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Tissues and organs provide the structural and biochemical landscapes upon which microbial pathogens and commensals function to regulate health and disease. While flat two-dimensional (2-D) monolayers composed of a single cell type have provided important insight into understanding host-pathogen interactions and infectious disease mechanisms, these reductionist models lack many essential features present in the native host microenvironment that are known to regulate infection, including three-dimensional (3-D) architecture, multicellular complexity, commensal microbiota, gas exchange and nutrient gradients, and physiologically relevant biomechanical forces (e.g., fluid shear, stretch, compression). A major challenge in tissue engineering for infectious disease research is recreating this dynamic 3-D microenvironment (biological, chemical, and physical/mechanical) to more accurately model the initiation and progression of host-pathogen interactions in the laboratory. Here we review selected 3-D models of human intestinal mucosa, which represent a major portal of entry for infectious pathogens and an important niche for commensal microbiota. We highlight seminal studies that have used these models to interrogate host-pathogen interactions and infectious disease mechanisms, and we present this literature in the appropriate historical context. Models discussed include 3-D organotypic cultures engineered in the rotating wall vessel (RWV) bioreactor, extracellular matrix (ECM)-embedded/organoid models, and organ-on-a-chip (OAC) models. Collectively, these technologies provide a more physiologically relevant and predictive framework for investigating infectious disease mechanisms and antimicrobial therapies at the intersection of the host, microbe, and their local microenvironments.
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Microambiente Celular , Interações Hospedeiro-Patógeno , Mucosa Intestinal/fisiologia , Técnicas de Cultura de Órgãos/métodos , Organoides , Engenharia Tecidual/métodos , História do Século XX , História do Século XXI , Humanos , Modelos Biológicos , Técnicas de Cultura de Órgãos/história , Engenharia Tecidual/históriaRESUMO
SUMMARYUnderstanding the dynamic adaptive plasticity of microorganisms has been advanced by studying their responses to extreme environments. Spaceflight research platforms provide a unique opportunity to study microbial characteristics in new extreme adaptational modes, including sustained exposure to reduced forces of gravity and associated low fluid shear force conditions. Under these conditions, unexpected microbial responses occur, including alterations in virulence, antibiotic and stress resistance, biofilm formation, metabolism, motility, and gene expression, which are not observed using conventional experimental approaches. Here, we review biological and physical mechanisms that regulate microbial responses to spaceflight and spaceflight analog environments from both the microbe and host-microbe perspective that are relevant to human health and habitat sustainability. We highlight instrumentation and technology used in spaceflight microbiology experiments, their limitations, and advances necessary to enable next-generation research. As spaceflight experiments are relatively rare, we discuss ground-based analogs that mimic aspects of microbial responses to reduced gravity in spaceflight, including those that reduce mechanical forces of fluid flow over cell surfaces which also simulate conditions encountered by microorganisms during their terrestrial lifecycles. As spaceflight mission durations increase with traditional astronauts and commercial space programs send civilian crews with underlying health conditions, microorganisms will continue to play increasingly critical roles in health and habitat sustainability, thus defining a new dimension of occupational health. The ability of microorganisms to adapt, survive, and evolve in the spaceflight environment is important for future human space endeavors and provides opportunities for innovative biological and technological advances to benefit life on Earth.
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The ability of bacteria to sense and respond to mechanical forces has important implications for pathogens during infection, as they experience wide fluid shear fluctuations in the host. However, little is known about how mechanical forces encountered in the infected host drive microbial pathogenesis. Herein, we combined mathematical modeling with hydrodynamic bacterial culture to profile transcriptomic and pathogenesis-related phenotypes of multidrug resistant S. Typhimurium (ST313 D23580) under different fluid shear conditions relevant to its transition from the intestinal tract to the bloodstream. We report that D23580 exhibited incremental changes in transcriptomic profiles that correlated with its pathogenic phenotypes in response to these progressive increases in fluid shear. This is the first demonstration that incremental changes in fluid shear forces alter stress responses and gene expression in any ST313 strain and offers mechanistic insight into how forces encountered by bacteria during infection might impact their disease-causing ability in unexpected ways.
