Vibrio cholerae is the bacterium that causes the diarrheal disease cholera, which is spread through the ingestion of contaminated food or water. Cholera endemics occur largely in developing countries that lack proper infrastructure to treat sewage and provide clean water. One environmental reservoir of V. cholerae is fish. Diarrheal symptoms similar to those seen in humans are seen in zebrafish, a natural host model, as early as 6 hours after exposure. Our understanding of basic zebrafish immunology is currently rudimentary, and no research has been done to date exploring the immune response of zebrafish to V. cholerae infection. Furthermore, the relationship between V. cholerae and select antimicrobial proteins has not been established.

Bacteria were grown in LB broth and were diluted in sterile 1× PBS to an infectious dose of 2.5 × 107 CFU/ml. One mL of bacterial inoculum was added to 400ml water with 4-5 adult wild-type ZDR zebrafish. RNA and protein were extracted from intestinal homogenates. A time-course study was performed to assess mRNA expression of neutrophils & neutrophil associated cytokines, as well as select neutrophilic antimicrobial proteins. Bacterial cultures were grown in the presence of purified antimicrobial proteins to assess inhibitory effects.

During the course of V. cholerae infection, large increases in neutrophils, neutrophil associated cytokines, and neutrophilic antimicrobial proteins were detected. Addition of purified antimicrobial proteins to bacterial culture significantly or completely inhibited growth. Protein variants with altered metal ion binding capacity highlight the importance of select metal ions in V. cholerae growth, as well as binding site dynamics.

Our study for the first time describes the neutrophil response in zebrafish to V. cholerae infection, as well as establishing relationships between select antimicrobial proteins and V. cholerae. These results provide valuable understanding of the natural life cycle of V. cholerae and its relationship with zebrafish, and help in understanding differences and similarities between the immune systems of zebrafish and our own.

The El Tor biotype of Vibrio cholerae is responsible for initiating and perpetuating the longest cholera pandemic in recorded history (1961-current). The Vibrio Seventh Pandemic Islands 1 and 2 (VSP-1 and -2), encoding a total of ~ 36 genes, are two genetic features that distinguish the El Tor biotype from strains of the classical biotype, known to be responsible for the previous two pandemics. VSP-1 contains a four gene operon (capV-dncV-vc0180-vc0181) that comprises an antiphage system termed CBASS (cyclic oligonucleotide-based antiphage signaling system). However, the functions of the majority of the remaining genes encoded in these two genomic islands are unknown. To understand their biological roles, we performed a bioinformatic analysis looking for the co-occurrence of VSP island gene products across bacterial genomes. This analysis predicted that vc0175, a VSP-1 encoded gene of unknown function renamed here as deoxycytidylate deaminase Vibrio (dcdV), is in a gene network with dncV, the CBASS cyclic GMP-AMP synthase. DcdV consists of two domains, a P-loop kinase (PLK) domain and a conserved deoxycytidylate deaminase (DCD) domain. Given that the APOBEC cytidine deaminase family of enzymes provides defense against viral infection in eukaryotes and the predicted association of DcdV with CBASS, we hypothesized that DcdV functioned to inhibit bacteriophage infection. Ectopic expression of DcdV in V. cholerae lacking VSP-1, but not in the wild type, results in a filamentous cell morphology, leading us to identify a region of 174 nucleotides 5’ of dcdV that encodes a novel noncoding RNA inhibitor of DcdV we named DifV. We show that DcdV deaminates both dCTP and dCMP and following ectopic expression of DcdV this activity significantly decreases the intracellular concentration of these nucleotide species. Finally, homologs of dcdV and difV are conserved in bacteria and eukaryotes and ectopic expression of DcdV and proteobacterial DcdV homologs inhibit phage infection in a heterologous host. Together, our results identify V. cholerae DcdV and DifV as the founding members of a previously undescribed bacterial phage defense system.

