Publications by authors named "Vincent Nieto"

12 Publications

  • Page 1 of 1

Nerve-associated transient receptor potential ion channels can contribute to intrinsic resistance to bacterial adhesion in vivo.

FASEB J 2021 10;35(10):e21899

Vision Science Program, University of California, Berkeley, California, USA.

The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1 more susceptible to P. aeruginosa adhesion while TRPV1 corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.
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http://dx.doi.org/10.1096/fj.202100874RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8486357PMC
October 2021

Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility.

Ocul Surf 2021 Jul 28;22:94-102. Epub 2021 Jul 28.

School of Optometry, University of California, Berkeley, CA, USA; Graduate Groups in Vision Science, Microbiology, and Infectious Diseases & Immunity, University of California, Berkeley, CA, USA. Electronic address:

Purpose: Previously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression.

Methods: RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 C). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure.

Results: Tear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 μg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 μg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca and Mg concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive.

Conclusion: P. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.
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http://dx.doi.org/10.1016/j.jtos.2021.07.004DOI Listing
July 2021

Martini 3: a general purpose force field for coarse-grained molecular dynamics.

Nat Methods 2021 04 29;18(4):382-388. Epub 2021 Mar 29.

Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Material, University of Groningen, Groningen, the Netherlands.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.
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http://dx.doi.org/10.1038/s41592-021-01098-3DOI Listing
April 2021

Triacylglycerols sequester monotopic membrane proteins to lipid droplets.

Nat Commun 2020 08 7;11(1):3944. Epub 2020 Aug 7.

Laboratoire de Physique de l'École Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université de Paris, F-75005, Paris, France.

Triacylglycerols (TG) are synthesized at the endoplasmic reticulum (ER) bilayer and packaged into organelles called lipid droplets (LDs). LDs are covered by a single phospholipid monolayer contiguous with the ER bilayer. This connection is used by several monotopic integral membrane proteins, with hydrophobic membrane association domains (HDs), to diffuse between the organelles. However, how proteins partition between ER and LDs is not understood. Here, we employed synthetic model systems and found that HD-containing proteins strongly prefer monolayers and returning to the bilayer is unfavorable. This preference for monolayers is due to a higher affinity of HDs for TG over membrane phospholipids. Protein distribution is regulated by PC/PE ratio via alterations in monolayer packing and HD-TG interaction. Thus, HD-containing proteins appear to non-specifically accumulate to the LD surface. In cells, protein editing mechanisms at the ER membrane would be necessary to prevent unspecific relocation of HD-containing proteins to LDs.
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http://dx.doi.org/10.1038/s41467-020-17585-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414839PMC
August 2020

Contact lens-related corneal infection: Intrinsic resistance and its compromise.

Prog Retin Eye Res 2020 05 20;76:100804. Epub 2019 Nov 20.

School of Optometry, University of California, Berkeley, CA, USA; College of Pharmacy, Touro University California, Vallejo, CA, USA.

Contact lenses represent a widely utilized form of vision correction with more than 140 million wearers worldwide. Although generally well-tolerated, contact lenses can cause corneal infection (microbial keratitis), with an approximate annualized incidence ranging from ~2 to ~20 cases per 10,000 wearers, and sometimes resulting in permanent vision loss. Research suggests that the pathogenesis of contact lens-associated microbial keratitis is complex and multifactorial, likely requiring multiple conspiring factors that compromise the intrinsic resistance of a healthy cornea to infection. Here, we outline our perspective of the mechanisms by which contact lens wear sometimes renders the cornea susceptible to infection, focusing primarily on our own research efforts during the past three decades. This has included studies of host factors underlying the constitutive barrier function of the healthy cornea, its response to bacterial challenge when intrinsic resistance is not compromised, pathogen virulence mechanisms, and the effects of contact lens wear that alter the outcome of host-microbe interactions. For almost all of this work, we have utilized the bacterium Pseudomonas aeruginosa because it is the leading cause of lens-related microbial keratitis. While not yet common among corneal isolates, clinical isolates of P. aeruginosa have emerged that are resistant to virtually all currently available antibiotics, leading the United States CDC (Centers for Disease Control) to add P. aeruginosa to its list of most serious threats. Compounding this concern, the development of advanced contact lenses for biosensing and augmented reality, together with the escalating incidence of myopia, could portent an epidemic of vision-threatening corneal infections in the future. Thankfully, technological advances in genomics, proteomics, metabolomics and imaging combined with emerging models of contact lens-associated P. aeruginosa infection hold promise for solving the problem - and possibly life-threatening infections impacting other tissues.
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http://dx.doi.org/10.1016/j.preteyeres.2019.100804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237316PMC
May 2020

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.

J Bacteriol 2019 12 6;202(1). Epub 2019 Dec 6.

Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

In and , the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of restores wild-type motor behavior in , indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In , c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in Instead, we found through reversion analysis that deletion of , which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σ for degradation, restored wild-type motility in the mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression. Flagellum-driven motility has been studied in and for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in , we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σ regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.
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http://dx.doi.org/10.1128/JB.00578-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932234PMC
December 2019

Potent LpxC Inhibitors with Activity against Multidrug-Resistant Pseudomonas aeruginosa.

Antimicrob Agents Chemother 2019 11 22;63(11). Epub 2019 Oct 22.

Achaogen Inc., South San Francisco, California, USA.

New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against We report here the antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in phase 1 clinical trials. In addition, we describe the profiles of three additional LpxC inhibitors that were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.
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http://dx.doi.org/10.1128/AAC.00977-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6811409PMC
November 2019

Type IV Pili Can Mediate Bacterial Motility within Epithelial Cells.

mBio 2019 08 20;10(4). Epub 2019 Aug 20.

