Influence of L-lactate and low glucose concentrations on the metabolism and the toxin formation of Clostridioides difficile

The virulence of Clostridioides difficile (formerly Clostridium difficile) is mainly caused by its two toxins A and B. Their formation is significantly regulated by metabolic processes. Here we investigated the influence of various sugars (glucose, fructose, mannose, trehalose), sugar derivatives (mannitol and xylitol) and L-lactate on toxin synthesis. Fructose, mannose, trehalose, mannitol and xylitol in the growth medium resulted in an up to 2.2-fold increase of secreted toxin. Low glucose concentration of 2 g/L increased the toxin concentration 1.4-fold compared to growth without glucose, while high glucose concentrations in the growth medium (5 and 10 g/L) led to up to 6.6-fold decrease in toxin formation. Transcriptomic and metabolic investigation of the low glucose effect pointed towards an inactive CcpA and Rex regulatory system. L-lactate (500 mg/L) significantly reduced extracellular toxin formation. Transcriptome analyses of the later process revealed the induction of the lactose utilization operon encoding lactate racemase (larA), electron confurcating lactate dehydrogenase (CDIF630erm_01321) and the corresponding electron transfer flavoprotein (etfAB). Metabolome analyses revealed L-lactate consumption and the formation of pyruvate. The involved electron confurcation process might be responsible for the also observed reduction of the NAD+/NADH ratio which in turn is apparently linked to reduced toxin release from the cell.

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Low Glucose Mediated Fluconazole Tolerance in Cryptococcus neoformans

Journal of Fungi ◽

10.3390/jof7060489 ◽

2021 ◽

Vol 7 (6) ◽

pp. 489

Author(s):

Somanon Bhattacharya ◽

Natalia Kronbauer Oliveira ◽

Anne G. Savitt ◽

Vanessa K. A. Silva ◽

Rachel B. Krausert ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Mitochondrial Function ◽

Efflux Pump ◽

Nile Red ◽

Fold Increase ◽

Growth Conditions ◽

Chromosome 1 ◽

Normal Glucose ◽

Low Glucose ◽

Synthetic Media

Chronic meningoencephalitis is caused by Cryptococcus neoformans and is treated in many parts of the world with fluconazole (FLC) monotherapy, which is associated with treatment failure and poor outcome. In the host, C. neoformans propagates predominantly under low glucose growth conditions. We investigated whether low glucose, mimicked by growing in synthetic media (SM) with 0.05% glucose (SMlowglu), affects FLC-resistance. A > 4-fold increase in FLC tolerance was observed in seven C. neoformans strains when minimum inhibitory concentration (MIC) was determined in SMlowglu compared to MIC in SM with normal (2%) glucose (SMnlglu). In SMlowglu, C. neoformans cells exhibited upregulation of efflux pump genes AFR1 (8.7-fold) and AFR2 (2.5-fold), as well as decreased accumulation (2.6-fold) of Nile Red, an efflux pump substrate. Elevated intracellular ATP levels (3.2-fold and 3.4-fold), as well as decreased mitochondrial reactive oxygen species levels (12.8-fold and 17-fold), were found in the presence and absence of FLC, indicating that low glucose altered mitochondrial function. Fluorescence microscopy revealed that mitochondria of C. neoformans grown in SMlowglu were fragmented, whereas normal glucose promoted a reticular network of mitochondria. Although mitochondrial membrane potential (MMP) was not markedly affected in SMlowglu, it significantly decreased in the presence of FLC (12.5-fold) in SMnlglu, but remained stable in SMlowglu-growing C. neoformans cells. Our data demonstrate that increased FLC tolerance in low glucose-growing C. neoformans is the result of increased efflux pump activities and altered mitochondrial function, which is more preserved in SMlowglu. This mechanism of resistance is different from FLC heteroresistance, which is associated with aneuploidy of chromosome 1 (Chr1).

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Kinetic analysis of N-acylphosphatidylserine accumulation and implications for membrane assembly in Rhodopseudomonas sphaeroides

Journal of Bacteriology ◽

10.1128/jb.152.2.607-615.1982 ◽

1982 ◽

Vol 152 (2) ◽

pp. 607-615

Author(s):

B D Cain ◽

T J Donohue ◽

S Kaplan

Keyword(s):

