Biology subjects

Harrison, G. A.

Publications and source records attributed to Harrison, G. A..

4 recordsLinked to original sources

bioRxiv2026

Defining the order of assembly of the Clostridioides difficile divisome complex

Cell division is the ancient pathway by which bacteria synthesize a septum of peptidoglycan, dividing the cell into two. Where all walled bacteria were previously thought to use FtsW-FtsI orthologs to synthesize septal peptidoglycan during division, we recently discovered that the major pathogen Clostridioides difficile is missing FtsW-FtsI and instead relies on the activity of the bifunctional Class A PBP called PBP1 to synthesize the septal peptidoglycan. Furthermore, C. difficile either does not encode or require the majority of canonical divisome proteins described in model bacteria aside from the divisome protein orthologs FtsZ, SepF, and ZapA. Indeed, unlike model systems, SepF and ZapA are essential in C. difficile, suggesting that they have evolved to have a critical function in cell division without the redundant mechanisms present in model organisms. Thus, C. difficile uses a fundamentally different division mechanism compared to previously studied bacteria. To understand how this unusual complex is assembled in C. difficile, we combine CRISPR interference (CRISPRi)-based knock-downs with fluorescent fusions to determine that the hierarchical order of assembly occurs in three phases: (i) FtsZ/ZapA, (ii) SepF, and (iii) PBP1. We further investigate the order of assembly of several non-essential mid-cell localizing proteins and discover that MldA, MldC, DivIVA, FtsK, and PBP3 depend on FtsZ, SepF, and PBP1 for localization, whereas MldB localizes independently of SepF and PBP1. Our work provides a model for divisome assembly in C. difficile and validates genetic and cytological tools that can be used to mechanistically dissect this pathway in the future. IMPORTANCEBacterial cell division has been extensively studied in model systems, but little is known about how this essential process occurs in the clinically important pathogen Clostridioides difficile. Studies in model systems have shown that cell division is carried out by a large multi-protein complex called the "divisome." While components of the divisome are widely conserved and can be traced back to the last bacterial common ancestor billions of years ago, C. difficile uses a unique mechanism of division that is independent of the majority of these canonical divisome genes. In the current study, we characterize the core, essential divisome comprised of FtsZ, ZapA, SepF, and PBP1, and build a model for the order of assembly of this unusual divisome complex.

microbiology↗

bioRxiv2025

Molecular dissection of Class A PBP function uncovers novel features of the non-canonical Clostridioides difficile divisome complex

Cell division in bacteria is mediated by the "divisome," a multiprotein complex that synthesizes the septal peptidoglycan needed to divide one cell into two. We recently showed that the major nosocomial pathogen Clostridioides difficile assembles a divisome that is fundamentally distinct from previously studied bacteria because it lacks functional orthologs of the septal peptidoglycan-synthesizing enzymes, FtsW and FtsI. While these enzymes were previously thought to mediate cell division in all walled bacteria, C. difficile instead uses the bifunctional Class A Penicillin Binding Protein PBP1 to mediate cell division. Here, we optimized a CRISPRi-based conditional expression system to define features within PBP1 that are critical for its essential functions. Our analyses identify a novel accessory domain that is required for PBP1 function and is conserved across Peptostreptococcaceae family PBP1 homologs. We further show that PBP1s glycosyltransferase and transpeptidase activities are both strictly required for bacterial growth. While PBP1 glycosyltransferase activity is required for septum synthesis during cell division, PBP1s transpeptidase activity is surprisingly dispensable for cell division, although TPase-deficient (PBP1TPase*) cells produce multiple aberrant septa. We demonstrate that the uncontrolled septum synthesis observed in PBP1TPase* cells depends on the non-essential Class B PBP, PBP3, but PBP3s catalytic activity is dispensable for this function. Since we also show that PBP3 is recruited to the divisome complex and forms a complex with PBP1, our analyses reveal a cryptic but important regulatory function for PBP3 in promoting C. difficile cell division. Author SummaryBacterial cell division is an ancient and essential process, but our molecular understanding of this process is primarily based on studies in a select few model systems. Recent work found that the major nosocomial pathogen Clostridioides difficile divides by a fundamentally distinct mechanism because homologs of the canonical septum synthesis complex used by virtually all walled bacteria are either missing from the C. difficile genome or have lost their function during vegetative division. This prior work revealed that septum synthesis in C. difficile is instead driven by the catalytic activity of the Class A Penicillin Binding Protein called PBP1, but the features of PBP1 that enable it to carry out this unusual function were unknown. In the current study, we perform detailed structure-function analyses to determine how C. difficile PBP1 has become specialized for this role. We find that PBP1 carries an unusual regulatory domain that is critical for its function. Our analyses also uncover an unexpected function for the non-essential enzyme PBP3 in cell division, identifying a new component of the C. difficile cell division complex. These analyses provide new insight into how bacteria can repurpose cell wall synthesis enzymes to fulfill essential functions in novel ways.

