Three orphan histidine kinases inhibit Clostridioides difficile sporulation

The ability of the anaerobic gastrointestinal pathogen, Clostridioides difficile, to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a conserved phosphotransfer protein, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional conserved orphan phosphotransfer proteins, CD2492, CD1579, and CD1949 which are hypothesized to regulate Spo0A phosphorylation. Disruption of the conserved phosphotransfer protein, CD2492, also increased sporulation frequency, similarly to the CD1492 mutant, and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of the conserved CD1579 phosphotransfer protein also variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492 and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant negative hypersporulation phenotype. Altogether, at least three phosphotransfer proteins, CD1492, CD2492 and CD1579 (herein, PtpA, PtpB and PtpC) repress C. difficile sporulation initiation by regulating activity of Spo0A.

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/β SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.

Infected Nonunion of the Tibia due to Paenibacillus turicensis in a Healthy Young Adult Following an All-terrain Vehicle Accident

We present the case of a 19-year-old man with an open fracture of the tibia and fibula secondary to an accident with an all-terrain vehicle. He underwent operative excisional irrigation, debridement and fixation on the day of injury. His course was complicated by nonunion of the tibia fracture. Infection is a common factor in fracture nonunion, even in patients who receive appropriate surgical and antimicrobial management. Paenibacillus turicensis, an organism adapted to survive in the environment via spore formation, was responsible for nonunion in our patient. A brief discussion of this unusual organism, fracture nonunion and the role of infection in etiology of nonunion follows.

Proteomic Analysis of the Parasitic Cnidarian Ceratonova shasta (Cnidaria: Myxozoa) Reveals Diverse Roles of Actin in Motility and Spore Formation

Myxozoans are widely distributed aquatic obligate endoparasites that were recently recognized as belonging within the phylum Cnidaria. They have complex life cycles with waterborne transmission stages: resistant, infectious spores that are unique to myxozoans. However, little is known about the processes that give rise to these transmission stages. To understand the molecular underpinnings of spore formation, we conducted proteomics on Ceratonova shasta, a highly pathogenic myxozoan that causes severe mortalities in wild and hatchery-reared salmonid fishes. We compared proteomic profiles between developmental stages from inside the fish host, and the mature myxospore, which is released into the water where it drifts passively, ready to infect the next host. We found that C. shasta contains 2,123 proteins; representing the first proteomic catalog of a myxozoan myxospore. Analysis of proteins differentially expressed between developing and mature spore stages uncovered processes that are active during spore formation. Our data highlight dynamic changes in the actin cytoskeleton, which provides myxozoan developmental stages with mobility through lamellipodia and filopodia, whereas in the mature myxospore the actin network supports F-actin stabilization that reinforces the transmission stage. These findings provide molecular insight into the myxozoan life cycle stages and, particularly, into the process of sporogenesis.

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation.

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/β SASPs, SspA and SspB, and two annotated SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated SASPs do not contribute to UV resistance. In addition, none of these SASPs contribute to the resistance of tested chemicals. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate / serve as a checkpoint for spore formation, a previously unreported function of SASPs.

Identification of a novel regulator of Clostridioides difficile cortex formation

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. C. difficile infections are transmitted by its metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers: a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. We used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness∼2-fold; the thinner cortex layer of ΔspoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile.ImportanceThe Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective anti-sporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the Peptostreptococcaceae family that regulates the formation of C. difficile’s protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.

Influence of Incubation Temperature and Total Dissolved Solids on Biofilm and Spore Formation by Dairy Isolates of Geobacillus stearothermophilus

