- Internal Medicine
PhD, Emory University, Laney Graduate School, Microbiology and Molecular Genetics (2017)
MD, Emory University, School of Medicine (2020)
BS, Rhodes College, Biochemistry and Molecular Biology (2012)
Cationic Homopolymers Inhibit Spore and Vegetative Cell Growth of Clostridioides difficile.
ACS infectious diseases
A wide range of synthetic polymers have been explored for antimicrobial activity. These materials usually contain both cationic and hydrophobic subunits because these two characteristics are prominent among host-defense peptides. Here, we describe a series of nylon-3 polymers containing only cationic subunits and their evaluation against the gastrointestinal, spore-forming pathogen Clostridioides difficile. Despite their highly hydrophilic nature, these homopolymers showed efficacy against both the vegetative and spore forms of the bacterium, including an impact on C.difficile spore germination. The polymer designated P34 demonstrated the greatest efficacy against C.difficile strains, along with low propensities to lyse human red blood cells or intestinal epithelial cells. To gain insight into the mechanism of P34 action, we evaluated several cell-surface mutant strains of C.difficile to determine the impacts on growth, viability, and cell morphology. The results suggest that P34 interacts with the cell wall, resulting in severe cell bending and death in a concentration-dependent manner. The unexpected finding that nylon-3 polymers composed entirely of cationic subunits display significant activities toward C.difficile should expand the range of other polymers considered for antibacterial applications.
View details for DOI 10.1021/acsinfecdis.0c00843
View details for PubMedID 33739823
Regulation and Anaerobic Function of the Clostridioides difficile beta-Lactamase
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
2020; 64 (1)
Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a β-lactamase, which is a common β-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a β-lactamase that was greatly induced in response to several classes of β-lactam antibiotics. An in-frame deletion of the operon abolished β-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the β-lactam ampicillin. We found that the activity of this β-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to β-lactams. These results provide support for the function of a β-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile β-lactamase activity.
View details for DOI 10.1128/AAC.01496-19
View details for Web of Science ID 000503839600047
View details for PubMedID 31611350
View details for PubMedCentralID PMC7187622
Examination of the Clostridioides (Clostridium) difficile VanZ ortholog, CD1240
ELSEVIER SCI LTD. 2018: 108–15
Clostridioides (Clostridium) difficile causes severe diarrheal disease that is directly associated with antibiotic use and resistance. Although C. difficile demonstrates intrinsic resistance to many antimicrobials, few genetic mechanisms of resistance have been characterized in this pathogen. In this study, we investigated the putative resistance factor, CD1240 (VanZ1), an ortholog of the teicoplanin resistance factor, VanZ, of Enterococcus faecium. In C. difficile, the vanZ1 gene is located within the skin element of the sporulation factor σK, which is excised from the mother cell compartment during sporulation. This unique localization enabled us to create a vanZ1 deletion mutant by inducing excision of the skin element. The Δskin mutant exhibited moderately decreased resistance to teicoplanin and had small effects on growth in some other cell-surface antimicrobials tested. Examination of vanZ1 expression revealed induction of vanZ1 transcription by the antimicrobial peptide LL-37; however, LL-37 resistance was not impacted by VanZ1, and none of the other tested antimicrobials induced vanZ1 expression. Further, expression of vanZ1 via an inducible promoter in the Δskin mutant restored growth in teicoplanin. These results demonstrate that like the E. faecium VanZ, C. difficile VanZ1 contributes to low-level teicoplanin resistance through an undefined mechanism.
View details for DOI 10.1016/j.anaerobe.2018.06.013
View details for Web of Science ID 000449900000016
View details for PubMedID 29940245
View details for PubMedCentralID PMC6309587
The C. difficile clnRAB operon initiates adaptations to the host environment in response to LL-37
2018; 14 (8): e1007153
To cause disease, Clostridioides (Clostridium) difficile must resist killing by innate immune effectors in the intestine, including the host antimicrobial peptide, cathelicidin (LL-37). The mechanisms that enable C. difficile to adapt to the intestine in the presence of antimicrobial peptides are unknown. Expression analyses revealed an operon, CD630_16170-CD630_16190 (clnRAB), which is highly induced by LL-37 and is not expressed in response to other cell-surface active antimicrobials. This operon encodes a predicted transcriptional regulator (ClnR) and an ABC transporter system (ClnAB), all of which are required for function. Analyses of a clnR mutant indicate that ClnR is a pleiotropic regulator that directly binds to LL-37 and controls expression of numerous genes, including many involved in metabolism, cellular transport, signaling, gene regulation, and pathogenesis. The data suggest that ClnRAB is a novel regulatory mechanism that senses LL-37 as a host signal and regulates gene expression to adapt to the host intestinal environment during infection.
View details for DOI 10.1371/journal.ppat.1007153
View details for Web of Science ID 000443296800008
View details for PubMedID 30125334
View details for PubMedCentralID PMC6117091
Ethanolamine is a valuable nutrient source that impacts Clostridium difficile pathogenesis
2018; 20 (4): 1419–35
Clostridium (Clostridioides) difficile is a gastrointestinal pathogen that colonizes the intestinal tract of mammals and can cause severe diarrheal disease. Although C. difficile growth is confined to the intestinal tract, our understanding of the specific metabolites and host factors that are important for the growth of the bacterium is limited. In other enteric pathogens, the membrane-derived metabolite, ethanolamine (EA), is utilized as a nutrient source and can function as a signal to initiate the production of virulence factors. In this study, we investigated the effects of ethanolamine and the role of the predicted ethanolamine gene cluster (CD1907-CD1925) on C. difficile growth. Using targeted mutagenesis, we disrupted genes within the eut cluster and assessed their roles in ethanolamine utilization, and the impact of eut disruption on the outcome of infection in a hamster model of disease. Our results indicate that the eut gene cluster is required for the growth of C. difficile on ethanolamine as a primary nutrient source. Further, the inability to utilize ethanolamine resulted in greater virulence and a shorter time to morbidity in the animal model. Overall, these data suggest that ethanolamine is an important nutrient source within the host and that, in contrast to other intestinal pathogens, the metabolism of ethanolamine by C. difficile can delay the onset of disease.
