John McKinney - Academia.edu (original) (raw)

Papers by John McKinney

Nature Communications, Dec 3, 2013

Mycobacterium tuberculosis (Mtb) makes 'first contact' with a host in the alveolar space, an inte... more Mycobacterium tuberculosis (Mtb) makes 'first contact' with a host in the alveolar space, an interaction largely inaccessible to experimental observation. We establish a lung-on-chip model for early tuberculosis and use time-lapse imaging to reveal the dynamics of host-Mtb interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models. By reconstituting host physiology in a modular manner, we probe the role of pulmonary surfactant secreted by alveolar epithelial cells (AECs) in early infection. This is difficult to study directly in animal models, as surfactant-deficient animals are either non-viable or develop acute lung pathologies. We demonstrate that surfactant deficiency results in rapid and uncontrolled Mtb growth in both macrophages and AECs. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins 1,2 from the bacterial cell surface and consistent with this mechanism of action, we show that attenuation of bacteria lacking the virulence-associated ESX-1 secretion system is independent of surfactant levels. These findings may partly explain why individuals with compromised surfactant function, such as smokers and elderly persons, are at increased risk of developing active tuberculosis. space 3 and the 'first contact' with a naïve host is by default a single-cell interaction between an Mtb bacillus and a host cell. There is some evidence that pulmonary surfactant plays hostprotective role in these early interactions, but a complete understanding of the role of surfactant is difficult to obtain from animal infection models owing to the lethality of surfactant deficiency. In addition, experiments in animal models 4 cannot provide information about the dynamics of host-Mtb interactions at this early stage with sufficient spatiotemporal resolution 5,6. A commonly-used in vitro model, infection of macrophages with Mtb 7 , has been used to probe the role of certain surfactant components, but these studies cannot address the role of native surfactant secreted by AECs at an air-liquid interface (ALI), a condition that has been reported to alter Mtb physiology 8. Organ-on-chip systems recreate tissue-level complexity in a modular fashion, allowing the number of cellular components, their identity, and environmental complexity to be tailored to mimic key aspects of the relevant physiology, such as an ALI in a lung-on-chip (LoC) 9. These systems have emerged as crucial tools for the replacement of animal models in drug development, toxicity testing, and personalised medicine 10,11. A far less-explored line of enquiry has been to use them as models to study the dynamics of host-pathogen interactions in a realistic physiological setting 12 , where they can combine key advantages of both simpler in vitro models and animal models 13. Here, we develop a LoC model of early TB infection and use time-lapse microscopy to study the infection dynamics for AECs and macrophages as independent sites of first contact, and the impact of surfactant on infection of AECs and macrophages under ALI conditions that mimic the alveolar environment in vivo. Lung-on-chip model of early tuberculosis .

bioRxiv (Cold Spring Harbor Laboratory), Jan 12, 2023

Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of small numbers of bacteri... more Mycobacterium tuberculosis (Mtb) infection is initiated by inhalation of small numbers of bacteria into lung alveoli, where they are phagocytosed by resident macrophages. Intracellular replication of Mtb leads to death of the infected macrophages, release of bacterial aggregates, and rapid growth of the extracellular aggregates on host-cell debris. Here, we show that extracellular Mtb aggregates can evade phagocytosis by killing macrophages in a contact-dependent but uptake-independent manner. We use single-cell time-lapse fluorescence microscopy to show that contact with extracellular Mtb aggregates triggers macrophage plasma membrane perturbation, cytoplasmic calcium accumulation, and pyroptotic cell death. These effects depend on the Mtb type VII secretion system ESX-1, however, this system alone cannot induce calcium accumulation and macrophage death in the absence of the Mtb surface-exposed lipid phthiocerol dimycocerosate. Unexpectedly, we found that ESX-1-mediated secretion of the EsxA/EsxB virulence factors is not required for uptake-independent killing of macrophages after contact with extracellular Mtb aggregates. In the absence of EsxA/EsxB secretion, killing is mediated by the 50-kDa isoform of the ESX-1-secreted protein EspB, while blocking secretion of both EsxA/EsxB and processed EspB reduces killing to background levels. Treatment with a small-molecule ESX-1 inhibitor reduces uptake-independent killing of macrophages by Mtb aggregates, suggesting that novel therapies targeting this antiphagocytic mechanism could prevent the propagation of extracellular bacteria within the lung. .

