Browsing by Subject "Gene Expression Regulation, Bacterial"
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Item Butyrate Sensing by Campylobacter jejuni Impacts Bacterial-Host Interactions(2020-08-01T05:00:00.000Z) Goodman, Kyle Nicholas; Winter, Sebastian E.; Hendrixson, David R.; Sperandio, Vanessa; Pfeiffer, Julie K.The intestinal microbial ecosystem aids the host in digestion and nutrition by breaking down goods and providing beneficial vitamins and metabolites, including short-chain fatty acids (SCFAs) and lactate. Due to the abundance and intestinal distribution of these metabolites, bacterial pathogens can use them as biogeographical cues to discriminate among different regions of the host intestines. Indeed, Campylobacter jejuni, a commensal bacterium of the lower intestinal tract of avian species and a leading cause of bacterial diarrheal disease in humans, recognizes intestinal niches that support growth by sensing molecular cues produced by the microbiota. How C. jejuni senses and responds to microbiota-generated SCFAs and organic acids is not understood. Herein, I identified and characterized the C. jejuni BumSR two-component signal transduction system (TCS) that specifically directs a response to butyrate. Deletion of either C. jejuni bumS or bumR abolishes butyrate-modulated transcriptional changes in gene expression. Analysis of ΔbumS and ΔbumR mutants in a chick model of commensalism indicated that bumR is important for early colonization. This contrasts with a human volunteer infection study that demonstrated bumR is essential for infection of humans. Mutational analyses of genes within the BumSR regulon in the natural avian host revealed additional colonization factors including peb3, a putative glycoprotein adhesin/substrate binding protein, and Cjj0580, a putative d- and tri-carboxylate transporter. Through multiple biochemical assays, I discovered that BumS lacks kinase activity in vitro but possesses specific phosphatase activity towards BumR. These activities are not directly influenced by butyrate, suggesting that other metabolites, perhaps resulting from butyrate catabolism, are the direct cues sensed by BumS to modulate butyrate-dependent responses. By site-directed mutagenesis, I identified residues in the conserved H box that are required for BumS phosphatase activity. Consistent with previous work, phospho-BumR exhibits enhanced binding of target promoters in electrophoretic mobility shift assays, indicating that phosphorylated BumR likely has higher affinity to bind DNA at target promoters in vivo to either enhance or repress gene expression. Overall, this highlights BumSR as a non-canonical and first-identified TCS that directs a response to butyrate to modulate colonization gene expression through a phosphatase-dependent mechanism.Item Characterization of the Activation of the FlgSR Two-Component System in Campylobacter Jejuni(2009-06-17) Joslin, Stephanie Nicole; Hendrixson, David R.Epidemiological studies indicate that Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide. This organism has the ability to live as a commensal or a pathogen, depending on the host with which it is associated. While colonization of the gastrointestinal tract of many avian and mammalian species results in a harmless commensal relationship, human infection can cause diarrheal disease. In both scenarios flagellar motility is crucial for promoting optimal host interactions, as non-motile C. jejuni colonize the gastrointestinal tracts of commensal hosts at levels significantly lower than motile isolates and are incapable of causing disease in humans. The means by which C. jejuni regulates flagellar gene transcription and assembly differ from the well-studied pathways in species of Salmonella, E. coli, and Vibrio. Previous studies found that C. jejuni requires the flagellar export apparatus, sigma54, and a two-component regulatory system comprised of the FlgS sensor kinase and the FlgR response regulator to activate transcription of the middle and late sigma54-dependent flagellar genes. The FlgR response regulator is an NtrC-like protein that can be divided into three domains: an N-terminal domain that is phosphorylated by FlgS, a central sigma54 interaction domain, and a C-terminal domain of unknown function. Characterization of FlgR was accomplished by generating constructs that lack the N- or C-terminal domains of the protein and the site of phosphorylation. Through genetic and biochemical analyses, we found that both the N- and C-terminal domains have suppressive functions that prevent FlgR activation of sigma54-dependent flagellar gene transcription in the absence of FlgS. Our data also indicate that unlike other NtrC-family proteins, the C-terminus of FlgR does not bind DNA and is dispensable for FlgR activity. The FlgS sensor kinase activates FlgR through