Publications by authors named "Melinda R Grosser"

9 Publications

  • Page 1 of 1

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

Analysis of a COVID-19 Clinical Trial to Emphasize Experimental Design and Quantitative Reasoning in an Introductory Biology Course.

J Microbiol Biol Educ 2021 31;22(1). Epub 2021 Mar 31.

Department of Biology, University of North Carolina Asheville, Asheville, NC 28804.

Increasing student exposure to primary literature in early biology coursework can enhance scientific literacy and quantitative reasoning skills. The efficacy of primary literature discussion is heavily impacted by article selection, as student engagement is optimal with material that is topical and has clear relevance to real world issues. During the COVID-19 pandemic, the prevalence of COVID-19-related scientific research in the mainstream media makes it an ideal topic for current discussion in entry-level biology courses. Here, we present an activity developed to facilitate a remote, synchronous discussion of an open access clinical trial publication on the experimental drug remdesivir in the treatment of COVID-19 (Beigel , 2020, N Engl J Med https://doi.org/10.1056/nejmoa2007764). The activity, which is amenable to adaptation for other research articles, emphasizes concepts in experimental design, statistical analysis, graphical interpretation, and the structure, content, and organization of typical sections of a primary research article. Importantly, the activity highlights the utility of the classroom response tool Pear Deck, a Google Slides add-on, for creating engaging literature discussions that can be readily adapted to a wide variety of teaching modalities.
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http://dx.doi.org/10.1128/jmbe.v22i1.2389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012038PMC
March 2021

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

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

Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence.

PLoS Pathog 2018 03 19;14(3):e1006907. Epub 2018 Mar 19.

Department of Microbiology and Molecular Genetics University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.
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http://dx.doi.org/10.1371/journal.ppat.1006907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884563PMC
March 2018

Expanded Glucose Import Capability Affords Staphylococcus aureus Optimized Glycolytic Flux during Infection.

mBio 2016 06 21;7(3). Epub 2016 Jun 21.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Unlabelled: Acquisition of numerous virulence determinants affords Staphylococcus aureus greater pathogenicity than other skin-colonizing staphylococci in humans. Additionally, the metabolic adaptation of S. aureus to nonrespiratory conditions encountered during infection (e.g., hypoxia, nitric oxide, iron chelation) has been implicated as contributing to S. aureus virulence. Specifically, S. aureus has been shown to ferment glycolytic substrates in nonrespiratory environments encountered within the host. Here, we show that S. aureus has acquired unique carbohydrate transporters that facilitate the maximal uptake of host sugars and serve to support nonrespiratory growth in inflamed tissue. The carbohydrate substrates of 11 S. aureus transporters were identified, and at least four of their genes encode S. aureus glucose transporters (glcA, glcB, glcC, and glcU). Moreover, two transporter genes (glcA and glcC) are unique to S. aureus and contribute disproportionately to the nonrespiratory growth of S. aureus on glucose. Targeted inactivation of sugar transporters reduced glucose uptake and attenuated S. aureus in a murine model of skin and soft tissue infections. These data expand the evidence for metabolic adaptation of S. aureus to invasive infection and demonstrate the specific requirement for the fermentation of glucose over all other available carbohydrates. Ultimately, acquisition of foreign genes allows S. aureus to adopt a metabolic strategy resembling that of infiltrating host immune cells: high glycolytic flux coupled to lactate excretion.

Importance: The bacterial pathogen Staphylococcus aureus causes a wide range of human infections that are costly and difficult to treat. S. aureus differs from closely related commensal staphylococci in its ability to flourish following the invasion of deeper tissue from the skin surface. There, S. aureus primarily uses glucose to grow under respiration-limiting conditions imposed by the immune system. It was previously unclear how S. aureus thrives in this environment when other Staphylococcus species cannot. Our results provide evidence that S. aureus has acquired an expanded repertoire of carbohydrate transporters. In particular, four glucose transporters contribute to efficient S. aureus growth during infection. Thus, S. aureus has evolved to maximize its glucose uptake abilities for enhanced glycolytic flux during tissue invasion. This dependence on glucose acquisition for S. aureus virulence may also explain links between serious infectious complications associated with diabetic patients exhibiting elevated blood glucose levels.
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http://dx.doi.org/10.1128/mBio.00296-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916373PMC
June 2016

Regulatory Requirements for Staphylococcus aureus Nitric Oxide Resistance.

