NOTE: Extended to 4/30/2022 per F. Hernandez/ARC (Ed., 7/27/21)

NOTE: Extended to 4/30/2021 per NSSC information (Ed., 7/22/2020)

NOTE: Extended to 4/30/2020 per NSSC information (Ed., 4/21/2020)

NOTE: Extended to 3/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/11/19)

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)

The overriding hypothesis for this project is that exposure of the yeast, C. albicans to microgravity will alter gene expression and morphology, consistent with a potential increase in virulence. More specifically, diminished fluid shear may result in alterations to the physical environment that contribute, directly or indirectly, to adaptations in the yeast cell surface resulting in increased virulence. Notably, these studies will further explore and document the genotypic and phenotypic parameters of C. albicans associated with pathogenicity, identify specific environmental influences on the physiological adaptation processes, and provide insight into mechanisms used by higher eukaryotes when adapting to spaceflight conditions.

To assess yeast responses to microgravity, the following flight experiments have been conducted in flight hardware provided by BioServe Space Technologies, Boulder, CO.

SpX CRS-16 • Fluid Processing Apparatus (FPA) in Group Activation Packs (GAP) with 10 GAPs (5 ea flight and ground) containing 80 FPAs (40 ea flight and ground) to assess the gas microenvironment. SpX CRS-17 • Fluorinated ethylene propylene (FEP) bags were used for serial cultivation and antifungal testing of yeast. • Human monocytes (THP-1) were cultured in 12-well BioCells and challenged with UV-killed C. albicans or sham inoculation. SpX CRS-21 (partial reflight) • FEP bags for serial cultivation of yeast • THP-1 cells cultured in 6 wells of a 12-well BioCell (no yeast challenge condition).

Temperature for flight samples was controlled through the use of an on-orbit incubator, SABL, at 4C, 30C, and 37C. Frozen samples were stored in an on-orbit freezer, GLACIER or equivalent, and transferred to Cold Stowage for return (< -32C). Temperature for ground controls was controlled by standard incubators/refrigerators set at 4C, 30C, and 37C. Freezing was achieved in a standard -80C freezer.

In preparation for these flight experiments, a full scale experiment verification test (EVT) was conducted in the Principal Investigator (PI) laboratory with BioServe personnel onsite. The science team consisted of the PI and teams of two (SpX CRS-16) or four (SpX CRS-17) students (one graduate student and 5 undergraduate students). SpX CRS-21 was conducted by the PI alone due to its smaller size and COVID-related precautions.

All ground controls were conducted near synchronously, with a time offset predetermined for each payload based on timing and complexity of operations, and established by real-time communication with BioServe. For SpX CRS-16, ground GAPs were stored on the horizontal and rotated 180° daily. When at 30C, the ground GAPs were laid on the horizontal and very slowly rocked lengthwise. For SpX CRS-17 and -21, FEP bags were maintained in a horizontal ‘flat’ orientation and flipped once during the incubation period. BioCell units were maintained in a habitat with the plate laying flat.

The main goal of the most recent payload, Micro-14A, was to serially culture C. albicans over a period of several days to establish whether exposure to microgravity over many generations had an impact on the yeast adaptation responses. Yeast Extract–Peptone–Dextrose (YPD) medium was launched preloaded in FEP bags and the yeast was launched in water-induced stasis. Once on orbit, the yeast was inoculated into the first-in-series FEP bag and cultured at 30C. The following day, an aliquot from the culture was diluted and used to inoculate the second-in-series FEP bag. This cycle continued through 5 days of growth (approx. 70 generations). Cells were frozen each day for analyses upon sample return. Cell density, viability, metabolic gas production, and antifungal resistance were each evaluated.

In aggregate, these studies have provided the rare opportunity to repeat experiments in different flight hardware and incrementally extend the studies.

NOTE: Extended to 4/30/2022 per F. Hernandez/ARC (Ed., 7/27/21)

NOTE: Extended to 4/30/2021 per NSSC information (Ed., 7/22/2020)

NOTE: Extended to 4/30/2020 per NSSC information (Ed., 4/21/2020)

NOTE: Extended to 3/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/11/19)

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)

The overriding hypothesis for this project is that exposure of the yeast, C. albicans, to microgravity will alter gene expression and morphology, consistent with a potential increase in virulence. More specifically, diminished fluid shear may result in alterations to the physical environment that contribute, directly or indirectly, to adaptations in the yeast cell surface, resulting in increased virulence. Notably, these studies will further explore and document the genotypic and phenotypic parameters of C. albicans associated with pathogenicity, identify specific environmental influences on the physiological adaptation processes, and provide insight into mechanisms used by higher eukaryotes when adapting to spaceflight conditions.

