The Morphogenetic Protein CotE Positions Exosporium Proteins CotY and ExsY during Sporulation of Bacillus cereus

ABSTRACT The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates the interactions of spores with their environment, modulates spore adhesion and germination, and has been implicated in pathogenesis. In Bacillus cereus, the exosporium consists of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, surrounded by a hairy nap composed of glycoproteins. The morphogenetic protein CotE is necessary for the integrity of the B. cereus exosporium, but how CotE directs exosporium assembly remains unknown. Here, we used super-resolution fluorescence microscopy to follow the localization of SNAP-tagged CotE, CotY, and ExsY during B. cereus sporulation and evidenced the interdependencies among these proteins. Complexes of CotE, CotY, and ExsY are present at all sporulation stages, and the three proteins follow similar localization patterns during endospore formation that are reminiscent of the localization pattern of Bacillus subtilis CotE. We show that B. cereus CotE guides the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process, as previously proposed, but occurs through the formation of two caps, as observed during B. subtilis coat morphogenesis, suggesting that a general principle governs the assembly of the spore surface layers of Bacillaceae. IMPORTANCE Spores of Bacillaceae are enveloped in an outermost glycoprotein layer. In the B. cereus group, encompassing the Bacillus anthracis and B. cereus pathogens, this layer is easily recognizable by a characteristic balloon-like appearance and separation from the underlying coat by an interspace. In spite of its importance for the environmental interactions of spores, including those with host cells, the mechanism of assembly of the exosporium is poorly understood. We used super-resolution fluorescence microscopy to directly visualize the formation of the exosporium during the sporulation of B. cereus, and we studied the localization and interdependencies of proteins essential for exosporium morphogenesis. We discovered that these proteins form a morphogenetic scaffold before a complete exosporium or coat is detectable. We describe how the different proteins localize to the scaffold and how they subsequently assemble around the spore, and we present a model for the assembly of the exosporium.

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The morphogenetic protein CotE drives exosporium formation by positioning CotY and ExsY during sporulation of Bacillus cereus

10.1101/2020.12.11.422311 ◽

2020 ◽

Author(s):

Armand Lablaine ◽

Monica Serrano ◽

Stéphanie Chamot ◽

Isabelle Bornard ◽

Frédéric Carlin ◽

...

Keyword(s):

Bacillus Subtilis ◽

Fluorescence Microscopy ◽

Bacillus Cereus ◽

Basal Layer ◽

Super Resolution ◽

Spore Surface ◽

Surface Layers ◽

Clostridium Species ◽

Localization Pattern ◽

Morphogenetic Protein

The exosporium is the outermost spore layer of some Bacillus and Clostridium species and related organisms. It mediates interactions of spores with their environment, modulates spore adhesion and germination and could be implicated in pathogenesis. The exosporium is composed of a crystalline basal layer, formed mainly by the two cysteine-rich proteins CotY and ExsY, and surrounded by a glycoprotein hairy nap. The morphogenetic protein CotE is necessary for Bacillus cereus exosporium integrity, but how CotE directs exosporium assembly remains unknown. Here, we followed the localization of SNAP-tagged CotE, -CotY and -ExsY during B. cereus sporulation, using super-resolution fluorescence microscopy and evidenced interactions among these proteins. CotE, CotY and ExsY are present as complexes at all sporulation stages and follow a similar localization pattern during endospore formation that is reminiscent of the localization of Bacillus subtilis CotE. We show that B. cereus CotE drives the formation of one cap at both forespore poles by positioning CotY and then guides forespore encasement by ExsY, thereby promoting exosporium elongation. By these two actions, CotE ensures the formation of a complete exosporium. Importantly, we demonstrate that the assembly of the exosporium is not a unidirectional process as previously proposed but it is performed through the formation of two caps, as observed during B. subtilis coat morphogenesis. It appears that a general principle governs the assembly of the spore surface layers of Bacillaceae.

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Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

Applied and Environmental Microbiology ◽

10.1128/aem.02626-15 ◽

2015 ◽

Vol 82 (1) ◽

pp. 232-243 ◽

Cited By ~ 13

Author(s):

Christelle Bressuire-Isoard ◽

Isabelle Bornard ◽

Adriano O. Henriques ◽

Frédéric Carlin ◽

Véronique Broussolle

Keyword(s):

Bacillus Cereus ◽

Optimal Growth ◽

Spore Surface ◽

Surface Layers ◽

Content Type ◽

Morphogenetic Protein ◽

Transmission Electron ◽

Wet Heat ◽

Uv C

ABSTRACTTheBacillus cereusspore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers ofB. cereusATCC 14579 spores and on the resulting spore properties. ThecotEdeletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotEspores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotEspores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotEspores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H2O2and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly ofB. cereusspore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.

