Browsing by Subject "Pseudomonas aeruginosa"

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Anchors away: Determining the role of outer membrane proteins in Pseudomonas aeruginosa vesicle formation
(2010) Liew, Jean; Marvin Whiteley
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes chronic infections in the lungs of individuals with cystic fibrosis (CF). Like many Gram-negatives, P. aeruginosa produces outer membrane vesicles (MVs), which have been shown to package numerous factors including antimicrobial quinolone molecules, toxins, DNA, antibiotic resistance determinants, and cell-cell signaling molecules. The mechanism for the formation of MVs has not been fully elucidated. The Gram-negative outer membrane (OM) contains associated proteins, which anchor it to the peptidoglycan, and keep the OM stable. We hypothesized that peptidoglycan-associated outer membrane lipoproteins OprF, OprL, and OprI contribute to MV formation in P. aeruginosa. In this study, we quantified MVs harvested from oprF, oprL, and oprI mutants. The MV levels produced by the oprL and oprI mutants were not significantly different from those produced by the wild type; however, the oprF mutant showed a three-fold increase in MV production. These data indicate that OprF plays a significant role in anchoring the outer membrane to the peptidoglycan and that, in its absence, more MVs are formed.
Asymmetry and Inequity in the Inheritance of a Bacterial Adhesive
(New Journal of Physics, 2016-04) Cooley, Benjamin J.; Dellos-Nolan, Sheri; Dhamani, Numa; Todd, Ross; Waller, William; Wozniak, Daniel; Gordon, Vernita D.; Gordon, Vernita D.
Pseudomonas aeruginosa is an opportunistic human pathogen that forms biofilm infections in a wide variety of contexts. Biofilms initiate when bacteria attach to a surface, which triggers changes in gene expression leading to the biofilm phenotype.Wehave previously shown, for the P. aeruginosa lab strain PAO1, that the self-produced polymer Psl is the most dominant adhesive for attachment to the surface but that another self-produced polymer, Pel, controls the geometry of attachment of these rod-shaped bacteria—strains that make Psl but not Pel are permanently attached to the surface but adhere at only one end (tilting up off the surface), whereas wild-type bacteria that make both Psl and Pel are permanently attached and lie down flat with very little or no tilting (Cooley et al 2013 Soft Matter 9 3871–6). Here we show that the change in attachment geometry reflects a change in the distribution of Psl on the bacterial cell surface. Bacteria that make Psl and Pel have Psl evenly coating the surface, whereas bacteria that make only Psl have Psl concentrated at only one end.Weshow that Psl can act as an inheritable, epigenetic factor. Rod-shaped P. aeruginosa grows lengthwise and divides across the middle.Wefind that asymmetry in the distribution of Psl on a parent cell is reflected in asymmetry between siblings in their attachment to the surface. Thus, Pel not only promotes P. aeruginosa lying down
Bacteria Use Type IV Pili to Walk Upright and Detach from Surfaces
(Science, 2010-10) Gibiansky, Maxsim L.; Conrad, Jacinta C.; Jin, Fan; Gordon, Vernita D.; Motto, Dominick A.; Mathewson, Margie A.
Bacterial biofilms are structured multicellular communities involved in a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near surfaces is crucial for understanding the transition between planktonic and biofilm phenotypes. By translating microscopy movies into searchable databases of bacterial behavior, we identified fundamental type IV pili–driven mechanisms for Pseudomonas aeruginosa surface motility involved in distinct foraging strategies. Bacteria stood upright and “walked” with trajectories optimized for two-dimensional surface exploration. Vertical orientation facilitated surface detachment and could influence biofilm morphology.
