scholarly journals Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Simonida Gencic ◽  
David A. Grahame
Keyword(s):  
Amino Acid ◽  
Clostridium Difficile ◽  
Central Component ◽  
Content Increase ◽  
Efficient System ◽  
Content Type ◽  
Atp Production ◽  
Cell Extracts ◽  
Complete Set

ABSTRACT Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO2 molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a ΔacsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD+ “acetobutyrogenesis,” requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile, explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon. IMPORTANCE Clostridium difficile is an anaerobic, multidrug-resistant, toxin-producing pathogen with major health impacts worldwide. It is the only widespread pathogen harboring a complete set of Wood-Ljungdahl pathway (WLP) genes; however, the role of the WLP in C. difficile is poorly understood. In other anaerobic bacteria and archaea, the WLP can operate in one direction to convert CO2 to acetic acid for biosynthesis or in either direction for energy conservation. Here, conditions are defined in which WLP levels in C. difficile increase markedly, functioning to support metabolism of carbohydrates. Amino acid nutritional requirements were better defined, with new insight into how the WLP and butyrate pathways act in concert, contributing significantly to energy metabolism by a mechanism that may have broad physiological significance within the group of nonclassical acetogens.

scholarly journals Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches inClostridium difficile

mSphere ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Robert W. McKee ◽  
Carissa K. Harvest ◽  
Rita Tamayo
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Biofilm Formation ◽  
Toxin Production ◽  
Intestinal Colonization ◽  
Cyclic Diguanylate ◽  
Signaling Molecule ◽  
Content Type ◽  
Surface Motility

ABSTRACTThe intracellular signaling molecule cyclic diguanylate (c-di-GMP) regulates many processes in bacteria, with a central role in controlling the switch between motile and nonmotile lifestyles. Recent work has shown that inClostridium difficile(also calledClostridioides difficile), c-di-GMP regulates swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we determined the transcriptional regulon of c-di-GMP inC. difficile,employing overexpression of a diguanylate cyclase gene to artificially manipulate intracellular c-di-GMP. Consistent with prior work, c-di-GMP regulated the expression of genes involved in swimming and surface motility. c-di-GMP also affected the expression of multiple genes encoding cell envelope proteins, several of which affected biofilm formationin vitro. A substantial proportion of the c-di-GMP regulon appears to be controlled either directly or indirectly via riboswitches. We confirmed the functionality of 11 c-di-GMP riboswitches, demonstrating their effects on downstream gene expression independent of the upstream promoters. The class I riboswitches uniformly functioned as “off” switches in response to c-di-GMP, while class II riboswitches acted as “on” switches. Transcriptional analyses of genes 3′ of c-di-GMP riboswitches over a broad range of c-di-GMP levels showed that relatively modest changes in c-di-GMP levels are capable of altering gene transcription, with concomitant effects on microbial behavior. This work expands the known c-di-GMP signaling network inC. difficileand emphasizes the role of the riboswitches in controlling known and putative virulence factors inC. difficile.IMPORTANCEInClostridium difficile, the signaling molecule c-di-GMP regulates multiple processes affecting its ability to cause disease, including swimming and surface motility, biofilm formation, toxin production, and intestinal colonization. In this study, we used RNA-seq to define the transcriptional regulon of c-di-GMP inC. difficile. Many new targets of c-di-GMP regulation were identified, including multiple putative colonization factors. Transcriptional analyses revealed a prominent role for riboswitches in c-di-GMP signaling. Only a subset of the 16 previously predicted c-di-GMP riboswitches were functionalin vivoand displayed potential variability in their response kinetics to c-di-GMP. This work underscores the importance of studying c-di-GMP riboswitches in a relevant biological context and highlights the role of the riboswitches in controlling gene expression inC. difficile.

scholarly journals Role of Leptin-Mediated Colonic Inflammation in Defense against Clostridium difficile Colitis

