Microbes have an arsenal of virulence factors that contribute to their pathogenicity. A number of challenges remain to fully understand disease transmission, fitness landscape, antimicrobial ...resistance and host heterogeneity. A variety of tools have been used to address diverse aspects of pathogenicity, from molecular host-pathogen interactions to the mechanisms of disease acquisition and transmission. Current gaps in our knowledge include a more direct understanding of host-pathogen interactions, including signaling at interfaces, and direct phenotypic confirmation of pathogenicity. Correlative microscopy has been gaining traction to address the many challenges currently faced in biomedicine, in particular the combination of optical and atomic force microscopy (AFM). AFM, generates high-resolution surface topographical images, and quantifies mechanical properties at the pN scale under physiologically relevant conditions. When combined with optical microscopy, AFM probes pathogen surfaces and their physical and molecular interaction with host cells, while the various modes of optical microscopy view internal cellular responses of the pathogen and host. Here we review the most recent advances in our understanding of pathogens, recent applications of AFM to the field, how correlative AFM-optical microspectroscopy and microscopy have been used to illuminate pathogenicity and how these methods can reach their full potential for studying host-pathogen interactions.
Delftia
is a diverse betaproteobacterial genus with many strains having agricultural and industrial relevance, including plant-growth promotion, bioremediation of hydrocarbon-contaminated soils, and ...heavy metal immobilization.
Delftia
spp. are broadly distributed in the environment, and have been isolated from plant hosts as well as healthy and diseased animal hosts, yet the genetic basis of this ecological versatility has not been characterized. Here, we present a phylogenomic comparison of published
Delftia
genomes and show that the genus is divided into two well-supported clades: one ‘
Delftia acidovorans
’ clade with isolates from soils and plant rhizospheres, and a second ‘
Delftia lacustris
and
Delftia tsuruhatensis
’ clade with isolates from humans and sludge. The pan-genome inferred from 61
Delftia
genomes contained over 28 000 genes, of which only 884 were found in all genomes. Analysis of industrially relevant functions highlighted the ecological versatility of
Delftia
and supported their role as generalists.
is a robust, easily adaptable and culturable bacterium
, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force - laser scanning ...confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in
WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests
cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.
There is a growing need to characterize the effects of environmental stressors at the molecular level on model organisms with the ever increasing number and variety of anthropogenic chemical ...pollutants. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), as one of the most widely applied pesticides in the world, is one such example. This herbicide is known to have non-targeted undesirable effects on humans, animals and soil microbes, but specific molecular targets at sublethal levels are unknown. In this study, we have used Rhizobium leguminosarum bv. viciae 3841 (Rlv) as a nitrogen fixing, beneficial model soil organism to characterize the effects of 2,4-D. Using metabolomics and advanced microscopy we determined specific target pathways in the Rlv metabolic network and consequent changes to its phenotype, surface ultrastructure, and physical properties during sublethal 2,4-D exposure. Auxin and 2,4-D, its structural analogue, showed common morphological changes in vitro which were similar to bacteroids isolated from plant nodules, implying that these changes are related to bacteroid differentiation required for nitrogen fixation. Rlv showed remarkable adaptation capabilities in response to the herbicide, with changes to integral pathways of cellular metabolism and the potential to assimilate 2,4-D with consequent changes to its physical and structural properties. This study identifies biomarkers of 2,4-D in Rlv and offers valuable insights into the mode-of-action of 2,4-D in soil bacteria.
There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser ...scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.
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•Biomarkers of sublethal 2,4-D exposure determined by a unique analytical platform.•E. coli strains were exposed to sublethal levels of 2,4-D.•Exhibit a filamentous phenotype that is ...reversible with polyamine treatment.•Have remodeled cell envelopes and altered vital metabolic pathways.•The majority of effects caused by 2,4-D exposure are related to oxidative stress.
The chlorophenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used extensively worldwide despite its known toxicity and our limited understanding of how it affects non-target organisms. Escherichia coli is a suitable model organism to investigate toxicity and adaptation mechanisms in bacteria exposed to xenobiotic chemicals. We developed a methodical platform that uses atomic force microscopy, metabolomics and biochemical assays to quantify the response of E. coli exposed to sublethal levels of 2,4-D. This herbicide induced a filamentous phenotype in E. coli BL21 and a similar phenotype was observed in a selection of genotypically diverse E. coli strains (A0, A1, B1, and D) isolated from the environment. The filamentous phenotype was observed at concentrations 1000 times below field levels and was reversible upon supplementation with polyamines. Cells treated with 2,4-D had more compliant envelopes, significantly remodeled surfaces that were rougher and altered vital metabolic pathways including oxidative phosphorylation, the ABC transport system, peptidoglycan biosynthesis, amino acid, nucleotide and sugar metabolism. Most of the observed effects could be attributed to oxidative stress, consistent with increases in reactive oxygen species as a function of 2,4-D exposure. This study provides direct evidence that 2,4-D at sublethal levels induces oxidative stress and identifies the associated metabolic changes in E. coli.
Post-translational modification expands the functionality of the proteome beyond genetic encoding, impacting many cellular processes. Cleavage of the carboxyl terminus is one of the many different ...ways proteins can be modified for functionality. Gel-electrophoresis and mass spectrometric-based techniques were used to identify proteins impacted by deficiency of a C-terminal protease, CtpA, in
bv.
3841. Predicted CtpA substrates from 2D silver stained gels were predominantly outer membrane and transport proteins. Proteins with altered abundance in the wild type and
(RL4692) mutant, separated by 2D difference gel electrophoresis, were selected for analysis by mass spectrometry. Of those identified, 9 were the periplasmic solute-binding components of ABC transporters, 5 were amino acid metabolic enzymes, 2 were proteins involved in sulfur metabolism, and 1 each was related to carbon metabolism, protein folding and signal transduction. Alterations to ABC-binding-cassette transporters, nutrient uptake efficiency and to amino acid metabolism indicated an impact on amino acid metabolism and transport for the
mutant, which was validated by measured amino acid levels.
Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in
The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp ...genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.
Antimicrobial resistance is a disseminated global health challenge because many of the genes that cause resistance can transfer horizontally between bacteria. Despite the central role of ...extrachromosomal DNA elements called plasmids in driving the spread of resistance, the detection and surveillance of plasmids remains a significant barrier in molecular epidemiology. We assessed two DNA sequencing platforms alone and in combination for laboratory diagnostics in Botswana by annotating antibiotic resistance genes and plasmids in extensively drug resistant bacteria from diarrhea in Botswana. Long-read Nanopore DNA sequencing and high accuracy basecalling effectively estimated the architecture and gene content of three plasmids in Escherichia coli HUM3355 and two plasmids in Klebsiella pneumoniae HUM7199. Polishing the assemblies with Illumina reads increased base calling precision with small improvements to gene prediction. All five plasmids encoded one or more antibiotic resistance genes, usually within gene islands containing multiple antibiotic and metal resistance genes, and four plasmids encoded genes associated with conjugative transfer. Two plasmids were almost identical to antibiotic resistance plasmids sequenced in Europe and North America from human infection and a pig farm. These One Health connections demonstrate how low-, middle-, and high-income countries collectively benefit from increased whole genome sequencing capacity for surveillance and tracking of infectious diseases and antibiotic resistance genes that can transfer between animal hosts and move across continents.
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