The catalytic mechanism of dimethylarginine dimethylaminohydrolase (DDAH) from pseudomonas aeruginosa
(2006) Stone, Everett Monroe; Fast, Walter
Dimethylarginine dimethylaminohydrolase (DDAH) catalyzes the hydrolysis of Nw–methyl–L–arginine (NMMA) and Nw,Nw –methyl–L–arginine (ADMA) to L-citrulline and methylamine or dimethylamine, respectively. ADMA and NMMA are endogenous inhibitors of nitric oxide synthase (NOS) in mammals. DDAH therefore partially regulates NOS activity, making it an attractive therapeutic target in disease states involving overproduction of nitric oxide. Understanding the mechanism of DDAH is important to inhibitor design and elucidating its physiological function. DDAH is a member of the amidinotransferase superfamily, and has conserved active–site residues including cysteine, histidine, and glutamate/aspartate that are integral to catalysis. In DDAH from Pseudomonas aeruginosa, the active–site Cys249 is activated as a nucleophile upon binding substrate, and forms a covalent intermediate concomitant with loss of the alkylamine leaving group. The active–site His162 has a dual role, first as a general acid in protonating the alkylamine leaving group and second as a general base in generating a hydroxide for attack on the covalent intermediate. The active–site Glu114 is essential for properly orienting and ionizing His162. The use of a substrate analog, S–methyl-L-thiocitrulline (SMTC), enabled development of a new method of continuously monitoring DDAH activity, allowing facile screening of inhibitors. Using this method, a haloacetamidine was identified as an active–site directed inactivator motif for DDAH, and other members of the amidinotransferase superfamily.
Drug delivery strategies to treat Pseudomonas aeruginosa biofilm infections
(2018-05) Bahamondez-Canas, Tania Francisca; Smyth, Hugh D. C.; Cui, Zhengrong; Ghosh, Debadyuti; Davies, Bryan W.
Bacteria growing as biofilms have gained recognition as an important therapeutic challenge due to their relation with the chronicity of infectious diseases. Biofilms are microbial communities that grow within a self-secreted polymeric matrix and represent the predominant growth form of bacteria in nature, as compared to free-floating bacteria (known as the planktonic growth). Biofilms can be thousands of times more resistant to antimicrobials than planktonic bacteria, which is explained, in part, by the shielding effect of the extracellular matrix and by the slow growth of subpopulation within the biofilm (also known as persister bacteria). Pseudomonas aeruginosa is an opportunistic pathogen and is reported to be the main organism responsible for the recurrent lung infections in cystic fibrosis (CF) patients. P. aeruginosa biofilms have been isolated from CF mucus samples, but also from chronic wounds and chronic rhinosinusitis. The objective of the work presented in this document was to investigate strategies to improve the activity of antibiotics against P. aeruginosa biofilm infections using drug delivery, pharmaceutical and material science approaches. First, we investigated via a high-throughput screening, potential excipients that could enhance the activity of tobramycin (Chapter 2). We then selected illustrative excipients that improved the activity of tobramycin against P. aeruginosa biofilms to develop three prototype dry powder formulations for pulmonary delivery (Chapter 3). These formulations reduced significantly the survival of P. aeruginosa biofilms in vitro under a treatment schedule that simulated tobramycin concentrations found in the airways after pulmonary delivery. In another approach, we tested PEG-conjugated antibiotics in an in vitro model that comprised two relevant barriers for drug delivery in lung infections: P. aeruginosa growing as biofilms and a CF-like mucus barrier. We found that the conjugation of tobramycin to PEG (Tob-PEG) (Chapter 4) and colistin to PEG (Col-PEG) (Chapter 5) significantly improved their antimicrobial activities against P. aeruginosa biofilms growing the in the mucus barrier model. Finally, we explored the complexation of ciprofloxacin with copper (CIP-Cu) as a strategy to reduce the lung-to-blood ciprofloxacin permeability and sustain high local concentrations after lung delivery. CIP-Cu retained the antimicrobial activity against P. aeruginosa biofilms in vitro (Appendix A) and the in vivo evaluation of CIP-Cu resulted in significant reduction of P. aeruginosa survival in a chronic lung infection model (Appendix B). Overall, these strategies have addressed different aspects of biofilms resistance to antibiotics with promising therapeutic potential for further development
Ecology of Pseudomonas aeruginosa infections
(2017-05) Dees, Justine Lynn; Whiteley, Marvin; Alper, Hal; Barrick, Jeffrey E.; Fast, Walter; Marcotte, Edward; Hunicke-Smith, Scott
The Gram-negative opportunistic pathogen Pseudomonas aeruginosa infects many different tissues types of immunocompromised individuals, especially the soft tissues and lungs. Despite the prevalence of P. aeruginosa in multiple infection environments, many of the mechanisms controlling this bacterium’s ability to thrive during infection remain unexplained. Therefore, I explored two facets of the ecology of P. aeruginosa infections: 1) the presence of co-infecting bacterial species and 2) the disturbance by antimicrobial treatment. The fitness-based genomic approach, Transposon sequencing (Tn-seq) was used to identify P. aeruginosa fitness determinants during chronic wound infection with the common co-infecting species, Staphylococcus aureus. The Tn-seq data revealed several genes that P. aeruginosa requires during co-infection with S. aureus. In particular, I demonstrated that the ability of P. aeruginosa to biosynthesize glutathione is a crucial determinant of P. aeruginosa fitness during chronic wound co-infection with S. aureus, potentially to relieve oxidative stress. To explore how the disturbance by antimicrobial treatment affects P. aeruginosa, I combined expression- (RNA sequencing) and fitness-based (Tn-seq) genomic techniques and identified genes involved in P. aeruginosa in vitro resistance to various antimicrobials.