School of Optometry, University of California, Berkeley, California, USA

is among bacterial pathogens capable of twitching motility, a form of surface-associated movement dependent on type IV pili (T4P). Previously, we showed that T4P and twitching were required for to cause disease in a murine model of corneal infection, to traverse human corneal epithelial multilayers, and to efficiently exit invaded epithelial cells. Here, we used live wide-field fluorescent imaging combined with quantitative image analysis to explore how twitching contributes to epithelial cell egress. Results using time-lapse imaging of cells infected with wild-type PAO1 showed that cytoplasmic bacteria slowly disseminated throughout the cytosol at a median speed of >0.05 μm s while dividing intracellularly. Similar results were obtained with flagellin () and flagellum assembly () mutants, thereby excluding swimming, swarming, and sliding as mechanisms. In contrast, mutants (lacking T4P) and mutants (twitching motility defective) appeared stationary and accumulated in expanding aggregates during intracellular division. Transmission electron microscopy confirmed that these mutants were not trapped within membrane-bound cytosolic compartments. For the wild type, dissemination in the cytosol was not prevented by the depolymerization of actin filaments using latrunculin A and/or the disruption of microtubules using nocodazole. Together, these findings illustrate a novel form of intracellular bacterial motility differing from previously described mechanisms in being directly driven by bacterial motility appendages (T4P) and not depending on polymerized host actin or microtubules. Host cell invasion can contribute to disease pathogenesis by the opportunistic pathogen Previously, we showed that the type III secretion system (T3SS) of invasive strains modulates cell entry and subsequent escape from vacuolar trafficking to host lysosomes. However, we also showed that mutants lacking either type IV pili (T4P) or T4P-dependent twitching motility (i) were defective in traversing cell multilayers, (ii) caused less pathology , and (iii) had a reduced capacity to exit invaded cells. Here, we report that after vacuolar escape, intracellular can use T4P-dependent twitching motility to disseminate throughout the host cell cytoplasm. We further show that this strategy for intracellular dissemination does not depend on flagellin and resists both host actin and host microtubule disruption. This differs from mechanisms used by previously studied pathogens that utilize either host actin or microtubules for intracellular dissemination independently of microbe motility appendages.
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http://dx.doi.org/10.1128/mBio.02880-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703432PMC
August 2019

Optimization of LpxC Inhibitors for Antibacterial Activity and Cardiovascular Safety.

ChemMedChem 2019 08 5;14(16):1560-1572. Epub 2019 Aug 5.

Nanosyn Inc., 3100 Central Expressway, Santa Clara, CA, 95051, USA.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL for parenteral administration and conversion into the active drug with a t of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.
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http://dx.doi.org/10.1002/cmdc.201900287DOI Listing
August 2019

A new player at the flagellar motor: FliL controls both motor output and bias.

mBio 2015 Feb 24;6(2):e02367. Epub 2015 Feb 24.

Department of Molecular Biosciences and Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA

Unlabelled: The bacterial flagellum is driven by a bidirectional rotary motor, which propels bacteria to swim through liquids or swarm over surfaces. While the functions of the major structural and regulatory components of the flagellum are known, the function of the well-conserved FliL protein is not. In Salmonella and Escherichia coli, the absence of FliL leads to a small defect in swimming but complete elimination of swarming. Here, we tracked single motors of these bacteria and found that absence of FliL decreases their speed as well as switching frequency. We demonstrate that FliL interacts strongly with itself, with the MS ring protein FliF, and with the stator proteins MotA and MotB and weakly with the rotor switch protein FliG. These and other experiments show that FliL increases motor output either by recruiting or stabilizing the stators or by increasing their efficiency and contributes additionally to torque generation at higher motor loads. The increased torque enabled by FliL explains why this protein is essential for swarming on an agar surface expected to offer increased resistance to bacterial movement.

Importance: FliL is a well-conserved bacterial flagellar protein whose absence leads to a variety of motility defects, ranging from moderate to complete inhibition of swimming in some bacterial species, inhibition of swarming in others, structural defects that break the flagellar rod during swarming in E. coli and Salmonella, and failure to eject the flagellar filament during the developmental transition of a swimmer to a stalk cell in Caulobacter crescentus. Despite these many phenotypes, a specific function for FliL has remained elusive. Here, we established a central role for FliL at the Salmonella and E. coli motors, where it interacts with both rotor and stator proteins, increases motor output, and contributes to the normal rotational bias of the motor.
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http://dx.doi.org/10.1128/mBio.02367-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358005PMC
February 2015

The c-di-GMP binding protein YcgR controls flagellar motor direction and speed to affect chemotaxis by a "backstop brake" mechanism.

Mol Cell 2010 Apr 25;38(1):128-39. Epub 2010 Mar 25.

Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.

We describe a mechanism of flagellar motor control by the bacterial signaling molecule c-di-GMP, which regulates several cellular behaviors. E. coli and Salmonella have multiple c-di-GMP cyclases and phosphodiesterases, yet absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di-GMP-binding protein, for motility inhibition. We demonstrate that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, most strongly in the presence of c-di-GMP. This interaction reduces the efficiency of torque generation and induces CCW motor bias. We present a "backstop brake" model showing how both effects can result from disrupting the organization of the FliG C-terminal domain, which interacts with the stator protein MotA to generate torque. Inhibition of motility and chemotaxis may represent a strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.
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http://dx.doi.org/10.1016/j.molcel.2010.03.001DOI Listing
April 2010
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