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Farmacorresistência Bacteriana Múltipla , Fenótipo , Salmonella typhimurium , Salmonella typhimurium/genética , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Salmonella/microbiologia , Infecções por Salmonella/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Hidrodinâmica , Transcriptoma , Estresse MecânicoRESUMO
The quorum sensing signal N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C(12) HSL), produced by Pseudomonas aeruginosa, exerts cytotoxic effects in macrophages in vitro, which is believed to affect host innate immunity in vivo. However, the medical significance of this finding to pulmonary disease remains unclear since the multicellular complexity of the lung was not considered in the assessment of macrophage responses to 3-oxo-C(12) HSL. We developed a novel three-dimensional co-culture model of alveolar epithelium and macrophages using the rotating wall vessel (RWV) bioreactor, by adding undifferentiated monocytes to RWV-derived alveolar epithelium. Our three-dimensional model expressed important architectural/phenotypic hallmarks of the parental tissue, as evidenced by highly differentiated epithelium, spontaneous differentiation of monocytes to functional macrophage-like cells, localization of these cells on the alveolar surface and a macrophage-to-epithelial cell ratio relevant to the in vivo situation. Co-cultivation of macrophages with alveolar epithelium counteracted 3-oxo-C(12) HSL-induced cytotoxicity via removal of quorum sensing molecules by alveolar cells. Furthermore, 3-oxo-C(12) HSL induced the intercellular adhesion molecule ICAM-1 in both alveolar epithelium and macrophages. These data stress the importance of multicellular organotypic models to integrate the role of different cell types in overall lung homeostasis and disease development in response to external factors.
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4-Butirolactona/análogos & derivados , Homosserina/análogos & derivados , Macrófagos/fisiologia , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/fisiologia , Percepção de Quorum , 4-Butirolactona/metabolismo , 4-Butirolactona/toxicidade , Técnicas de Cocultura , Células Epiteliais/imunologia , Células Epiteliais/fisiologia , Citometria de Fluxo , Homosserina/metabolismo , Homosserina/toxicidade , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Pulmão/imunologia , Microscopia Confocal , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Mucosa Respiratória/imunologia , Transdução de Sinais , Células U937RESUMO
The discovery that biomechanical forces regulate microbial virulence was established with the finding that physiological low fluid shear (LFS) forces altered gene expression, stress responses, and virulence of the enteric pathogen Salmonella enterica serovar Typhimurium during the log phase. These log phase LFS-induced phenotypes were independent of the master stress response regulator, RpoS (σS). Given the central importance of RpoS in regulating stationary-phase stress responses of S. Typhimurium cultured under conventional shake flask and static conditions, we examined its role in stationary-phase cultures grown under physiological LFS. We constructed an isogenic rpoS mutant derivative of wild-type S. Typhimurium and compared the ability of these strains to survive in vitro pathogenesis-related stresses that mimic those encountered in the infected host and environment. We also compared the ability of these strains to colonize (adhere, invade, and survive within) human intestinal epithelial cell cultures. Unexpectedly, LFS-induced resistance of stationary-phase S. Typhimurium cultures to acid and bile salts stresses did not rely on RpoS. Likewise, RpoS was dispensable for stationary-phase LFS cultures to adhere to and survive within intestinal epithelial cells. In contrast, the resistance of these cultures to challenges of oxidative and thermal stresses, and their invasion into intestinal epithelial cells was influenced by RpoS. These findings expand our mechanistic understanding of how physiological fluid shear forces modulate stationary-phase S. Typhimurium physiology in unexpected ways and provide clues into microbial mechanobiology and nuances of Salmonella responses to microenvironmental niches in the infected host. IMPORTANCE Bacterial pathogens respond dynamically to a variety of stresses in the infected host, including physical forces of fluid flow (fluid shear) across their surfaces. While pathogens experience wide fluctuations in fluid shear during infection, little is known about how these forces regulate microbial pathogenesis. This is especially important for stationary-phase bacterial growth, which is a critical period to understand microbial resistance, survival, and infection potential, and is regulated in many bacteria by the general stationary-phase stress response protein RpoS. Here, we showed that, unlike conventional culture conditions, several stationary-phase Salmonella pathogenic stress responses were not impacted by RpoS when bacteria were cultured under fluid shear conditions relevant to those encountered in the intestine of the infected host. These findings offer new insight into how physiological fluid shear forces encountered by Salmonella during infection might impact pathogenic responses in unexpected ways that are relevant to their disease-causing ability.