Kinetic Analysis ◽

Growth Medium ◽

Metabolic Pathway ◽

Fold Increase ◽

Phospholipid Content ◽

Membrane Biogenesis ◽

Rhodopseudomonas Sphaeroides ◽

Intracytoplasmic Membrane ◽

Phospholipid Transfer ◽

Cellular Phospholipid

The accumulation of N-acylphosphatidylserine (NAPS) in response to the inclusion of Tris in the growth medium of Rhodopseudomonas sphaeroides strain M29-5 has been examined. In the accompanying paper (Donohue et al., J. Bacteriol. 152:000--000, 1982), we show that in response to Tris, NAPS accumulated to as much as 40% of the total cellular phospholipid content. NAPS accumulation began immediately upon addition of Tris and was reflected as an abrupt 12-fold increase in the apparent rate of NAPS accumulation. We suggest that Tris altered the flow of metabolites through a preexisting and previously unknown metabolic pathway. NAPS accumulation ceased immediately upon the removal of Tris; however, accumulated NAPS remained largely metabolically stable. Importantly, under conditions in which NAPS was not accumulated, the intracytoplasmic membrane was shown to be virtually devoid of newly synthesized NAPS. The significance of this observation is discussed in terms of its physiological implications on phospholipid transfer and membrane biogenesis in R. sphaeroides.

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Metabolic Engineering of Saccharomyces cerevisiae for Conversion of d-Glucose to Xylitol and Other Five-Carbon Sugars and Sugar Alcohols

Applied and Environmental Microbiology ◽

10.1128/aem.02707-06 ◽

2007 ◽

Vol 73 (17) ◽

pp. 5471-5476 ◽

Cited By ~ 23

Author(s):

Mervi H. Toivari ◽

Laura Ruohonen ◽

Andrei N. Miasnikov ◽

Peter Richard ◽

Merja Penttilä

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Engineering ◽

Growth Medium ◽

Fold Increase ◽

Pichia Stipitis ◽

Xylitol Dehydrogenase ◽

Sugar Alcohols ◽

Pentose Sugar ◽

Encoding Gene ◽

Pentose Sugars

ABSTRACT Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar d-ribose from d-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated d-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (d-ribose, d-ribulose, and d-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted.

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Characterization of a β-lactamase that contributes to intrinsic β-lactam resistance inClostridioides difficile

10.1101/630020 ◽

2019 ◽

Cited By ~ 1

Author(s):

Brindar K. Sandhu ◽

Adrianne N. Edwards ◽

Sarah E. Anderson ◽

Emily C. Woods ◽

Shonna M. McBride

Keyword(s):

Membrane Protein ◽

Unknown Function ◽

Signal Sequence ◽

Bacterial Species ◽

Resistance Mechanism ◽

Regulatory System ◽

Gram Positive ◽

Clostridioides Difficile ◽

Antibiotic Associated Diarrhea

ABSTRACTClostrididioides difficilecauses severe antibiotic-associated diarrhea and colitis.C. difficileis an anaerobic, Gram-positive spore former that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance ofC. difficileto β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance inC. difficileare not fully understood. Our data reinforce prior evidence thatC. difficileproduces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. We identified an operon encoding a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity inC. difficilestrain 630Δermand resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, herein named BlaD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a co-transcribed lipoprotein, BlaX, aids in BlaD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of theblaXDoperon in response to β-lactams. These results provide support for the function of a β-lactamase inC. difficileantibiotic resistance, and reveal the unique roles of a co-regulated lipoprotein and reducing environment in β-lactamase activity.IMPORTANCEClostridioides difficileis an anaerobic, gastrointestinal human pathogen. One of the highest risk factors for contractingC. difficileinfection is antibiotic treatment, which causes microbiome dysbiosis.C. difficileis resistant to β-lactam antibiotics, the most commonly prescribed class of antibiotics.C. difficileproduces a recently discovered β-lactamase, which cleaves and inactivates numerous β-lactams. In this study, we report the contribution of this anaerobic β-lactamase to ampicillin resistance inC. difficile, as well as the transcriptional regulation of the gene,blaD, by a BlaRI system. In addition, our data demonstrate co-transcription ofblaDwithblaX, which encodes a membrane protein of previously unknown function. Furthermore, we provide evidence that BlaX enhances β-lactamase activity in a portion ofC. difficilestrains. This study demonstrates a novel interaction between a β-lactamase and a membrane protein in a Gram-positive pathogen, and due to the anaerobic nature of the β-lactamase activity, suggests that more β-lactamases are yet to be identified in other anaerobes.

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Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence

PLoS Pathogens ◽

10.1371/journal.ppat.1009817 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009817

Author(s):

Megan G. Taggart ◽

William J. Snelling ◽

Patrick J. Naughton ◽

Roberto M. La Ragione ◽

James S. G. Dooley ◽

...