phosphorylation, but little was known about its activation prior to these studies. We have identified the site of FlgS autophosphorylation and demonstrated that formation of the flagellar export apparatus and the presence of at least one other flagellum-associated protein is required for autoactivation of this protein. This study provides insight into the unusual regulation of the FlgSR two-component system and its role in activating sigma54-dependent flagellar gene transcription.Item Dynamics of Cell Fate Decision Making Between Sporulation and Competence in Bacillus Subtilis(2013-01-02) Kuchina, Anna 1985-; Altschuler, Steven J.; Alto, Neal; Graff, Jonathan M.; Süel, Gürol M.During multipotent differentiation cells must reliably make a cell fate decision under a variety of conditions, yet remain sensitive to changes in extracellular environment. It is unclear how the cells reconcile these seemingly contradictory requirements. To complicate the issue, the cells often face a decision between multiple fates mediated by the respective differentiation programs which could become active at once. How cells make a specific cell fate choice when presented with several possibilities is a fundamental, yet poorly resolved question. To study cell-fate decision-making dynamics, I utilized the soil bacterium Bacillus subtilis which under stress can either become competent for DNA uptake or undergo sporulation. The master regulator of sporulation is the transcription factor Spo0A. Single cell measurements of Spo0A dynamics along with activities of stage-specific sporulation reporters Spo0F, SpoIIE and SpoIIR revealed the reversible and noisy progression of sporulation up until the final irreversible decision point. Mathematical modeling suggested that such strategy might be advantageous for coping with unpredictable environment. The alternative cell fate of competence is controlled by the transcription factor ComK. Using time-lapse fluorescence microscopy, I quantitatively measured the activities of Spo0A and ComK, along with other cross-regulatory genes, simultaneously in single B. subtilis cells. I found that, surprisingly, sporulation and competence progressed independently in the same cell without cross-regulation up to the final decision point. This finding was confirmed by the discovery of cells in a conflicted state that progressed to sporulation despite the expression of ComK. Measurements of gene expression dynamics in these cells revealed key differences in the relative timing of differentiation programs. To investigate the importance of relative timing, I altered it by engineering artificial cross-regulatory links between the sporulation and competence genetic circuits. Results favor a simple model for cellular decision-making that does not require intricate cross-regulation prior to the decision. Rather, cell fate choice appears to be the outcome of a "molecular race" between independently progressing differentiation programs. This temporal competition mechanism provides a simple, yet efficient way to generate mutually exclusive cell fates. Investigation of the benefits and limitations of such strategy opens a promising venue for future studies.Item Post-Transcriptional Regulation of Virulence Genes by GlmY and GlmZ in Enterohemorrhagic E. Coli(2014-04-14) Gruber, Charley C.; Hooper, Lora V.; Sperandio, Vanessa; Gardner, Kevin H.; Conrad, NicholasEnterohemorrhagic E. coli O157:H7 (EHEC) is a major cause of foodborne illness and hemolytic uremic syndrome (HUS) throughout the world. One of the major virulence factors in this pathogen is a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). EHEC uses this proteinaceous needle to inject effector proteins into host cells to hijack various host cellular processes, as well as the translocation and insertion of the translocated intimin receptor (Tir) into the host cell membrane. The bacterial adhesin intimin then binds to Tir, allowing EHEC to tightly adhere to the host cell’s membrane (109). Tir also indirectly recruits another bacterial effector EspFu, which induces actin polymerization. This causes the formation of the characteristic pedestal that cups the bacterial cell. This process from the assembly of the needle apparatus to the formation of the pedestal must be tightly regulated both transcriptionally and post-transcriptionally. The two two-component systems QseEF and QseBC have been previously shown to regulate various virulence genes. We established that these systems both regulate the transcription of the small RNA (sRNA) glmY. GlmY is known to stabilize another sRNA, GlmZ, which activates the translation of glucosamine synthetase (GlmS) (72). Here we show that GlmY and GlmZ are also important players in the post-transcriptional regulation of virulence genes in EHEC. The transcription factor QseF is required for the expression of EspFu and