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Salmonella typhimurium , Fator sigma , Ácidos/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Salmonella typhimurium/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Virulência/genéticaRESUMO
Physical forces associated with spaceflight and spaceflight analogue culture regulate a wide range of physiological responses by both bacterial and mammalian cells that can impact infection. However, our mechanistic understanding of how these environments regulate host-pathogen interactions in humans is poorly understood. Using a spaceflight analogue low fluid shear culture system, we investigated the effect of Low Shear Modeled Microgravity (LSMMG) culture on the colonization of Salmonella Typhimurium in a 3-D biomimetic model of human colonic epithelium containing macrophages. RNA-seq profiling of stationary phase wild type and Δhfq mutant bacteria alone indicated that LSMMG culture induced global changes in gene expression in both strains and that the RNA binding protein Hfq played a significant role in regulating the transcriptional response of the pathogen to LSMMG culture. However, a core set of genes important for adhesion, invasion, and motility were commonly induced in both strains. LSMMG culture enhanced the colonization (adherence, invasion and intracellular survival) of Salmonella in this advanced model of intestinal epithelium using a mechanism that was independent of Hfq. Although S. Typhimurium Δhfq mutants are normally defective for invasion when grown as conventional shaking cultures, LSMMG conditions unexpectedly enabled high levels of colonization by an isogenic Δhfq mutant. In response to infection with either the wild type or mutant, host cells upregulated transcripts involved in inflammation, tissue remodeling, and wound healing during intracellular survival. Interestingly, infection by the Δhfq mutant led to fewer transcriptional differences between LSMMG- and control-infected host cells relative to infection with the wild type strain. This is the first study to investigate the effect of LSMMG culture on the interaction between S. Typhimurium and a 3-D model of human intestinal tissue. These findings advance our understanding of how physical forces can impact the early stages of human enteric salmonellosis.
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Biomimética , Voo Espacial , Animais , Técnicas de Cocultura , Interações Hospedeiro-Patógeno , Humanos , Mamíferos , Salmonella typhimurium/genéticaRESUMO
Salmonella enterica serovar Typhimurium possesses a stimulon of genes that are differentially regulated in response to conditions of low fluid shear force that increase bacterial virulence and alter other phenotypes. In this study, we show that a previously uncharacterized member of this stimulon, ydcI or STM1625, encodes a highly conserved DNA binding protein with related homologs present in a range of gram-negative bacterial genera. Gene expression analysis shows that ydcI is expressed in different bacterial genera and is involved in its autoregulation in S. Typhimurium. We demonstrate that purified YdcI protein specifically binds a DNA probe consisting of its own promoter sequence. We constructed an S. Typhimurium ΔydcI mutant strain and show that this strain is more sensitive to both organic and inorganic acid stress than is an isogenic WT strain, and this defect is complemented in trans. Moreover, our data indicate that ydcI is part of the rpoS regulon related to stress resistance. The S. Typhimurium ΔydcI mutant was able to invade cultured cells to the same degree as the WT strain, but a strain in which ydcI expression is induced invaded cells at a level 2.8 times higher than that of the WT. In addition, induction of ydcI expression in S. Typhimurium resulted in the formation of a biofilm in stationary-phase cultures. These data indicate the ydcI gene encodes a conserved DNA binding protein involved with aspects of prokaryotic biology related to stress resistance and possibly virulence.
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Proteínas de Bactérias/metabolismo , Sequência Conservada , DNA Bacteriano/metabolismo , Salmonella typhimurium/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biofilmes , Regulação Bacteriana da Expressão Gênica/fisiologia , Anotação de Sequência Molecular , Mutação , Salmonella typhimurium/genética , Estresse FisiológicoRESUMO
The opportunistic pathogen Staphylococcus aureus encounters a wide variety of fluid shear levels within the human host, and they may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. By using rotating-wall vessel bioreactors to create a physiologically relevant, low-fluid-shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low-fluid-shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear-cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole-genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed downregulation of the RNA chaperone Hfq, which parallels low-fluid-shear responses of certain Gram-negative organisms. This is the first study to report an Hfq association with fluid shear in a Gram-positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low-fluid-shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during the initial host-pathogen interaction.