Recently, we described type IV pilus (T4P)-dependent attachment to plant polysaccharides by the extremely thermophilic cellulolytic Caldicellulosiruptor bescii. Additionally, two genes encoding for tāpirin proteins, which are also implicated in cell attachment to cellulose, are located directly downstream of the T4P locus in the C. bescii genome. Interestingly, there is no transcription terminator between the T4P locus and the tāpirin encoding genes suggesting that this entire region is transcribed as an operon. Based on their genomic proximity, and role in cell attachment to cellulose, our hypothesis is that the tāpirins incorporate into the T4P acting as a ‘molecular bridge’ connecting C. bescii cells to cellulose. Recombinant forms of the major pilin (rCbPilA) and both tāpirins (rCbTāpirin1, rCbTāpirin2) from C. bescii, with truncated N-termini, were produced and their interaction was studied using the surface plasmon resonance (SPR) technique. To confirm that the N-terminal truncation of rCbPilA does not affect its ability to interact with other proteins we first tested the ability of rCbPilA to self-assemble. Kinetic evaluation of SPR data indicate that rCbPilA retained the ability to self-assemble (KD= 4 µM) indicating that this N-terminally truncated protein would also retain its ability to interact with other proteins. Additionally, rCbPilA has an affinity for both recombinant CbTāpirins in the micromolar range (KD, 2.1 – 2.9 µM). moreover, recombinant CbTāpirins are capable of completely disrupting C. bescii cell binding to cellulose in response to xylan as a growth substrate. Overall, our results demonstrate for the first time an interaction between Gram positive T4 pilins and T4P accessory proteins encoded collinear in the T4P locus, and support a adhesin-like role for similarly arranged proteins in related clostridia.

Shigella flexneri is a gram-negative human pathogen that causes bacillary dysentery. This bacterium targets the colonic epithelium, resulting in bloody diarrhea. There is no vaccine for the prevention or treatment of Shigella infection, and antibiotic resistance is on the rise, making it a high priority target for antibacterial therapy development. Before Shigella initiates infection in the colon, it utilizes bile salt exposure in small intestine to promote biofilm formation and as a signal to modulate virulence gene expression to intensify infection; however, the mechanism underpinning the regulation of biofilm formation is largely unknown.

Bacterial nucleotide-based signalling system, cyclic di-guanosine monophosphate (c-di-GMP), regulates many behavioral changes in bacteria including biofilm formation. The levels of this second messenger are determined by two classes of enzymes: diguanylate cyclases (DGC) and phosphodiesterases (PDE). In many bacteria, high intercellular levels of c-di-GMP levels promote biofilm formation, while low levels promote motility. Shigella flexneri encodes 4 DGC’s, namely dgcP, dgcC, dgcI and dgcF; however, there have been no studies examining the role of c-di-GMP signaling in Shigella.

Here, we want to study if the virulence phenotypes in S. flexneri are mediated by c-di-GMP levels. To answer this question, we expressed a Vibrio cholerae DGC, VCA0956 in S. flexneri. Our results suggest that overexpression of an active DGC increases c-di-GMP levels, biofilm formation and decreased virulence. We further characterized each of S. flexneri’s native DGC’s by studying their role in biofilm formation and virulence. As DGC’s regulate biofilm formation in other enteric and pathogenic bacteria, we hypothesize that individual DGC mutants will show significant decrease in biofilm formation and increased virulence. Our result show that knocking our dgcC and dgcF regulate S. flexneri biofilm and invasion capacity and dgcF mutant also regulate plaque phenotype.

There is still a knowledge gap on the scope of c-di-GMP signaling in S. flexneri pathogenesis, as it is involved in many functions; with this study, we will characterize the role of DGC’s in S. flexneri pathogenesis.

Background: Severe Acute Respiratory Syndrome (SARS) coronavirus (CoV) infections are an increasingly critical public health threat through their pandemic spread. This work is the first to perform a meta-analysis of mRNA expression data to identify gene expression and pathway activity changes from SARS (SARS-CoV, MERS-CoV, and SARS-CoV2) infections by comparing gene or pathway signatures.