2013 ◽  
Vol 82 (1) ◽  
pp. 341-349 ◽  
Author(s):  
Rajat Madan ◽  
Xiaoti Guo ◽  
Caitlin Naylor ◽  
Erica L. Buonomo ◽  
Donald Mackay ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Murine Model ◽  
Leptin Receptor ◽  
Mucosal Immune Response ◽  
Infectious Colitis ◽  
Content Type ◽  
Bacterial Peritonitis ◽  
Stat3 Signaling ◽  
Increased Risk

ABSTRACTThe role of leptin in the mucosal immune response toClostridium difficilecolitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due toEntamoeba histolyticaas well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the sameLEPRQ223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk ofC. difficileinfection (odds ratio, 3.03;P= 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance ofC. difficilefrom the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin inC. difficilecolitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in theLEPRQ223R mutation. Identification of the role of leptin in protection fromC. difficileoffers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.

scholarly journals Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

2015 ◽  
Vol 197 (15) ◽  
pp. 2600-2609 ◽  
Author(s):  
Revathi Govind ◽  
Leah Fitzwater ◽  
Rebekah Nichols
Keyword(s):  
Amino Acids ◽  
Clostridium Difficile ◽  
Cell Viability ◽  
Virulence Factors ◽  
Start Codon ◽  
Nosocomial Pathogen ◽  
Content Type ◽  
Antibiotic Associated Diarrhea ◽  
Toxin Secretion

ABSTRACTClostridium difficileis a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenicC. difficilestrains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation.C. difficiletoxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms onC. difficilecell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166and TcdE142had a profound effect on cell viability compared to the TcdE140isoform. Similarly, TcdE166and TcdE142facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed.IMPORTANCEC. difficileis a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains ofC. difficileproduce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are secreted from bacterial cells is not yet clear but involves TcdE, a holin-like protein. In this work, we show that TcdE could be translated from three different start codons, resulting in the production of three TcdE isoforms. Furthermore, we investigated the role of these isoforms in toxin release and cell lysis inC. difficile. An understanding of TcdE-dependent toxin secretion may be helpful for the development of strategies for preventing and treatingC. difficileinfections.

scholarly journals Genetic Basis forIn VitroandIn VivoResistance to Lincosamides, Streptogramins A, and Pleuromutilins (LSAP Phenotype) in Enterococcus faecium

2013 ◽  
Vol 57 (9) ◽  
pp. 4463-4469 ◽  
Author(s):  
Christophe Isnard ◽  
Brigitte Malbruny ◽  
Roland Leclercq ◽  
Vincent Cattoir
Keyword(s):  
Amino Acid ◽  
Molecular Mechanism ◽  
Enterococcus Faecium ◽  
Full Genome Sequence ◽  
Comparative Genomic ◽  
Content Type ◽  
Abc Protein

ABSTRACTAs opposed toEnterococcus faecalis, which is intrinsically resistant to lincosamides, streptogramins A, and pleuromutilins (LSAP phenotype) by production of the ABC protein Lsa(A),Enterococcus faeciumis naturally susceptible. Since this phenotype may be selected forin vivoby quinupristin-dalfopristin (Q-D), the aim of this study was to investigate the molecular mechanism of acquired LSAP resistance inE. faecium. Six LSAP-resistantin vitromutants ofE. faeciumHM1070 as well as three different pairs of clinical isolates (pre- and postexposure to Q-D) were studied. The full genome sequence of anin vitromutant (E. faeciumUCN90B) was determined by using 454 sequencing technology and was compared with that of the parental strain. Single-nucleotide replacement was carried out to confirm the role of this mutation. By comparative genomic analysis, a point mutation was found within a 1,503-bp gene coding for an ABC homologue showing 66% amino acid identity with Lsa(A). This mutation (C1349T) led to an amino acid substitution (Thr450Ile). An identical mutation was identified in allin vitroandin vivoresistant strains but was not present in susceptible strains. The wild-type allele was namedeat(A) (forEnterococcusABCtransporter), and its mutated allelic variant was namedeat(A)v. The introduction ofeat(A)vfrom UCN90B into HM1070 conferred the LSAP phenotype, whereas that ofeat(A) from HM1070 into UCN90B restored susceptibility entirely. This is the first description of the molecular mechanism of acquired LSAP resistance inE. faecium. Characterization of the biochemical mechanism of resistance and the physiological role of this ABC protein need further investigations.