Elucidating the LPS modification repertoire of Pseudomonas aeruginosa
(2015-05) Nowicki, Emily Marie; Trent, Michael Stephen; Whiteley, Marvin; Upton, Jason; Davies, Bryan W.; Kirisits, Mary Jo
Gram-negative bacteria enhance their survival in harmful environments by outer membrane remodeling, particularly at the lipid A moiety of LPS. We recently identified a functional ortholog of the lipid A kinase, lpxT, in Pseudomonas aeruginosa. LpxT[subscript Pa] is unique from previously characterized LpxT enzymes in that it is able to phosphorylate both lipid A phosphate groups as well as generate a novel 1-triphosphate species. Low Mg²⁺ results in modulation of LpxT[subscript Pa] activity and is influenced by transcription of lipid A aminoarabinose (L-Ara4N) transferase ArnT, which is induced when Mg²⁺ is limiting (Nowicki et al., Mol Micro, 2014). We have also revealed the identity of a functional phosphoethanolamine (pEtN) transferase, EptA[subscript Pa], in P. aeruginosa, and the first report of pEtN-modified lipid A in this organism. EptA[subscript Pa] adds pEtN strictly to the non-canonical position of lipid A. Transcription of EptA[subscript Pa] is regulated by Zn²⁺ via the ColRS twocomponent system, contrasting from EptA regulation in enteric bacteria such as Salmonella enterica and Escherichia coli. Further, although L-Ara4N modification readily occurs at the same site of pEtN addition under several environmental conditions, Zn²⁺exclusively induces pEtN addition to lipid A and downregulates transcription of the L-Ara4N transferase gene (Nowicki et al., Mol Micro, 2015). The existence and specificity of these modification enzymes suggests that coordinated regulation of P. aeruginosa outer membrane remodeling occurs to permit adaptation to a changing environment.
Flagella and Pili-Mediated Near-Surface Single-Cell Motility Mechanisms in P. aeruginosa
(2011-04) Conrad, Jacinta C.; Gibiansky, Maxsim L.; Jin, Fan; Gordon, Vernita D.; Motto, Dominick A.
Bacterial biofilms are structured multicellular communities that are responsible for a broad range of infections. Knowing how free-swimming bacteria adapt their motility mechanisms near a surface is crucial for understanding the transition from the planktonic to the biofilm phenotype. By translating microscopy movies into searchable databases of bacterial behavior and developing image-based search engines, we were able to identify fundamental appendage-specific mechanisms for the surface motility of Pseudomonas aeruginosa. Type IV pili mediate two surface motility mechanisms: horizontally oriented crawling, by which the bacterium moves lengthwise with high directional persistence, and vertically oriented walking, by which the bacterium moves with low directional persistence and high instantaneous velocity, allowing it to rapidly explore microenvironments. The flagellum mediates two additional motility mechanisms: near-surface swimming and surface-anchored spinning, which often precedes detachment from a surface. Flagella and pili interact cooperatively in a launch sequence whereby bacteria change orientation from horizontal to vertical and then detach. Vertical orientation facilitates detachment from surfaces and thereby influences biofilm morphology.
Investigating prokaryotic communities : group activities and physiological heterogeneity
(2013-12) Wessel, Aimee Katherine; Whiteley, Marvin
Bacterial communities engage in social activities, exhibiting behaviors such as communicating with small signaling molecules (quorum sensing [QS]) and building antibiotic-resistant biofilms. The opportunistic human pathogen Pseudomonas aeruginosa produces both freely diffusible QS molecules, as well as a QS molecule that is packaged or transported across cell membranes via the production of outer membrane vesicles. Despite the ubiquity of vesicle production in bacteria, the mechanism of outer membrane vesicle production has not been fully elucidated. In addition, most of our understanding of QS and biofilm formation arises from in vitro studies of bacterial communities containing large numbers of cells, often with greater than 10⁸ bacteria. However, many bacterial communities are comprised of small, densely packed aggregates of cells (≤10⁵ bacteria), and it is unclear how group behaviors and chemical interactions take place in densely packed, small populations. This dissertation has two main goals: i) to provide insights into the mechanism of bacterial membrane vesicle production, and ii) to understand how population size and the spatial distribution of cells affect cell-cell interactions and the nutritional microenvironment within a small (≤10⁵ bacteria) prokaryotic community.