Methods: Gene signatures (T-score ranked gene lists) for 37 comparisons of human or mouse lung cultures or mouse samples 48hrs post SARS and mock infection are generated. Gene signatures represent 29 SARS-CoV infections across seven different strains with varying virulence (icSARS, Urbani, MA15, ΔORF6, BAT-SRBD, ΔNSP16, and ExoNI), five MERS-CoV infections, and three SARS-CoV2 infections. Gene signatures are converted to pathway signatures (pathway lists ranked by normalized enrichment score) through Gene Set Enrichment Analysis (GSEA) with 7573 Gene Ontology Biological Process gene sets. For both gene and pathway signatures, positive and negative panels of genes or pathways, respectively, are defined from the leading-edge identified by GSEA between two icSARSvsmock signatures. These icSARS panels are compared to remaining signatures using GSEA, and meta-analysis is completed on identified leading-edges to find common genes and pathways.

Results: For both gene and pathway signatures, enrichment is observed consistently between icSARS positive gene and pathway panels and all SARS signatures (GSEA p<0.001). Inconsistent enrichment is observed for the icSARS negative panels. Identified leading-edges from icSARS positive panel comparisons find five common genes and 11 common pathways across SARS signatures regardless of strain or virulence, suggesting these genes and pathways are associated with SARS infection. Identified genes and pathways are involved in immune response.

Conclusion: This GSEA-based meta-analysis identifies genes and pathways with and without reported associations with SARS infections, highlighting this approach’s predictability and usefulness in identifying molecular changes associated with SARS infections that may have therapeutic potential.

ZupT is a divalent cation transporter from Escherichia coli responsible for the uptake of Zn²⁺, Fe²⁺, and Mn²⁺. It is a member of the ZIP (zinc-regulated, iron-regulated like protein) family of transporters that are ubiquitous in living systems. Both prokaryotic and eukaryotic pathogens require transition metals for virulence and ZIP transporters have been shown to play a critical role. Despite their importance, ZIP transporters lack detailed molecular characterization. An integral part of the transport process is metal recruitment. ZupT, like all ZIP transporters, is an 8 transmembrane domain protein with a characteristic ZIP metal-binding motif in transmembrane domains 4 and 5. Within the ZIP motif it is unclear which amino acids are responsible for coordinating its different substrates. To probe this question recombinant ZupT was expressed in the pBAD vector in LMG194 E. coli cells and purified with a Strep-tag. Purified protein was assayed for metal binding by intrinsic fluorescence tryptophan quenching. Site-directed mutagenesis was used to identify important metal binding residues. Results: Within the ZIP motif, Zn²⁺, Fe²⁺, and Mn²⁺ bind to a unique subset of residues. This pattern allows Zn²⁺ and Fe²⁺ to bind simultaneously while Mn²⁺ competes for ligands with both metals. These findings explain activity assay results indicating Mn²⁺ is a competitive inhibitor with respect to Zn²⁺ transport, but Fe²⁺ is not. An additional important ligand for Fe²⁺ and Mn²⁺ was also confirmed in transmembrane domain 6, which is unique to a ZIP subfamily that includes prokaryotic ZIPs and human ZIP11. This work helps explain the binding and transport mechanism of an important class of proteins for many pathogens with focus on the relevant substrates. It also provides insight into the metal preference of ZIP transporters from pathogenic microorganisms based on their ZIP metal binding motif.

Misrepair of DNA double-stranded breaks (DSB) can result in genomic rearrangements and cancer. SGS1 is a helicase that unwinds DNA for DSB repair. To test its role in break repair, I analyzed the frequency of various DSB repairs in yeast cells with the mutation sgs1-FD. Given this mutation disrupts an interaction between SGS1 and a critical protein, Rad51, I hypothesized mutant cells would have more error-prone repair than high fidelity canonical BIR repair. Genome sequence and chromosome III size were examined in sgs1-FD cultures that underwent DSB repair. No difference was found in the frequency of error-prone repair, indicating the mutation did not affect the repair process.

