R-3 Repository :: Browsing by Author "Shamoo, Yousif"

Browsing by Author "Shamoo, Yousif"

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A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci
(2017-04-17) Wang, Xu; Shamoo, Yousif; Olson, John
Antimicrobial resistance is a serious and growing threat to public health. The Centers for Disease Control estimates that at least 2 million people contract serious infections each year resulting in at least 23,000 deaths each year in the United States alone. To develop more effective treatments, it is important to understand the fundamental biology underlying the mechanisms of antibiotic resistance. Substitutions in the LiaFSR membrane stress response pathway are frequently associated with emergence of antimicrobial peptide resistance in both Enterococcus faecalis and Enterococcus faecium. Cyclic di-AMP (c-di-AMP) is an important signal molecule that affects many aspects of bacterial physiology including stress response. We have previously identified a mutation in a gene (designated yybT) of E. faecalis that was associated with development of daptomycin resistance. The adaptive mutation produced a change at position 440 in the predicted protein (yybTI440S). Here, we show that intracellular cyclic di-AMP signaling is present in enterococci and, based upon in vitro physicochemical characterization, we show that E. faecalis yybT encodes a cyclic dinucleotide phosphodiesterase of the GdpP family that exhibits specific activity toward c-di-AMP by hydrolyzing it to 5’pApA. The E. faecalis GdpPI440S substitution reduces cyclic di-AMP phosphodiesterase activity more than 11-fold leading to further increases in cyclic di-AMP levels. Additionally, deletions of liaR (encoding the response regulator of the LiaFSR system) that lead to daptomycin hypersusceptibility in both E. faecalis and E. faecium also resulted in increased cyclic-di-AMP levels suggesting that changes in the LiaFSR stress response pathway are linked to broader physiological changes. Taken together, our data show that modulation of cyclic di-AMP pools is strongly associated with antibiotic-induced cell membrane stress response via changes in GdpP activity and signaling through the LiaFSR system.
A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin
(Oxford University Press, 2015) Davlieva, Milya; Shi, Yiwen; Leonard, Paul G.; Johnson, Troy A.; Zianni, Michael R.; Arias, Cesar A.; Ladbury, John E.; Shamoo, Yousif
LiaR is a ‘master regulator’ of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaRD191N increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.
Acinetobacter baumanniiﾠ Repeatedly Evolves a Hypermutator Phenotype in Response to Tigecycline That Effectively Surveys Evolutionary Trajectories to Resistance
(Public Library of Science, 2015) Hammerstrom, Troy G.; Beabout, Kathryn; Clements, Thomas P.; Saxer, Gerda; Shamoo, Yousif
The evolution of hypermutators in response to antibiotic treatment in both clinical and laboratory settings provides a unique context for the study of adaptive evolution. With increased mutation rates, the number of hitchhiker mutations within an evolving hypermutator population is remarkably high and presents substantial challenges in determining which mutations are adaptive. Intriguingly however, hypermutators also provide an opportunity to explore deeply the accessible evolutionary trajectories that lead to increased organism fitness, in this case the evolution of antibiotic resistance to the clinically relevant antibiotic tigecycline by the hospital pathogen Acinetobacter baumannii. Using a continuous culture system, AB210M, a clinically derived strain of A. baumannii, was evolved to tigecycline resistance. Analysis of the adapted populations showed that nearly all the successful lineages became hypermutators via movement of a mobile element to inactivate mutS. In addition, metagenomic analysis of population samples revealed another 896 mutations that occurred at a frequency greater than 5% in the population, while 38 phenotypically distinct individual colonies harbored a total of 1712 mutations. These mutations were scattered throughout the genome and affected ~40% of the coding sequences. The most highly mutated gene wasadeS, a known tigecycline-resistance gene; however, adeS was not solely responsible for the high level of TGC resistance. Sixteen other genes stood out as potentially relevant to increased resistance. The five most prominent candidate genes (adeS, rpsJ, rrf, msbA, andgna) consistently re-emerged in subsequent replicate population studies suggesting they are likely to play a role in adaptation to tigecycline. Interestingly, the repeated evolution of a hypermutator phenotype in response to antibiotic stress illustrates not only a highly adaptive strategy to resistance, but also a remarkably efficient survey of successful evolutionary trajectories.