scholarly journals A Novel Role of Fungal Type I Myosin in Regulating Membrane Properties and Its Association with D-Amino Acid Utilization in Cryptococcus gattii

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Ami Khanal Lamichhane ◽  
H. Martin Garraffo ◽  
Hongyi Cai ◽  
Peter J. Walter ◽  
Kyung J. Kwon-Chung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Membrane Permeability ◽  
Cryptococcus Gattii ◽  
Membrane Properties ◽  
Type I ◽  
Content Type ◽  
Myosin I ◽  
Amino Acid Utilization

ABSTRACT We found a novel role of Myo5, a type I myosin (myosin-I), and its fortuitous association with d-amino acid utilization in Cryptococcus gattii. Myo5 colocalized with actin cortical patches and was required for endocytosis. Interestingly, the myo5Δ mutant accumulated high levels of d-proline and d-alanine which caused toxicity in C. gattii cells. The myo5Δ mutant also accumulated a large set of substrates, such as membrane-permeant as well as non-membrane-permeant dyes, l-proline, l-alanine, and flucytosine intracellularly. Furthermore, the efflux rate of fluorescein was significantly increased in the myo5Δ mutant. Importantly, the endocytic defect of the myo5Δ mutant did not affect the localization of the proline permease and flucytosine transporter. These data indicate that the substrate accumulation phenotype is not solely due to a defect in endocytosis, but the membrane properties may have been altered in the myo5Δ mutant. Consistent with this, the sterol staining pattern of the myo5Δ mutant was different from that of the wild type, and the mutant was hypersensitive to amphotericin B. It appears that the changes in sterol distribution may have caused altered membrane permeability in the myo5Δ mutant, allowing increased accumulation of substrate. Moreover, myosin-I mutants generated in several other yeast species displayed a similar substrate accumulation phenotype. Thus, fungal type I myosin appears to play an important role in regulating membrane permeability. Although the substrate accumulation phenotype was detected in strains with mutations in the genes involved in actin nucleation, the phenotype was not shared in all endocytic mutants, indicating a complicated relationship between substrate accumulation and endocytosis. IMPORTANCE Cryptococcus gattii, one of the etiological agents of cryptococcosis, can be distinguished from its sister species Cryptococcus neoformans by growth on d-amino acids. C. gattii MYO5 affected the growth of C. gattii on d-amino acids. The myo5Δ cells accumulated high levels of various substrates from outside the cells, and excessively accumulated d-amino acids appeared to have caused toxicity in the myo5Δ cells. We provide evidence on the alteration of membrane properties in the myo5Δ mutants. Additionally, alteration in the myo5Δ membrane permeability causing higher substrate accumulation is associated with the changes in the sterol distribution. Furthermore, myosin-I in three other yeasts also manifested a similar role in substrate accumulation. Thus, while fungal myosin-I may function as a classical myosin-I, it has hitherto unknown additional roles in regulating membrane permeability. Since deletion of fungal myosin-I causes significantly elevated susceptibility to multiple antifungal drugs, it could serve as an effective target for augmentation of fungal therapy.

Tunisian National Innovation System futures: an actors’ analysis focus

foresight ◽  
2020 ◽  
Vol 22 (3) ◽  
pp. 273-286
Author(s):  
Refaat Chaabouni ◽  
Lotfi Bouzaiane
Keyword(s):  
Social Inclusion ◽  
Innovation System ◽  
Economic Transformation ◽  
National Innovation System ◽  
Efficient System ◽  
Content Type ◽  
Current Trends ◽  
Key Players ◽  
Specific Project