Biofilms are microbial communities encased in a self-produced extracellular matrix. Cells in the biofilm state are difficult to combat in part because of increased production of quiescent cells within the biofilm tower. Myxococcus xanthus produces a specialized biofilm involving segregation of cells into three distinct fates: programmed cell death, production of multicellular fruiting bodies filled with spores, and persister-like cells termed peripheral rods. Peripheral rods and fruiting bodies are spatially distinct and the proportion of cells in these two fates depends on environmental signals. Thus, M. xanthus is an excellent model system to investigate regulatory mechanisms controlling production of distinct quiescent states. We hypothesize that cell fate segregation is controlled by the EspAC signaling system in response to environmental signals. EspA and EspC are sensory kinases that function together to regulate accumulation of MrpC, a transcription factor that is necessary to induce fruiting body formation, but is absent from peripheral rods. Our current working model is that the EspAC signaling system is fully functional specifically within cells destined to become peripheral rods and we hypothesize that EspA and EspC are differentially expressed within the biofilm population. To understand the basis of this differential expression we are investigating the cis and trans regulatory elements necessary for espA and espC expression. We have demonstrated that MrpC is necessary for full espA and partial espC expression leading to a composite negative feedback loop. We have identified one and two MrpC binding sites in the promoters for espA and espC, respectively. We are currently identifying other regulatory elements involved in controlling EspAC heterogenous expression. Our data suggests distinct quiescent states are controlled by a novel regulatory network that couples production of different quiescent states to distinct environmental conditions.

Candida albicans is a naturally occurring fungus in the gut microflora that can become deadly through the formation of biofilms on medical implants in immunosuppressed patients. Biofilms form in four steps, adherence, where yeast cells adhere to a medical implant or device, initiation, where the yeast cells begin to clump and create larger colonies as well as begin to filament, maturation, where filamentation continues and an extra cellular matrix forms resulting in resistance to host defenses and antifungal treatment, and dispersal, where yeast cells break off from the hyphal cells and begin the cycle again. Our research is focused on finding specific cell wall mutations as possible targets for drugs, by assaying a library of insertion mutants of C. albicans for biofilm formation. Cell wall genes are used as drug target due to the fact that humans have no existing homologous genes, as well as the cell wall being vital for the fungus to survive. Here we report that orf19.5267, a gene of previously unknown function, is essential for biofilm formation in vitro, showing defects in adherence, filamentation, and growth.

Candida albicans is a pathogenic fungus that lives inside the human biological system. This fungus is known to live on mucous surfaces inside the human body, such as the gastrointestinal tract and the urinary tract. The primary mechanism of infection is C. albicans ability to produce a biofilm. Biofilms are known to promote antibiotic resistance when induced in C. albicans. This type of infection has a ~30% mortality rate. The formation of biofilms is dependent on two types of cells, yeast cells (blastopores) and hyphal cells (long, tubular structures); this coupled with the extracellular matrix secreted by C. albicans. Biofilms proliferate when foreign devices are placed into the body, such as catheters and pacemakers. The in vitro model assay is used to screen for unknown mutant strains by using Day 286 as the wild-type strain and BCR 1 -/- as the established biofilm defective mutant. These screenings strain PGA17 has been established as a mutant strain as it could not form a complete biofilm on the catheter square. Using PCR, the mutant gene responsible for the incomplete formation of the biofilm can be purified, extracted, and studied. The mutation in these biofilms can then be used to help solve Candida albicans infections more efficiently.

