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

Abstract

Clostridium difficile is the leading cause of hospital-acquired, antibiotic-associated diarrhea, and is the only wide-spread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO2 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-CoA synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of an 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 co-localization 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 wide-spread 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. Herein, 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 non-classical acetogens.

What’s a Biofilm?—How the Choice of the Biofilm Model Impacts the Protein Inventory of Clostridioides difficile

“…The WLP, also known as the reductive Acetyl-CoA pathway, is able to generate acetyl-CoA from CO 2 ( Stupperich et al, 1983 ; Köpke et al, 2013 ). Moreover, the WLP has recently been suggested as an electron sink to maintain cell homeostasis in the absence of Stickland acceptors ( Gencic and Grahame, 2020 ). Taken together, the higher levels of proteins from the oxidative branch of Stickland fermentation, of butyrate fermentation and of the V-type ATPase discussed above as well of WLP proteins in colony biofilms strongly support the hypothesis that the WLP plays an important role in maintaining cell growth in environments depleted in reductive equivalents and potentially maintains the membrane potential in concert with the coupled V-type ATPase ( Gencic and Grahame, 2020 ).…”

“…Moreover, the WLP has recently been suggested as an electron sink to maintain cell homeostasis in the absence of Stickland acceptors ( Gencic and Grahame, 2020 ). Taken together, the higher levels of proteins from the oxidative branch of Stickland fermentation, of butyrate fermentation and of the V-type ATPase discussed above as well of WLP proteins in colony biofilms strongly support the hypothesis that the WLP plays an important role in maintaining cell growth in environments depleted in reductive equivalents and potentially maintains the membrane potential in concert with the coupled V-type ATPase ( Gencic and Grahame, 2020 ). Of note, Poquet et al (2018) reported a downregulation of WLP, glycine cleavage system and V-type ATPase genes in aggregate biofilms compared to planktonic cells which is in line with results of this proteome analysis.…”

AMiGA: software for automated Analysis of Microbial Growth Assays

“…It is also capable of using amino acids as its sole energy and biomass source (18), which explains its ability to grow on the no-carbon control and may explain its biphasic growth on ribose and sorbitol. C. difficile may initially ferment amino acids via the Stickland pathway until they are depleted then transition to growth on sugars present in the environment (18, 19). However, it is less clear why rapid growth on certain monosaccharides was swiftly followed by rapid death.…”

“…In our Biolog phenotyping, we noticed that certain monosaccharides promoted an atypical growth curve characterized by rapid rise followed by a rapid decay in optical density. Substantial death in gram-positive bacteria may be explained by autolysis due to rapid loss of energy-generating substrates (19) or sugar-driven phage induction that leads to lysis (20). Because the substrate concentration in Biolog PM plates is proprietary information and thus unknown, we wanted to see if this phenomenon occurs at different concentrations of these substrates.…”

The selenophosphate synthetase,selD, is important forClostridioides difficilephysiology

“…C. difficile has multiple metabolic pathways that overlap to ensure generation of key metabolites. The Wood-Ljungdahl pathway was recently found to be not as active in C. difficile as in other acetogens, but this pathway can be used in conjunction with butyrate formation to regenerate NAD + in the absence of Stickland metabolism (13). Carbon metabolism includes breaking down sugars such as glucose and mannitol to generate pyruvate and acetyl-CoA for glycolysis and the tricarboxylic acid cycle, although the latter is incomplete.…”