ABSTRACT Geobacillus species are important contaminants in the dairy industry, and their presence is often considered an indicator of poor plant hygiene with the potential to cause spoilage. They can form heat-resistant spores that adhere to surfaces of processing equipment and germinate to form biofilms. Therefore, strategies aimed toward preventing or controlling biofilm formation in the dairy industry are desirable. In this study, we demonstrated that the preferred temperature for biofilm and spore formation among Geobacillus stearothermophilus A1, D1, P3, and ATCC 12980 was 65°C. Increasing the total dissolved milk solid concentration to 20% (wt/vol) caused an apparent delay in the onset of biofilm and spore formation to detectable concentrations among all the strains at 55°C. Compared to the onset time of the biofilm formation of A1 in 10% (wt/vol) reconstituted skim milk, addition of milk protein (whey protein and sodium caseinate) caused an apparent delay in the onset of biofilm formation to detectable concentrations by an average of 10 h at 55°C. This study proposes that temperature and total dissolved solid concentration have a cumulative effect on biofilm and spore formation by G. stearothermophilus A1, D1, P3, and ATCC 12980. In addition, the findings from this study may indicate that preconditioning of stainless steel surfaces with adsorbed milk proteins may delay the onset of biofilm and spore formation by thermophilic bacteria during milk powder manufacture. IMPORTANCE The thermophilic bacillus Geobacillus stearothermophilus is a predominant spoilage bacterium in milk powder manufacturing plants. If its numbers exceed the accepted levels, financial losses may be incurred because of the need to lower the price of the end product. Furthermore, G. stearothermophilus bacilli can form heat-resistant spores which adhere to processing surfaces and can germinate to form biofilms. Previously conducted research had highlighted the variation in the spore and biofilm formation among three specific strains of G. stearothermophilus isolated from a milk powder manufacturing plant in New Zealand. The significance of our research is in demonstrating the effects of two abiotic factors, namely, temperature and total dissolved solid concentration, on biofilm and spore formation by these three dairy isolates, leading to modifications in the thermal processing steps aimed toward controlling biofilm and spore formation by G. stearothermophilus in the dairy industry.

How Do Smut Fungi Use Plant Signals to Spatiotemporally Orientate on and In Planta?

Smut fungi represent a large group of biotrophic plant pathogens that cause extensive yield loss and are also model organisms for studying plant–pathogen interactions. In recent years, they have become biotechnological tools. After initial penetration of the plant epidermis, smut fungi grow intra—and intercellularly without disrupting the plant-plasma membrane. Following the colonialization step, teliospores are formed and later released. While some smuts only invade the tissues around the initial penetration site, others colonize in multiple plant organs resulting in spore formation distal from the original infection site. The intimate contact zone between fungal hyphae and the host is termed the biotrophic interaction zone and enables exchange of signals and nutrient uptake. Obviously, all steps of on and in planta growth require fine sensing of host conditions as well as reprogramming of the host by the smut fungus. In this review, we highlight selected examples of smut fungal colonization styles, directional growth in planta, induction of spore formation, and the signals required, pointing to excellent reviews for details, to draw attention to some of the open questions in this important research field.

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.

Biological features and conditions of surface cultivating of a strain-producer of microbiopreparation Т-1 Trichoderma sp. against pathogen causing fusarium on oil flax

To develop technological regimen for production of microbiopreparations in a preparation form ‘wetting powder’ we studied biological features and conditions of surface cultivating of a strain-producer Т-1 Trichoderma sp. – an antagonist of pathogen Fusarium oxysporum Schlecht. emend. Shyd. et Hans. var. orthoceras (App. еt Wr.) Bilai and Fusarium poae (Peck) Wollenw., Lewis on oil flax. To study cultural and physiological qualities of the strainproducer we used agar and liquid mediums. Surface cultivation of a fungus on agar and liquid Rudakov’s medium at a temperature 25–30 оС was the most favorable for mycelium growth and spore formation. Stationary fungus cultivation on liquid medium with рН from 3 to 6 provided maximal mycelium growth with spore formation and the highest dry mass. Addition of starch into the Chapek’s nutrient medium caused maximal growth of fungus mycelium and increase of its dry mass. The best source of nitrogen for a fungus strain was corn extract. Rudakov’s and No1 mediums are optimal compound liquid nutrient mediums for a surface cultivation of the strain-producer. Optimal period of the surface cultivation of the fungal strain Т-1 Trichoderma sp. on liquid Rudakov’s nutrient medium was 10 days, and a volume of sowing culture to a nutrient medium – 2.0%.