Purpose This paper aims to focus on the Tunisian National Innovation System (NIS) and the conditions that might foster its aptitude to support socio-economic transformation. Design/methodology/approach The paper adopts an original twofold foresight methodology combining the analysis of key players with the exploration of scenarios. While usually, a foresight exercise considers only one of these two futures studies approaches. Findings The “Dynamic Islands System” scenario reflects the present situation and current trends. It shows that all expected components of an efficient system are in place, but they are evolving more or less independently. “Connected System” is the most desirable option because it reflects the potential to improve strongly productivity, competitiveness and social inclusion. The “Dislocated System” is a third possible but risky scenario. Despite the interesting insights provided through futures imaging, the scenario approach is not sufficient in providing indications of how and which actors can make happen the changes needed to move towards the desired state. So, a second step of the adopted twofold foresight approach addresses the role of key players. The analysis aims to reveal which actors are in a position to implement the required changes and thereby support the transition from the present to the desired scenario. Practical implications The challenges ahead are then pointed out, namely, that the private sector and firms would have to take the lead in the future; and that the less influential players would have to work side by side to be able to direct the NIS to the “Connected scenario”. Originality/value The clue in this approach is to transform a sub-optimal reality to achieve a desired end-state by understanding the trend scenario. The actors’ analysis helps to clarify the respective attitudes and concerns of the players vis-à-vis the required changes. In this two-step foresight approach, the desired scenario is considered as a specific project.

scholarly journals ERG11 Polymorphism in Voriconazole-Resistant Candida tropicalis: Weak Role of ERG11 Expression, Ergosterol Content, and Membrane Permeability

2020 ◽  
Vol 65 (1) ◽  
pp. e00325-20
Author(s):  
Patricia Navarro-Rodríguez ◽  
Loida López-Fernández ◽  
Adela Martin-Vicente ◽  
Josep Guarro ◽  
Javier Capilla
Keyword(s):  
Amino Acid ◽  
Membrane Permeability ◽  
Candida Tropicalis ◽  
Gene Sequencing ◽  
Missense Mutations ◽  
Sterol Content ◽  
Content Type ◽  
Ergosterol Content ◽  
Amino Acid Alignment

ABSTRACTMutations in ERG11 were detected by gene sequencing and amino acid alignment in 18 Candida tropicalis strains with different degrees of sensitivity to voriconazole (VRC). ERG11 expression, sterol content, and membrane permeability were also evaluated. We report three missense mutations in ERG11 that resulted in resistance to VRC. The transcriptional levels of ERG11 as well as the ergosterol content and membrane permeability demonstrated no correlation to only a slight correlation with the obtained MIC values, but the data did suggest a tendency toward such a correlation.

scholarly journals Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype

2015 ◽  
Vol 53 (8) ◽  
pp. 2575-2580 ◽  
Author(s):  
Rossella Baldan ◽  
Alberto Trovato ◽  
Valentina Bianchini ◽  
Anna Biancardi ◽  
Paola Cichero ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Genetic Relatedness ◽  
Public Health Problem ◽  
Antibiotic Consumption ◽  
Transmission Capacity ◽  
Predominant Genotype ◽  
Content Type ◽  
Molecular Features ◽  
Severity Of The Disease

Clostridium difficileinfection (CDI) became a public health problem for the global spreading of the so-called hypervirulent PCR ribotypes (RTs) 027 and 078, associated with increases in the transmission and severity of the disease. However, especially in Europe, several RTs are prevalent, and the concept of hypervirulence is currently debated. We investigated the toxin and resistance profiles and the genetic relatedness of 312C. difficilestrains isolated in a large Italian teaching hospital during a 5-year period. We evaluated the role of CDI-related antibiotic consumption and infection control practices on the RT predominance in association with their molecular features and transmission capacity. Excluding secondary cases due to nosocomial transmission, RT018 was the predominant genotype (42.4%) followed by RT078 (13.6%), while RT027 accounted for 0.8% of the strains. RT078 was most frequently isolated from patients in intensive care units. Its prevalence significantly increased over time, but its transmission capacity was very low. In contrast, RT018 was highly transmissible and accounted for 95.7% of the secondary cases. Patients with the RT018 genotype were significantly older than those with RT078 and other RTs, indicating an association between epidemic RT and age. We provide here the first epidemiological evidence to consider RT018 as a successful epidemic genotype that deserves more attention in clinical practice.