Adaptation of Enterococcus faecalis to Daptomycin Reveals an Ordered Progression to Resistance
(American Society for Microbiology, 2013) Miller, Corwin A.; Kong, Jiayi; Tran, Truc T.; Arias, Cesar A.; Saxer, Gerda; Shamoo, Yousif
With increasing numbers of hospital-acquired antibiotic resistant infections each year and staggering health care costs, there is a clear need for new antimicrobial agents, as well as novel strategies to extend their clinical efficacy. While genomic studies have provided a wealth of information about the alleles associated with adaptation to antibiotics, they do not provide essential information about the relative importance of genomic changes, their order of appearance, or potential epistatic relationships between adaptive changes. Here we used quantitative experimental evolution of a single polymorphic population in continuous culture with whole-genome sequencing and allelic frequency measurements to study daptomycin (DAP) resistance in the vancomycin-resistant clinical pathogen Enterococcus faecalis S613. Importantly, we sustained both planktonic and nonplanktonic (i.e., biofilm) populations in coculture as the concentration of antibiotic was raised, facilitating the development of more ecological complexity than is typically observed in laboratory evolution. Quantitative experimental evolution revealed a clear order and hierarchy of genetic changes leading to resistance, the signaling and metabolic pathways responsible, and the relative importance of these mutations to the evolution of DAP resistance. Despite the relative simplicity of this ex vivo approach compared to the ecological complexity of the human body, we showed that experimental evolution allows for rapid identification of clinically relevant adaptive molecular pathways and new targets for drug design in pathogens.
An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State
(Elsevier, 2016) Davlieva, Milya; Tovar-Yanez, Angel; DeBruler, Kimberly; Leonard, Paul G.; Zianni, Michael R.; Arias, Cesar A.; Shamoo, Yousif
The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated E. faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials.
Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence in Enterococcus faecalis
(National Academy of Sciences, 2019) Khan, Ayesha; Davlieva, Milya; Panesso, Diana; Rincon, Sandra; Miller, William R.; Diaz, Lorena; Reyes, Jinnethe; Cruz, Melissa R.; Pemberton, Orville; Nguyen, April H.; Siegel, Sara D.; Planet, Paul J.; Narechania, Apurva; Latorre, Mauricio; Rios, Rafael; Singh, Kavindra V.; Ton-That, Hung; Garsin, Danielle A.; Tran, Truc T.; Shamoo, Yousif; Arias, Cesar A.
Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistant Enterococcus faecalis, an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in the Caenorhabditis elegans model, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host–pathogen interactions.
Biochemical and crystallographic studies of bacteriophage RB69 DNA polymerase and single-stranded DNA binding protein interactions
(2006) Sun, Siyang; Shamoo, Yousif
The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. The DNA replication process in RB69 (a T4-like bacteriophage) is similar to the processes in eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. In order to examine protein-protein interactions at the DNA replication fork, solution conditions have been established for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA and RB69 single-stranded DNA binding protein (gp32) using equilibrium fluorescence and light scattering. The interaction between DNA polymerase and single-stranded DNA binding protein has been characterized by fluorescence titrations and results in a 60-fold increase in the overall affinity of RB69 SSB for template-strand DNA in the presence of DNA polymerase. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site-of-action. A functional domain of RB69 single-stranded DNA-binding protein previously suggested to be the site of RB69 DNA polymerase:SSB interactions has been shown to be dispensable. The data from these studies have been used to model the RB69 DNA polymerase:SSB interaction at the primer-template junction. Fusion of RB69 SSB with its cognate DNA polymerase via a short six amino acid linker increases affinity for primer-template DNA by 6-fold and increases processivity by 7-fold while maintaining fidelity. The crystal structure of this fusion protein was solved by a combination of multiwavelength anomalous diffraction and molecular replacement to 3.2 A resolution and shows that RB69 SSB is positioned proximal to the N-terminal domain of RB69 DNA polymerase near the template strand entry channel. The structural and biochemical data suggest that SSB interactions with DNA polymerase are transient and flexible, consistent with models of a dynamic replisome during elongation.