Candida albicans is the most prevalent fungal infection for humans. It is the root cause of many infections including urinary tract infection, genital yeast infection, and oral thrush. The intimacy of the human relationship with Candida albicans is showcased in the estimated 9.4 million cases of infection of hospital implants by Candida albicans each year. This fungus, when systematic, is the 3rd most prevalent secondary infection in hospitals, with a mortality rate of ~30%, which it achieves through producing a biofilm. Our laboratory aims to understand and identify genes that are required for biofilm formation on medical implants. To do so, we have conducted in vitro tests using catheter squares assaying the ability of wild type and mutated yeast isolates to form a biofilm. We focus on cell wall defects and adherence issues of yeast and filamentous cells through PCR DNA manipulation of yeast cells, observation of biofilm after being stressed, weighing of the biofilms, and observing drop tests and dilutions of specific yeast strains. Because human cells do not have cell wall proteins, finding a way to target the cell wall of a yeast or filamentous biofilm forming cells would be the safest and most beneficial form of treatment.

BACKGROUND: Few studies have documented in vitro oral biofilm development over time. The aim of this work is to enhance our previously published image analysis software called BAIT (Biofilm Architecture Inference Tool) to quantitatively study biofilm development over time.

METHODS: Using a hybrid microplate-based in vitro biofilm system that facilitates media exchange, biofilms of S. mutans 3209/pVMCherry that constitutively expressed mCherry were developed in filter-sterilized pooled human saliva with or without 0.1% sucrose. Biofilm development was monitored continuously (with intervals of approximately 3.5 minutes) over 20 h and the resulting four-dimensional dataset was visually inspected and quantified for architectural differences during biofilm development.

RESULTS: Spatiotemporal differences in biofilm architecture were observed between biofilms developed in the presence or absence of sucrose. Notably, sucrose supplemented S. mutans biofilms formed plump microcolonies whereas in the absence of sucrose, S. mutans biofilms were fibrous in appearance. This visual observation was supported quantitatively by using our modified software called BAIT-T (Biofilm Architecture Inference Tool – Time). For example, in saliva without sucrose supplementation, biofilm fluffiness and convex hull porosity were consistently higher during biofilm development as compared to biofilm development in saliva supplemented with sucrose. Analysis of changes in biofilm biovolume over time indicated comparable rates of development of S. mutans biofilms for approximately four hours, after which, the sucrose-supplemented biofilms continued to increase while the biovolume of the non-sucrose developed biofilms plateaued.

CONCLUSIONS: Using BAIT-T, differences in architectural changes in biofilms developed in pooled saliva with or without sucrose supplementation were quantified over time.

Yersinia pestis, the causative agent of plague, is a deadly bacterial pathogen that must combat cells of the innate immune system by deployment of a Type III Secretion System (T3SS) for delivery of cytotoxic Yop protein effectors, upon entry into the host via inhalation or flea bite. Neutrophils are one of the first cells encountered in tissues, a phagocytic cell-type designed to ingest and kill invading pathogens. Studies with pathogens and human neutrophils are hampered by the short life-span of neutrophils, about 1 day, such that experiments involving manipulation of neutrophils are limited. The goal of this study was to assess the ability of a neutrophil-like cell line, HL-60, to replicate typical neutrophil/Y. pestis interactions. Using HEp-2 cells (a non-phagocytic epithelial cell line) and HL-60 cells, we found Y. pestis adhesins such as Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) contribute to Yop delivery with both cell types. However, when bacteria were coated with human serum (as would occur during human infections), the role of adhesins on Yop delivery to HL-60 cells was diminished. Furthermore, pre-induced expression of the T3SS also reduced the dependence of Yop delivery to HL-60 cells on Y. pestis adhesins. Yop delivery via the T3SS was critical for preventing phagocytosis by HL-60 cells (a 40-fold reduction) as found previously with freshly isolated phagocytes. Finally, delivery of Yops via T3SS prevented degranulation of HL-60 neutrophil-like cells as demonstrated by reduced surface exposure of the degranulation marker CD63 when comparing wild-type Y. pestis (KIM5) to a T3SS mutant (ΔyopB). This prevention of degranulation resulted in ~100-fold increased survival of KIM5 relative to the ΔyopB mutant upon interaction with HL-60 cells. These studies demonstrate the potent antimicrobial properties of HL-60 cells for Y. pestis and are comparable to studies performed with freshly isolated human neutrophils. Thus, we have a cell line amenable to genetic and transcriptional manipulation for future studies to determine critical cellular pathways that contribute to Y. pestis control.