Cell Reports, Jul 1, 2021

Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutro... more Early invasion of the bladder wall by solitary bacteria protects UPEC from antibiotics and neutrophil swarms in an organoid model Graphical abstract Highlights d A bladder organoid model reproduces key features of urinary tract infections (UTIs) d UPEC forms intracellular bacterial communities (IBCs) in bladder organoids d QIR-like bacteria appear concurrently with and independently of IBCs d QIR-like bacteria are protected from clearance by antibiotics and host immune cells

Molecular and Cellular Biology, Jul 1, 1994

Molecular and Cellular Biology, Jun 1, 1996

The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate... more The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate during the cell cycle. In the absence of a functional Ste12 transcription factor, both the levels and the cell cycle pattern of expression of these genes were affected. FUS1 and AGA1 levels, which are maximally expressed only in G 1-phase cells, were strongly reduced in ste12 ؊ cells. The cell cycle transcription pattern for FAR1 was changed in ste12 ؊ cells: the gene was still significantly expressed in G 2 /M, but transcript levels were strongly reduced in G 1 phase, resulting in a lack of Far1 protein accumulation. G 2 /M transcription of FAR1 was dependent on the transcription factor Mcm1, and expression of a gene with Mcm1 fused to a strong transcriptional activation domain resulted in increased levels of FAR1 transcription. The pattern of cell cycle-regulated transcription of FAR1 could involve combinatorial control by Ste12 and Mcm1. Forced G 1 expression of FAR1 from the GAL1 promoter restored the ability to arrest in response to pheromone in ste12 ؊ cells. This indicates that transcription of FAR1 in the G 1 phase is essential for accumulation of the protein and for pheromone-induced cell cycle arrest.

Genes & Development, May 1, 1993

In budding yeast, a switch between the mutually exclusive pathways of cell cycle progression and ... more In budding yeast, a switch between the mutually exclusive pathways of cell cycle progression and conjugation is controlled at Start in late Gt phase. Mating pheromones promote conjugation by arresting cells in G1 phase before Start. Pheromone-induced cell cycle arrest requires a functional FAR1 gene. We have found that FAR1 transcription and protein accumulation are regulated independently during the cell cycle. FAR1 RNA and protein are highly expressed in early G1, but decline sharply at Start. Farl is phosphorylated just before it disappears at Start, suggesting that modification may target Farl for degradation. Although FAR1 mRNA levels rise again during late S or G 2 phase, reaccumulation of Farl protein to functional levels is restricted until after nuclear division.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resour... more Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

Nature microbiology, Jun 26, 2017

Cell division is tightly controlled in space and time to maintain 2 cell size and ploidy within n... more Cell division is tightly controlled in space and time to maintain 2 cell size and ploidy within narrow bounds. In bacteria, the 3 canonical Minicell (Min) and nucleoid occlusion (Noc) systems 4 together ensure that division is restricted to midcell after 5 completion of chromosome segregation 1. It is unknown how 6 division site selection is controlled in bacteria that lack homo-7 logues of the Min and Noc proteins, including mycobacteria 8 responsible for tuberculosis and other chronic infections 2. 9 Here, we use correlated optical and atomic force microscopy 3,4 10 to demonstrate that morphological landmarks (waveform 11 troughs) on the undulating surface of mycobacterial cells 12 correspond to future sites of cell division. Newborn cells 13 inherit wave troughs from the (grand)mother cell and ultimately 14 divide at the centre-most wave trough, making these morpho-15 logical features the earliest known landmark of future division 16 sites. In cells lacking the chromosome partitioning (Par) 17 system, missegregation of chromosomes is accompanied by 18 asymmetric cell division at off-centre wave troughs, resulting 19 in the formation of anucleate cells. These results demonstrate 20 that inherited morphological landmarks and chromosome 21 positioning together restrict mycobacterial division to the 22 midcell position. 23 Atomic force microscopy (AFM) has been used previously for 24 static 5,6 or Q2 short-term time-lapse 7 imaging of mycobacteria, 25 primarily to study the impact of antibiotics and antimicrobial 26 peptides on nanoscale features of the mycobacterial cell surface. 27 Here, we use long-term time-lapse AFM to track cell growth and 28 division over multiple generations in Mycobacterium smegmatis, a 29 non-pathogenic relative of Mycobacterium tuberculosis (Fig. 1a, 30 Supplementary Videos 1 and 2 and Supplementary Figs 1 and 2). 31 Unexpectedly, we found that the cell surface undulates along the 32 long axis (Fig. 1b,c) in a roughly repeating waveform pattern with 33 an average wavelength of ∼1.8 µm (Supplementary Fig. 3). These 34 morphological features are too small in amplitude (∼100 nm from 35 wave crest to wave trough) to resolve by conventional optical 36 microscopy and they are morphologically distinct from the 37 previously described 'division scars' 8 (Supplementary Fig. 4, last 38 panel, black arrow). Cell elongation is accompanied by an increase 39 in wave trough number, as cells, on average, are born with three 40 wave troughs and divide with four wave troughs after elongating 41 by 2 µm (Supplementary Table 1). In contrast, the distance 42 between wave troughs does not scale with increasing cell 43 length. Cells filamented with ciprofloxacin exhibit a greater 44 number of wave troughs as a function of increased cell length 45 (Supplementary Fig. 6). Conversely, blocking cell elongation 46 with isoniazid (Supplementary Fig. 2a) prevents the formation of 47 new wave troughs (Supplementary Fig. 7). While the undulating surface morphology is maintained in isoniazid-treated cells, height LETTERS