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Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/biossíntese , Staphylococcus aureus/fisiologia , Reatores Biológicos , Perfilação da Expressão Gênica , Análise em Microsséries , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/patogenicidade , Estresse Fisiológico , Fatores de Virulência/biossínteseRESUMO
Assessing bacterial behavior in microgravity is important for risk assessment and prevention of infectious diseases during spaceflight missions. Furthermore, this research field allows the unveiling of novel connections between low-fluid-shear regions encountered by pathogens during their natural infection process and bacterial virulence. This study is the first to characterize the spaceflight-induced global transcriptional and proteomic responses of Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat. P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq as a global transcriptional regulator. Since Hfq was also differentially regulated in spaceflight-grown Salmonella enterica serovar Typhimurium, Hfq represents the first spaceflight-induced regulator acting across bacterial species. The major P. aeruginosa virulence-related genes induced in spaceflight were the lecA and lecB lectin genes and the gene for rhamnosyltransferase (rhlA), which is involved in rhamnolipid production. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data for this organism grown in microgravity analogue conditions using the rotating wall vessel (RWV) bioreactor. Interesting similarities were observed, including, among others, similarities with regard to Hfq regulation and oxygen metabolism. While RWV-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight presumably adopted an anaerobic mode of growth, in which denitrification was most prominent. Whether the observed changes in pathogenesis-related gene expression in response to spaceflight culture could lead to an alteration of virulence in P. aeruginosa remains to be determined and will be important for infectious disease risk assessment and prevention, both during spaceflight missions and for the general public.
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Fator Proteico 1 do Hospedeiro/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Voo Espacial , Ausência de Peso , Adesinas Bacterianas/genética , Proteínas de Bactérias/genética , Desnitrificação , Sistemas Ecológicos Fechados , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Hexosiltransferases/genética , Lectinas/genética , Dados de Sequência Molecular , Oxigênio/metabolismo , Proteômica , Pseudomonas aeruginosa/patogenicidade , Salmonella enterica/genética , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/metabolismo , Transcrição Gênica , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Spaceflight uniquely alters the physiology of both human cells and microbial pathogens, stimulating cellular and molecular changes directly relevant to infectious disease. However, the influence of this environment on host-pathogen interactions remains poorly understood. Here we report our results from the STL-IMMUNE study flown aboard Space Shuttle mission STS-131, which investigated multi-omic responses (transcriptomic, proteomic) of human intestinal epithelial cells to infection with Salmonella Typhimurium when both host and pathogen were simultaneously exposed to spaceflight. To our knowledge, this was the first in-flight infection and dual RNA-seq analysis using human cells.
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The long-term response of microbial communities to the microgravity environment of space is not yet fully understood. Of special interest is the possibility that members of these communities may acquire antibiotic resistance. In this study, Escherichia coli cells were grown under low-shear modeled microgravity (LSMMG) conditions for over 1,000 generations (1000G) using chloramphenicol treatment between cycles to prevent contamination. The results were compared with data from an earlier control study done under identical conditions using steam sterilization between cycles rather than chloramphenicol. The sensitivity of the final 1000G-adapted strain to a variety of antibiotics was determined using Vitek analysis. In addition to resistance to chloramphenicol, the adapted strain acquired resistance to cefalotin, cefuroxime, cefuroxime axetil, cefoxitin, and tetracycline. In fact, the resistance to chloramphenicol and cefalotin persisted for over 110 generations despite the removal of both LSMMG conditions and trace antibiotic exposure. Genome sequencing of the adapted strain revealed 22 major changes, including 3 transposon-mediated rearrangements (TMRs). Two TMRs disrupted coding genes (involved in bacterial adhesion), while the third resulted in the deletion of an entire segment (14,314 bp) of the genome, which includes 14 genes involved with motility and chemotaxis. These results are in stark contrast with data from our earlier control study in which cells grown under the identical conditions without antibiotic exposure never acquired antibiotic resistance. Overall, LSMMG does not appear to alter the antibiotic stress resistance seen in microbial ecosystems not exposed to microgravity.IMPORTANCE Stress factors experienced during space include microgravity, sleep deprivation, radiation, isolation, and microbial contamination, all of which can promote immune suppression (1, 2). Under these conditions, the risk of infection from opportunistic pathogens increases significantly, particularly during long-term missions (3). If infection occurs, it is important that the infectious agent should not be antibiotic resistant. Minimizing the occurrence of antibiotic resistance is, therefore, highly desirable. To facilitate this, it is important to better understand the long-term response of bacteria to the microgravity environment. This study demonstrated that the use of antibiotics as a preventive measure could be counterproductive and would likely result in persistent resistance to that antibiotic. In addition, unintended resistance to other antimicrobials might also occur as well as permanent genome changes that might have other unanticipated and undesirable consequences.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Estresse Mecânico , Ausência de Peso , Adaptação Biológica , Cloranfenicol/farmacologia , Elementos de DNA Transponíveis , Rearranjo Gênico , Tetraciclina/farmacologia , Sequenciamento Completo do Genoma , beta-Lactamas/farmacologiaRESUMO
Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.
