Biology subjects

McNellis, M. E.

Publications and source records attributed to McNellis, M. E..

4 recordsLinked to original sources

bioRxiv2025

The CspC:CspA heterodimer transduces germinant and co-germinant signals during Clostridioides difficile spore germination.

The clinically significant pathogen Clostridioides difficile, which lacks the transmembrane nutrient germinant receptors conserved in almost all spore-forming bacteria, initiates spore germination by a unique mechanism that requires two signals: a bile acid germinant and an amino acid or divalent cation co-germinant signal. While two soluble pseudoproteases, CspC and CspA, were initially identified as the germinant and co-germinant receptors, respectively, in C. difficile, we previously identified residues in an unstructured region of CspC that regulate the sensitivity of C. difficile spores to both signals. However, the mechanism by which CspC transduces these signals remained unclear. Here, we demonstrate that CspC forms a stable complex with CspA and determine the crystal structure of the CspC:CspA heterodimer. The structure reveals extensive interactions along the binding interface, including direct interactions between the unstructured region of CspC and CspA. Using structure-function analyses, we identify CspC:CspA interactions that regulate the sensitivity of C. difficile spores to germinant signals and show for the first time that CspA regulates C. difficile response to not only co-germinant but also germinant signals. While we also show that CspA can form a homodimer and determine its crystal structure, CspA homodimerization appears unimportant for C. difficile spore germination. Collectively, our analyses establish the CspC:CspA heterodimer, rather than its individual constituents, as a critical signaling node for sensing both germinant and co-germinant signals. They also suggest a new mechanistic model for how C. difficile transduces germinant signals, which could guide the development of therapeutics against this important pathogen.

microbiology↗

bioRxiv2025

Increased proportion of growth-arrested bacilli in acidic pH adaptation promotes Mycobacterium tuberculosis treatment survival

The ability of Mycobacterium tuberculosis (Mtb) to adapt its growth behavior in response to host environments promotes survival against immune and drug stressors. However, how these behaviors shift at the single-cell level remains poorly understood. Here, we show that Mtb adapts to acidic conditions by increasing the proportion of bacteria in a growth-arrested state, rather than uniformly slowing the growth rate of the entire population. This non-growing subpopulation exhibits greater tolerance to ethambutol. Clinical strains displayed higher proportions of growth-arrested cells under both neutral and acidic conditions, suggesting that growth arrest serves as a bet-hedging strategy during infection. Though the PhoPR two-component system contributes to regulating this non-growing state, we show that it is a partial regulator of the non-growing bacterial subpopulation and that additional transcriptional regulators are involved. Our study demonstrates that non-growing subpopulations of Mtb provide fitness benefits and are an active adaptation to environmental cues and not a passive consequence of stressors.

microbiology↗

bioRxiv2024

Penicillin-binding proteins exhibit catalytic redundancy during asymmetric cell division in Clostridioides difficile

Peptidoglycan synthesis is an essential driver of bacterial growth and division. The final steps of this crucial process involve the activity of the SEDS family glycosyltransferases that polymerize glycan strands and the class B penicillin-binding protein (bPBP) transpeptidases that cross-link them. While many bacteria encode multiple bPBPs to perform specialized roles during specific cellular processes, some bPBPs can play redundant roles that are important for resistance against certain cell wall stresses. Our understanding of these compensatory mechanisms, however, remains incomplete. Endospore-forming bacteria typically encode multiple bPBPs that drive morphological changes required for sporulation. The sporulation-specific bPBP, SpoVD, is important for synthesizing the asymmetric division septum and spore cortex peptidoglycan during sporulation in the pathogen Clostridioides difficile. Although SpoVD catalytic activity is essential for cortex synthesis, we show that it is unexpectedly dispensable for SpoVD to mediate asymmetric division. The dispensability of SpoVDs catalytic activity requires the presence of its SEDS partner, SpoVE, and is facilitated by another sporulation-induced bPBP, PBP3. Our data further suggest that PBP3 interacts with components of the asymmetric division machinery, including SpoVD. These findings suggest a possible mechanism by which bPBPs can be functionally redundant in diverse bacteria and facilitate antibiotic resistance. ImportancePeptidoglycan synthesis requires the transpeptidase activity of penicillin-binding proteins (PBPs), which can have specialized functions during cellular growth, division, and differentiation. However, many bacteria produce PBPs with overlapping functions, and this functional redundancy can increase antibiotic resistance. The spore-forming pathogen, Clostridioides difficile, encodes four PBPs: two are essential for growth and division and another, SpoVD, is essential for spore formation. Here, we show that the transpeptidase activity of SpoVD is dispensable during the first morphological step of sporulation, asymmetric division, because a sporulation-induced PBP, PBP3, partially substitutes for SpoVDs function during this early stage. We find that SpoVD and PBP3 interact during sporulation, suggesting a mechanism by which bPBPs can confer functional redundancy and potentially contribute to antibiotic resistance.

microbiology↗

bioRxiv2020

Morphological profiling of tubercule bacilli identifies drug pathways of action

Morphological profiling is a method to classify target pathways of antibacterials based on how bacteria respond to treatment through changes to cellular shape and spatial organization. Here, we utilized the cell-to-cell variation in morphological features of Mycobacterium tuberculosis bacilli to develop a rapid profiling platform called Morphological Evaluation and Understanding of Stress (MorphEUS). MorphEUS classified 94% of tested drugs correctly into broad categories according to modes of action previously identified in the literature. In the other 6%, MorphEUS pointed to key off-target or secondary bactericidal activities. We observed cell-wall damaging activity induced by bedaquiline and moxifloxacin through secondary effects downstream from their main target pathways. We implemented MorphEUS to correctly classify three compounds in a blinded study and identified an off-target effect for one compound that was not readily apparent in previous studies. We anticipate that the ability of MorphEUS to rapidly identify pathways of drug action and the proximal cause of bactericidal activity in tubercule bacilli will make it applicable to other pathogens and cell types where morphological responses are subtle and heterogeneous. Significance StatementTuberculosis is a leading cause of death in the world and requires treatment with an arduous multidrug regimen. Many new tuberculosis drugs are in development, and the drug development pipeline would benefit from more rapid methods to learn drug mechanism of action and off-target effects. Here, we describe a high throughput imaging method for rapidly classifying drugs into categories based on the primary and secondary cellular damage called Morphological Evaluation and Understanding of drug-Stress (MorphEUS). We anticipate that MorphEUS will assist in rapidly pinpointing causes of cellular death in response to drug treatment in tuberculosis and other bacterial pathogens.

microbiology↗