thus pedestal formation. This defect can be complemented by overexpression of either GlmY or GlmZ and is not at the transcriptional level. Instead, the expression of espFu requires a processing event that is QseF dependent. We have shown that GlmZ also post-transcriptionally regulates two of the operons of the LEE, LEE4 and LEE5. Both of these operons are transcribed from a single promoter, but there is a processing event that separates the first gene of the operon from the rest that requires the endoribonuclease RNase E. Overexpression of either sRNA results in the downregulation of the latter fragment of both the LEE4 and LEE5 operons. In the case of LEE4, this is through direct binding of GlmZ to a region within the LEE4 mRNA. We also investigated the global role of GlmY and GlmZ in EHEC through microarrays and RNA sequencing of the knockout strains. Aside from the LEE, GlmZ also regulates curli which are used to facilitate bacteria attachment to host cells. These data show that GlmZ has been co-opted into being an important regulator of virulence genes in EHEC.Item The Regulation of Flagellar Biosynthesis and Cell Division in Campylobacter jejuni(2016-04-05) Gulbronson, Connor James; Hansen, Eric J.; Hendrixson, David R.; Norgard, Michael V.; Alto, NealFlagellar biosynthesis is one of the rare processes known to be spatially and numerically regulated in polarly-flagellated bacteria. Polar flagellates must spatially and numerically regulate flagellar biogenesis to create flagellation patterns for each species that are ideal for motility. FlhG ATPases numerically regulate polar flagellar biogenesis, yet FlhG orthologs are diverse in motif composition. We discovered that Campylobacter jejuni FlhG is at the center of a multipartite mechanism that likely influences a flagellar biosynthetic step to control flagellar number for amphitrichous flagellation, rather than suppressing activators of flagellar gene transcription as in Vibrio and Pseudomonas species. FlhG also influences spatial regulation of division, which is essential for viability and is typically regulated by the Min system in most bacteria. However, C. jejuni lacks the Min system, but appears to utilize FlhG and components of the flagellar MS and C ring to influence spatial regulation of division. We utilized a variety imaging techniques to quantify the in vivo effects of mutations in C. jejuni and used purified proteins to assay the in vitro enzymatic activity of FlhG and FlhF (a GTPase) to determine the influence these factors have on both regulation of flagellar biogenesis and spatial regulation of division. We found that unlike other FlhG orthologs, the FlhG ATPase domain was not required to regulate flagellar number in C. jejuni instead, other regions of C. jejuni FlhG were discovered to be involved in numerical regulation of flagellar biogenesis. Mutations in the α6 and α7 helices of FlhG were found to influence aspects of FlhG biology, spatial regulation of division, and numerical regulation of flagellar biogenesis. We also found that C. jejuni FlhG influences FlhF GTPase activity, which may mechanistically contribute to flagellar number regulation. In this work, we propose a model in which FlhF in a GTP-bound ('active') state promotes the formation of the MS and C rings at the aflagellated pole after a division event. We then hypothesize that MS and C ring proteins influence FlhG localization to stimulate FlhF GTPase activity and, by extension, numerical regulation of flagellar biogenesis and spatial regulation of division at poles. Although some aspects of this model have yet to be fully tested, our data could potentially be applied in other polar flagellates to gain a better understanding of numerical regulation of flagellar biogenesis and spatial regulation of division in these organisms.Item The Role of Host Hormones and Metabolites in the Regulation of Virulence in Enterohemorrhagic Escherichia coli (EHEC)(2012-07-17) Njoroge, Jacqueline W.; Sperandio, VanessaGastrointestinal bacteria, including the enteric pathogen enterohemorrhagic Escherichia coli O157:H7 (EHEC) that causes hemorrhagic colitis, sense diverse environmental signals, and use them as cues for differential gene regulation and niche adaptation. This allows for a temporal and energy efficient up-regulation of EHEC virulence factors that is essential for successful colonization and infection of the host. These virulence factors include motility genes, Shiga toxin, and attaching and effacing (AE) lesion formation on colonic epithelial cells. AE lesion formation is primarily regulated by a pathogenicity island (PI) known as the locus of enterocyte effacement (LEE). One of the signals sensed by EHEC to activate virulence is the mammalian hormone epinephrine. We investigated the extent of epinephrine regulation in EHEC through transcriptome studies. The