Investigation and engineering of PvdQ, a Pseudomonas aeruginosa enzyme at the nexus of quorum sensing and iron uptake pathways
(2014-12) Clevenger, Kenneth David; Fast, Walter L.; Whitman, Christian P; Whiteley, Marvin; Keatinge-Clay, Adrian; Zhang, Jessie Y
The gram-negative human pathogen Pseudomonas aeruginosa is a widespread global health concern. Two key pathways of P. aeruginosa that are involved in its infection and/or survival within mammalian hosts are the pyoverdine biosynthetic pathway, which is necessary for iron acquisition, and the N-acyl-homoserine lactone (AHL) quorum sensing (QS) system, which is necessary for P. aeruginosa virulence and infection. The P. aeruginosa enzyme PvdQ is unique in that it is proposed to play roles in both these pathways. To investigate the role of PvdQ in P. aeruginosa, the enzyme’s in vitro substrate profile was characterized and found to favor substrates with unsubstituted myristic acyl groups, supporting a role for PvdQ in pyoverdine biosynthesis, but challenging its proposed role in regulating P. aeruginosa QS. Knowledge of PvdQ’s substrate preference and mechanism enabled rational inhibitor design, leading to the discovery of long chain (≥ 10 carbons) n-alkylboronic acids as highly potent transition state analog inhibitors of PvdQ, with 1-tridecylboronic acid having a Ki of 200 ± 40 pM and having the ability to recapitulate the growth phenotype of a pvdQ transposon insertion strain of P. aeruginosa under certain experimental conditions. Investigation of PvdQ inhibition by alkylboronic acids was extended to short- and medium-chain alkylboronic acids and revealed an eight order of magnitude span of affinity that is linearly related to alkyl chain length. Through structural studies, short-chain alkylboronic acids (≤ 4 carbons) were found to adopt an alternate binding conformation relative to other alkylboronic acids, which suggests a mechanism by which PvdQ excludes short substrates such as P. aeruginosa QS molecule N-butyroyl-homoserine lactone. Finally, a circular permutation of PvdQ (cpPvdQ) was designed and constructed in order to uncouple the catalytic and self-processing activities of PvdQ and to facilitate the production of mutants that affect catalysis and specificity. The cpPvdQ monomer recapitulates the catalytic and structural features of PvdQ. A catalytically impaired cpPvdQ mutant that is shown to maintain affinity for PvdQ’s native substrate, the pyoverdine precursor, but does not hydrolyze the substrate, has been produced and will be a valuable tool for future efforts to trap a PvdQ derivative in complex with the pyoverdine precursor.
Investigations into the role of aromatic amino acids in quorum sensing-mediated virulence in Pseudomonas aeruginosa
(2012-08) Palmer, Gregory Charles; Whiteley, Marvin; Ellington, Andrew; Kirisits, Mary Jo; Payne, Shelley; Meyer, Richard
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is a primary constituent of chronic, polymicrobial infections in the lungs of individuals with cystic fibrosis (CF). A significant consequence of CF is production of thick mucus along epithelial surfaces. In the lungs, this mucus collects and serves as an excellent growth substrate for a range of bacteria including. CF lung fluids (sputum) also enhance the virulence of P. aeruginosa, as production of a signaling molecule critical for virulence, the Pseudomonas quinolone signal (PQS), is enhanced in the presence of phenylalanine and tyrosine in CF sputum. The goal of this dissertation is to better understand how phenylalanine and tyrosine affect PQS production and ultimately P. aeruginosa virulence. To address this, I use transcriptome profiling to determine that genes for phenylalanine and tyrosine catabolism, PQS biosynthesis, and a transcriptional regulator called PhhR are up-regulated in the presence of phenylalanine and tyrosine. I determine that PhhR regulates genes for aromatic amino acid catabolism but not genes for PQS biosynthesis. The PhhR regulon is further characterized by mapping of PhhR-regulated promoters with primer extension, and evidence for direct regulation is presented. To explain enhanced production of PQS in CF sputum, I favor a model in which flux of a shared metabolic precursor, chorismate, toward PQS biosynthesis is enhanced when phenylalanine and tyrosine are present. I investigate this model by examining the first step in PQS biosynthesis, conversion of chorismate to anthranilate by an anthranilate synthase (AS). P. aeruginosa possesses two AS enzymes encoded by the trpEG and phnAB genes, with the former generating anthranilate specifically for tryptophan biosynthesis while the latter generates anthranilate for PQS biosynthesis. I investigate the evolutionary origins of these two enzymes and generate unmarked deletion mutants to dissect their roles in tryptophan and PQS biosynthesis. The ability of PhnAB to compensate for loss of TrpEG at high cell densities is documented, and a model explaining anthranilate sequestering is developed. Knowledge gained from these studies will be useful in developing novel therapeutic strategies.