Cellular fitness as a proxy to determine the physicochemical parameters of an antibiotic efflux pump
(2016-12-02) Perez, Anisha M; Shamoo, Yousif; Olson, John S
Determining the quantitative link between protein function and cellular fitness can be challenging as even the modestly sized genome of Escherichia coli is comprised of thousands of genes. Using an appropriate survey of fitness across a range of selective conditions, we can reduce the complexity of this system by tightly linking cellular fitness to the function of one protein essential for growth within that selective environment. Several model proteins have been studied in this fashion whereby in vitro protein parameters are used to predict cellular fitness as a function of selection strength. Underlying this approach, however, is the idea that the reverse relationship is also true: analysis of cellular fitness can be used to predict protein physicochemical properties. In this study I present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the tetracycline efflux pump, TetB, and a family of strategically chosen single amino acid variants. TetB is a member of the Major Facilitator Superfamily (MFS) of transporters which have a conserved protein fold and for which we have a general understanding of how protein structure relates to function. We first performed growth rate analysis on our host strain without tet(B) at a wide range of drug concentrations to obtain global parameters that describe the baseline response of our cellular system. Growth analysis was also performed on strains expressing a chromosomal copy of tet(B) or variant allowing for a quantitative measurement of the fitness effects produced by TetB. Using both sets of fitness data and in vivo protein concentration, our model was able to predict physicochemical pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants which match the current knowledge of how MFS transporter structure influences function. Taken together, this study shows that cellular fitness in strong selective conditions can be used to characterize efflux pumps, a class of proteins which are classically challenging to characterize using classical in vitro biochemistry techniques. Additionally, this analysis opens up the possibility of characterizing protein libraries from high-throughput growth rate assays.
Characterization of Proteins Involved in Membrane Fusion- Atlastin and Munc18c
(2013-09-16) Verma, Avani; McNew, James A.; Braam, Janet; Raphael, Robert M.; Shamoo, Yousif; Wagner, Daniel S.
Membranes provide a barrier to cells and organelles, and allow the selective transport of molecules between compartments. Membrane fusion is essential for organelle biogenesis as well as trafficking of molecules between cellular compartments. Membrane fusion is also required for the formation of the branched network of tubules that make up the Endoplasmic Reticulum (ER). One protein implicated in ER fusion is Atlastin, a dynamin like GTPase. Mutations in Atlastin-1, among others, cause Hereditary Spastic Paraplegias (HSP), a group of neurological disorders that cause progressive weakness of lower extremities. We have shown that the C-terminal tail of atlastin is necessary for membrane fusion. The requirement of the C-terminal tail can be partially abrogated in an unstable lipid environment. This implies that the C-terminal tail of Atlastin plays a role in perturbing the lipid bilayer to allow membrane fusion. Understanding the molecular details of how Atlastin drives membrane fusion may help elucidate the pathogenesis of HSP. Intracellular fusion at the plasma membrane is SNARE mediated and regulated by Sec1p/Munc18 (SM) proteins. Increased rate of glucose transport into fat and muscles cells by translocation of glucose transporter GLUT4 in response to insulin is a SNARE regulated fusion process. Recent reports have linked Munc18c and Syntaxin4 with obesity and Type 2 diabetes. We characterized the function of Munc18c, an SM protein, in regulating GLUT-4 containing vesicle fusion with the plasma membrane. We have shown that Munc18c directly inhibits membrane fusion by interacting with its cognate SNARE complexes. Characterization of membrane fusion in a minimal system as the in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function.
