scholarly journals Clostridioides difficile SpoVAD and SpoVAE interact and are required for DPA packaging into spores

2021 ◽  
Author(s):  
Marko Baloh ◽  
Joseph A. Sorg

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g., dry heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is packaged into the spore core, thereby replacing the majority of water. DPA is synthesized by the mother cell and its packaging into the spore core is regulated by the spoVA operon that has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile. In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA packaging into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC / SpoVAD / SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA packaging into the spore

2021 ◽  
Author(s):  
Marko Baloh ◽  
Joseph A. Sorg

Clostridioides difficile spores, like the spores from most endospore-forming organisms, are a metabolically dormant stage of development with a complex structure that conveys considerable resistance to environmental conditions, e.g. , wet heat. This resistance is due to the large amount of dipicolinic acid (DPA) that is taken up by the spore core, preventing rotational motion of the core proteins. DPA is synthesized by the mother cell and its packaging into the spore core is mediated by the products of the spoVA operon, which has a variable number of genes, depending on the organism. C. difficile encodes 3 spoVA orthologues, spoVAC, spoVAD, and spoVAE. Prior work has shown that C. difficile SpoVAC is a mechanosensing protein responsible for DPA release from the spore core upon the initiation of germination. However, the roles of SpoVAD and SpoVAE remain unclear in C. difficile . In this study we analyzed the roles of SpoVAD and SpoVAE and found that they are essential for DPA uptake into the spore, similar to SpoVAC. Using split luciferase protein interaction assays we found that these proteins interact, and we propose a model where SpoVAC/SpoVAD/SpoVAE proteins interact at or near the inner spore membrane, and each member of the complex is essential for DPA uptake into the spore core. Importance C. difficile spore heat resistance provides an avenue for it to survive the disinfection protocols in hospital and community settings. The spore heat resistance is mainly the consequence of the high DPA content within the spore core. By elucidating the mechanism by which DPA is taken up by the spore core, this study may provide insight in how to disrupt the spore heat resistance with the aim of making the current disinfection protocols more efficient at preventing the spread of C. difficile in the environment.


2017 ◽  
Vol 25 (4) ◽  
Author(s):  
E. A. Belyakov ◽  
A. G. Lapirov ◽  
O. A. Lebedeva

This article examines the ecology of germination and the features of ontogenesis of the floating mat-forming hygrogelophyte Calla palustris L. in the territory of some regions in the central part of European Russia and the Republic of Belarus under laboratory conditions. It has been found that in the surveyed territory in the dense ear-like collective fruit of C. palustris, 40.7 ± 6.4 fruits, juicy berries, are formed and the number of seeds produced (actual seed productivity), averages 164.0 ± 89.3. Widely varying data on the number of berries in the collective fruit, as well as a variable number of ovules in them, shows that the seed productivity of C. palustris depends on a whole range of endogenous and exogenous factors, including the effectiveness of pollination of flowers by insects. In laboratory experiments, various storage periods (2 and 12 months) and methods of presowing seed treatment (stratification, drying, ice-freezing) were applied to simulate the ecological conditions of the growth of white alder. It is shown that freshly harvested seeds do not germinate at once, and wet cold stratification makes it possible to achieve maximum values of laboratory germination (from 84.4 to 99.0) and germination energy (from 66.6 to 88.3). Given that the features of germination are indicators of dormancy, it is demonstrated that seeds of C. palustris are in a state of shallow physiological dormancy, conditioned by the physiological mechanism of inhibition. Along with dry storage, wet cold stratification is the main way for seeds to enter a non-dormant state. Such mechanisms are consistent with the climatic features of the regions in which the species grows. It is found that C. palustris seeds, in common with many other species of hygrogelophytes, can float on the water surface for a long time (more than 30 days), spreading with water flow (hydrochoria). Seeds of C. palustris are photosensitive, germination is observed in a wide range of temperatures – from 10–14 to 30 ºС (at constant humidity), type of germination – underground (hypogeal). It is found that ontogeny of individuals of generative origin of C. palustris in the laboratory is terminated (the plants died after passing the juvenile ontogenetic state). The formation of C. palustris seedlings under laboratory conditions lasts 23–25 days and is characterized by the appearance of the main organs of the plant and the anisotropic growth of the shoot axis. At the final stage of development, the seedling is represented by a uniaxial monopodial and anisotropic growing rosette shoot with shortened internodes. The juvenile ontogenetic state in laboratory conditions lasts up to 7 months, after which the plants die off. In the framework of ontomorphogenesis, the stages of ontogenetic development under study (the seedling and the juvenile plant) correspond to the phase of the primary uniaxial rosette shoot. The plant in this period is represented by a uniaxial monopodially growing anchorage shoot. The detection of virgin plants in natural conditions indicates the possibility of their further development from the rudiments of generative origin. The main way of the species reproduction is vegetative, characteristic of most aquatic and semi-aquatic plants. In the course of ontogenesis, progressive features of development such as cotyledon greening and early death of the radicle root have been revealed.