A mechanism for interspecies competition and virulence in Pseudomonas aeruginosa-containing polymicrobial infections
(2012-08) Korgaonkar, Aishwarya Kiran, 1983-; Whiteley, Marvin; Trent, Michael S.; Payne, Shelley; Meyer, Richard; McLean, Robert J.
Pseudomonas aeruginosa is a ubiquitous bacterium that is commonly isolated from soil and water. Additionally, this bacterium can cause infections in individuals with compromised immune systems and in those with underlying debilitating conditions. Individuals with cystic fibrosis, burn wounds, AIDS and diabetes are more likely to being infected by P. aeruginosa than healthy individuals. In individuals with CF, there is a marked increase in the accumulation of lung mucus that serves as a source of nutrition for P. aeruginosa and other bacterial species resulting in chronic and often fatal infections. While CF lung infections are initially caused by more than one species of bacteria, over time P. aeruginosa emerges as the dominant species. P. aeruginosa also causes chronic infections in association with other bacteria in wounds. Microbes within these infections are engaged in complex interactions with each other. Often, these interactions are synergistic resulting in infections that are recalcitrant to antimicrobial therapy. While many studies have documented the occurrence of synergistic polymicrobial infections, little is known about the molecular mechanisms prevailing in these infections. Interestingly, production of virulence factors by P. aeruginosa has been shown to correlate with the presence of specific nutrients in their growth environment. Expanding on the idea of available nutrients affecting virulence, I demonstrate the ability of N-Acetylglucosamine (GlcNAc) and GlcNAc-containing polymers such as peptidoglycan to induce production of virulence factors in P. aeruginosa. Peptidoglycan shed by Gram-positive bacteria acts as a cue for P. aeruginosa in polymicrobial environments, to enhance production of virulence factors. In the context of a polymicrobial infection, this results in enhanced pathogenesis. Here, I provide insights into mechanisms influencing such interspecies interactions between the opportunistic pathogen Pseudomonas aeruginosa and S.aureus.
Mechanosensing and early biofilm development in Pseudomonas aeruginosa
(2017-05) Rodesney, Christopher Anania; Gordon, Vernita Diane; Florin, Ernst-Ludwig; Marder, Michael; Fink, Manfred; Whiteley, Marvin
Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically-identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface, as this attachment triggers intracellular signaling to change gene expression of individual bacteria from the planktonic to the biofilm phenotype. However the initial cues leading allowing bacteria to sense a surface, as well as the role of spatial structure in biofilm development, are not well known. This dissertation has two main parts, the first presenting a method for growing biofilms from initiating cells whose positions are controlled with single-cell precision using laser trapping. Biofilm infections are notoriously intractable, in part due to changes in the bacterial phenotype that result from spatial structure. Understanding the role of structure in biofilm development requires methods to control the spatial structure of biofilms. The native growth, motility, and surface adhesion of positioned microbes are preserved, as we show for model organisms Pseudomonas aeruginosa and Staphylococcus aureus. We demonstrate that laser-trapping and placing bacteria on surfaces can reveal the e↵ects of spatial structure on bacterial growth in early biofilm development. In the second part we show that mechanical shear acts as a cue for surface adhesion in P. aeruginosa. For P. aeruginosa, it has long been known that intracellular levels of the signaling molecule cyclic-di-GMP increase upon surface adhesion and that increased cyclic-di-GMP is required to begin biofilm development. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional Type IV pili are required mechanosensory elements. Finally, we propose an analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides, describing our data
Microbial Population Structure Impacts Antibiotic Resistance
(2015-03) Ratnayeke, Nalin; Gordon, Vernita