bacterial adrenergic kinases QseC and QseE both respond to epinephrine to regulate the LEE PI positively and negatively respectively. We also demonstrated for the first time that co-incubation with epinephrine increases the formation of AE lesions, and that QseC and QseE are the only sensors of epinephrine in EHEC. Epinephrine is not the only host hormone sensed by EHEC. We showed that another human hormone, serotonin is sensed by EHEC, Citrobacter rodentium and uropathogenic E.coli. In EHEC and C.rodentium we showed that serotonin inhibits the transcription of the LEE PI. We also determined that the mechanism of LEE PI inhibition by serotonin is through the reduction of autophosphorylation of the bacterial sensor kinase CpxA, which is itself an activator of the LEE PI. In addition to chemical signaling, nutrient availability plays an important role in bacterial gene regulation. We investigated the role that carbon nutrients play in the regulation of EHEC virulence. We showed that the LEE PI is activated under gluconeogenic conditions, which has been shown to be important for the maintenance of colonization in vivo, and inhibited under glycolytic conditions. We also identified a novel glucose concentration dependent regulator of the LEE PI, Cra. These findings enhanced our understanding of the role that epinephrine plays in virulence, and introduced two other signals, serotonin and glucose which are both important for the regulation of EHEC virulence genes.Item Signal Transduction Pathways That Impact Polar Flagellar Biogenesis(2020-12-01T06:00:00.000Z) Burnham, Peter Michael; Sperandio, Vanessa; Orth, Kim; Winter, Sebastian E.; Hendrixson, David R.Bacterial flagella are rotating nanomachines required for motility. Flagellar gene expression and protein secretion are coordinated for efficient flagellar biogenesis. Polar flagellates, unlike peritrichous bacteria, commonly order flagellar rod and hook gene transcription as a separate step after production of the MS ring, rotor, and flagellar type III secretion system (fT3SS) core proteins. This thesis describes two different ways MS ring-rotor-fT3SS assembly regulates flagellar gene expression. MS ring-rotor-fT3SS assembly stimulates expression of the next stage of flagellar genes establishing a unique polar flagellar transcriptional program. Conserved regulatory mechanisms in diverse polar flagellates to create this polar flagellar transcriptional program centered on MS ring-rotor-fT3SS assembly have not been thoroughly examined. Using in silico and genetic analyses and our previous findings in Campylobacter jejuni as a foundation, we observed that a large subset of Gram-negative bacteria with the FlhF/FlhG regulatory system for polar flagellation also possess flagellum-associated two-component signal transduction systems (TCS). I present data supporting a general theme in polar flagellates where MS ring, rotor, and fT3SS proteins contribute to a regulatory checkpoint during polar flagellar biogenesis. I demonstrated that Vibrio cholerae and Pseudomonas aeruginosa require the formation of this regulatory checkpoint for the TCS to directly activate subsequent rod and hook gene transcription, which are hallmarks of the polar flagellar transcriptional program. By reprogramming transcription in V. cholerae to more closely follow the peritrichous flagellar transcriptional program, I discovered a link between the polar flagellar transcription program and the activity of FlhF and FlhG flagellar biogenesis regulators in which the transcriptional program allows polar flagellates to continue to produce flagella for motility when FlhF or FlhG activity may be altered. I discovered a second mechanism by which the MS ring-rotor-fT3SS regulates polar flagellar gene expression as V. cholerae MS ring-rotor-fT3SS mutants increased expression of flrB, the sensor kinase of flagellar FlrBC TCS in V. cholerae. This suggested that MS ring-rotor-fT3SS formation may act as a feedback inhibition mechanism to repress the activity of the master flagellar regulator, FlrA. I examined if this effect was on flrA transcription or FlrA activity and found that early flagellar formation appears to impact V. cholerae FlrA activity. I hypothesized that early flagellar formation may repress FlrA activity through c-di-GMP in a FlhG-independent or dependent manner. I then examined the effect of DGC and PDE mutants that either 1) increased c-di-GMP levels in a FlhA mutant or 2) were known to affect V. cholerae motility to identify DGC or PDE that may link early flagellar formation to FlrA activity. I found evidence for two different early flagellar formation feedback inhibition mechanisms: a possibly c-di-GMP-independent mechanism through FlhA, and a c-di-GMP-related mechanism through FlhG, CdgE, and RocS. Although more characterization is needed, our data suggests a complex previously