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The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00050-15 ◽

2015 ◽

Vol 79 (4) ◽

pp. 437-457 ◽

Cited By ~ 62

Author(s):

George C. Stewart

Keyword(s):

Bacillus Cereus ◽

Multilayered Structure ◽

Structure And Function ◽

Bacterial Spore ◽

Spore Surface ◽

Surface Layers ◽

Content Type ◽

Molecular Aspects ◽

And Function ◽

Structure Assembly

SUMMARYMuch of what we know regarding bacterial spore structure and function has been learned from studies of the genetically well-characterized bacteriumBacillus subtilis. Molecular aspects of spore structure, assembly, and function are well defined. However, certain bacteria produce spores with an outer spore layer, the exosporium, which is not present onB. subtilisspores. Our understanding of the composition and biological functions of the exosporium layer is much more limited than that of other aspects of the spore. Because the bacterial spore surface is important for the spore's interactions with the environment, as well as being the site of interaction of the spore with the host's innate immune system in the case of spore-forming bacterial pathogens, the exosporium is worthy of continued investigation. Recent exosporium studies have focused largely on members of theBacillus cereusfamily, principallyBacillus anthracisandBacillus cereus. Our understanding of the composition of the exosporium, the pathway of its assembly, and its role in spore biology is now coming into sharper focus. This review expands on a 2007 review of spore surface layers which provided an excellent conceptual framework of exosporium structure and function (A. O. Henriques and C. P. Moran, Jr., Annu Rev Microbiol61:555–588, 2007,http://dx.doi.org/10.1146/annurev.micro.61.080706.093224). That review began a process of considering outer spore layers as an integrated, multilayered structure rather than simply regarding the outer spore components as independent parts.

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Orthologues ofBacillus subtilisSpore Crust Proteins Have a Structural Role in theBacillus megateriumQM B1551 Spore Exosporium

Applied and Environmental Microbiology ◽

10.1128/aem.01734-18 ◽

2018 ◽

Vol 84 (20) ◽

Cited By ~ 3

Author(s):

Julia Manetsberger ◽

Abhinaba Ghosh ◽

Elizabeth A. H. Hall ◽

Graham Christie

Keyword(s):

Bacillus Cereus ◽

Bacillus Megaterium ◽

Bacterial Species ◽

Basal Layer ◽

Structural Components ◽

Content Type ◽

Indigenous Plasmids ◽

Crust Layer ◽

Air Water Interface ◽

Spore Forming Bacteria

ABSTRACTThe exosporium ofBacillus megateriumQM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of theBacillus megateriumexosporium. Here, we provide evidence that pBM600-encoded orthologues of theBacillus subtilisCotW and CotX proteins, which form the crust layer in spores of that species, are structural components of theBacillus megateriumQM B1551 spore exosporium. The introduction of plasmid-bornecotWand orthologouscotXgenes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in theBacillus megateriumQM B1551 exosporium.IMPORTANCEWhen starved of nutrients, some bacterial species develop metabolically dormant spores that can persist in a viable state in the environment for several years. The outermost layers of spores are of particular interest since (i) these represent the primary site for interaction with the environment and (ii) the protein constituents may have biotechnological applications. The outermost layer, or exosporium, inBacillus megateriumQM B1551 spores is of interest, as it is morphologically distinct from the exosporia of spores of the pathogenicBacillus cereusfamily. In this work, we provide evidence that structurally important protein constituents of theBacillus megateriumexosporium are different from those in theBacillus cereusfamily. We also show that one of these proteins, when purified, can assemble to form sheets of exosporium-like material. This is significant, as it indicates that spore-forming bacteria employ different proteins and mechanisms of assembly to construct their external layers.

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A spatial multi-scale fluorescence microscopy toolbox discloses entry checkpoints of SARS-CoV-2 variants in VeroE6 cells

10.1101/2021.03.31.437907 ◽

2021 ◽

Author(s):

Barbara Storti ◽

Paola Quaranta ◽

Cristina Di Primio ◽

Nicola Clementi ◽

Pier Giorgio Spezia ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Super Resolution ◽

Host Cells ◽

Pseudotyped Virus ◽

Microscopy Imaging ◽

S Protein ◽

Multi Scale ◽

Late Entry ◽

Kinetics Of

We developed a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy (Airyscan, STORM, and STED) and easily matches the spatial scale of single virus-cell checkpoints. We deployed this toolbox to characterize subtle issues related to the entry phase of SARS-CoV-2 variants in VeroE6 cells. Our results suggest that the variant of concern B.1.1.7, currently on the rise in several countries by a clear transmission advantage, in these cells outcompetes its ancestor B.1 in terms of a much faster kinetics of entry. Given the molecular scenario (entry by the late pathway and similar fraction of pre-cleaved S protein for B.1.1.7 and B.1), the faster entry of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike RBD with ACE2. Remarkably, we also observed directly the significant role of clathrin as mediator of late entry endocytosis, which had been previously suggested in analogy with other CoVs and from experiments on pseudotyped virus models. On overall, we believe that our fluroescence microscopy-based approach is valuable for future studies addressing of how SARS-CoV-2 and its variants interact with cells.