Journal of Bacteriology, Apr 21, 2021

Mycobacteria have unique cell envelopes, surface properties and growth dynamics, which all play a... more Mycobacteria have unique cell envelopes, surface properties and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate and to resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometre-scale ultrastructural and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unravelled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope and cellular processes leading to division. The unique capabilities of AFM, which include sub-diffraction limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties and growth dynamics of this important class of pathogens.

Biomedical Physics & Engineering Express, 2017

Real-time, quantitative characterization of cells at single-cell resolution, particularly while m... more Real-time, quantitative characterization of cells at single-cell resolution, particularly while maintaining their intrinsic properties and without affecting cellular processes, is of primary importance in modern biological assays. Dielectrophoresis is a label-free, real-time, and quantitative technique, and is amenable to integration with other techniques, thus providing new and powerful tools for biology and medicine. In this study we present dielectrophoresis as a characterization tool for Mycobacterium smegmatis single cells. Understanding how phenotypically variant M. smegmatis cells respond dielectrophoretically when subject to the same electric field, could reveal underlying membrane altering mechanisms related to cell death, drug-tolerance, and drug-resistance. In this study, we dielectrophoretically characterized live, heat-treated and antibiotic-treated bacteria. Our results present quantifications of cellular behaviors associated with membrane-specific cell damages and demonstrate adequacy of dielectrophoretic devices in point-of-care diagnostic and monitoring for bacterial infections.

Nature Physics, Oct 21, 2019

Mechanisms to control cell division are essential for cell proliferation and survival 1. Bacteria... more Mechanisms to control cell division are essential for cell proliferation and survival 1. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes 2-4 to maintain mechanical integrity of the cell wall 5. Recent studies suggest that cell separation is governed by mechanical forces 6,7. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here, we use a combination of atomic force microscope (AFM) imaging, nanomechanical mapping, and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with AFM. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division. Marked morphological changes occur when a microbial cell divides to form two daughter cells 1. In Escherichia coli this process involves gradual constriction of the cell envelope and structural remodelling of the new cell poles 2-4. In contrast, other microbial species build a septum without gradual constriction of the cell envelope 8,9. Instead, the cell wall connecting Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are notoriously d... more Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are notoriously difficult to treat due to the ability of UPEC to adhere to and invade urothelial bladder cells. UPEC strains encode a variety of adhesins whose roles in adhesion and invasion are not fully elucidated. Using a transposon insertion library derived from the UPEC clinical isolate CFT073, we developed a high-content screening assay to identify UPEC mutants with defects in early adhesion to human bladder epithelial cells. Of a total of 8,184 mutants screened, we recovered 82 (1.0%) and 54 (0.7%) mutants with decreased and increased adhesion, respectively. Surprisingly, nine low-adhesion hits mapped to the two P pili operons encoded by CFT073, which are usually thought to mediate adhesion to kidney cells rather than bladder cells. These results were reinforced by examination of six high-adhesion hits mapping to the operon coding for F1C pili, where disruption of F1C pili function resulted in inc...

Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to... more Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report a new virulence mechanism by which mycobacteria alter the mechanical stiffness of their cell surface to become refractory to killing during infection. Long-Term Time-Lapse Atomic Force Microscopy was used to reveal a process of “mechanical morphotype switching” in mycobacteria exposed to host intracellular stress. A “soft” mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Genetic manipulation, by deletion of uvrA, or pharmacological treatment, with bedaquiline, locked mycobacteria into a “soft” mechanical morphotype state, enhancing survival in macrophages. Our study proposes microbial mechanical adaptation as a new axis for surviving host-mediated stressors.One-Sentence SummaryBacteria alter their cell sur...