Comparative Genomics of Cephalochordates
(2015-04-23) Yue, Jiaxing; Kohn, Michael H.; Nakhleh, Luay K.; Shamoo, Yousif; Guerra, Rudy; Putnam, Nicholas H.
Cephalochordates, commonly known as lancelets or amphioxus, represent an ancient chordate lineage falling at the boundary between invertebrates and vertebrates. They are considered the best living proxy for the common ancestor of all chordate animals and hold the key for understanding chordate evolution. Despite such great importance, current studies on cephalochordates are generally limited to the Branchiostoma genus, leaving the other two genera, Asymmetron and Epigonichthys largely unexplored. In this dissertation, I set out to fill this gap by developing an array of genomic resources for the Bahama cephalochordate, Asymmetron lucayanum, by both RNA-Seq and whole-genome shotgun (WGS) sequencing. The transcriptome and genome of this representative cephalochordate species were assembled and characterized via the state-of-arts comparative genomics approach. By comparing its transcriptome and genome sequences with those of a distant related cephalochordate species, Branchiostoma floridae, as well as with several representative vertebrate species, many aspects of their genome biology were illuminated, which includes lineage-specific molecular evolution rate, fast-evolving genes, evolution time frame, conserved non-coding elements, and germline-related genes. The raw genomic resources, technical pipelines and biological results and insights generated by this dissertation work will benefit the whole cephalochordate research community by providing a powerful guide for formulating new hypotheses and designing new experiments towards a better understanding about the biology and evolution of cephalochordates, as well as the evolutionary transition from invertebrates to vertebrates.
Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of Cell Membrane Phospholipid Content
(Public Library of Science, 2012) Mishra, Nagendra N.; Bayer, Arnold S.; Tran, Truc T.; Shamoo, Yousif; Mileykovskaya, Eugenia; Dowhan, William; Guan, Ziqiang; Arias, Cesar A.
Background: The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. Methodology: Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. Principal Findings: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM. Conclusion: Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids
(American Society for Microbiology, 2013) Tran, Truc T.; Panesso, Diana; Mishra, Nagendra N.; Mileykovskaya, Eugenia; Guan, Ziqiang; Munita, Jose M.; Reyes, Jinnethe; Diaz, Lorena; Weinstock, George M.; Murray, Barbara E.; Shamoo, Yousif; Dowhan, William; Bayer, Arnold S.; Arias, Cesar A.
Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calciumdependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.
Data publication with the structural biology data grid supports live analysis
(Springer Nature, 2016) Meyer, Peter A.; Socias, Stephanie; Key, Jason; Ransey, Elizabeth; Tjon, Emily C.; Buschiazzo, Alejandro; Lei, Ming; Botka, Chris; Withrow, James; Neau, David; Rajashankar, Kanagalaghatta; Anderson, Karen S.; Baxter, Richard H.; Blacklow, Stephen C.; Boggon, Titus J.; Bonvin, Alexandre M.J.J.; Borek, Dominika; Brett, Tom J.; Caflisch, Amedeo; Chang, Chung-I; Chazin, Walter J.; Corbett, Kevin D.; Cosgrove, Michael S.; Crosson, Sean; Dhe-Paganon, Sirano; Di Cera, Enrico; Drennan, Catherine L.; Eck, Michael J.; Eichman, Brandt F.; Fan, Qing R.; Ferré-D'Amaré, Adrian R.; Fromme, J.Christopher; Garcia, K.Christopher; Gaudet, Rachelle; Gong, Peng; Harrison, Stephen C.; Heldwein, Ekaterina E.; Jia, Zongchao; Keenan, Robert J.; Kruse, Andrew C.; Kvansakul, Marc; McLellan, Jason S.; Modis, Yorgo; Nam, Yunsun; Otwinowski, Zbyszek; Pai, Emil F.; Pereira, Pedro José Barbosa; Petosa, Carlo; Raman, C.S.; Rapoport, Tom A.; Roll-Mecak, Antonina; Rosen, Michael K.; Rudenko, Gabby; Schlessinger, Joseph; Schwartz, Thomas U.; Shamoo, Yousif; Sondermann, Holger; Tao, Yizhi Jane; Tolia, Niraj H.; Tsodikov, Oleg V.; Westover, Kenneth D.; Wu, Hao; Foster, Ian; Fraser, James S.; Maia, Filipe R.N.C.; Gonen, Tamir; Kirchhausen, Tom; Diederichs, Kay; Crosas, Mercè; Sliz, Piotr
Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.
Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background
(American Society for Microbiology, 2015) Panesso, Diana; Reyes, Jinnethe; Gaston, Elizabeth; Deal, Morgan; Londoño, Alejandra; Nigo, Masayuki; Munita, Jose M.; Miller, William R.; Shamoo, Yousif; Tran, Truc T.; Arias, Cesar A.
We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaRnot only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.
Determining Daptomycin Resistance Mechanisms in Enterococcus faecium
(2019-08-05) Prater, Amy G; Shamoo, Yousif; Silberg, Joff
The ascent of antibiotic resistance poses a serious threat to human health. Left unaddressed, we could enter a “post-antibiotic” era where everyday infections prove fatal. As such, new ways of treating these infections or prolonging current antibiotic efficacy are needed. One method for prolonging antibiotic efficacy is to design “anti-evolution” co-therapies that target and inhibit the primary resistance pathway evolved against the antibiotic. Administering this co-drug alongside the antibiotic could then delay the onset of resistance. Previously, our lab found that the clinically important pathogen, Enterococcus faecalis, evolved resistance to the rescue-drug, daptomycin (DAP), via activating mutations to the LiaFSR membrane-stress-response pathway and cardiolipin synthase (cls) that redistribute membrane phospholipids and divert DAP binding away from vulnerable septal targets. While LiaFSR is heavily implicated in Enterococcus faecium DAP resistance, several clinical isolates containing activated liaFSR alleles are DAP tolerant and do not display the redistribution phenotype; similarly, several DAP resistant E. faecium isolates possess mutations in alternative pathways to LiaFSR. To identify the prominent DAP resistance mechanisms in E. faecium and further understand the role of LiaFSR, this thesis evaluated how clinical E. faecium evolved DAP resistance A) in the presence of activated LiaFSR alleles, and B) when liaR was deleted from the genome. We found that unlike E. faecalis, E. faecium evolved multiple and different resistance strategies that were heavily influenced by the environment within which they were adapted and that DAP repulsion from the cell surface was the more common mode of resistance. We also found that deletion of liaR significantly delayed the onset of resistance and that the resulting mechanisms were quite complex and varied, suggesting that this deletion significantly hindered the bacterial population which then struggled to find adaptive mutations. Regardless of environment or the presence of liaR, evolution converged on mutations to cls, highlighting the importance of membrane modification in enterococcal DAP resistance. While E. faecium can use multiple pathways to resist DAP, the increased timeline to DAP resistance when liaR is deleted supports the hypothesis for developing a small molecule inhibitor of this system to increase DAP efficacy against enterococcal infections.