microbiology↗

bioRxiv2023

Inducing vulnerability to InhA inhibition restores isoniazid susceptibility in drug resistant Mycobacterium tuberculosis

Of the approximately 10 million cases of Mycobacterium tuberculosis (Mtb) infections each year, over 10% are resistant to the frontline antibiotic isoniazid (INH). INH resistance is predominantly caused by mutations that decrease the activity of the bacterial enzyme KatG, which mediates conversion of the pro-drug INH to its active form INH-NAD. We previously discovered an inhibitor of Mtb respiration, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a collection of INH-resistant mutants to INH through an unknown mechanism. To investigate the mechanism of action of C10, we exploited the toxicity of high concentrations of C10 to select for resistant mutants. We discovered two mutations that confer resistance to the disruption of energy metabolism and allow for growth of Mtb in high C10 concentrations, indicating that growth inhibition by C10 is associated with inhibition of respiration. Using these mutants as well as direct inhibitors of the Mtb electron transport chain, we provide evidence that inhibition of energy metabolism by C10 is neither sufficient nor necessary to potentiate killing by INH. Instead, we find that C10 acts downstream of INH-NAD synthesis, causing Mtb to become particularly sensitive to inhibition of the INH-NAD target, InhA, without changing the concentration of INH-NAD or the activity of InhA, the two predominant mechanisms of potentiating INH. Our studies revealed that there exists a vulnerability in Mtb that can be exploited to render Mtb sensitive to otherwise subinhibitory concentrations of InhA inhibitor. SignificanceIsoniazid (INH) is a critical frontline antibiotic to treat Mycobacterium tuberculosis (Mtb) infections. INH efficacy is limited by its suboptimal penetration of the Mtb-containing lesion and by the prevalence of clinical INH-resistance. We previously discovered a compound, C10, that enhances the bactericidal activity of INH, prevents the emergence of INH-resistant mutants, and re-sensitizes a set of INH-resistant mutants to INH. Resistance is typically mediated by katG mutations that decrease the activation of INH, which is required for INH to inhibit the essential enzyme InhA. Our current work demonstrates that C10 re-sensitizes INH-resistant katG-hypomorphs without enhancing the activation of INH. We furthermore show that C10 causes Mtb to become particularly vulnerable to InhA inhibition without compromising InhA activity on its own. Therefore, C10 represents a novel strategy to curtail the development of INH resistance and to sensitize Mtb to sub-lethal doses of INH, such as those achieved at the infection site.

microbiology↗

bioRxiv2019

Dual role of auxin in regulating plant defense and bacterial virulence gene expression during Pseudomonas syringae PtoDC3000 pathogenesis.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen Pseudomonas syringae strain PtoDC3000 produces the plant hormone auxin (Indole-3-acetic acid, or IAA) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth, demonstrating that host auxin signaling is required for normal susceptibility to PtoDC3000, and this phenotype was dependent on SA-mediated defenses. However, despite exhibiting decreased auxin perception, tir1 afb1 afb4 afb5 quadruple mutant plants lacking four of the six known auxin co-receptors supported increased levels of bacterial growth. This mutant also exhibited elevated IAA levels, suggesting that the increased IAA in these plants may promote PtoDC3000 growth independent of host auxin signaling, perhaps through a direct effect on the pathogen. In support of this, we found that IAA directly impacted the pathogen, by modulating expression of bacterial virulence genes, both in liquid culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

plant biology↗