Expanding the CRISPR Toolbox in P. patens Using SpCas9-NG Variant and Application for Gene and Base Editing in Solanaceae Crops.

Genome editing has become a major tool for both functional studies and plant breeding in several species. Besides generating knockouts through the classical CRISPR-Cas9 system, recent development of CRISPR base editing holds great and exciting opportunities for the production of gain-of-function mutants. The PAM requirement is a strong limitation for CRISPR technologies such as base editing, because the base substitution mainly occurs in a small edition window. As precise single amino-acid substitution can be responsible for functions associated to some domains or agronomic traits, development of Cas9 variants with relaxed PAM recognition is of upmost importance for gene function analysis and plant breeding. Recently, the SpCas9-NG variant that recognizes the NGN PAM has been successfully tested in plants, mainly in monocotyledon species. In this work, we studied the efficiency of SpCas9-NG in the model moss Physcomitrella patens and two Solanaceae crops (Solanum lycopersicum and Solanum tuberosum) for both classical CRISPR-generated gene knock-out and cytosine base editing. We showed that the SpCas9-NG greatly expands the scope of genome editing by allowing the targeting of non-canonical NGT and NGA PAMs. The CRISPR toolbox developed in our study opens up new gene function analysis and plant breeding perspectives for model and crop plants.

Identification of potential antivirulence agents by substitution-oriented screening for inhibitors of Streptococcus pyogenes sortase A.

Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10 µM and a Kd of 60 µM. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.

Deciphering molecular mechanisms of arginine deiminase-based therapy - Comparative response analysis in paired human primary and recurrent glioblastomas.

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (» cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations. Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

An Approach for Identification of Novel Drug Targets in Streptococcus pyogenes SF370 Through Pathway Analysis.

Streptococcus pyogenes is one of the most important pathogens as it is involved in various infections affecting upper respiratory tract and skin. Due to the emergence of multidrug resistance and cross-resistance, S. Pyogenes is becoming more pathogenic and dangerous. In the present study, an in silico comparative analysis of total 65 metabolic pathways of the host (Homo sapiens) and the pathogen was performed. Initially, 486 paralogous enzymes were identified so that they can be removed from possible drug target list. The 105 enzymes of the biochemical pathways of S. pyogenes from the KEGG metabolic pathway database were compared with the proteins from the Homo sapiens by performing a BLASTP search against the non-redundant database restricted to the Homo sapiens subset. Out of these, 83 enzymes were identified as non-human homologous while 30 enzymes of inadequate amino acid length were removed for further processing. Essential enzymes were finally mined from remaining 53 enzymes. Finally, 28 essential enzymes were identified in S. pyogenes SF370 (serotype M1). In subcellular localization study, 18 enzymes were predicted with cytoplasmic localization and ten enzymes with the membrane localization. These ten enzymes with putative membrane localization should be of particular interest. Acyl-carrier-protein S-malonyltransferase, DNA polymerase III subunit beta and dihydropteroate synthase are novel drug targets and thus can be used to design potential inhibitors against S. pyogenes infection. 3D structure of dihydropteroate synthase was modeled and validated that can be used for virtual screening and interaction study of potential inhibitors with the target enzyme.

Arginine deprivation by arginine deiminase of Streptococcus pyogenes controls primary glioblastoma growth in vitro and in vivo.

Arginine auxotrophy constitutes a weak point of several tumors, among them glioblastoma multiforme (GBM). Hence, those tumors are supposed to be sensitive for arginine-depleting substances, such as arginine deiminase (ADI). Here we elucidated the sensitivity of patient-individual GBM cell lines toward Streptococcus pyogenes-derived ADI. To improve therapy, ADI was combined with currently established and pre-clinical cytostatic drugs. Additionally, effectiveness of local ADI therapy was determined in xenopatients. Half of the GBM cell lines tested responded well toward ADI monotherapy. In those cell lines, viability decreased significantly (up to 50%). Responding cell lines were subjected to combination therapy experiments to test if any additive or even synergistic effects may be achieved. Such promising results were obtained in 2/3 cases. In cell lines HROG02, HROG05 and HROG10, ADI and Palomid 529 combinations were most effective yielding more than 70% killing after 2 rounds of treatment. Comparable boosted antitumoral effects were observed after adding chloroquine to ADI (>60% killing). Apoptosis, as well as cell cycle dysregulation were found to play a minor role. In some, but clearly not all cases, (epi-) genetic silencing of arginine synthesis pathway genes (argininosuccinate synthetase 1 and argininosuccinate lyase) explained obtained results. In vivo, ADI as well as the combination of ADI and SAHA efficiently controlled HROG05 xenograft growth, whereas adding Palomid 529 to ADI did not further increase the strong antitumoral effect of ADI. The cumulative in vitro and in vivo results proved ADI as a very promising candidate therapeutic, especially for development of adjuvant GBM combination treatments.

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequent cause of hospital-acquired infection, which manifests as surgical site infections, bacteremia, and sepsis. Due to drug-resistance, prophylaxis of MRSA infection with antibiotics frequently fails or incites nosocomial diseases such as Clostridium difficile infection. Sortase A is a transpeptidase that anchors surface proteins in the envelope of S. aureus, and sortase mutants are unable to cause bacteremia or sepsis in mice. Here we used virtual screening and optimization of inhibitor structure to identify 3-(4-pyridinyl)-6-(2-sodiumsulfonatephenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole and related compounds, which block sortase activity in vitro and in vivo. Sortase inhibitors do not affect in vitro staphylococcal growth yet protect mice against lethal S. aureus bacteremia. Thus, sortase inhibitors may be useful as antiinfective therapy to prevent hospital-acquired S. aureus infection in high-risk patients without the side effects of antibiotics.

Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice.

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors of bacterial DNA polymerase III.

High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication results in bacterial cell death. The synthesis, structure-activity relationships and functional activity are described.

PolC-type polymerase III of Streptococcus pyogenes and its use in screening for chemical inhibitors.

The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag. The purified recombinant enzyme showed an apparent molecular mass of 160 kDa on SDS-PAGE and a specific activity of 3.5 nmol/min/mg when assayed in the presence of calf thymus DNA and the four deoxyribonucleoside triphosphates. This activity was inhibited by TMAU, a specific inhibitor of PolC. To facilitate kinetic studies, and high-throughput assays, a double-stranded oligo DNA primer/template was used as a substrate. The minimum requirement for the length of the substrate was a 20-base oligo primer annealed to a 35-base template. PolC activity was detected either by a filter-binding format or by a novel homogeneous scintillation proximity assay (SPA). Sensitivity to inhibition by anilinouracil analogs was improved by incorporating three deoxycytidines in the template strand as the first 3 bases to be copied by the polymerase. Inhibition of PolC activity by trimethyleneanilinouracil by the filtration and SPA methods gave comparable results, but the SPA assay uses less radioactive label, is less time-consuming, and is amenable to high-throughput formatting.

The group A streptococcal dipeptide permease (Dpp) is involved in the uptake of essential amino acids and affects the expression of cysteine protease.

The majority of characterized bacterial dipeptide permeases (Dpp) are membrane-associated complexes of five proteins belonging to the ABC-transporter family. They have been found to be involved in the uptake of essential amino acids, haem production, chemotaxis and sporulation. A 5.8 kb genomic DNA fragment of the serotype M49 group A streptococcal (GAS) strain CS101 was sequenced and found to contain five putative GAS Dpp genes (dppA to dppE). Deduced amino acid sequences exhibited 17-54% similarity to corresponding ABC-transporter sequences. The operon organization of the five genes was confirmed by transcriptional analysis, and a shorter, more abundant, dppA-only transcript was detected similar to that found in the GAS oligopeptide permease (Opp) system. Insertional inactivation was used to create serotype M2 and M49 strains that did not express the dppD and dppEATPase genes or nearly the entire operon. In feeding experiments with di- to hexapeptides, the wild-type strain grew with each peptide tested. The dpp mutants were unable to grow on dipeptides, whereas hexapeptides did not sustain the growth of opp mutants. Expression of the dpp operon was induced approximately fourfold in late exponential growth phase. In addition, a striking increase in the dppA to dppA-E ratio from 5:1 to more than 20:1 occurred during late exponential growth phase in complex medium. Growth in chemically defined medium (CDM) supplemented with various dipeptides specifically induced the expression of dpp and reduced both the dppA to dppA-E and oppA to oppA-F mRNA ratios. Expression of the virulence factor SpeB (major cysteine protease) was reduced eightfold in dpp mutants, whereas dpp expression was decreased about fourfold in a Mga virulence regulator mutant. Taken together, these data indicate a correlation between levels of intracellular essential amino acids and the regulation of virulence factor expression.

Treatment of Group A streptococcal pharyngitis in children. Results of a prospective, randomized study of four antimicrobial agents.

Penicillin V, benzathine/procaine penicillin G, cefadroxil monohydrate, and erythromycin estolate were randomly assigned for therapy of group A streptococcal pharyngitis in 198 children. All patients improved with in 24 hours of initiating therapy. Reinfection with a new group A streptococcal serotype occurred in 13 patients, 12 developing 7 to 12 days after stopping therapy and 11 becoming symptomatic. Relapse with the same organism occurred in 16 patients, only 5 (31%) of whom were symptomatic. Antibody titer rises, antibiotic resistance of group A organisms, presence of penicillinase-producing staphylococci, and lack of compliance were not related to recurrent infections. There were no significant differences between the failure rates of the four test drugs: penicillin V, 12%; benzathine/procaine penicillin G, 12%; cefadroxil monohydrate, 5%; and erythromycin, 2%.

Chemical analysis of changes in membrane composition during growth of Streptococcus pyogenes.

Changes in the structural components of the Streptococcus pyogenes membrane between exponential and early stationary phases of growth are reported. The overall protein composition ranged from 70 to 73% of the dry weight of the membranes, irrespective of the phase of growth from which they were isolated. Amino acid analyses of membranes isolated from streptococci in either the exponential or stationary phase of growth demonstrated that two amino acids, cysteine and tryptophan, were absent. Further analysis of the membrane proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis demonstrated that there were proteins unique to a particular phase of growth as well as differences in the amount of specific proteins from the various growth phases. In addition, membranes isolated from exponential-phase cultures contained a higher percentage of peripheral protein than did stationary-phase membranes. There also appeared to be an increase in the amount of outer surface proteins during this growth phase. The phosphorus content of the membranes increased during the stationary phase of growth, whereas the sugar composition remained constant. The only sugar found under various conditions of growth in any of the strains was glucose. Total fatty acid content and the mole percent composition of various fatty acids did not change in the different phases of growth. However, the mole percent composition of fatty acids in the membranes of various group A streptococci did differ between strains. Therefore, these results provide evidence that the composition of membranes of S. pyogenes does not remain constant throughout the growth phases of the culture.