2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Willie Taylor ◽  
Emily Camilleri ◽  
D. Levi Craft ◽  
George Korza ◽  
Maria Rocha Granados ◽  
...  

ABSTRACT This study examined the microbicidal activity of 222-nm UV radiation (UV222), which is potentially a safer alternative to the 254-nm UV radiation (UV254) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV222 and at lower fluences than with UV254. B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV222, as were spores lacking their DNA-protective proteins, the α/β-type small, acid-soluble spore proteins. The spore cores’ large amount of Ca2+-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV222, while spores’ proteinaceous coat may have given some slight protection against UV222. Survivors among B. subtilis spores treated with UV222 acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV222 resistance. UV222-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV222 does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV222. IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis. Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV254) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV222) may be less harmful to people than UV254 yet may still kill bacteria and at lower fluences than UV254. The present work has identified the damage by UV222 that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV222 also inactivates a herpesvirus.


2020 ◽  
Vol 8 (6) ◽  
pp. 901
Author(s):  
Martina Masarikova ◽  
Ivana Simkova ◽  
Martin Plesko ◽  
Veronika Eretova ◽  
Marcela Krutova ◽  
...  

To investigate a possible Clostridioides difficile reservoir in the Czech Republic, we performed a study in 297 calves from 29 large-scale dairy farms. After enrichment, faecal samples were inoculated onto selective agar for C. difficile. From the 297 samples, 44 C. difficile isolates were cultured (prevalence of 14.8%, 10 farms). The Holstein breed and use of digestate were associated with C. difficile colonisation (p ˂ 0.05). C. difficile isolates belonged to the ribotype/sequence type: RT033/ST11 (n = 37), RT126/ST11 (n = 6) and RT046/ST35 (n = 1). A multiple-locus variable-number tandem-repeat analysis revealed four clonal complexes of RT033 isolates and one clonal complex of RT126 isolates. All isolates were sensitive to amoxicillin, metronidazole and vancomycin. Forty isolates were resistant to ciprofloxacin, twenty-one to clindamycin, seven to erythromycin, seven to tetracycline and six to moxifloxacin. Moxifloxacin resistant isolates revealed an amino-acid substitution Thr82Ile in the GyrA. In conclusion, the calves of Holstein breed from farms using digestate as a product of bio-gas plants are more likely to be colonised by clonally-related C. difficile of ST 11 represented by ribotypes 033 and 126. The identified resistance to moxifloxacin with a Thr82Ile substitution in the GyrA highlights the need for further monitoring by the “One health approach”.


Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 802-802 ◽  
Author(s):  
Sten Eirik W. Jacobsen ◽  
Robert Mansson ◽  
Anne Hultquist ◽  
Mikael Sigvardsson ◽  
Natalija Buza-Vidas ◽  
...  

Abstract We recently identified a novel Lin−Sca-1+c-kithiCD34+Flt3hi (LSKCD34+Flt3hi) lymphoid-primed multipotent progenitor (LMPP) in adult mouse bone marrow which, although possessing a combined lymphoid (B and T cell) and myeloid (granulocyte-monocyte; GM) differentiation potential, have little or no ability to adopt erythroid (E) and megakaryocyte (MK) lineage fates (Adolfsson et al, Cell121:295, 2005). The identification of this lineage restricted lymphomyeloid progenitor implicates the existence of alternative roadmaps for lineage commitment of pluripotent hematopoietic stem cells (HSCs), distinct from the classical model suggesting that the first HSC commitment step results in a strict separation into common lymphoid and myeloid progenitors. Herein we provide further, genetic evidence for such a model. Affymetrix global gene profiling, quantitative PCR, and multiplex single cell PCR analysis of LSKCD34−Flt3− long-term (LT)-HSCs, LSKCD34+Flt3− short-term (ST)-HSCs and LSKCD34+Flt3hi LMPPs, demonstrate that LMPPs in contrast to LT-HSCs and ST-HSCs down-regulate or turn off a number of genes critically involved in MkE lineage development, including GATA-1 and the receptors for erythropoietin and thrombopoietin. In contrast, a number of genes specific for early lymphoid development, including Rag-1, sterile Ig and IL-7 receptor are upregulated in LMPPs but absent in LT-HSCs and ST-HSCs. Importantly, within the LMPP, these lymphoid genes are typically co-expressed with a number of GM associated genes such as G-CSF receptor and MPO, but virtually never co-expressed with MkE associated genes. Investigating fetal liver day 14.5 we also provide evidence for existence of the LSKCD34+Flt3hi LMPPs at this early stage of development, and using a single cell clonal assay promoting combined B, T and myeloid lineage development, we demonstrate that a large fraction of fetal LMPPs lacking MkE potential possess a combined GM, B and T cell potential. Thus, evaluation at the single cell level of combined lineage potentials and multilineage gene expression provide compelling evidence for lymphoid-priming within the HSC compartment being preceeded by a loss of MkE potential, but occurring prior to loss of GM potential.


