. Results: Of 234 women enrolled, 221 had specimens available for analysis. The proportion of follow-up visits with detectable quantities was lower in the PPT arm versus the placebo arm for the following bacteria: BVAB1, BVAB2, Atopobium vaginae, Leptotrichia/Sneathia, and Megasphaera. The magnitude of reductions was greater among Kenyan participants as compared to US participants. Conclusions: Use of monthly PPT for 1 year reduced colonization with several bacteria strongly associated with BV. The role of PPT to improve vaginal health should be considered, and efforts to improve the impact of PPT regimens are warranted.