Infections caused by antibiotic-resistant organisms pose a global health threat. Concurrently, antibiotic discovery, critical to treating such infections, has stagnated due to scientific hurdles. In their natural environments, many soil microbes produce antimicrobial compounds to compete with proximal microbes. Our research aims to utilize microbial ecology to trigger the expression of antibiotic metabolites under laboratory conditions. Recent studies suggest that when available carbon is restricted to complex polysaccharides, microbes respond by increasing antimicrobial production. By varying the polysaccharide source, we observed distinct differences in antimicrobial production.

Plant pathogenic fungi are known to cause devastating loss to crops for several years. Developing countries including India face a serious concern in the agriculture sectors due to plant diseases. For years, synthetic fungicides are known to effectively control mycelium growth and protect the crops from diseases. The repeated use of these chemical constituents causes a long-term harmful effect not only on living beings and food quality but disrupts the whole ecosystem. To reduce chemical fungicides, natural plant extracted botanical fungicides have been studied as antifungal agents. Botanicals being natural have eco-friendly nature with a rich source of metabolites that reduce the negative impact of microbes and promote positive responses by the plants. In this context, three medicinal plants namely, Catharanthus roseus, Calotropis procera, and Ocimum sanctum were evaluated in 100% concentration against Alternaria alternata, Fusarium oxysporum, and Curvularia inaequalis against phytopathogens isolated from Aegle marmelos. We studied commercial fungicide fluconazole for comparative study. Disc diffusion technique assay was used to access the antifungal activity of three different solvents with three replicates each. Our results showed that methanolic stem extract of C. procera has the highest antifungal property against the isolated pathogen C. inaequalis having 25±2 mm mycelium inhibition. Whereas ethanolic stem extracts of C. procera and O. sanctum showed the highest antifungal potential against Alternaria and Fusarium species having 22±2 mm and 23±1 mm of the inhibitory zone.

The three medicinal plants exhibited a greater percentage of mycelium inhibition compared to fluconazole and therefore, can be used as a potential alternative to fungicides for crop protection and development.

Objective: To identify the bacteriophage resistance mechanism(s) employed by S. mutans using ΦAPCM01 sensitive and resistant strains isolated from saliva samples.

Methods: First, we tested to see if the first step of infection, binding to cell receptors, was altered in phage-resistant strains by performing adsorption assays in order to compare ΦAPCM01 adsorption to phage-sensitive strains with adsorption to phage-resistant strains. We then considered if phage resistance is a result of phage immunity provided by chromosomal prophage, using lysogen induction assays with the chemical, Mitomycin C. Lastly, analysis of CRISPR spacer sequences was done using PCR-based amplification and sequencing of the CRISPR arrays to determine if phage-resistant strains contained spacers with identity to ΦAPCM01 sequence.

Results: There is no adsorption defect of ΦAPCM01 to the different serotype e strains regardless of susceptibility to infection.

Next, MMC induction assays indicate the likelihood of prophage presence in phage resistant strains, 72-02 and 88-01 due to the marked decrease in growth observed over a specific time interval. Finally, we were able to detect whether certain strains of S. mutans possessed CRISPR arrays with spacer sequences as well as compare the homology of CRISPR spacers with the sequence of ɸAPCM01 with only one strain, 88-01, containing one perfect match for the ɸAPCM01 genome. All other mismatching may be enough to still allow the bacteriophage to infect and kill the strain.

Conclusions: Multiple resistance mechanisms may play a role in S. mutans resistance to phage; however, elucidation of the mechanism may provide the key to successful bacteriophage therapy against the bacterium.