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Advanced Static and Dynamic Fluorescence Microscopy Techniques to Investigate Drug Delivery Systems

Pharmaceutics ◽

10.3390/pharmaceutics13060861 ◽

2021 ◽

Vol 13 (6) ◽

pp. 861

Author(s):

Jacopo Cardellini ◽

Arianna Balestri ◽

Costanza Montis ◽

Debora Berti

Keyword(s):

Drug Delivery ◽

Fluorescence Microscopy ◽

Drug Delivery Systems ◽

Laser Scanning ◽

Super Resolution ◽

Laser Scanning Confocal Microscopy ◽

Delivery Systems ◽

Scanning Confocal Microscopy ◽

Biological Phenomena

In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments.

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Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽

10.3390/pathogens10060772 ◽

2021 ◽

Vol 10 (6) ◽

pp. 772

Author(s):

Isao Murakami ◽

Takashi Iwata ◽

Tohru Morisada ◽

Kyoko Tanaka ◽

Daisuke Aoki

Keyword(s):

Life Cycle ◽

Cultured Cells ◽

Basal Layer ◽

Nucleosome Positioning ◽

Viral Life Cycle ◽

Human Papillomaviruses ◽

Host Cells ◽

Micrococcal Nuclease ◽

Hpv Dna ◽

Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

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Intracellular localization of the mycobacterial stressosome complex

Scientific Reports ◽

10.1038/s41598-021-89069-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Malavika Ramesh ◽

Ram Gopal Nitharwal ◽

Phani Rama Krishna Behra ◽

B. M. Fredrik Pettersson ◽

Santanu Dasgupta ◽

...

Keyword(s):

Bacillus Subtilis ◽

Fluorescence Microscopy ◽

Bacillus Cereus ◽

Intracellular Localization ◽

Sigma Factors ◽

Alternative Sigma Factors ◽

Expression Of Genes ◽

Molecular Machinery ◽

Stress Signals ◽

Activation Pathways

AbstractMicroorganisms survive stresses by alternating the expression of genes suitable for surviving the immediate and present danger and eventually adapt to new conditions. Many bacteria have evolved a multiprotein "molecular machinery" designated the "Stressosome" that integrates different stress signals and activates alternative sigma factors for appropriate downstream responses. We and others have identified orthologs of some of the Bacillus subtilis stressosome components, RsbR, RsbS, RsbT and RsbUVW in several mycobacteria and we have previously reported mutual interactions among the stressosome components RsbR, RsbS, RsbT and RsbUVW from Mycobacterium marinum. Here we provide evidence that "STAS" domains of both RsbR and RsbS are important for establishing the interaction and thus critical for stressosome assembly. Fluorescence microscopy further suggested co-localization of RsbR and RsbS in multiprotein complexes visible as co-localized fluorescent foci distributed at scattered locations in the M. marinum cytoplasm; the number, intensity and distribution of such foci changed in cells under stressed conditions. Finally, we provide bioinformatics data that 17 (of 244) mycobacteria, which lack the RsbRST genes, carry homologs of Bacillus cereus genes rsbK and rsbM indicating the existence of alternative σF activation pathways among mycobacteria.

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hfqPlays Important Roles in Virulence and Stress Adaptation in Cronobacter sakazakii ATCC 29544

Infection and Immunity ◽

10.1128/iai.03161-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 2089-2098 ◽

Cited By ~ 21

Author(s):

Seongok Kim ◽

Hyelyeon Hwang ◽

Kwang-Pyo Kim ◽

Hyunjin Yoon ◽

Dong-Hyun Kang ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Soft Agar ◽

Host Cells ◽

Neonatal Meningitis ◽

Animal Cells ◽

Content Type ◽

Flagellar Biosynthesis

Cronobacterspp. are opportunistic pathogens that cause neonatal meningitis and sepsis with high mortality in neonates. Despite the peril associated withCronobacterinfection, the mechanisms of pathogenesis are still being unraveled. Hfq, which is known as an RNA chaperone, participates in the interaction with bacterial small RNAs (sRNAs) to regulate posttranscriptionally the expression of various genes. Recent studies have demonstrated that Hfq contributes to the pathogenesis of numerous species of bacteria, and its roles are varied between bacterial species. Here, we tried to elucidate the role of Hfq inC. sakazakiivirulence. In the absence ofhfq,C. sakazakiiwas highly attenuated in disseminationin vivo, showed defects in invasion (3-fold) into animal cells and survival (103-fold) within host cells, and exhibited low resistance to hydrogen peroxide (102-fold). Remarkably, the loss ofhfqled to hypermotility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyperflagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagellar biosynthesis in a strain lackinghfq. Together, these data strongly suggest thathfqplays important roles in the virulence ofC. sakazakiiby participating in the regulation of multiple genes.

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Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato

Applied and Environmental Microbiology ◽

10.1128/aem.01722-17 ◽

2017 ◽

Vol 83 (24) ◽

Author(s):

Alistair H. Bishop

Keyword(s):

Bacillus Cereus ◽

Large Scale ◽

Germination Rate ◽

Dependent Manner ◽

Clostridium Sporogenes ◽

Specific Chemical ◽

Content Type ◽

Ph Range ◽

Significant Discovery ◽

The Impact

ABSTRACT Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficile. IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of “germinate to decontaminate” strategy may thus become more practicable.

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