Antibiotics, 2021

To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be adv... more To reveal rare phenotypes in bacterial populations, conventional microbiology tools should be advanced to generate rapid, quantitative, accurate, and high-throughput data. The main drawbacks of widely used traditional methods for antibiotic studies include low sampling rate and averaging data for population measurements. To overcome these limitations, microfluidic-microscopy systems have great promise to produce quantitative single-cell data with high sampling rates. Using Mycobacterium smegmatis cells, we applied both conventional assays and a microfluidic-microscopy method to reveal the antibiotic tolerance mechanisms of wild-type and msm2570::Tn mutant cells. Our results revealed that the enhanced antibiotic tolerance mechanism of the msm2570::Tn mutant was due to the low number of lysed cells during the antibiotic exposure compared to wild-type cells. This is the first study to characterize the antibiotic tolerance phenotype of the msm2570::Tn mutant, which has a transposon inse...

ABSTRACTA DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of dr... more ABSTRACTA DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded “mycobacterial mutasome” – minimally comprising DnaE2 polymerase and ImuA′ and ImuB accessory proteins – remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA ...

Nature Communications, 2020

Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This p... more Mycobacteria grow by inserting new cell wall material in discrete zones at the cell poles. This pattern implies that polar growth zones must be assembled de novo at each division, but the mechanisms that control the initiation of new pole growth are unknown. Here, we combine time-lapse optical and atomic force microscopy to measure single-cell pole growth in mycobacteria with nanometer-scale precision. We show that single-cell growth is biphasic due to a lag phase of variable duration before the new pole transitions from slow to fast growth. This transition and cell division are independent events. The difference between the lag and interdivision times determines the degree of single-cell growth asymmetry, which is high in fast-growing species and low in slow-growing species. We propose a biphasic growth model that is distinct from previous unipolar and bipolar models and resembles “new end take off” (NETO) dynamics of polar growth in fission yeast.

Biophysical Journal, 2019

characterize the interplay between tumor cells and angiogenesis, as well as their proliferation, ... more characterize the interplay between tumor cells and angiogenesis, as well as their proliferation, phenotypic transitions, and death. We use this model to predictearly in course of neoadjuvant therapy-the eventual response of the individual patient. Success in this endeavor would enable replacing an ineffective treatment with an alternative regimen, thereby potentially improving outcomes and curtailing unnecessary toxicities. Furthermore, our approach can be applied to any disease site for which neoadjuvant therapy is indicated and the requisite data is accessible.

Microbiology, 2007

Catabolism of odd-chain-length fatty acids yields acetyl-CoA and propionyl-CoA. A common pathway ... more Catabolism of odd-chain-length fatty acids yields acetyl-CoA and propionyl-CoA. A common pathway of propionyl-CoA metabolism in microorganisms is the methylcitrate cycle, which includes the dedicated enzymes methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). The methylcitrate cycle is essential for propionate metabolism in Mycobacterium tuberculosis. Unusually, M. tuberculosis lacks an MCL orthologue and this activity is provided instead by two isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL1 and ICL2). These bifunctional (ICL/MCL) enzymes are jointly required for propionate metabolism and for growth and survival in mice. In contrast, the non-pathogenic species Mycobacterium smegmatis encodes a canonical MCL enzyme in addition to ICL1 and ICL2. The M. smegmatis gene encoding MCL (prpB) is clustered with genes encoding MCS (prpC) and MCD (prpD). Here we show that deletion of the M. smegmatis prpDBC locus reduced but did not eliminate MCL activity in cell-free extracts. The residual MCL activity was abolished by deletion of icl1 and icl2 in the DprpDBC background, suggesting that these genes encode bifunctional ICL/MCL enzymes. A DprpB Dicl1 Dicl2 mutant was unable to grow on propionate or mixtures of propionate and glucose. We hypothesize that incomplete propionyl-CoA metabolism might cause toxic metabolites to accumulate. Consistent with this idea, deletion of prpC and prpD in the DprpB Dicl1 Dicl2 background paradoxically restored growth on propionate-containing media. These observations suggest that the marked attenuation of ICL1/ICL2-deficient M. tuberculosis in mice could be due to the accumulation of toxic propionyl-CoA metabolites, rather than inability to utilize fatty acids per se.

eLife, 2020

We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynam... more We establish a murine lung-on-chip infection model and use time-lapse imaging to reveal the dynamics of host-Mycobacterium tuberculosis interactions at an air-liquid interface with a spatiotemporal resolution unattainable in animal models and to probe the direct role of pulmonary surfactant in early infection. Surfactant deficiency results in rapid and uncontrolled bacterial growth in both macrophages and alveolar epithelial cells. In contrast, under normal surfactant levels, a significant fraction of intracellular bacteria are non-growing. The surfactant-deficient phenotype is rescued by exogenous addition of surfactant replacement formulations, which have no effect on bacterial viability in the absence of host cells. Surfactant partially removes virulence-associated lipids and proteins from the bacterial cell surface. Consistent with this mechanism, the attenuation of bacteria lacking the ESX-1 secretion system is independent of surfactant levels. These findings may partly explain...