To assess yeast responses to microgravity, the following flight experiments have been conducted in flight hardware provided by BioServe Space Technologies, Boulder, CO.

SpX CRS-16 • Fluid Processing Apparatus (FPA) in Group Activation Packs (GAP) with 10 GAPs (5 ea flight and ground) containing 80 FPAs (40 ea flight and ground) to assess the gas microenvironment. SpX CRS-17 • Fluorinated ethylene propylene (FEP) bags were used for serial cultivation and antifungal testing of yeast. • Human monocytes (THP-1) were cultured in 12-well BioCells and challenged with UV-killed C. albicans or sham inoculation. SpX CRS-21 (partial reflight) • FEP bags for serial cultivation of yeast • THP-1 cells cultured in 6 wells of a 12-well BioCell (no yeast challenge condition).

Temperature for flight samples was controlled through the use of an on-orbit incubator, SABL, at 4C, 30C, and 37C. Frozen samples were stored in an on-orbit freezer, GLACIER or equivalent, and transferred to Cold Stowage for return (< -32C). Temperature for ground controls was controlled by standard incubators/refrigerators set at 4C, 30C, and 37C. Freezing was achieved in a standard -80C freezer.

In preparation for these flight experiments, a full scale experiment verification test (EVT) was conducted in the Principal Investigator (PI) laboratory with BioServe personnel on site. The science team consisted of the PI and teams of two (SpX CRS-16) or four (SpX CRS-17) students (one graduate student and 5 undergraduate students). SpX CRS-21 was conducted by the PI alone due to its smaller size and COVID-related precautions.

All ground controls were conducted near synchronously, with a time offset predetermined for each payload based on timing and complexity of operations, and established by real time communication with BioServe. For SpX CRS-16, ground GAPs were stored on the horizontal and rotated 180° daily. When at 30C, the ground GAPs were laid on the horizontal and very slowly rocked lengthwise. For SpX CRS-17 and -21, FEP bags were maintained in a horizontal ‘flat’ orientation and flipped once during the incubation period. BioCell units were maintained in a habitat with the plate laying flat.

The main goal of the most recent payload, Micro-14A, was to serially culture C. albicans over a period of several days to establish whether exposure to microgravity over many generations had an impact on the yeast adaptation responses. Yeast Extract–Peptone–Dextrose (YPD) medium was launched preloaded in FEP bags and the yeast was launched in water-induced stasis. Once in orbit, the yeast was inoculated into the first-in-series FEP bag and cultured at 30C. The following day, an aliquot from the culture was diluted and used to inoculate the second-in-series FEP bag. This cycle continued through 5 days of growth (approx. 70 generations). Cells were frozen each day for analyses upon sample return. Cell density, viability, metabolic gas production, and antifungal resistance were each evaluated. In addition, RNA was isolated from each of the samples and submitted for gene expression analysis. The data are currently being analyzed.

In aggregate, these and previous flight studies have provided the rare opportunity to repeat experiments in different flight hardware, and to then compare the results to that obtained in one type of ground-based simulator. An initial analysis has been published: Growth and antifungal resistance of the pathogenic yeast, Candida albicans, in the microgravity environment of the International Space Station: an aggregate of multiple flight experiences. Nielsen S et al. "Growth and antifungal resistance of the pathogenic yeast, Candida albicans, in the microgravity environment of the International Space Station: An aggregate of multiple flight experiences." [Ed. Note. Reference: Nielsen S, White K, Preiss K, Peart D, Gianoulias K, Juel R, Sutton J, McKinney J, Bender J, Pinc G, Bergren K, Gans W, Kelley J, McQuaid M. "Growth and antifungal resistance of the pathogenic yeast, Candida albicans, in the microgravity environment of the International Space Station: An aggregate of multiple flight experiences." Life (Basel). 2021 Mar 27;11(4):283. https://doi.org/10.3390/life11040283.]

NOTE: Extended to 4/30/2021 per NSSC information (Ed., 7/22/2020)

NOTE: Extended to 4/30/2020 per NSSC information (Ed., 4/21/2020)

NOTE: Extended to 3/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/11/19)

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)

From terrestrial experiments and previous flights, we have seen alterations in Candida albicans that appear to be induced by microgravity/low fluid shear. Specifically, the increases in filamentous cell morphology, resistance to antifungal agents, and altered colony morphology are also consistent with increased virulence. Due to the absence of fluid shear in microgravity, we propose that through metabolic activity yeast are influencing their local environment by depleting the oxygen and elevating the carbon dioxide levels. The Micro-14 FPA/GAP experiment was designed to specifically analyze the effects of an altered gas environment and inform whether cells are adapting to these changes during flight. The experiments were built in FPAs with medium that had been equilibrated with carbon dioxide and/or nitrogen (hypoxia) or oxygen to determine whether the effects of microgravity could be augmented or diminished, respectively.

