Recognition of extracellular DNA by type IV pili promotes biofilm formation by \u3ci\u3eClostridioides difficile\u3c/i\u3e

Clostridioides difficile is a Gram-positive bacillus, which is a frequent cause of gastrointestinal infections triggered by the depletion of the gut microbiome. Because of the frequent recurrence of these infections after antibiotic treatment, mechanisms of C. difficile persistence and recurrence, including biofilm formation, are of increasing interest. Previously, our group and others found that type IV pili, filamentous helical appendages polymerized from protein subunits, promoted microcolony and biofilm formation in C. difficile. In Gram-negative bacteria, the ability of type IV pili to mediate bacterial self-association has been explained through interactions between the pili of adjacent cells, but type IV pili from several Gram-negative species are also required for natural competence through DNA uptake. Here, we report the ability of two C. difficile pilin subunits, PilJ and PilW, to bind to DNA in vitro, as well as the defects in biofilm formation in the pilJ and pilW gene-interruption mutants. Additionally, we have resolved the X-ray crystal structure of PilW, which we use to model possible structural mechanisms for the formation of C. difficile biofilm through interactions between type IV pili and the DNA of the extracellular matrix. Taken together, our results provide further insight into the relationship between type IV pilus function and biofilm formation in C. difficile and, more broadly, suggest that DNA recognition by type IV pili and related structures may have functional importance beyond DNA uptake for natural competence

A Mechanistic Study of Biofilm Formation in Clostridioides Difficile

Clostridioides difficile is a leading cause of nosocomial infections in the United States, with an estimated healthcare burden of over $1 billion. One of the chief difficulties in treating C. difficile infections is their recurrence after treatment with antimicrobials, which occurs in 20-30% of patients. The mechanism for C. difficile persistence and reoccurrence remains unclear though recently, biofilm formation has been hypothesized to be a significant contributing factor. Our group and others have identified type IV pili (T4P) as essential for in vitro biofilm formation by C. difficile. T4P are extracellular helical fibers composed of protein subunits (pilins). These appendages have diverse functions in various bacterial species, including surface (twitching) motility, cellular adhesion, horizontal gene transfer, and biofilm formation. Using gene-interruption mutants of T4P subunits, we found that pilA1 (the major subunit) resulted in a total loss of T4P assembly and a significant reduction in biofilm formation. To probe the mechanism of biofilm formation through T4P, we measured biofilm formation by two mutants, pilJ and pilW (minor subunits), which showed an intermediate phenotype with significantly less biofilm than the wild type. We hypothesized that interactions between T4P and extracellular DNA (eDNA) contributed to biofilm stability, as eDNA is a substantial component of the biofilm extracellular matrix (ECM). We found that PilJ and PilW (but not PilA1) could bind to dsDNA using Electrophoretic Mobility Shift Assays, with PilJ having stronger affinity and less specificity than PilW. We are investigating potential DNA binding sites using our recently determined x-ray crystal structure of PilW and our previously determined structure of PilJ. These results suggest that interactions between C. difficile T4P and eDNA stabilize biofilm, mediating the attachment of bacterial cells to the extracellular matrix and that minor pilin subunits (those incorporated at low abundance) are potential targets for biofilm dispersal

The structure of PilA from \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e AB5075 suggests a mechanism for functional specialization in \u3ci\u3eAcinetobacter\u3c/i\u3e type IV pili

Type IV pili (T4P) are bacterial appendages composed of protein subunits, called pilins, noncovalently assembled into helical fibers. T4P are essential, in many bacterial species, for processes as diverse as twitching motility, natural competence, biofilm or microcolony formation, and host cell adhesion. The genes encoding type IV pili are found universally in the Gram-negative, aerobic, nonflagellated, and pathogenic coccobacillus Acinetobacter baumannii, but there is considerable variation in PilA, the major protein subunit, both in amino acid sequence and in glycosylation patterns. Here we report the X-ray crystal structure of PilA from AB5075, a recently characterized, highly virulent isolate, at 1.9 Å resolution and compare it to homologues from A. baumannii strains ACICU and BIDMC57, which are C-terminally glycosylated. These structural comparisons revealed that PilAAB5075 exhibits a distinctly electronegative surface chemistry. To understand the functional consequences of this change in surface electrostatics, we complemented a ΔpilA knockout strain with divergent pilA genes from ACICU, BIDMC57, and AB5075. The resulting transgenic strains showed differential twitching motility and biofilm formation while maintaining the ability to adhere to epithelial cells. PilAAB5075 and PilAACICU, although structurally similar, promote different characteristics, favoring twitching motility and biofilm formation, respectively. These results support a model in which differences in pilus electrostatics affect the equilibrium of microcolony formation, which in turn alters the balance between motility and biofilm formation in Acinetobacter

Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

Calsequestrin 1 is the principal Ca(2+) storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, whereas its M87T "variant" is weakly associated with malignant hyperthermia. We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca(2+) binding exhibited by wild type calsequestrin 1 at high Ca(2+) concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca(2+) concentrations lacks two Ca(2+) ions normally present in wild type that weakens the hydrophobic core of Domain II. D244G crystallized in high Ca(2+) concentrations regains its missing ions and Domain II order but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations

Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregate Myopathy

Calsequestrin 1 is the principal Ca(2+) storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, whereas its M87T “variant” is weakly associated with malignant hyperthermia. We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca(2+) binding exhibited by wild type calsequestrin 1 at high Ca(2+) concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca(2+) concentrations lacks two Ca(2+) ions normally present in wild type that weakens the hydrophobic core of Domain II. D244G crystallized in high Ca(2+) concentrations regains its missing ions and Domain II order but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations