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At least 37 records · Page 2

Identification of small-molecule protein–protein interaction inhibitors for NKG2D

NKG2D (natural-killer group 2, member D) is a homodimeric transmembrane receptor that plays an important role in NK, γδ + , and CD8 + T cell-mediated immune responses to environmental stressors such as viral or bacterial infections and oxidative stress. However, aberrant NKG2D signaling has also been associated with chronic inflammatory and autoimmune diseases, and as such NKG2D is thought to be an attractive target for immune intervention. Here, in this study, we describe a comprehensive small-molecule hit identification strategy and two distinct series of protein–protein interaction inhibitors of NKG2D. Although the hits are chemically distinct, they share a unique allosteric mechanism of disrupting ligand binding by accessing a cryptic pocket and causing the two monomers of the NKG2D dimer to open apart and twist relative to one another. Leveraging a suite of biochemical and cell-based assays coupled with structure-based drug design, we established tractable structure–activity relationships with one of the chemical series and successfully improved both the potency and physicochemical properties. Together, we demonstrate that it is possible, albeit challenging, to disrupt the interaction between NKG2D and multiple protein ligands with a single molecule through allosteric modulation of the NKG2D receptor dimer/ligand interface.

59 BASIC BIOLOGICAL SCIENCES↗

Bioorthogonal Click Chemistry for Antibody-Free Profiling of Acetylation, Propionylation, and Butyrylation in Pseudomonas aeruginosa and Methicillin-Resistant Staphylococcus aureus

Lysine acylation is a posttranslational modification (PTM) conserved in all domains of life and is essential for regulating diverse biological processes. Traditional methods for investigating acylation rely on anti-acyl-lysine antibodies, which are costly and time-consuming and often exhibit variable affinity. To remedy these pitfalls, we developed an antibody-free method for bacterial acylome enrichment using bioorthogonal click chemistry coupled with tandem mass spectrometry. We applied this approach to the pathogens Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to explore the biological significance of acylation in each organism. We characterized the acetylome, propionylome, and butyrylome in P. aeruginosa UCBPP-PA14 and the acetylome and propionylome in MRSA. Comparative analyses revealed unique PTM dynamics showing that acylation regulated a wide range of cellular functions, including metabolism, antibiotic resistance, virulence, and stress response. This work establishes the first antibody-free enrichment method for defining bacterial acylomes and provides new insight into global lysine acylation networks in pathogenic bacteria.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Low-Shear modeled microgravity alters the Salmonella enterica serovar typhimurium stress response in an RpoS-independent manner

We have previously demonstrated that low-shear modeled microgravity (low-shear MMG) serves to enhance the virulence of a bacterial pathogen, Salmonella enterica serovar Typhimurium. The Salmonella response to low-shear MMG involves a signaling pathway that we have termed the low-shear MMG stimulon, though the identities of the low-shear MMG stimulon genes and regulatory factors are not known. RpoS is the primary sigma factor required for the expression of genes that are induced upon exposure to different environmental-stress signals and is essential for virulence in mice. Since low-shear MMG induces a Salmonella acid stress response and enhances Salmonella virulence, we reasoned that RpoS would be a likely regulator of the Salmonella low-shear MMG response. Our results demonstrate that low-shear MMG provides cross-resistance to several environmental stresses in both wild-type and isogenic rpoS mutant strains. Growth under low-shear MMG decreased the generation time of both strains in minimal medium and increased the ability of both strains to survive in J774 macrophages. Using DNA microarray analysis, we found no evidence of induction of the RpoS regulon by low-shear MMG but did find that other genes were altered in expression under these conditions in both the wild-type and rpoS mutant strains. Our results indicate that, under the conditions of these studies, RpoS is not required for transmission of the signal that induces the low-shear MMG stimulon. Moreover, our studies also indicate that low-shear MMG can be added to a short list of growth conditions that can serve to preadapt an rpoS mutant for resistance to multiple environmental stresses.

Non-NASA Center↗

Salinity Stress and the Holobiont: Investigating Algal-Bacterial Interactions in Chrysochromulina tobinii

Algae and bacteria form a symbiotic partnership, known as a ”holobiont”, in which both partners provide critical nutrients to each other. Previous research indicates that the composition and structure of the holobiont plays a crucial role in the algal response to environmental stressors, with expression trends varying under stress conditions. Given the increasing prevalence of salinity shifts due to climate change, investigating the responses of these aquatic organisms to salt stress is essential. This is especially true because algae are ecologically and economically important, serving as a nutritive food source for a large range of eco-cohorts and holding potential for biofuel and nutraceutical applications. This study aimed to examine how holobiont community structure is altered in response to salinity shifts and to assess the corresponding changes in gene expression. Metagenomic and metatranscriptomic approaches were utilized to analyze the interactions between the algae, Chrysochromulina tobinii, and its associated bacterial community under varying salinity conditions. Our findings provide insight into the metabolic adaptations of the holobiont and enhance understanding of the survival mechanisms employed in response to osmotic stress.

59 BASIC BIOLOGICAL SCIENCES↗

Challenges and physiological implications of wood feeding in termites

Termites are fascinating insects for a number of reasons, one of which being their specialization on diets of wood lignocellulose. The goal of this review is to consider stress-inducing characteristics of wood and apparent molecular-physiological adaptations in termite guts to overcome these stressors. Defensive factors present in wood include extractive secondary plant metabolites, lignin and related phenolics, crystalline cellulose, and low nitrogen content. Molecular-physiological adaptations of the termite gut to deal with these factors include robust detoxification and antioxidant machinery, the production of a peritrophic matrix and a wide range of cellulases from host and symbiotic sources, and creation of niches available to nitrogen-fixing bacterial symbionts. Considering termite gut physiology and symbioses in the context of stress-response has applied implications. Overall, these outcomes can include development of efficient biomass breakdown strategies, protection of microbes during industrial processing applications, and safeguarding wooden structures from termite damage.

54 ENVIRONMENTAL SCIENCES↗

Phosphate Availability Modulates Root Exudate Composition and Rhizosphere Microbial Community in a Teosinte and a Modern Maize Cultivar

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability.

59 BASIC BIOLOGICAL SCIENCES↗

Identification of a broadly conserved family of enzymes that hydrolyze (p)ppApp

Bacteria produce a variety of nucleotide second messengers to adapt to their surroundings. Although chemically similar, the nucleotides guanosine penta- and tetraphosphate [(p)ppGpp] and adenosine penta- and tetraphosphate [(p)ppApp] have distinct functions in bacteria. (p)ppGpp mediates survival under nutrient-limiting conditions and its intracellular levels are regulated by synthetases and hydrolases belonging to the RelA-SpoT homolog (RSH) family of enzymes. By contrast, (p)ppApp is not known to be involved in nutrient stress responses and is synthesized by RSH-resembling toxins that inhibit the growth of bacterial cells. However, it remains unclear whether there exists a family of hydrolases that specifically act on (p)ppApp to reverse its toxic effects. Here, we present the structure and biochemical characterization of adenosine 3'-pyrophosphohydrolase 1 (Aph1), the founding member of a monofunctional (p)ppApp hydrolase family of enzymes. Our work reveals that Aph1 adopts a histidine-aspartate (HD)-domain fold characteristic of phosphohydrolase metalloenzymes and its activity mitigates the growth inhibitory effects of (p)ppApp-synthesizing toxins. Using an informatic approach, we identify over 2,000 putative (p)ppApp hydrolases that are widely distributed across bacterial phyla and found in diverse genomic contexts, and we demonstrate that 12 representative members hydrolyze ppApp. In addition, our in silico analyses reveal a unique molecular signature that is specific to (p)ppApp hydrolases, and we show that mutation of two residues within this signature broadens the specificity of Aph1 to promiscuously hydrolyze (p)ppGpp in vitro. Overall, our findings indicate that like (p)ppGpp hydrolases, (p)ppApp hydrolases are widespread in bacteria and may play important and underappreciated role(s) in bacterial physiology.

(p)ppApp↗

Biomedical Science and Technology on the International Space Station - Benefits for Humanity (Ciencia Y Tecnologia Biomedica En La Estacion Espacial Internacional - Beneficios Para La Humanidad)

The International Space Station is a unique laboratory for performing investigations that affect human health both in space and on Earth. During its time in orbit, the space station has enabled research that is providing a better understanding of many aspects of human health including aging, trauma, disease and environmental impacts. Driven by the need to support astronaut health, several biological and human physiological investigations have yielded important results that we on Earth can also benefit from. These results include new ways to mitigate bone loss, insights into bacterial behavior, and innovative wound-healing techniques. Advances in telemedicine, disease models, psychological stress response systems, nutrition and cell behavior are just a few more examples of the benefits that have been gained from applying studies in orbit to human health back on Earth.

Love, John E.↗

Ecological and genomic responses of soil microbiomes to high-severity wildfire: linking community assembly to functional potential

Increasing wildfire severity, which is common throughout the western United States, can have deleterious effects on plant regeneration and large impacts on carbon (C) and nitrogen (N) cycling rates. Soil microbes are pivotal in facilitating these elemental cycles, so understanding the impact of increasing fire severity on soil microbial communities is critical. Here, we assess the long-term impact of high-severity fires on the soil microbiome. We find that high-severity wildfires result in a multi-decadal (>25 y) recovery of the soil microbiome mediated by concomitant differences in aboveground vegetation, soil chemistry, and microbial assembly processes. Our results depict a distinct taxonomic and functional successional pattern of increasing selection in post-fire soil microbial communities. Changes in microbiome composition corresponded with changes in microbial functional potential, specifically altered C metabolism and enhanced N cycling potential, which related to rates of potential decomposition and inorganic N availability, respectively. Based on metagenome-assembled genomes, we show that bacterial genomes enriched in our earliest site (4 y since fire) harbor distinct traits such as a robust stress response and a high potential to degrade pyrogenic, polyaromatic C that allow them to thrive in post-fire environments. Taken together, these results provide a biological basis for previously reported process rate measurements and explain the temporal dynamics of post-fire biogeochemistry, which ultimately constrains ecosystem recovery.

59 BASIC BIOLOGICAL SCIENCES↗

Spaceflight-Induced Changes in Microbial Virulence and the Impact to the Host Immune Response

Many microbial pathogens have repeatedly exhibited unexpected responses relevant to infectious disease when grown in microgravity and microgravity analogue environments, including changes in final cell concentration, biofilm production, stress resistance, antibiotic sensitivity, gene expression, host-pathogen interactions, and virulence. Notably, the classic foodborne pathogen Salmonella enterica serovar Typhimurium displayed increased virulence in animals when cultured in either the spaceflight analogue or true spaceflight environment. Recently, Serratia marcescens also was shown to increase virulence when cultured in the spaceflight environment. In parallel, astronaut studies have characterized a persistent spaceflight-induced dysregulation of the human immune system at multiple levels, which suggests an increased risk of infectious diseases. Moreover, astronauts have some degree of clinical infectious disease incidence. However, the contribution of the microgravity environment on host-pathogen interactions and potential for clinical disease remains understudied and poorly characterized. The goal of this study is to gain insight into the breadth of other medically significant microbial pathogens that may exhibit altered virulence and pathogenesis-related responses when cultured in spaceflight analogue conditions. Specifically, we are characterizing the effect of spaceflight analogue culture (Low Shear Modeled Microgravity/LSMMG) on microbial pathogenesis-related stress responses, in vitro host-pathogen interactions, gene expression, and virulence potential in animals using five important model bacterial pathogens, Salmonella enterica Enteritidis, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus pneumoniae, and enterohemorrhagic Escherichia coli. The information to date is providing a better understanding into the potential impact of microgravity on alterations in microbial virulence and associated infectious disease risk to crew health during spaceflight missions.

C M Ott↗

Drought Shifts Sorghum Root Metabolite and Microbiome Profiles and Enriches for Pipecolic Acid

Plant-associated microbial communities shift in composition as a result of environmental perturbations, such as drought. It has been shown that Actinobacteria are enriched in plant roots and rhizospheres during drought stress. However, the correlations between microbiome dynamics and plant response to drought are poorly understood. Here we apply a combination of bacterial community composition analysis and plant metabolite profiling in Sorghum bicolor roots, rhizospheres, and soil during drought and drought recovery to investigate potential contributions of host metabolism to shifts in bacterial composition. Our results provide a detailed view of metabolic shifts across the plant root during drought and show that the response to rewatering differs between root and soil; additionally, we identify drought-responsive metabolites that are highly correlated with the observed changes in Actinobacteria abundance. Furthermore, we find that pipecolic acid is a drought-enriched metabolite in sorghum roots, and that exogenous application of pipecolic acid inhibits root growth. Finally, we show that this activity functions independent of the systemic acquired resistance pathway and has the potential to impact Actinobacterial taxa within the root microbiome.

165 RRNA↗

Mechanical morphotype switching as an adaptive response in mycobacteria

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 the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction 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. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

59 BASIC BIOLOGICAL SCIENCES↗

Desiccation induces varied responses within a soil bacterial genus

Abstract Desiccation impacts a suite of physiological processes in microbes by elevating levels of damaging reactive oxygen species and inducing DNA strand breaks. In response to desiccation‐induced stress, microbes have evolved specialized mechanisms to help them survive. Here, we performed a 128‐day lab desiccation experiment on nine strains from three clades of an abundant soil bacterium, Curtobacterium . We sequenced RNA from each strain at three time points to investigate their response. Curtobacterium was highly resistant to desiccation, outlasting both Escherichia coli and a famously DNA damage‐resistant bacterium, Deinococcus radiodurans . However, within the genus, there were also 10‐fold differences in survival rates among strains. Transcriptomic profiling revealed responses shared within the genus including up‐regulation of genes involved in DNA damage repair, osmolyte production, and efflux pumps, but also up‐regulation of pathways and genes unique to the three clades. For example, trehalose synthesis gene otsB , the chaperone groEL , and the oxygen scavenger katA were all found in either one or two clades but not the third. Here, we provide evidence of considerable variation in closely related strains, and further elucidation of the phylogenetic conservation of desiccation tolerance remains an important goal for microbial ecologists.

59 BASIC BIOLOGICAL SCIENCES↗

Targeting the Bet-Hedging Strategy with an Inhibitor of Bacterial Efflux Capacity Enhances Antibiotic Efficiency and Ameliorates Bacterial Persistence In Vitro

Persistence is a bet-hedging strategy in bacterial populations that increases antibiotic tolerance and leads to the establishment of latent infections. In this study, we demonstrated that a synthetic non-toxic taxane-based reversal agent (tRA), developed as an inhibitor of ABC transporter systems in mammalian cancer cells, enhanced antibiotic killing of persister populations from different pathogens, including Burkholderia, Pseudomonas, Francisella, and Yersinia. Acting as an inhibitor of bacterial efflux at 100 nM, tRA99020 enhanced antibiotic efficiency and suppressed the production of natural products of Burkholderia species polyketide synthase (PKS) function. We demonstrate that the metabolites produced by PKS in response to stress by different antibiotics act as inhibitors of mammalian histone deacetylase activity and stimulate cell death. Applying a single-molecule fluorescence in situ hybridization (smFISH) assay, we analyzed on a single-cell level the activation profiles of the persistence regulating pks gene in Burkholderia thailandensis treated with tRA99020 and antibiotics. We posit that a multi-pronged approach encompassing antibiotic therapies and inhibition of efflux systems and fatty acid catabolism will be required for efficient eradication of persistent bacterial populations.

59 BASIC BIOLOGICAL SCIENCES↗

CRAGE-RB-PI-seq reveals transcriptional dynamics of plant-associated bacteria during root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because of low bacterial biomass and interference from plant RNA. To overcome this, we developed a randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) combined with chassis-independent recombinase-assisted genome engineering (CRAGE). Using Pseudomonas simiae WCS417 as a model rhizobacterium, this method enabled targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during Arabidopsis root colonization. Our analysis revealed temporally resolved transcriptional regulation, including those associated with cell growth, chemotaxis, plant immune suppression, biofilm formation, and stress responses, reflecting the coordinated physiological adaptation to the root environment. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor is crucial for evading plant immune systems. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

59 BASIC BIOLOGICAL SCIENCES↗

Effects of Spaceflight Relevant Carbon Dioxide Levels on Pathogenesis Related Microbial Characteristics