View details for DOI 10.1111/1462-2920.14048
View details for Web of Science ID 000430171900009
View details for PubMedID 29349925
View details for PubMedCentralID PMC5903940
Regulation of antimicrobial resistance by extracytoplasmic function (ECF) sigma factors.
Microbes and infection
2017; 19 (4-5): 238–48
Extracytoplasmic function (ECF) sigma factors are a subfamily of σ70 sigma factors that activate genes involved in stress-response functions. In many bacteria, ECF sigma factors regulate resistance to antimicrobial compounds. This review will summarize the ECF sigma factors that regulate antimicrobial resistance in model organisms and clinically relevant pathogens.
View details for DOI 10.1016/j.micinf.2017.01.007
View details for PubMedID 28153747
View details for PubMedCentralID PMC5403605
The Phosphotransfer Protein CD1492 Represses Sporulation Initiation in Clostridium difficile
INFECTION AND IMMUNITY
2016; 84 (12): 3434–44
The formation of spores is critical for the survival of Clostridium difficile outside the host gastrointestinal tract. Persistence of C. difficile spores greatly contributes to the spread of C. difficile infection (CDI), and the resistance of spores to antimicrobials facilitates the relapse of infection. Despite the importance of sporulation to C. difficile pathogenesis, the molecular mechanisms controlling spore formation are not well understood. The initiation of sporulation is known to be regulated through activation of the conserved transcription factor Spo0A. Multiple regulators influence Spo0A activation in other species; however, many of these factors are not conserved in C. difficile and few novel factors have been identified. Here, we investigated the function of a protein, CD1492, that is annotated as a kinase and was originally proposed to promote sporulation by directly phosphorylating Spo0A. We found that deletion of CD1492 resulted in increased sporulation, indicating that CD1492 is a negative regulator of sporulation. Accordingly, we observed increased transcription of Spo0A-dependent genes in the CD1492 mutant. Deletion of CD1492 also resulted in decreased toxin production in vitro and in decreased virulence in the hamster model of CDI. Further, the CD1492 mutant demonstrated effects on gene expression that are not associated with Spo0A activation, including lower sigD and rstA transcription, suggesting that this protein interacts with factors other than Spo0A. Altogether, the data indicate that CD1492 negatively affects sporulation and positively influences motility and virulence. These results provide further evidence that C. difficile sporulation is regulated differently from that of other endospore-forming species.
View details for DOI 10.1128/IAI.00735-16
View details for Web of Science ID 000390128700017
View details for PubMedID 27647869
View details for PubMedCentralID PMC5116721
The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σV in Response to Lysozyme.
Infection and immunity
2016; 84 (6): 1902–16
Clostridium difficile (also known as Peptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary for C. difficile to cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yet C. difficile is able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition of d-alanine to teichoic acids, is important for C. difficile resistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that a dlt null mutant is severely attenuated for growth in lysozyme and that expression of the dltDABC operon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σ(V) does not induce dlt expression in response to lysozyme, indicating that σ(V) is required for regulation of lysozyme-dependent d-alanylation of the cell wall. Using reporter gene fusions and 5' RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independent dlt expression. In addition, we observed that both a sigV mutant and a dlt mutant are more virulent in a hamster model of infection. These findings demonstrate that cell wall d-alanylation in C. difficile is induced by lysozyme in a σ(V)-dependent manner and that this pathway impacts virulence in vivo.
View details for DOI 10.1128/IAI.00207-16
View details for PubMedID 27068095
View details for PubMedCentralID PMC4907151
An alkaline phosphatase reporter for use in Clostridium difficile
2015; 32: 98–104
Clostridium difficile is an anaerobic, Gram-positive pathogen that causes severe gastrointestinal disease in humans and other mammals. C. difficile is notoriously difficult to work with and, until recently, few tools were available for genetic manipulation and molecular analyses. Despite the recent advances in the field, there is no simple or cost-effective technique for measuring gene transcription in C. difficile other than direct transcriptional analyses (e.g., quantitative real-time PCR and RNA-seq), which are time-consuming, expensive and difficult to scale-up. We describe the development of an in vivo reporter assay that can provide qualitative and quantitative measurements of C. difficile gene expression. Using the Enterococcus faecalis alkaline phosphatase gene, phoZ, we measured expression of C. difficile genes using a colorimetric alkaline phosphatase assay. We show that inducible alkaline phosphatase activity correlates directly with native gene expression. The ability to analyze gene expression using a standard reporter is an important and critically needed tool to study gene regulation and design genetic screens for C. difficile and other anaerobic clostridia.
View details for DOI 10.1016/j.anaerobe.2015.01.002
View details for Web of Science ID 000353427200018
View details for PubMedID 25576237
View details for PubMedCentralID PMC4385412