Keyword(s):

Biofilm Formation ◽

Gastrointestinal Disease ◽

Regulatory System ◽

Disease Recurrence ◽

The United States ◽

Extracellular Dna ◽

Published Data ◽

Bacterial Cells ◽

Healthcare Associated Infection ◽

Clostridioides Difficile

Clostridiodes difficile (C. difficile) was ranked an “urgent threat” by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rod-shaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilm-forming ability between C. difficile strains such as 630 and the “hypervirulent” strain, R20291, make it difficult to assign a “one size fits all” mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

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“If the glove fits”: Hospital-wide universal gloving is associated with improved hand hygiene and may reduce Clostridioides difficile infection

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.1422 ◽

2021 ◽

pp. 1-5

Author(s):

Paritosh Prasad ◽

Lynne Brown ◽

Shiyang Ma ◽

Andrew McDavid ◽

Andrew Rudmann ◽

...

Keyword(s):

Hand Hygiene ◽

Hand Hygiene Compliance ◽

Fold Increase ◽

Credible Interval ◽

Clostridioides Difficile ◽

Before And After ◽

Clostridioides Difficile Infection ◽

Quasi Experimental ◽

Hygiene Compliance ◽

Hospital Acquired

Abstract Objective: To determine whether a hospital-wide universal gloving program resulted in increased hand hygiene compliance and reduced inpatient Clostridioides difficile infection (CDI) rates. Design: We carried out a multiple-year before-and-after quasi-experimental quality improvement study. Gloving and hand hygiene compliance data as well as hospital-acquired infection rates were prospectively collected from January 1, 2015, to December 31, 2017, by secret monitors. Settings: The University of Rochester Strong Memorial Hospital, an 849-bed quaternary-care teaching hospital. Patients: All adult inpatients with the exception of patients in the obstetrics unit. Interventions: A hospital-wide universal gloving protocol was initiated on January 1, 2016. Results: Hand hygiene compliance increased from 68% in 2015 reaching an average of 88% by 2017 (P < .0002). A 10% increase in gloving per unit was associated with a 1.13-fold increase in the odds of hand hygiene (95% credible interval, 1.12–1.14). The rates of CDI decreased from 1.05 infections per 1,000 patient days in 2015 to 0.74 in 2017 (P < .04). Conclusion: A universal gloving initiative was associated with a statistically significant increase in both gloving and hand hygiene compliance. CDI rates decreased during this intervention.

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Low-Level Pilin Expression Allows for Substantial DNA Transformation Competence in Neisseria gonorrhoeae

Infection and Immunity ◽

10.1128/iai.71.11.6279-6291.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6279-6291 ◽

Cited By ~ 49

Author(s):

Cynthia D. Long ◽

Deborah M. Tobiason ◽

Matthew P. Lazio ◽

Kimberly A. Kline ◽

H. Steven Seifert

Keyword(s):

Growth Medium ◽

Posttranslational Modifications ◽

Regulatory System ◽

Dependent Manner ◽

Dna Transformation ◽

Dna Uptake ◽

Transformation Competence ◽

Pilin Protein ◽

Different Levels ◽

Pilus Assembly

ABSTRACT The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.

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The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation

Journal of Bacteriology ◽

10.1128/jb.00574-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6559-6567 ◽

Cited By ~ 11

Author(s):

Tomas Fiedler ◽

Maren Mix ◽

Uta Meyer ◽

Stefan Mikkat ◽

Michael O. Glocker ◽

...

Keyword(s):

Clostridium Acetobutylicum ◽

Response Regulator ◽

Specific Binding ◽

Expression Patterns ◽

Regulatory System ◽

Fold Increase ◽

Growth Conditions ◽

Two Dimensional Gel Electrophoresis ◽

Two Component

ABSTRACT The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in Pi-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of Pi. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

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Layered dynamic regulation for improving metabolic pathway productivity inEscherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716920115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 2964-2969 ◽

Cited By ~ 70

Author(s):

Stephanie J. Doong ◽

Apoorv Gupta ◽

Kristala L. J. Prather

Keyword(s):

Regulatory System ◽

Value Added ◽

Fold Increase ◽

Product Formation ◽

Glycolytic Flux ◽

Glucaric Acid ◽

Dynamic Regulation ◽

Fourfold Increase ◽

Product Titer ◽

K 12

Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from “growth” to “production” mode. The second strategy, developed in this work, uses a biosensor formyo-inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date inEscherichia coliK-12 strains.

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Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2861-2866.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2861-2866 ◽

Cited By ~ 50

Author(s):

Eoin Barrett ◽

Catherine Stanton ◽

Oskar Zelder ◽

Gerald Fitzgerald ◽

R. Paul Ross

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Sole Carbon Source ◽

Fold Increase ◽

Negative Control ◽

Lactobacillus Delbrueckii ◽

Lactose Permease ◽

Genetic Determinants ◽

Lactose Utilization ◽

Galactose Utilization

ABSTRACT The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.

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