undescribed feedback inhibition mechanism that links completion of the MS ring-rotor-fT3SS complex to both repress FlrA activity and stimulate flagella-associated TCS.Item Signals and Sensory Mechanisms that Impact Campylobacter jejuni-Host Interactions(2015-05-21) Luethy, Paul Michael; Sperandio, Vanessa; Hendrixson, David R.; Winter, Sebastian E.; Michael, AnthonyCampylobacter jejuni is a leading cause of bacterial diarrheal disease worldwide and a frequent commensal organism of the intestinal tract of poultry and other agriculturally-important animals. Upon infection of the avian host, C. jejuni likely responds to external stimuli present within the intestinal tract to establish commensalism. The sensing mechanisms and subsequent physiological responses by C. jejuni can be crucial for initial growth and colonization and long-term persistence within the infected host. However, how many of the signals and sensing mechanisms affecting C. jejuni biology are not fully understood. In this work, I explored signal transduction mechanisms and possible in vivo signals that may influence the colonization capacity of C. jejuni. One method C. jejuni employs to monitor environmental stimuli are two-component regulatory systems (TCSs). I analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Through transcriptome analysis, I discovered that the regulons of the Cjj1484 histidine kinase and the Cjj1483 response regulator contain many common genes, which suggests these proteins likely form a cognate TCS. I found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. I observed that the Cjj1483 response regulator binds target promoters both in unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. I propose that this TCS may sense signals found in the host intestinal tract, wherein repression of genes may be relieved. In addition to characterizing the Cjj1484/Cjj1483 TCS, I explored the role of metabolites that are commonly found in the intestines -- organic acids and short chain fatty acids (SCFAs) -- in C. jejuni commensal colonization. C. jejuni has both acetate and lactate utilization pathways, as well as for acetate production. I observed that acetogenesis mutants incapable of producing acetate were deficient for colonization of the avian intestinal tract early during infection, but not at later points during infection. Furthermore, I found that an acetogenesis mutant was impaired during growth in a defined media containing solely amino acids and organic acids as carbon sources. Transcriptome analysis of the acetogenesis mutant identified the SCFA-induced regulon which contains metabolically important genes, many of which have been implicated in C. jejuni colonization and virulence. In addition, I found that peb1C, which was downregulated in the acetogenesis mutant, was important for colonization of the chick ceca. I further confirmed in vitro that physiological concentrations of the SCFAs acetate and butyrate activated expression of the SCFA-induced regulon whereas the organic acid lactate repressed these genes. I found that in vivo expression of the SCFA-induced regulon was highest in regions of the intestinal tract where SCFAs are present in the greatest concentration. Furthermore, butyrate counteracted the inhibitory effects of lactate when the two compounds were combined in culture in vitro. I propose that C. jejuni senses the concentration of SCFAs and organic acids to discriminate between different regions of the intestinal tract and to coordinate expression of colonization genes in the preferred niche for colonization. In effect, SCFA sensing and signaling allows C. jejuni to home to appropriate sites of the host for colonization and long-term persistence.Item Structural and Mechanistic Roles of Novel Chemical Ligands on Quorum Sensing Transcription Regulator SdiA in Enterohemorrhagic Escherichia coli(2014-11-18) Nguyen, Y. Nhu; Alto, Neal; Sperandio, Vanessa; Hooper, Lora V.; Winter, Sebastian E.Microorganisms and their eukaryotic hosts have co-evolved for millions of years. How bacteria sense and adapt to different environments is still unclear. Most Gram-negative bacteria use the LuxR family of transcription factors to regulate gene expression to coordinate population behavior by sensing endogenously produced chemical signaling molecules, acyl-homoserine lactones (AHLs) [1]. However, some bacteria such as Escherichia coli (E. coli) do not produce AHLs and, therefore, their quorum sensing LuxR-type proteins are thought to be regulated by AHLs from other bacteria [2-4]. These sub-family of LuxR proteins known as LuxR solos can also regulate and detect non-AHL signals to regulate gene expression independently of AHLs [5,6]. This AHL-dependent and -independent regulation of transcription is still unknown. Here we present several structures of one such solo LuxR-type protein, SdiA, from