CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus ...pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
Group A Streptococcus (GAS) M protein is an important virulence factor and potential vaccine antigen, and constitutes the basis for strain typing (emm-typing). Although >200 emm-types are ...characterized, structural data were obtained from only a limited number of emm-types. We aim to evaluate the sequence diversity of near-full-length M proteins from worldwide sources and analyse their structure, sequence conservation and classification. GAS isolates recovered from throughout the world during the last two decades underwent emm-typing and complete emm gene sequencing. Predicted amino acid sequence analyses, secondary structure predictions and vaccine epitope mapping were performed using MUSCLE and Geneious software. A total of 1086 isolates from 31 countries were analysed, representing 175 emm-types. emm-type is predictive of the whole protein structure, independent of geographical origin or clinical association. Findings of an emm-type paired with multiple, highly divergent central regions were not observed. M protein sequence length, the presence or absence of sequence repeats and predicted secondary structure were assessed in the context of the latest vaccine developments. Based on these global data, the M6 protein model is updated to a three representative M protein (M5, M80 and M77) model, to aid in epidemiological analysis, vaccine development and M protein-related pathogenesis studies.
We applied an emm cluster typing system to group A Streptococcus strains in New Zealand, including those associated with acute rheumatic fever (ARF). We observed few so-called rheumatogenic emm types ...but found a high proportion of emm types previously associated with pyoderma, further suggesting a role for skin infection in ARF.
The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal ...regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which “pain is out of proportion” to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections.
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•S. pyogenes produces pain during bacterial invasion through streptolysin S (SLS)•SLS induces TRPV1 neurons to release the neuropeptide CGRP in infected tissues•Neurons suppress neutrophil recruitment and bactericidal activity•BoNT/A and CGRP antagonist block neural suppression of immunity to treat infection
Flesh-eating bacteria hijack a pain response that suppresses host defense, and blocking this neuro-immune communication holds potential for treatment.
Group A
(GAS) is the etiologic agent of numerous high-morbidity and high-mortality diseases. Infections are typically highly proinflammatory. During the invasive infection necrotizing fasciitis, this ...is in part due to the GAS protease SpeB directly activating interleukin-1β (IL-1β) independent of the canonical inflammasome pathway. The upper respiratory tract is the primary site for GAS colonization, infection, and transmission, but the host-pathogen interactions at this site are still largely unknown. We found that in the murine nasopharynx, SpeB enhanced IL-1β-mediated inflammation and the chemotaxis of neutrophils. However, neutrophilic inflammation did not restrict infection and instead promoted GAS replication and disease. Inhibiting IL-1β or depleting neutrophils, which both promote invasive infection, prevented GAS infection of the nasopharynx. Mice pretreated with penicillin became more susceptible to GAS challenge, and this reversed the attenuation from neutralization or depletion of IL-1β, neutrophils, or SpeB. Collectively, our results suggest that SpeB is essential to activate an IL-1β-driven neutrophil response. Unlike during invasive tissue infections, this is beneficial in the upper respiratory tract because it disrupts colonization resistance mediated by the microbiota. This provides experimental evidence that the notable inflammation of strep throat, which presents with significant swelling, pain, and neutrophil influx, is not an ineffectual immune response but rather is a GAS-directed remodeling of this niche for its pathogenic benefit.
Background. Group A streptococcus (GAS) commonly colonizes the oropharynx and nonintact skin. However, colonization has been little studied and the role of biofilm formation is unclear, as biofilm ...experiments to date have not been conducted under conditions that mimic the host environment. Methods. In this study we grew GAS biofilms on human keratinocytes under various environmental conditions and used this model to evaluate colonization, invasive disease and natural transformation. Results. GAS grown on epithelial cells, but not biofilms grown on abiotic surfaces, produced biofilms with characteristics similar to in vivo colonization. These biofilm bacteria showed a 100-fold higher bacterial burden of nasalassociated lymphoid tissue in mice than broth-grown bacteria, and were not virulent during septic infection, which was attributed in part to down-regulation of genes typically involved in localized and invasive disease. We also showed for the first time that GAS were naturally transformable when grown in biofilms and during colonization of NALT in vivo. Conclusions. These findings provide novel model systems to study biofilm formation of GAS in vitro and in vivo, suggest an important role for biofilm formation during GAS colonization, and provide an explanation for the known genome diversity within the GAS population.
M-type 28 (M28) Streptococcus pyogenes (group A Streptococcus GAS) strains are highly associated with life-threatening puerperal infections. Genome sequencing has revealed a large mobile genetic ...element, RD2, present in most M28 GAS isolates but not found widely in other serotypes. Previous studies have linked RD2 to the ability of M28 GAS to colonize the vaginal tract. A new study by Roshika and colleagues (R. Roshika, I. Jain, J. Medicielo, J. Wächter, J. L. Danger, P. Sumby, Infect Immun 89:e00722-20, 2021, https://doi.org/10.1128/IAI.00722-20) used gain-of-function mutants in three different GAS serotypes to help determine why RD2 appears to have a serotype preference and what that could mean for GAS mucosal colonization and pathogenesis.
A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the ...pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, Staphylococcus aureus and Streptococcus pyogenes. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th) response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining "core genome" was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species' 'core genome', common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.
We employ the CRISPR-Cas system of Streptococcus pyogenes as programmable RNA-guided endonucleases (RGENs) to cleave DNA in a targeted manner for genome editing in human cells. We show that complexes ...of the Cas9 protein and artificial chimeric RNAs efficiently cleave two genomic sites and induce indels with frequencies of up to 33%.
Scarlet fever notifications surged across the United Kingdom in spring 2014. Molecular epidemiologic investigation of Streptococcus pyogenes infections in North-West London highlighted increased emm4 ...and emm3 infections coincident with the upsurge. Unlike outbreaks in other countries, antimicrobial resistance was uncommon, highlighting an urgent need to better understand the drivers of scarlet fever activity.
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