J Bacteriol 2016 08 13;198(15):2043-55. Epub 2016 Jul 13.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Unlabelled: The ability of Staphylococcus aureus to resist host innate immunity augments the severity and pervasiveness of its pathogenesis. Nitric oxide (NO˙) is an innate immune radical that is critical for the efficient clearance of a wide range of microbial pathogens. Exposure of microbes to NO˙ typically results in growth inhibition and induction of stress regulons. S. aureus, however, induces a metabolic state in response to NO˙ that allows for continued replication and precludes stress regulon induction. The regulatory factors mediating this distinctive response remain largely undefined. Here, we employ a targeted transposon screen and transcriptomics to identify and characterize five regulons essential for NO˙ resistance in S. aureus: three virulence regulons not formerly associated with NO˙ resistance, SarA, CodY, and Rot, as well as two regulons with established roles, Fur and SrrAB. We provide new insights into the contributions of Fur and SrrAB during NO˙ stress and show that the S. aureus ΔsarA mutant, the most sensitive of the newly identified mutants, exhibits metabolic dysfunction and widespread transcriptional dysregulation following NO˙ exposure. Altogether, our results broadly characterize the regulatory requirements for NO˙ resistance in S. aureus and suggest an intriguing overlap between the regulation of NO˙ resistance and virulence in this well-adapted human pathogen.

Importance: The prolific human pathogen Staphylococcus aureus is uniquely capable of resisting the antimicrobial radical nitric oxide (NO˙), a crucial component of the innate immune response. However, a complete understanding of how S. aureus regulates an effective response to NO˙ is lacking. Here, we implicate three central virulence regulators, SarA, CodY, and Rot, as major players in the S. aureus NO˙ response. Additionally, we elaborate on the contribution of two regulators, SrrAB and Fur, already known to play a crucial role in S. aureus NO˙ resistance. Our study sheds light on a unique facet of S. aureus pathogenicity and demonstrates that the transcriptional response of S. aureus to NO˙ is highly pleiotropic and intrinsically tied to metabolism and virulence regulation.
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http://dx.doi.org/10.1128/JB.00229-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4944221PMC
August 2016

Method for Preparation and Electroporation of S. aureus and S. epidermidis.

Methods Mol Biol 2016 ;1373:51-7

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 125 Mason Farm Rd, Marsico Hall 6209, Chapel Hill, NC, 27599, USA.

For bacterial species that are not known to be naturally competent, such as Staphylococcus aureus and Staphylococcus epidermidis, electroporation is an efficient method for introducing genetic material into the cell. The technique utilizes electrical pulses to transiently permeabilize bacterial cell membranes, which allows for the passage of plasmid DNA across the membranes. Here, we describe methods for preparing electrocompetent S. aureus and S. epidermidis cells and outline a procedure for electroporation of the prepared competent cells.
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http://dx.doi.org/10.1007/7651_2014_183DOI Listing
August 2016

Commensal microbiota stimulate systemic neutrophil migration through induction of serum amyloid A.

Cell Microbiol 2014 Jul 24;16(7):1053-67. Epub 2014 Jan 24.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC, 27599, USA.

Neutrophils serve critical roles in inflammatory responses to infection and injury, and mechanisms governing their activity represent attractive targets for controlling inflammation. The commensal microbiota is known to regulate the activity of neutrophils and other leucocytes in the intestine, but the systemic impact of the microbiota on neutrophils remains unknown. Here we utilized in vivo imaging in gnotobiotic zebrafish to reveal diverse effects of microbiota colonization on systemic neutrophil development and function. The presence of a microbiota resulted in increased neutrophil number and myeloperoxidase expression, and altered neutrophil localization and migratory behaviours. These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to injury. Genetic analysis identified the microbiota-induced acute phase protein serum amyloid A (Saa) as a host factor mediating microbial stimulation of tissue-specific neutrophil migratory behaviours. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB, and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression. These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signalling axis in mediating neutrophil migratory responses.
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http://dx.doi.org/10.1111/cmi.12257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364439PMC
July 2014
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