E. coli, in the presence and absence of AHL. Our study demonstrated that without AHL, SdiA is actually not in an apo-state, but regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life, and serve as energy sources, signaling molecules, and substrates for membrane biogenesis. While exogenous AHL renders SdiA much higher stability and DNA binding affinity, we propose that OCL may function as a chemical chaperone placeholder in the absence of AHL and stabilizes SdiA as a dimer, allowing for some basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides some crucial mechanistic insights into the ligand regulation of SdiA transcription activity. Understanding the role of ligand binding on the function SdiA is important for elucidating how SdiA regulates expression of virulence genes in the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7. Although EHEC causes foodborne infections worldwide that result in bloody diarrhea and hemolytic uremic syndrome (HUS), cattle is the major reservoir of EHEC. In cattle, EHEC colonizes predominately at the recto-anal junction (RAJ). Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses AHLs produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine whether the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica (Y. enterocolitica) AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI⁺ EHEC produces endogenous AHLs, and has a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI⁺ EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI⁺ EHEC. Although the yenI⁺ EHEC colonized the RAJ with equal efficiency to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI⁺ EHEC. The findings demonstrate that the regulation of EHEC in cattle is complex. Other factors such as fimbriae [161] may also contribute the colonization of EHEC in cattle. Identifying new factors and mechanisms of EHEC regulation is crucial for developing a better preventive approach against EHEC survival and colonization in cattle and subsequent EHEC contamination in the environment.Item Synergy of AcpP PPMO and Piperacillin/Tazobactam in the Breakdown of Pseudomonas aeruginosa PA01 Biofilms(2018-01-23) Wallace, Ashley; Sturge, Carolyn R.; Pybus, Christine; Greenberg, David E.INTRODUCTION: Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium and one of the most common causes of hospital-acquired infection, especially in immunocompromised patients. It is particularly pathogenic because of its ability to form biofilm, an extra-cellular matrix that makes it more resistant to host defenses and antibiotic therapies. Combination therapies have proven to be more effective at clearing biofilms because they target different processes or cell populations, but concerns about toxicity and antibiotic resistance have led to the exploration of alternative therapies that target biofilm formation at a genetic level. One such alternative is the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which are sequence-specific antisense oligomers that target the mRNA of bacterial genes and prevent translation of particular proteins. HYPOTHESIS: PPMOs targeting the essential gene AcpP will act synergistically with the antibiotic Piperacillin/Tazobactam (Pip/Tazo) in the breakdown of Pseudomonas aeruginosa PA01 biofilms in vitro. METHODS: To test synergy between the PPMO and antibiotic, PA01 biofilms were grown in filtered Mueller Hinton broth II (MHII) on minimum biofilm eradication concentration (MBEC) plates. MBEC plates are 96-well plates that have pegs attached to the lid to provide additional surface area for biofilm growth. After 24 hours of growth, the biofilm-covered pegs were switched to another 96-well plate containing fresh media with different combinations of antibiotic and PPMO in each well. Three such doses were administered every 8 hours, and at 48 hours of total growth, the biofilm remaining on the pegs was analyzed by one of four methods: 1) crystal violet assay, 2) resazurin assay, 3) CFU count, and 4) confocal microscopy. RESULTS: Crystal violet and resazurin assays demonstrated that Pip/Tazo and AcpP PPMO were potentially synergistic in biofilm breakdown for Pip/Tazo 0.5-0.0625 ug/mL and AcpP 5-0.625 uM. For several combinations, CFU measurements yielded 2-log or 3-log reduction in CFU compared to the control and synergistic effects on biofilm breakdown, particularly in cases where the antibiotic alone had no effect. While confocal microscopy demonstrated a decrease in viable bacteria with antibiotic treatment and PPMO treatment, the most significant eradication of biofilm occurred with combined treatment. CONCLUSION: The synergy demonstrated in vitro between AcpP PPMO and Pip/Tazo in the breakdown of P. aeruginosa PA01 biofilms is promising for in vivo studies as well, since PPMOs have the potential to increase biofilm sensitivity to lower doses of traditional antibiotics.