scholarly journals A Novel, WidespreadqacAAllele Results in Reduced Chlorhexidine Susceptibility inStaphylococcus epidermidis

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Amin Addetia ◽  
Alexander L. Greninger ◽  
Amanda Adler ◽  
Shuhua Yuan ◽  
Negar Makhsous ◽  
...  
Keyword(s):  
Amino Acid ◽  
Staphylococcus Epidermidis ◽  
Sequence Data ◽  
Methicillin Resistance ◽  
Single Amino Acid ◽  
Data Sets ◽  
The Novel ◽  
Content Type

ABSTRACTChlorhexidine gluconate (CHG) is a topical antiseptic widely used in health care settings. InStaphylococcusspp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneousStaphylococcusisolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed that 63 (6%) of these isolates had elevated CHG MICs (≥4 μg/ml). Screening of all 1,050 isolates for theqacA/Bgene (the sameqacgene with A or B allele) by restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novelqacA/BRFLP pattern,qacA/B273. The CHG MIC was significantly higher forqacA/B273-positive isolates (MIC50, 4 μg/ml; MIC range, 0.5 to 4 μg/ml) than for otherqacgroups:qacA-positive isolates (n = 559; MIC50, 1 μg/ml; MIC range, 0.5 to 4 μg/ml),qacB-positive isolates (n = 17; MIC50, 1 μg/ml; MIC range, 0.25 to 2 μg/ml), andqacA/B-negative isolates (n = 418, MIC50, 1 μg/ml; MIC range, 0.125 to 2 μg/ml) (P = 0.001). A high proportion of theqacA/B273-positive isolates also displayed methicillin resistance (96.4%) compared to the otherqacgroups (24.9 to 61.7%) (P = 0.001). Whole-genome sequencing revealed thatqacA/B273-positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, namedqacA4, was carried on the novel plasmid pAQZ1. TheqacA4-carrying isolates belonged to the highly resistantStaphylococcus epidermidissequence type 2 clone. By searching available sequence data sets, we identified 39 additionalqacA4-carryingS. epidermidisstrains from 5 countries. Curing an isolate ofqacA4resulted in a 4-fold decrease in the CHG MIC, confirming the role ofqacA4in the elevated CHG MIC. Our results highlight the importance of further studyingqacA4and its functional role in clinical staphylococci.

scholarly journals Enhanced survival of Drosophila Akt1 hypomorphs during amino-acid starvation requires foxo

Genome ◽  
2016 ◽  
Vol 59 (2) ◽  
pp. 87-93 ◽  
Author(s):  
Jennifer D. Slade ◽  
Brian E. Staveley
Keyword(s):  
Amino Acid ◽  
Disordered Eating ◽  
Amino Acid Starvation ◽  
Central Component ◽  
Receptor Signalling ◽  
Drosophila Model ◽  
Insulin Receptor Signalling ◽  
Severe Reduction ◽  
Mutant Lines

Disordered eating includes any pattern of irregular eating that may lead to either extreme weight loss or obesity. The conserved insulin receptor signalling pathway acts to regulate energy balance and nutrient intake, and its central component Akt1 and endpoint effector foxo are pivotal for survival during nutritional stress. Recently generated Akt1 hypomorphic mutant lines exhibit a moderate decrease in lifespan when aged upon standard media, yet show a considerable increase in survival upon amino-acid starvation media. While the loss of foxo function significantly reduces the survival response to amino-acid starvation, a combination of these Akt1 hypomorphs and a null foxo mutation reveal a synergystic and severe reduction in lifespan upon standard media, and an epistatic relationship when undergoing amino-acid starvation. Evaluation of survivorship upon amino-acid starvation media of these double mutants indicate a phenotype similar to the original foxo mutant demonstrating the role of foxo in this Akt1 phenotype. These results indicate that the subtle manipulation of foxo through Akt1 can enhance survival during adverse nutrient conditions to model the ability of individuals to tolerate nutrient deprivation. Ultimately, we believe that a Drosophila model of disordered eating could generate new avenues to develop potential therapies for related human conditions.

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