Biology subjects

Rohlfing, A. E.

Publications and source records attributed to Rohlfing, A. E..

3 recordsLinked to original sources

bioRxiv2026

Adaptation of the Geometric Viability assay as a high throughput assay to measure Clostridioides difficile spore germination

Clostridioides difficile is the leading cause of hospital acquired infections and gastroenteritis associated death in the United States. As an obligate anaerobe, C. difficile relies on the metabolically dormant, aerotolerant spore form to transmit infection. C. difficile infection begins when spores germinate in the small intestine in response to specific bile acids and a co-germinant signal (either amino acids or divalent cations). As germination is required to initiate infection, understanding how spores sense germinants and outgrow into toxin-producing cells is an active area of research. One of the fundamental assays used to study C. difficile germination is a resource and time intensive assay, similar to a bacterial CFU assay, that relies on serial dilutions of a spore sample plated on agar media in triplicate to determine the number of viable spores in a sample. As this assay is similar to assays used to enumerate viable bacteria in a sample, we hypothesized that a recently developed high-throughput technique to measure bacterial CFUs published by Meyers et al., the Geometric viability assay (GVA), could be modified to measure the number of viable spores in a sample. We compared measurements of viable spores per mL from the conventional spore viability assay and the modified GVA protocol and found that the two measurements were highly consistent with each other (Pearson r=0.991, R2=0.9821). The modified GVA protocol was also able to measure differences in sensitivity to germination signals in strains of C. difficile hypersensitive to co-germinants and measure low levels of spontaneous germination. The GVA protocol is less resource and time intensive than the conventional assay, was easy adapt, and likely would be feasible to study germination in a wide range of spore-formers.

microbiology↗

bioRxiv2019

Differential Effects of 'Resurrecting' Cap Pseudoproteases during Clostridioides difficile Spore Germination

Clostridioides difficile is a spore-forming bacterial pathogen that is the leading cause of hospital-acquired gastroenteritis. C. difficile infections begin when its spore form germinates in the vertebrate gut upon sensing bile acids. These germinants induce a proteolytic signaling cascade controlled by three members of the subtilisin-like serine protease family, CspA, CspB, and CspC. Notably, even though CspC and CspA are both pseudoproteases, they are nevertheless required to sense germinants and activate the protease, CspB. Thus, CspC and CspA are part of a growing list of pseudoenzymes that play important roles in regulating cellular processes. However, despite their importance, the structural properties of pseudoenzymes that allow them to function as regulators remain poorly understood. Our recently determined crystal structure of CspC revealed that its degenerate site residues align closely with the catalytic triad of CspB, so in this study we tested whether the ancestral protease activity of the CspC and CspA pseudoproteases could be "resurrected." Restoring the catalytic triad to these pseudoproteases failed to resurrect their protease activity, although the mutations differentially affected the stability and function of these pseudoproteases. Degenerate site mutations destabilized CspC and impaired spore germination without impacting CspA stability or function. Thus, our results surprisingly reveal that the presence of a catalytic triad does not necessarily predict protease activity. Since close homologs of C. difficile CspA occasionally carry an intact catalytic triad, our results imply that bioinformatics predictions of enzyme activity may overlook pseudoenzymes in some cases.

biochemistry↗

bioRxiv2018

The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile uses the soluble pseudoprotease, CspC, to detect bile salt germinants. To gain insight into CspCs unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile salt germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile salt-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since the mechanism by which C. difficile spores sense co-germinants is unknown, our study provides the first evidence that CspC senses distinct classes of co-germinants in addition to bile salts. Since we observed that specific residues control CspCs responsiveness to these different signals, CspC is critical for regulating C. difficile germination in response to multiple environmental signals.

microbiology↗