Developing Microdroplet Emulsions as a System for the Study of Microbial Social Interactions
(2023-04-18) Song, Xinhao; Shamoo, Yousif
The evolution and maintenance of social behaviors, especially cooperative interactions, is an intensively-studied field in ecology. Social microbes that interact through the secretion of diffusible chemical signals are ideal model organisms for both fundamental and translational studies of this fascinating process. In this thesis, I investigate the social interactions among soil-dwelling Gram-positive bacteria Streptomyces spp. Streptomyces species have contributed to more than two-thirds of current antibiotics and have a remarkable capability for the synthesis of secondary metabolites. Moreover, in their natural soil habitats, neighboring Streptomyces species can form complex social interaction networks that are mediated by diffusible secondary metabolites, further adding to their qualities as model organisms for the study of microbial social interactions. One of the key factors for the evolution and maintenance of social interactions among microbes is spatially structured environments. Such environments limit the diffusion of “public good” molecules such as beneficial metabolites and chemical signals and may favor social traits with indirect fitness benefits. In this thesis, I used microdroplet emulsions to construct artificial and configurable spatially segregated environments for the investigation of nascent social interactions. Two comprehensive case studies of inhibition and cooperative Streptomyces pairs were conducted. First, a collection of Streptomyces environmental isolates was obtained and characterized both phenotypically and genomically. From this collection, isolates T4-11 and AMS-5 were identified as inhibitive and cooperative partner strains for the model strain S. venezuelae. Next, using both plasmid-based and genome integration approaches, various RFP and GFP fluorescence reporter strains were constructed to facilitate the investigation of social interactions. Subsequently, interactions between T4-11/S. venezuelae and AMS-5/S. venezuelae pairs were thoroughly investigated using conditioned medium and co-culture assays in both liquid suspension and microdroplet environments. The inhibition of T4-11 on S. venezuelae was potent and reproducible across various conditions, whereas the interactions between AMS-5 and S. venezuelae varied significantly in different environments. To expand further the set of tools available to study social interactions, a volatile gas-based S. venezuelae reporter strain was constructed and characterized. It is well known that fluorescence reporters suffer from limitations when used in microdroplets, such as inaccurate readings due to strong background or limited sensitivity during early growth stages. To overcome such limitations, I showed that volatile gas produced by S. venezuelae gas reporters could be quantified by GC-MS in situ and could serve as an alternative non-optical detection approach for microbes encapsulated microdroplets. In addition, a 100-1000 fold improvement in sensitivity was observed for the gas reporter when compared to an RFP fluorescence reporter.
Dynamic Self-Stiffening in Liquid Crystal Elastomers
(Nature Publishing Group, 2013) Agrawal, Aditya; Chipara, Alin C.; Shamoo, Yousif; Patra, Prabir K.; Carey, Brent J.; Ajayan, Pulickel M.; Chapman, Walter G.; Verduzco, Rafael
Biological tissues have the remarkable ability to remodel and repair in response to disease, injury and mechanical stresses. Synthetic materials lack the complexity of biological tissues, and man-made materials that respond to external stresses through a permanent increase in stiffness are uncommon. Here we report that polydomain nematic liquid crystal elastomers increase in stiffness by up to 90% when subjected to a low-amplitude (5%), repetitive (dynamic) compression. Elastomer stiffening is influenced by liquid crystal content, the presence of a nematic liquid crystal phase and the use of a dynamic as opposed to static deformation. Through rheological and X-ray diffraction measurements, stiffening can be attributed to a mobile nematic director, which rotates in response to dynamic compression. Stiffening under dynamic compression has not been previously observed in liquid crystal elastomers and may be useful for the development of self-healing materials or for the development of biocompatible, adaptive materials for tissue replacement.