Multiple stressors in the spaceflight environment, including microgravity and radiation, have the potential to alter microbial pathogenesis and virulence characteristics. Understanding how these and other factors may exacerbate the risk of infectious disease for astronauts is important to ensure the success of the mission. One understudied question in our assessment of infectious disease risk is the contribution carbon dioxide (CO2) at atmospheric concentrations higher than would be observed in terrestrial settings. Previous terrestrial studies have shown altered gene expression, selective bacterial inhibition, increased growth and diversity, and increased antibiotic resistance of bacterial communities individually and in biofilms when exposed to increased levels of CO2. While many of these findings are based on levels of CO2 higher than would be found during spaceflight exploration missions, the potential for subtle changes in CO2 levels to exacerbate infectious disease risks warrants an evaluation of pathogen responses to spaceflight CO2 conditions. To address this question, we are evaluating the effects of elevated atmospheric CO2 on microbial concentration, diversity, and pathogenesis-related characteristics using medically significant microorganisms cultured at CO2 levels representing terrestrial atmospheric concentration (≈0.04 %), ISS elevated concentration (0.4 %), and an unexpectedly elevated concentration (1 %) to represent a worst-case scenario. To determine if microbial responses to these CO2 concentrations may be exacerbated in response to the spaceflight environment, microorganisms are being cultured under static conditions and both spaceflight analogue and control conditions using the Rotating Wall Vessel (RWV) bioreactor (Synthecon, Houston, TX). For these studies, we will evaluate the response of Staphylococcus aureus, Streptococcus pneumoniae, and Enterobacter aerogenes, which are opportunistic bacterial pathogens that are of medical significance and have been or are likely to be found aboard human-inhabited spacecraft. Bacteria will be evaluated individually and together as a consortium. The following Specific Aims are be investigated: Aim 1. Characterize the growth, diversity, and pathogenesis-related stress responses of medically significant microorganisms grown under multiple CO2 concentrations under static growth in a liquid medium. Stress responses include acid stress, oxidative stress, and thermal stress. Aim 2. Characterize the growth, diversity, and pathogenesis-related stress responses of medically significant microorganisms grown under multiple CO2 concentrations in both spaceflight analogue and control conditions. Stress responses include acid stress, oxidative stress, and thermal stress.

S G Thornhill↗

2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice

Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis. The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes. Female BDF1 mice were injected with 2-DG (500 mg/kg b. wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L. monocytogenes. Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection. In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge. The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments. First, 2-DG did not inhibit listeria replication in trypticase soy broth. Second, replication of L. monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG. Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L. monocytogenes. These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens.

Non-NASA Center↗

Constraints on the Metabolic Activity of Microorganisms in Atacama Surface Soils Inferred from Refractory Biomarkers: Implications for Martian Habitability and Biomarker Detection

Dryness is one of the main environmental challenges to microbial survival. Understanding the threshold of microbial tolerance to extreme dryness is relevant to better constrain the environmental limits of life on Earth and critically evaluate long-term habitability models of Mars. Biomolecular proxies for microbial adaptation and growth were measured in Mars-like hyper-arid surface soils in the Atacama Desert that experience only a few millimeters of precipitation per decade, and in biologically active soils a few hundred kilometers away that experience two- to fivefold more precipitation. Diversity and abundance of lipids and other biomolecules decreased with increasing dryness. Cyclopropane fatty acids (CFAs), which are indicative of adaptive response to environmental stress and growth in bacteria, were only detected in the wetter surface soils. The ratio of trans to cis isomers of an unsaturated fatty acid, another bacterial stress indicator, decreased with increasingly dry conditions. Aspartic acid racemization ratios increased from 0.01 in the wetter soils to 0.1 in the driest soils, which is indicative of racemization rates comparable to de novo biosynthesis over long timescales (10,000 years). The content and integrity of stress proteins profiled by immunoassays were additional indicators that biomass in the driest soils is sin situ microbial growth in the driest surface soils of the Atacama, and any metabolic activity is likely to be basal for cellular repair and maintenance only. Our data add to a growing body of evidence that the driest Atacama surface soils represent a threshold for long-term habitability (i.e., growth and reproduction). These results place constraints on the potential for extant life on the surface of Mars, which is 100–1000 times drier than the driest regions in the Atacama.

Wilhelm, Mary Beth↗