2006 ◽  
Vol 188 (11) ◽  
pp. 3740-3747 ◽  
Author(s):  
Barbara Setlow ◽  
Swaroopa Atluri ◽  
Ryan Kitchel ◽  
Kasia Koziol-Dube ◽  
Peter Setlow

ABSTRACT Dipicolinic acid (DPA) comprises ∼10% of the dry weight of spores of Bacillus species. Although DPA has long been implicated in spore resistance to wet heat and spore stability, definitive evidence on the role of this abundant molecule in spore properties has generally been lacking. Bacillus subtilis strain FB122 (sleB spoVF) produced very stable spores that lacked DPA, and sporulation of this strain with DPA yielded spores with nearly normal DPA levels. DPA-replete and DPA-less FB122 spores had similar levels of the DNA protective α/β-type small acid-soluble spore proteins (SASP), but the DPA-less spores lacked SASP-γ. The DPA-less FB122 spores exhibited similar UV resistance to the DPA-replete spores but had lower resistance to wet heat, dry heat, hydrogen peroxide, and desiccation. Neither wet heat nor hydrogen peroxide killed the DPA-less spores by DNA damage, but desiccation did. The inability to synthesize both DPA and most α/β-type SASP in strain PS3664 (sspA sspB sleB spoVF) resulted in spores that lost viability during sporulation, at least in part due to DNA damage. DPA-less PS3664 spores were more sensitive to wet heat than either DPA-less FB122 spores or DPA-replete PS3664 spores, and the latter also retained viability during sporulation. These and previous results indicate that, in addition to α/β-type SASP, DPA also is extremely important in spore resistance and stability and, further, that DPA has some specific role(s) in protecting spore DNA from damage. Specific roles for DPA in protecting spore DNA against damage may well have been a major driving force for the spore's accumulation of the high levels of this small molecule.


1980 ◽  
Vol 26 (12) ◽  
pp. 1386-1391 ◽  
Author(s):  
James P. Yeggy ◽  
Donald P. Stahly

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potential inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funneling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.


Author(s):  
Raudah Raudah ◽  
Tien Zubaidah ◽  
Imam Santoso

Abstract: Effectiveness Of Sterilization Method Of Dry Heat At The Wound Care Medical Devices Hospital Room Dr. H. Soemarno Sosroatmodjo Kuala Kapuas. Hospital Dr. H. Soemarno Sosroatmodjo is a Type C hospitals that provide 24-hour medical services.Bed capacity of 145 with the number BOR (Bed Occupancy Rate) 51.36%.Efforts to prevent the occurrence of nosocomial infections from medical devices one of which is the process of sterilization with dry heat heating process.The purpose of this study was to determine the effectiveness of dry heat sterilization methods on medical devices in the Hospital Dr. H. Soemarno Sosroatmodjo. This type of research is quasi-experimental.Type variables used are the independent variables (temperature variation with the dry heat oven), the dependent variable (number of bacteria).Samples taken medication is a tool set consisting of a pair of tweezers and tub instruments. Analysis of data use Oneway Anova test. Based on the results of the examination, in the highest number of bacteria known medical tool tweezers is the average number of bacteria colonies 1.33 / cm at 125oC, and the highest number of bacteria of medical tools like instrument that is the average number of bacteria colonies 0.66 / cm at the temperature of 125oC. Statistical data analysis for medical devices tweezers with Oneway Anova test, after sterilized generate significant value of 0.09, which sig> 0.05 so that Ho is accepted, meaning there is no significant difference and statistical analysis for medical devices like instrument with Oneway Anova test produces the significant value of 0.40, which sig> 0.05 means that there is no significant difference. Keywords: Hospitals; medical device; numbers germs; sterilization.


1910 ◽  
Vol 7 (11) ◽  
pp. 481-483 ◽  
Author(s):  
R. M. Brydone

Zoarium always unilaminate, nearly always adherent.Zoœcia broad, length ·65–·8 mm., breadth ·45–·55 mm., aperture primarily semicircular but tending to quadrangular by thickening of the upper lip, and with a semicircular collar, the two ends of which are expanded into large perforated spine bases: the arched front wall springs from the inside of the side walls and is pierced along its edge by a row of large pores, and at a short distance inside this row by a row of smaller pores in fairly close correspondence with the outer row; the centre of the front wall is pierced by a very variable number of fine pores, which sometimes give clear indications of a third row corresponding to the two outer ones; the zoœcia always have common side walls of considerable width; in immature stages (Fig. 1) the front walls rise high above the side walls, and small beak-shaped avicularia are scattered irregularly between them; in a further stage of development (Fig. 2) the side walls are greatly thickened and raised above the front walls, absorbing the avicularia, and the perforated spine bases at the corners of the aperture grow inwards until they meet, and are fused into a stout bar across the aperture, so qualifying the species for admission to the Steginoporidæ; in the stage of maximum development (Fig. 3) the bar develops a stout denticle in the middle of the lower edge.


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