Temperature recorders were included in several GAPs and representative tracings suggest the ground samples were exposed to a lower ambient temperature than were the flight samples. The impacts of this difference were minimal since the cells were in stasis during the storage phase. The post-termination storage temperature was also lower in ground samples, but they didn’t freeze so viability was retained.

Several outcome measures have been evaluated. Cells were returned viable such that colony structure could be evaluated. Cells in both flight and ground conditions grew comparably, although there was significant variability depending on the gas environment in which they were cultured, with cells growing poorly in hypoxic conditions and the combination of elevated CO2 and hypoxia. Cell viability was determined by colony formation and varied from a low of 5% in mixed gas to a high of 63% in atmospheric conditions. Terrestrial samples retained a greater level of viability throughout the conditions as compared to the flight samples. Variations in colony morphology were not observed.

Previous studies evaluating the adaptation responses of C. albicans to microgravity have also demonstrated changes in sensitivity to the antifungal agent, Amphotericin B. Although the cells grown in variable gas conditions could not be tested for antifungal drug sensitivity during flight, due to technical constraints, samples were returned and subjected to post-flight growth in various concentrations of the drug. Cells cultured in microgravity exhibited increased resistance to Amphotericin B, particularly the higher drug concentrations, as compared to the terrestrial controls. This data is consistent with that obtained in previous payloads and ground-based bioreactor studies.

The experiment flown on Space X-17 had several components, including an analysis of the interaction between human monocytes and yeast, when one or the other was cultured in space. Importantly, we also had the opportunity to dilute and subculture the yeast allowing an analysis of long term culture for the first time. Unfortunately, there were some sample treatment inconsistencies that prevented us from analyzing these samples to the extent required. A reflight opportunity has been approved and we are currently preparing for the upcoming payload.

NOTE: Extended to 4/30/2019 per F. Hernandez/ARC (Ed., 11/2/17)

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)

Our overriding hypothesis is that exposure of C. albicans to microgravity will alter gene expression and morphology, consistent with a potential increase in virulence. More specifically, we suggest that diminished fluid shear results in alterations to the physical environment that contribute, directly or indirectly, to adaptations in the yeast cell surface resulting in increased virulence. Notably, these studies will further explore and document the genotypic and phenotypic parameters of C. albicans associated with pathogenicity, identify specific environmental influences on the physiological adaptation processes, and provide insight into mechanisms used by higher eukaryotes when adapting to spaceflight conditions.

Objectives/Aims of Investigation

• Evaluate the microgravity-induced alterations in biosynthetic regulation, cellular content, and subcellular localization of ergosterol and b-glucans.

• Delineate the contributions of fluid shear, oxygen depletion, and carbon dioxide enrichment in the microenvironment to cellular adaptation responses.

• Characterize the effect of spaceflight on C. albicans virulence using a human monocyte host.

The experiments included in the payload that flew on Space X CRS-16 specifically addressed Aim #2 (internally the payload is being referred to Micro-14alpha). Additional experiments flew on Space X CRS-17 and will address the other aims listed above (Micro-14beta). Initial reporting for both aspects of this payload will occur mid-summer 2019.

A full scale experiment verification test (EVT) (including all hardware components for the full Micro-14 payload) was initiated on 9/24/18 with BioServe Space Technologies personnel on site.

The 6-well BioCell and FEP (fluorinated ethylene-propylene) bag experiments were carried to completion as planned and on schedule. The Fluid Processing Apparatus (FPA) in experiments required some final adaptations.

Ultimately, each phase of the FPA experiment was completed, albeit with overlapping timelines. Selection for Flight permission was granted on 11/14/18, with some reservations due to the compartmentalized nature of the FPA EVT.

Experiment preparation:

FPA loading spanned 11/29/18 (L-5) through 12/1/18 (L-3, including a reload of one condition – N2). FPAs were handed over to BioServe on 12/2/18 at 8 am for incorporation into Group Activation Packs (GAP). GAPs were turned over to CMC on 12/3/18 at 8 am. Launch was delayed 24 hrs to 12/5/18 at 1:16 pm, without implications for the Micro-14a payload.

On-orbit Operations:

Spaceflight time line -- all ground controls were conducted with a 1-hr offset based on real time communication with BioServe. Ground GAPs were stored on the horizontal and rotated 180° daily. When at 30°C, the ground GAPs were laid on the horizontal and very slowly rocked length wise.

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)

NOTE: End date changed to 10/31/2017 per NSSC information (Ed., 11/29/16)