Antibiotic resistance is an urgent and growing concern, and developing novel therapeutic approaches to overcome antibiotic-resistant infections has assumed immense significance. Recently, it has been shown that heterogeneity in a microbial environment can accelerate the development of antibiotic resistance. However, the effect of the spatial distribution of the microbial population itself remains to be investigated. Using the human pathogen Pseudomonas aeruginosa, we show that increasing cell density can drastically impact the survival and growth of antibiotic-resistant mutants in the presence of aminoglycoside antibiotics in a density-dependent manner. Increasing density confers both positive and negative effects to mutant survival, which we term “protection” and “inhibition”, respectively. Using a combination of microbiological assays and biophysical modeling, we find that inhibition is mediated by a low-molecular weight, native by-product of bacterial metabolism that acts in conjunction with aminoglycosides through an effected increase in pH. A wide range of bacterial species are capable of producing inhibition, which is related to their growth by amino acid catabolism. We additionally develop a stochastic model of inhibition in homogeneous environments, which indicates that local density fluctuations on the scale of individual bacteria can significantly alter bacterial survival when colonizing a new environment. This work raises the possibility that the manipulation of population structure and the nutrient environment of microbes in conjunction with the use existing antibiotics could provide novel therapeutic approaches to combat antibiotic resistance.
Microfabricated environments for the study of bacterial group behavior
(2015-12) Fitzpatrick, Mignon Denise; Shear, Jason B.; Whiteley, Marvin
This thesis describes the application of micro three dimensional printing (µ3DP) techniques to create protein microstructures for the study of bacterial group behavior in small populations. Studies involving aggregates of ~10^1 to ~10^5 cells have shown extensive and complex communication and spatial organization. Multiphoton lithography (MPL) provides a means to quickly design and execute the fabrication of microscale structures with submicron resolution from a variety of biocompatible polymers. Using this technique, intricate spatial arrangements of bacteria can be achieved while maintaining small population sizes at high cell density (≥10^8 cells/mL), providing in vitro culture conditions which better simulate in vivo settings. As a result, valuable information can be obtained about bacterial social interactions through the coupling of additional analytical techniques to detect the presence or absence of extracellular signaling molecules. While quorum sensing (QS) remains the most extensively studied means of bacterial communication, it is becoming increasingly apparent that additional factors are necessary to effect certain changes in population-wide genetic expression which can lead to increased virulence, pathogenicity, and the development of antibiotic resistance. The work presented in this thesis addresses the influences of cell density, chemical heterogeneity of the environment within cell aggregates, and level of cell surface attachment as mechanisms to induce or influence the development of antibiotic resistance. Building upon previous work presented by members of the Shear lab, BSA-gelatin protein microstructures were used to study the behavior and response of the opportunistic pathogen Pseudomonas aeruginosa under these conditions. Antibiotic resistance was observed in low cell number/high density populations in agreement with previous work presented by the Shear lab. In addition, it was found that localized regions of oxygen depletion do not correlate directly with antibiotic resistance development, as the population size required for depletion far exceeded that for development of resistance. Finally, a new technique directed at simultaneous biofilm inhibition and cell removal from solution was explored.
Modulating the crosslinking of a hydrogel impacts cyclic-di-GMP signaling of Pseudomonas aeruginosa
(2020-05-09) Blacutt, Jacob Matthew; Gordon, Vernita Diane; Contreras, Lydia M.