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Bactérias/crescimento & desenvolvimento , Ausência de Peso , Adaptação Fisiológica , Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/microbiologia , Infecções Bacterianas/fisiopatologia , Reatores Biológicos , Células Cultivadas , Previsões , Regulação Bacteriana da Expressão Gênica , Mecanotransdução Celular , Voo Espacial , Estresse MecânicoRESUMO
Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a three-dimensional (3D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells, as has been demonstrated in non-neuronal cell lines. In our studies comparing 3D versus two-dimensional (2D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA-binding protein HuD was decreased in 3D culture as compared to standard 2D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of the two culture types, and indicated that alterations in the G1/S cell-cycle progression contributed to the diminished doubling rate in the 3D-cultured SY cells. These results demonstrate that a 3D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.
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Neurônios/citologia , Neurônios/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Técnicas de Cultura de Células/métodos , Proteínas de Ciclo Celular/genética , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular Transformada , Proliferação de Células , Forma Celular/fisiologia , Proteínas ELAV/genética , Proteína Semelhante a ELAV 4 , Perfilação da Expressão Gênica , Genes cdc/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos/métodos , Células PC12 , Fenótipo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RatosRESUMO
Recent studies have established that dysregulation of the human immune system and the reactivation of latent herpesviruses persists for the duration of a 6-month orbital spaceflight. It appears certain aspects of adaptive immunity are dysregulated during flight, yet some aspects of innate immunity are heightened. Interaction between adaptive and innate immunity also seems to be altered. Some crews experience persistent hypersensitivity reactions during flight. This phenomenon may, in synergy with extended duration and galactic radiation exposure, increase specific crew clinical risks during deep space exploration missions. The clinical challenge is based upon both the frequency of these phenomena in multiple crewmembers during low earth orbit missions and the inability to predict which specific individual crewmembers will experience these changes. Thus, a general countermeasure approach that offers the broadest possible coverage is needed. The vehicles, architecture, and mission profiles to enable such voyages are now under development. These include deployment and use of a cis-Lunar station (mid 2020s) with possible Moon surface operations, to be followed by multiple Mars flyby missions, and eventual human Mars surface exploration. Current ISS studies will continue to characterize physiological dysregulation associated with prolonged orbital spaceflight. However, sufficient information exists to begin consideration of both the need for, and nature of, specific immune countermeasures to ensure astronaut health. This article will review relevant in-place operational countermeasures onboard ISS and discuss a myriad of potential immune countermeasures for exploration missions. Discussion points include nutritional supplementation and functional foods, exercise and immunity, pharmacological options, the relationship between bone and immune countermeasures, and vaccination to mitigate herpes (and possibly other) virus risks. As the immune system has sentinel connectivity within every other physiological system, translational effects must be considered for all potential immune countermeasures. Finally, we shall discuss immune countermeasures in the context of their individualized implementation or precision medicine, based on crewmember specific immunological biases.
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BACKGROUND: Extra-cellular shear force is an important environmental parameter that is significant both medically and in the space environment. Escherichia coli cells grown in a low-shear modeled microgravity (LSMMG) environment produced in a high aspect rotating vessel (HARV) were subjected to transcriptional and physiological analysis. RESULTS: Aerobic LSMMG cultures were grown in rich (LB) and minimal (MOPS + glucose) medium with a normal gravity vector HARV control. Reproducible changes in transcription were seen, but no specific LSMMG responsive genes were identified. Instead, absence of shear and a randomized gravity vector appears to cause local extra-cellular environmental changes, which elicit reproducible cellular responses. In minimal media, the majority of the significantly up- or down-regulated genes of known function were associated with the cell envelope. In rich medium, most LSMMG down-regulated genes were involved in translation. No observable changes in post-culture stress responses and antibiotic sensitivity were seen in cells immediately after exposure to LSMMG. Comparison with earlier studies of Salmonella enterica serovar Typhimurium conducted under similar growth conditions, revealed essentially no similarity in the genes that were significantly up- or down-regulated. CONCLUSION: Comparison of these results to previous studies suggests that different organisms may dramatically differ in their responses to medically significant low-shear and space environments. Depending on their specific response, some organisms, such as Salmonella, may become preadapted in a manner that predisposes them to increased virulence.