Future Directions: Future experiments can be designed to delete the essential cas genes in phage-resistant strains in order to determine whether it is CRISPR providing the immunity from φAPCM01.

Maintaining a heathy oral cavity is an important aspect to the overall wellbeing of individuals. Poor oral health has known associations to various systemic health illnesses. One of the major and known oral health conditions that affects millions worldwide are dental caries/cavities also known as tooth decay. Dental caries occurs when bacterial biofilm (dental plaque) on the tooth surface remains undisturbed and becomes composed primarily of acid-producing bacteria. One of the most well-studied bacterial agents associated with the initiation of the transition to an acidogenic plaque biofilm is Streptococcus mutans. Today, some of the efficient methods of maintaining low plaque accumulation is through mechanical disruption using a toothbrush and dentifrice (toothpaste). Flossing and mouth rinses have also contributed to the killing of bacteria – aiding the mechanical use of toothbrush with dentifrice; however, a very common natural toothbrush used in the East is rarely known to the Western society. With immigration, people with different western cultural upbringing have brought some of their cultural belongings to the West and utilize them for religious (Islamic), cultural, and medicinal practices. An example of this is the Salvadora persica toothbrush sticks, also known as Miswak, native to the Arabian Peninsula and Africa. The roots of S. persica have been used in the East for thousands of years to maintain oral health. Studies have shown the inhibitory effects of S. persica on various oral pathogens such as S. mutans and Candida albicans. Additionally, Miswak has been shown to possess analgesic, whitening, anti-viral, and anti-oxidative properties. Therefore, S. persica sticks may be a potential dental hygiene tool in the West.

A biofilm is a collection of microscopic organisms and extracellular substances that adhere to various surfaces. Biofilm-associated bacteria, due to the architecture and composition of the biofilm, have increased tolerance to host defense mechanisms and antimicrobials. One example of bacterial biofilms is dental plaque. Plaque is the buildup of bacterial biofilm on tooth surfaces, which if undisturbed, can progress towards a pathogenic composition associated with dental disease such as caries, gingivitis, and periodontitis. Use of mouthrinses during routine oral hygiene practices is sometimes advised to reduce bacterial burden in the oral cavity. Generally, these contain ethanol as the active ingredient, but sometimes use other antimicrobial substances, natural products, or combinations. To start my first ever research experience, I was completely remote, never having stepped foot in the lab. During this time of remote research, my goal was to research the antimicrobial and antibiofilm properties of various compositions of mouthwashes to learn what different components are used and their antimicrobial role. Active ingredients in mouthrinses ranged from alcohol to cetylpyridinium chloride, chlorhexidine, essential oils, fluoride, and saliva substitutes. After literature research, I found some other natural ingredients that helped with biofilm killing and removal such as curcumin and melaleuca. However, they were not always tested in complex biofilm models. So, I then explored the diversity of oral biofilm models such as mono-, dual-, and multi- species biofilms. My future directions, once in-person, include comparing previous monospecies biofilm results from the lab with killing and biofilm removal of dual and multispecies biofilms. Also, I plan on experimenting with a dual targeting approach where one active ingredient is aimed at the matrix of the biofilm and the second is aimed at killing the bacteria.

Myxococcus xanthus is a model organism to study signaling systems necessary to regulate complex behavior. Under nutrient limiting conditions, these bacteria produce a specialized biofilm (aka a developmental program) wherein cells segregate into different fates: aggregation into mounds followed by differentiation into environmentally resistant spores, differentiation into a persister like state called peripheral rods, or programmed cell death.