The growth of bacterial biofilms on medical devices is a persistent cause of device failure, necessitating removal, and infections that harm patients. Therefore, much effort has been expended on a variety of approaches to developing materials that resist biofilm development. However, to date the effects of varying the solid mechanics of the device material have not been tested. Biofilm development is initiated when bacteria attach to a surface, sense the surface, and begin the transition into the biofilm phenotype. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, that transition is controlled by the second messenger cyclic-di-GMP. Here, we allow P. aeruginosa to attach to PEGDA gels and use a green fluorescent protein (GFP) reporter and laser-scanning confocal microscopy to measure the dynamics of the cyclic-di-GMP response in the first three hours after an initial hour-long attachment period. PEGDA gels are widely used in biomedical applications, in part because their mechanical properties are very tunable. We find that wild-type P. aeruginosa increase production of cyclic-di-GMP more quickly when they attach to a stiffer PEGDA gel, with elastic modulus about 4000 kPa, than when they attach to a softer PEGDA gel, with elastic modulus about 50 kPa. Upon measuring the skewness and kurtosis of the per-cell GFP brightness distributions, we find that population’s cyclic-di-GMP average is more heavily affected by a few strong responders, which upregulate cyclic-di-GMP production more quickly, on the softer gel than on the stiffer gel. Use of a mutant strain that does not make envelope protein PilY1, which has previously been suggested as a possible mechanosensor, shows that the WT’s increased signaling speed on the stiffer surface is dependent on PilY1. Thus, the work presented here both contributes to the emerging field of bacterial mechanosensing and, speculatively, suggests that tuning the surface mechanics of medical devices might be a new approach to hindering biofilm development.
Overcoming antibiotic resistance in microbial populations : an interdisciplinary perspective
(2015-08) Kaushik, Karishma Surendra; Gordon, Vernita Diane
Antibiotic resistance is a major public health problem. The increase in antibiotic-resistant bacteria and decline in the approval of newer antibiotics has prompted the need for novel therapeutic approaches. In the environment and in the human body, microbes are exposed to varying spatial landscapes. Further, bacteria assemble into multicellular assemblies called biofilms, which possess intricate spatial structure. Inherent to this spatial structure, microbial communities also possess population structure, characterized by cell density, spatial organization, and different cell types. This dissertation has three main goals: i) to study the effect of microbial population structure on the survival of antibiotic resistant mutants, using P. aeruginosa, an opportunistic human pathogen as a model organism, ii) to evaluate the therapeutic potential of using bicarbonate to enhance the efficacy of aminoglycoside antibiotics, first-line agents for P. aeruginosa infections, and develop an improved method of analysis of drug interactions iii) to develop a low-cost, hands-on, educational module to characterize antimicrobial compounds using an interdisciplinary, biophysical approach.
The Pel Polysaccharide Can Serve a Structural and Protective Role in the Biofilm Matrix of Pseudomonas aeruginosa
(Public Library of Science, 2011-01-27) Colvin, Kelly M.; Gordon, Vernita D.; Murakami, Keiji; Borlee, Bradley R.; Wozniak, Daniel J.; Wong, Gerard C. L.; Parsek, Matthew R.
Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.
Physical characterization of bacterial biofilm polymer networks to determine the role of mechanics in infection and treatment
(2018-11-29) Kovach, Kristin N.; Gordon, Vernita Diane; Florin, Ernst-Ludwig; Marder, Michael P; Smyth, Hugh D; Lynd, Nathaniel
Biofilms are communities of microorganisms that produce a matrix of extracellular polymers to surround and protect themselves from external forces in their environment. This communal lifestyle is incredibly beneficial for microorganism survival. Characterization of the mechanical properties of biofilms is a vital and understudied component of fully understanding these biological systems. In this dissertation, we break down the mechanical response of the Pseudomonas aeruginosa biofilm by its constituent polymers. These bacteria produce unique polymers to resist a variety of stresses. In the first part of this dissertation, using oscillatory bulk rheology, we characterize the viscoelasticity of biofilm polymer networks. Using genetically manipulated lab strains of P. aeruginosa, we isolate the mechanical response of each polymer by analyzing biofilms comprised primarily of one type of polymer. We find that the polymers have unique mechanical properties: some increase the yield strain and others increase elastic modulus. In strains of P. aeruginosa isolated from chronic infections, we find that the bacteria evolve to increase production of polymers that maximize the energy required to yield the matrix. In the second part of this dissertation, we work to mechanically compromise each of the polymers in the matrix. By attacking different matrix components, we learn more about the structural properties that give rise to mechanical properties as well as identify the most promising therapeutic treatments to break down biofilm infections. We find that specific enzymes are useful for decreasing yield strain of biofilms and increasing the diffusivity of the matrix. Decrease in yield strain means that biofilms will take less deformation before losing mechanical integrity, and the increase in matrix diffusivity means that current treatments such as antibiotics are more effective as the antibiotics can more easily reach the bacteria in the matrix to effectively kill them. This dissertation treats biofilms as polymer networks, divorcing the analysis from biological responses, in an attempt to well-characterize the understudied mechanical properties of biofilms. By approaching these systems from a physical standpoint, we are able to learn more about biofilms by breaking the mechanical response into constituent components, as well as learn about how enzymatic treatments alter biofilm properties.