Dynamics of a Synthetic Microbial Community in a Structured Droplet Environment
(2021-12-03) Ganiga Prabhakar, Ramya; Shamoo, Yousif
In nature, microbes live in multispecies communities where they compete for nutrients within a localized and sometimes structured environment. Outcomes of such microbial interactions are dynamic and can vary in response to environmental cues. To study community dynamics in laboratory conditions, the choice of culturing environments is therefore crucial. Studying microbial interactions using suspension cultures is challenging because it favors fast-growers over high-yielding cells. To this end, we developed emulsion droplets as a structured culturing method to investigate the population dynamics of a synthetic microbial community. We also developed a mathematical model to provide insights into the key parameters shaping the structure and function of such communities in droplets. In the first part of this project, to encapsulate competitive sub-communities we built microfluidic devices to generate water-in-oil droplets as discrete, picolitre-volume culture vessels. The ideal population sizes and culturing conditions for optimum bacterial growth in droplets were determined. We also engineered a microfluidic module for efficient droplet sorting based on fluorescence. Next, we test the dynamics of a synthetic microbial community within droplets. Here, we have rationally engineered competitive interactions among Producer, Non-producer, and the sensitive Receivers in Escherichia coli to study competitive outcomes in a well-mixed environment versus spatially structured conditions. The Producer strain kills a competing Receiver strain, mediated through secreted small molecules. The Non-producer strain is a phenotypic (and genotypic) variant of the Producer that cannot kill the Recipient strain as the gene for metabolite production is disrupted. We found that the secreted molecules diffuse between the droplets and therefore the droplets afford confinement of only the cells but not the secreted metabolite. Even in this condition, by encapsulating the competitive co-cultures in droplets, we successfully enriched the Producers from a large population of Non-producers and Receivers. We found that the metabolite concentration, the duration of bacterial incubation, the initial population sizes, and the droplet carrying capacity affect the Producer dynamics in the population. The final part of this thesis focuses on applying the insights from the population dynamics of the model community to enrich for novel antibiotic-producing Streptomyces roseosporus, by rationally competing against Enterococcus faecalis using the microfluidic platform. As daptomycin is ineffective against a resistant variant of E. faecalis, S roseosporus must trigger activation of a cryptic pathway or produce daptomycin-variant that is effective against E. faecalis for success in the population. Preliminary work in this project shows that the growth rates of the two populations must be further optimized for testing experimental evolution in droplets. Overall, in this thesis, we have established the microfluidic platform, validated the synthetic community dynamics, and set up initial work towards applying the platform for wild strains of bacteria. These insights can guide design principles to study the microbial community dynamics of natural and synthetic multispecies communities in the laboratory.
EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde
(Public Library of Science, 2021) Bazurto, Jannell V.; Nayak, Dipti D.; Ticak, Tomislav; Davlieva, Milya; Lee, Jessica A.; Hellenbrand, Chandler N.; Lambert, Leah B.; Benski, Olivia J.; Quates, Caleb J.; Johnson, Jill L.; Patel, Jagdish Suresh; Ytreberg, F.Marty; Shamoo, Yousif; Marx, Christopher J.
Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.
Elucidating the Roles of PEX19 and Prenylation in Arabidopsis Peroxisomes
(2012-09-05) Stoddard, Jerrad; Bartel, Bonnie; Shamoo, Yousif; Farach-Carson, Cindy; Braam, Janet; Rudgers, Jennifer A.
Peroxisomes are organelles originating from the endoplasmic reticulum. Peroxisome biogenesis requires multiple peroxins, including PEX19, a prenylated protein that helps deliver peroxisomal membrane proteins in yeast and mammals. Arabidopsis thaliana PEX19 is encoded by two isogenes, PEX19A and PEX19B. I demonstrate that pex19A and pex19B insertional mutants lack obvious abberant physiological phenotypes. I provide evidence that pex19A pex19B double mutants are inviable, that PEX19B is more abundant than PEX19A in young seedlings, that Arabidopsis PEX19 is farnesylated in vivo, and that YFP-PEX19 predominantly associates with what appears to be a subcellular membrane regardless of its prenylation state. I show that farnesyltransferase mutants apparently contain only non-prenylated PEX19 and lack phenotypes that would indicate inefficient peroxisome activity. My analysis of PEX19 suggests that PEX19 prenylation is dispensable for peroxisome biogenesis, and has generated tools for future studies of the earliest steps in peroxisome biogenesis in plants.

Browsing by Author "Shamoo, Yousif"

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