TodK, an orphan histidine protein kinase, has been previously proposed to control the developmental program by modulating activation of FruA, major transcription factor necessary for induction of aggregation and sporulation. The mechanism by which TodK controls FruA activation is not known. In this study, we utilize TodK mutants deficient in kinase-, phosphatase-, or dual kinase/phosphatase-activity to delineate the TodK signaling mechanism. We demonstrate that overexpression of functional TodK dampens FruA accumulation, which prevents induction of aggregation. Disruption of TodK kinase and/or phosphatase activity allows FruA accumulation and induces aggregation onset. Interestingly, however, TodK kinase activity is necessary to delay sporulation onset, while phosphatase activity antagonizes kinase activity. We propose a model in which TodK fine-tunes sporulation onset to environmental conditions. We are currently exploring whether TodK activity additionally controls cell fate segregation between peripheral rods and fruiting bodies.

Smoking related illnesses have been estimated to contribute to over 300 billion dollars in annual healthcare costs in the US. It is well established that smoking is associated with poor oral health. Besides tobacco smoke, it is known that dynamic, multispecies biofilms also play a role in oral health and disease progression. While both oral biofilms and smoking have been implicated as detriments to oral health, little research has been done to examine the potential impact tobacco smoke exposure has on oral biofilm development. We hypothesized that in vitro oral biofilms grown in pooled human saliva treated with tobacco smoke might exhibit significantly different architectural features compared to biofilms grown without exposure to tobacco smoke extract. To explore this hypothesis, 24-well glass-bottomed SensoPlatesTM were used to develop multi-species biofilms derived from unfiltered, pooled human saliva. Biofilms were grown in 25% filter-sterilized human saliva that was either treated with tobacco smoke (tCFS), or not treated (CFS). These biofilms were incubated in 5% CO2 at 37°C for 22 hours. Resulting biofilms were subsequently treated with commercially available LIVE/DEADTM viability assay and imaged using Confocal Laser Scanning Microscopy (CLSM). Biofilm images were rendered in 3D using Imaris, and BAIT (Biofilm Architecture Inference Tool) was used to analyze biofilm architecture. Biofilms grown in tCFS exhibited reduced biovolume, number of objects, surface area and connectivity, while biofilm fluffiness was higher. Moreover, microcolony development was found to be sparse in wells with tCFS compared to CFS. Based upon LIVE/DEADTM staining, there was no significant difference in cell viability. These results aligned with visual inspections of CLSM 3D renders of the biofilms. Our observations suggest that smoking tobacco may alter the architecture of oral multi-species biofilms. Better understanding this relationship between smoking and oral biofilm development may prove valuable in understanding how smoking might influence biofilm-associated diseases in the human oral cavity.

Cyclic di-nucleotide (cdN) second messenger molecules modulate global pathways imperative for cellular adaptation in bacteria and eukaryotes. cGAS and DncV synthesize structural isomers of cyclic GMP-AMP in eukaryotes and bacteria respectively, and recent studies have shown it makes up a novel class of cdN synthases that are structurally related. The nucleotide signals synthesized by this third class of synthases and the phenotypes they regulate is poorly understood. To better understand these signaling systems, we began to characterize the mechanism and function of CD-Ntases and their associated nuclease effector domains from a variety of Gram-negative organisms. These effectors encode a SAVED domain, a cdN receptor fused to a putative nuclease domain. To explore the functions of these signaling systems, we constructed inducible plasmid vectors to express putative CD-Ntases and their neighboring putative SAVED containing nuclease effectors from Escherichia coli, Vibrio cholerae, and Pseudomonas fluorescens. Overexpression of the P. fluorescens CD-Ntase and associated nuclease decreased its viability, suggesting this signaling module is active. We are also examining the ability of these effector/CD-Ntase system to provide protection against phage infection. We have begun to purify these proteins to determine what nucleotide is synthesized by these CD-Ntase enzymes and test if these nucleotides regulate the associated SAVED nucleases. We hypothesize that CD-Ntase have a global signaling role in P. fluorescens affecting cell growth, adaptation and defense in the environment. CD-Ntase/SAVED pairs are a novel nucleotide signaling system conserved in many species of bacteria and our studies will help to delineate their signaling mechanisms and function.