The Pseudomonas Aeruginosa PSL Polysaccharide is a Social but Non-Cheatable Trait in Biofilms
(bioRxiv, 2016-05) Irie, Yasuhiko; Roberts, Aled E. L.; Kragh, Kasper N.; Gordon, Vernita D.; Hutchison, Jamie; Allen, Rosalind J.; Bjarnsholt, Thomas; West, Stuart A.; Diggle, Stephen P.; Gordon, Vernita D.
Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment and which are important for surface attachment and maintaining structural integrity within biofilms. They have been suggested to be metabolically costly to produce, and because they are secreted, to act as co-operative shared resources within biofilm communities. These assumptions have, however, not been experimentally well-examined. Here we empirically test the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa. We show that: (1) PSL provides population level benefits in biofilms, for both growth and antibiotic tolerance; (2) the benefits of PSL production are social and are shared with other cells; (3) the benefits of PSL production appear to be preferentially directed towards cells which produce PSL; (4) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively non-exploitable trait, and that growth within biofilms selects for PSL-producing strains, even when multiple strains can interact (low relatedness). The growth and proliferative success of many bacteria, including human pathogens, depends upon their ability to form biofilms in their respective environmental niches. Biofilms are multicellular three dimensional biomass structures, held together by extracellular matrix molecules that encapsulate cells and cause them to aggregate. These extracellular polysaccharides (EPS) which are secreted by the bacteria, typically function as adhesins that are used to attach to a surface and to maintain the three-dimensional biofilm structure, and sometimes aid in protection against a variety of stresses, including dehydration, antibiotics, and predators. The production of EPS represents a problem from an evolutionary perspective 3, because it appears to be a type of co-operative behaviour that can potentially provide a benefit to all cells in the community, and not just to those that produce EPS. Consequently, the question arises: “what prevents the invasion of potential cheats that do not produce EPS?”. A possible solution to this problem is that EPS production may not be an exploitable co32 operative trait. Xavier & Foster showed, in an individual based simulation, that if the production of EPS facilitated growth into areas where nutrient availability was greater, then EPS producing lineages could spatially smother and outcompete non-producers. In this case, EPS production was costly, but the benefits were preferentially provided to nearby cells, which were likely to be EPS-producing clone mates. Some empirical support for this particular mechanism has been demonstrated in Vibrio cholerae, where it has been shown that EPS producing lineages are able to displace non-producers. In addition, EPS producers in V. cholerae are also able to share other secreted public goods with each other, which provides another benefit that is preferentially directed towards other EPS producers. However, the generality of these explanations for the evolutionary stability of EPS production remains unclear, and the work on V. cholerae represents the only empirical study to measure the social costs and benefits of EPS production. In addition, EPS produced by other bacterial species can vary greatly in both their chemical structure and the biological roles they play within biofilms. Furthermore, many species produce more than one type of EPS that are sometimes but not necessarily co-regulated. This means that there may be differences in the social nature of different types of EPS, and for different bacterial species. Pseudomonas aeruginosa is an opportunistic pathogen that causes various biofilm infections such as chronic respiratory infections of cystic fibrosis, keratitis, and chronic wound infections. P. aeruginosa is known to produce at least three different types of EPS as major components of its biofilm matrix: alginate, PEL, and PSL polysaccharides. Alginate production is inversely regulated with PSL and is not expressed to high levels in the majority of non-CF isolates. In contrast, PSL is expressed in most P. aeruginosa natural and clinical isolates. PSL is a crucial adhesive scaffolding component of the biofilm matrix, promoting both cell-to-cell interactions and surface attachment. Here we test the social nature of PSL and find that (1) PSL production is not metabolically costly to P. aeruginosa cells; (2) PSL+ strains are significantly fitter than PSL- strains in mixed culture biofilms and PSL- strains cannot act as social cheats; (3) the benefit of producing PSL is enhanced when there are many PSL- cells present; (4) biofilms containing a high proportion of PSL- cells are more susceptible to antibiotics; (5) relatedness in biofilms does not matter since PSL+ strains are favoured in conditions of high and low relatedness. More generally we highlight that not all components of the biofilm matrix should be considered as shared resources.