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Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Ausência de Peso , Aerobiose , Antibacterianos/farmacologia , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Microorganisms impact spaceflight in a variety of ways. They play a positive role in biological systems, such as waste water treatment but can be problematic through buildups of biofilms that can affect advanced life support. Of special concern is the possibility that during extended missions, the microgravity environment will provide positive selection for undesirable genomic changes. Such changes could affect microbial antibiotic sensitivity and possibly pathogenicity. To evaluate this possibility, Escherichia coli (lac plus) cells were grown for over 1000 generations on Luria Broth medium under low-shear modeled microgravity conditions in a high aspect rotating vessel. This is the first study of its kind to grow bacteria for multiple generations over an extended period under low-shear modeled microgravity. Comparisons were made to a non-adaptive control strain using growth competitions. After 1000 generations, the final low-shear modeled microgravity-adapted strain readily outcompeted the unadapted lac minus strain. A portion of this advantage was maintained when the low-shear modeled microgravity strain was first grown in a shake flask environment for 10, 20, or 30 generations of growth. Genomic sequencing of the 1000 generation strain revealed 16 mutations. Of the five changes affecting codons, none were neutral. It is not clear how significant these mutations are as individual changes or as a group. It is concluded that part of the long-term adaptation to low-shear modeled microgravity is likely genomic. The strain was monitored for acquisition of antibiotic resistance by VITEK analysis throughout the adaptation period. Despite the evidence of genomic adaptation, resistance to a variety of antibiotics was never observed.
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Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.
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In vitro cell culture models used to study how Salmonella initiates disease at the intestinal epithelium would benefit from the recognition that organs and tissues function in a three-dimensional (3-D) environment and that this spatial context is necessary for development of cultures that more realistically resemble in vivo tissues/organs. Our aim was to establish and characterize biologically meaningful 3-D models of human colonic epithelium and apply them to study the early stages of enteric salmonellosis. The human colonic cell line HT-29 was cultured in 3-D and characterized by immunohistochemistry, histology, and scanning electron microscopy. Wild-type Salmonella typhimurium and an isogenic SPI-1 type three secretion system (TTSS) mutant derivative (invA) were used to compare the interactions with 3-D cells and monolayers in adherence/invasion, tissue pathology, and cytokine expression studies. The results showed that 3-D culture enhanced many characteristics normally associated with fully differentiated, functional intestinal epithelia in vivo, including better organization of junctional, extracellular matrix, and brush-border proteins, and highly localized mucin production. Wild-type Salmonella demonstrated increased adherence, but significantly lower invasion for 3-D cells. Interestingly, the SPI-I TTSS mutant showed wild-type ability to invade into the 3-D cells but did not cause significant structural changes to these cells. Moreover, 3-D cells produced less interleukin-8 before and after Salmonella infection. These results suggest that 3-D cultures of human colonic epithelium provide valuable alternative models to study human enteric salmonellosis with potential for novel insight into Salmonella pathogenesis.
Assuntos
Técnicas de Cultura de Células , Colo/microbiologia , Mucosa Intestinal/microbiologia , Organoides/microbiologia , Salmonella typhimurium/patogenicidade , Aderência Bacteriana , Colo/citologia , Citoplasma/microbiologia , Células HT29 , Humanos , Imuno-Histoquímica , Interleucina-8/biossíntese , Mucosa Intestinal/citologia , Microscopia Eletrônica de Varredura , Organoides/química , Organoides/citologia , Organoides/ultraestrutura , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologiaRESUMO
Salmonella enterica serovar Typhimurium strains belonging to sequence type ST313 are a major cause of fatal bacteremia among HIV-infected adults and children in sub-Saharan Africa. Unlike "classical" non-typhoidal Salmonella (NTS), gastroenteritis is often absent during ST313 infections and isolates are most commonly recovered from blood, rather than from stool. This is consistent with observations in animals, in which ST313 strains displayed lower levels of intestinal colonization and higher recovery from deeper tissues relative to classic NTS isolates. A better understanding of the key environmental factors regulating these systemic infections is urgently needed. Our previous studies using dynamic Rotating Wall Vessel (RWV) bioreactor technology demonstrated that physiological levels of fluid shear regulate virulence, gene expression, and stress response profiles of classic S. Typhimurium. Here we provide the first demonstration that fluid shear alters the virulence potential and pathogenesis-related stress responses of ST313 strain D23580 in a manner that differs from classic NTS.