Looking for the X Factor in Bacterial Pathogenesis: Association of orfX-p47 Gene Clusters with Toxin Genes in Clostridial and Non-Clostridial Bacterial Species

The botulinum neurotoxin (BoNT) has been extensively researched over the years in regard to its structure, mode of action, and applications. Nevertheless, the biological roles of four proteins encoded from a number of BoNT gene clusters, i.e., OrfX1-3 and P47, are unknown. Here, we investigated the diversity of orfX-p47 gene clusters using in silico analytical tools. We show that the orfX-p47 cluster was not only present in the genomes of BoNT-producing bacteria but also in a substantially wider range of bacterial species across the bacterial phylogenetic tree. Remarkably, the orfX-p47 cluster was consistently located in proximity to genes coding for various toxins, suggesting that OrfX1-3 and P47 may have a conserved function related to toxinogenesis and/or pathogenesis, regardless of the toxin produced by the bacterium. Our work also led to the identification of a putative novel BoNT-like toxin gene cluster in a Bacillus isolate. This gene cluster shares striking similarities to the BoNT cluster, encoding a bont/ntnh-like gene and orfX-p47, but also differs from it markedly, displaying additional genes putatively encoding the components of a polymorphic ABC toxin complex. These findings provide novel insights into the biological roles of OrfX1, OrfX2, OrfX3, and P47 in toxinogenesis and pathogenesis of BoNT-producing and non-producing bacteria

Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures

Funding Information: The work was funded by the European Research Council (ERC-CoG 683099), Academy of Finland (299700), Marie Skłodowska Curie Innovative Training Network (CLOSPORE 642068), the Doctoral Programmes in Microbiology and Biotechnology and in Food Chain and Health of the University of Helsinki, Fundação para a Ciência e a Tecnologia, Portugal (PEst-OE/EQB/LA0004/2011), FEDER funds through COMPETE2020 “Programa Operacional Competitividade e Internacionalização” (LISBOA-01-0145-FEDER-007660), and the national funds from OE-"Orçamento de Estado" and European funds from FEDER "Fundo Europeu de Desenvolvimento Regional" (PPBI-POCI-01-0145-FEDER-022122). Funding Information: We warmly thank Nigel Minton, University of Nottingham, for the generous provision of vectors and bacterial strains required in mutagenesis. Mikael Niku is thanked for technical advice with fluorescence microscopy, and Hanna Korpunen is thanked for technical assistance. The work was funded by the European Research Council (ERC-CoG 683099), Academy of Finland (299700), Marie Skłodowska Curie Innovative Training Network (CLOSPORE 642068), the Doctoral Programmes in Microbiology and Biotechnology and in Food Chain and Health of the University of Helsinki, Fundação para a Ciência e a Tecnologia, Portugal (PEst-OE/EQB/LA0004/2011), FEDER funds through COMPETE2020 “Programa Operacional Competitividade e Internacionalização” (LISBOA-01-0145-FEDER-007660), and the national funds from OE-"Orçamento de Estado" and European funds from FEDER "Fundo Europeu de Desenvolvimento Regional" (PPBI-POCI-01-0145-FEDER-022122). A.M., G.M., A.O.H., H.K., and M.L. conceived and designed the study; A.M., G.M., and M.N. performed the laboratory experiments; A.M., G.M., and A.O.H. performed the image analysis; A.M. and M.L. analyzed the data and wrote the manuscript; and all authors contributed to the final manuscript. Funder EC | European Research Council (ERC) Academy of Finland (AKA) Marie Sk&łodowska-Curie Innovative Training Network CLOSPORE University of Helsinki, Doctoral Programme in Food Chain and Health University of Helsinki, Doctoral Programme in Microbiology and Biotechnology Fundação para a Ciência e a Tecnologia, Portugal Orcamento de Estado/Fundo Europeu de Desenvolvimento Regional Fundo Europeu de Desenvolvimento Regional Grant(s) 683099 299700 642068 PEst-OE/EQB/ LA0004/2011 PPBI-POCI-01-0145-FEDER-022122 PPBI-POCI-01-0145-FEDER-022122 Author(s) Miia Lindstrom Miia Lindstrom Miia Lindstrom Anna Mertaoja Maria B. Nowakowska Anna Mertaoja Adriano O. Henriques Adriano O. Henriques Adriano O. Henriques Publisher Copyright: Copyright © 2023 Mertaoja et al.Toxin production and sporulation are key determinants of pathogenesis in Clostridia. Clostridium botulinum produces the most potent toxin known, the botulinum neurotoxin (BoNT), which blocks neurotransmission and causes a life-threatening paralysis called botulism. BoNT production and sporulation share a common regulator Spo0A, which suggests coordination of the two traits. Describing the relationship between toxin production and sporulation is fundamental toward understanding the evolutionary and mechanistic logic and further control of clostridial pathogenesis. Here, we provide the first single-cell resolution analysis of BoNT production and sporulation in C. botulinum type E cultures by using a fluorescent reporter to follow the activation of the BoNT gene promoter. BoNT was expressed by a subpopulation of cells and was released through Spo0A-mediated autolysis of vegetative cells or upon release of mature spores. All possible combinations of toxin production and sporulation resided in wild-type C. botulinum type E cultures, indicating neither tight co-regulation nor strict independence of the two traits. The population structure and the degree of overall heterogeneity were affected by growth phase and environmental conditions, with cold temperature inducing large diversity and cultural stability, in line with adaptation to fluctuating temperatures that C. botulinum type E strains likely encounter in nature. We also observed Spo0A-independent BoNT production by a small cell subpopulation of the spo0A-null strain. Our observation of toxin gene activation in the forespore invites speculation on possible alternative biological roles for toxin production by vegetative and sporulating cells and reflection on the evolutionary rationale of toxin production with respect to the ecology of spore-forming pathogens.publishersversionpublishe

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.Peer reviewe

Crystal structures of OrfX1, OrfX2, and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.Peer reviewe

Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

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Updated clinical practice recommendations for managing children with 22q11.2 deletion syndrome

This review aimed to update the clinical practice guidelines for managing children and adolescents with 22q11.2 deletion syndrome (22q11.2DS). The 22q11.2 Society, the international scientific organization studying chromosome 22q11.2 differences and related conditions, recruited expert clinicians worldwide to revise the original 2011 pediatric clinical practice guidelines in a stepwise process: (1) a systematic literature search (1992-2021), (2) study selection and data extraction by clinical experts from 9 different countries, covering 24 subspecialties, and (3) creation of a draft consensus document based on the literature and expert opinion, which was further shaped by survey results from family support organizations regarding perceived needs. Of 2441 22q11.2DS-relevant publications initially identified, 2344 received full-text reviews, including 1545 meeting criteria for potential relevance to clinical care of children and adolescents. Informed by the available literature, recommendations were formulated. Given evidence base limitations, multidisciplinary recommendations represent consensus statements of good practice for this evolving field. These recommendations provide contemporary guidance for evaluation, surveillance, and management of the many 22q11.2DS-associated physical, cognitive, behavioral, and psychiatric morbidities while addressing important genetic counseling and psychosocial issues

CRISPR-Cas9-Based Toolkit for Clostridium botulinum Group II Spore and Sporulation Research

The spores of Clostridium botulinum Group II strains pose a significant threat to the safety of modern packaged foods due to the risk of their survival in pasteurization and their ability to germinate into neurotoxigenic cultures at refrigeration temperatures. Moreover, spores are the infectious agents in wound botulism, infant botulism, and intestinal toxemia in adults. The identification of factors that contribute to spore formation is, therefore, essential to the development of strategies to control related health risks. Accordingly, development of a straightforward and versatile gene manipulation tool and an efficient sporulation-promoting medium is pivotal. Our strategy was to employ CRISPR-Cas9 and homology-directed repair (HDR) to replace targeted genes with mutant alleles incorporating a unique 24-nt “bookmark” sequence that could act as a single guide RNA (sgRNA) target for Cas9. Following the generation of the sporulation mutant, the presence of the bookmark allowed rapid generation of a complemented strain, in which the mutant allele was replaced with a functional copy of the deleted gene using CRISPR-Cas9 and the requisite sgRNA. Then, we selected the most appropriate medium for sporulation studies in C. botulinum Group II strains by measuring the efficiency of spore formation in seven different media. The most effective medium was exploited to confirm the involvement of a candidate gene in the sporulation process. Using the devised sporulation medium, subsequent comparisons of the sporulation efficiency of the wild type (WT), mutant and “bookmark”-complemented strain allowed the assignment of any defective sporulation phenotype to the mutation made. As a strain generated by complementation with the WT gene in the original locus would be indistinguishable from the parental strain, the gene utilized in complementation studies was altered to contain a unique “watermark” through the introduction of silent nucleotide changes. The mutagenesis system and the devised sporulation medium provide a solid basis for gaining a deeper understanding of spore formation in C. botulinum, a prerequisite for the development of novel strategies for spore control and related food safety and public health risk management.Peer reviewe