In parallel to the observations regarding microgravity exposure and bacterial virulence, multiple studies onboard ISS have documented and characterized the dysregulation of the human immune system associated with spaceflight. The phenomenon is generally characterized by altered leukocyte reductions in T and Natural Killer (NK) cell function, altered plasma cytokine profiles, and the reactivation of latent herpesviruses. Most of these studies were generally performed by returning astronaut biosamples for evaluation. Akin to microbial studies, spaceflight analogue cell culture is also well established as a terrestrial analogue that mimics key aspects of microgravity on immune cell activation. For example, both T and NK cells exhibit inhibited activation during spaceflight analogue cell culture.

Unknown, and almost completely uninvestigated, is the possible synergistic effect between increased microbial virulence and reductions in immune cell function during microgravity/spaceflight conditions. Should virulence increase for multiple pathogens during crewed deep space missions, synergy with diminished immunity could increase crew health risk for infectious diseases during pending missions of exploration. The NASA Human Research Program created a specific ‘Knowledge Gap’ in their Integrated Research Plan regarding this issue, but to date, no study has provided an integrated systematic approach to address this Gap.

Using spaceflight analogue technology, the proposed studies are incorporating microbial and mammalian cell culture, animal studies, and ISS crew immune cell studies in an integrated systematic approach to better understand how these systems shape the dynamics of the host-pathogen interaction that lead to infectious disease in microgravity conditions. Our hypothesis is that the incidence of higher virulence observed in both spaceflight and spaceflight analogue culture for the foodborne pathogen S. Typhimurium is not limited to this organism, and that multiple bacteria will exhibit similar increases in virulence when cultured under spaceflight analogue conditions. We further hypothesize that spaceflight-induced alterations in crew immune cell function will lead to compromised defenses against pathogen infection, which when combined with alterations in microbial virulence, will lead to a synergistic response that will reflect greater risk to crew health.

Accordingly, the goal of this study is to gain an understanding of medically important microorganisms relevant to crew health that exhibit altered virulence and pathogenesis-related properties and the impact of those changes on the crew immune cell response using spaceflight analogue culture conditions. This study is incorporating relevant ISS bacterial pathogens that have either been identified from operational microbial monitoring activities or that have a clear route of infection for the crew, including potential foodborne pathogens applicable to future development of bioregenerative food systems.

Insufficient time for solicitation: Continued delays in initiating the proposed study will continue to impact the schedule and decrease our likelihood of gaining the knowledge needed to close the risk. Note that the delay in this work may impact the Path to Risk Reduction (PRR) color change from yellow to green and put the studies outside of the window for use of the International Space Station (ISS). Two prior solicitations have been released (in 2009 and 2014) for ground-based proposals to understand microbial responses to simulated microgravity. Even though the prior solicitations were written clearly, the selected studies did not focus on identifying the microbial alterations that would gain the understanding needed to inform the risk, and they did not produce the needed ground-based investigations on mechanisms. The 2009 selection addresses collective changes of organisms within the human microbiome, and the 2014 selection addresses viral reactivation. The selected studies will provide information applicable to the gaps Micro-101 to better understand the potential impact of microgravity on microbial virulence and Micro-201 to better understand the contribution of these changes on adverse health events. Completion of the proposed work will provide clear evidence as to the operational applicability of these original microbial virulence data to a variety of microorganisms and will include measurements of host immune responses to microbial challenge.

Access to Previous Crew Data: This proposed study will leverage previous microbiology operational and research data as well as previously published immunology research data to provide a better understanding of impacts of microbial changes to the host and to determine the need for countermeasure evaluation as outlined in our PRR.

Salmonella Enteritidis: • Growth kinetics, stress responses, infection of 2-D monolayers and 3-D tissue culture models have been completed. One virulence trial in mice has been completed, with a second trial planned in October 2023. Transcriptomic studies are ongoing. Manuscript in preparation.

Pseudomonas aeruginosa: • Growth kinetics and stress responses have been completed. 3-D tissue culture infections and transcriptomic studies are ongoing.

Burkholderia cepacia: • Growth kinetics and several stress responses have been completed. 3-D tissue culture infections and transcriptomic studies are ongoing.

Streptococcus pneumoniae: • Obtained a new S. pneumoniae strain due to limited survival characteristics of the original strain. Growth conditions and media requirements are being optimized. Enterohemorrhagic E. coli: • Growth kinetics completed. Stress responses have been completed. Two trials of 2-D macrophages infections and 3-D tissue culture infections have been completed with triplicate trials to be completed shortly. Transcriptomic studies are ongoing. One virulence trial in mice has been completed, with a second trial planned in October 2023.

In parallel to the observations regarding microgravity exposure and bacterial virulence, multiple studies onboard ISS have documented and characterized the dysregulation of the human immune system associated with spaceflight. The phenomenon is generally characterized by altered leukocyte reductions in T and Natural Killer (NK) cell function, altered plasma cytokine profiles, and the reactivation of latent herpesviruses. Most of these studies were generally performed by returning astronaut biosamples for evaluation. Akin to microbial studies, spaceflight analogue cell culture is also well established as a terrestrial analogue that mimics key aspects of microgravity on immune cell activation. For example, both T and NK cells exhibit inhibited activation during spaceflight analogue cell culture.

Unknown, and almost completely uninvestigated, is the possible synergistic effect between increased microbial virulence and reductions in immune cell function during microgravity/spaceflight conditions. Should virulence increase for multiple pathogens during crewed deep space missions, synergy with diminished immunity could increase crew health risk for infectious diseases during pending missions of exploration. The NASA Human Research Program created a specific ‘Knowledge Gap’ in their Integrated Research Plan regarding this issue, but to date, no study has provided an integrated systematic approach to address this Gap.

Using spaceflight analogue technology, the proposed studies are incorporating microbial and mammalian cell culture, animal studies, and ISS crew immune cell studies in an integrated systematic approach to better understand how these systems shape the dynamics of the host-pathogen interaction that lead to infectious disease in microgravity conditions. Our hypothesis is that the incidence of higher virulence observed in both spaceflight and spaceflight analogue culture for the foodborne pathogen S. Typhimurium is not limited to this organism, and that multiple bacteria will exhibit similar increases in virulence when cultured under spaceflight analogue conditions. We further hypothesize that spaceflight-induced alterations in crew immune cell function will lead to compromised defenses against pathogen infection, which when combined with alterations in microbial virulence, will lead to a synergistic response that will reflect greater risk to crew health.

Accordingly, the goal of this study is to gain an understanding of medically important microorganisms relevant to crew health that exhibit altered virulence and pathogenesis-related properties and the impact of those changes on the crew immune cell response using spaceflight analogue culture conditions. This study is incorporating relevant ISS bacterial pathogens that have either been identified from operational microbial monitoring activities or that have a clear route of infection for the crew, including potential foodborne pathogens applicable to future development of bioregenerative food systems.

Insufficient time for solicitation: Continued delays in initiating the proposed study will continue to impact the schedule and decrease our likelihood of gaining the knowledge needed to close the risk. Note that the delay in this work may impact the Path to Risk Reduction (PRR) color change from yellow to green and put the studies outside of the window for use of the International Space Station (ISS). Two prior solicitations have been released (in 2009 and 2014) for ground-based proposals to understand microbial responses to simulated microgravity. Even though the prior solicitations were written clearly, the selected studies did not focus on identifying the microbial alterations that would gain the understanding needed to inform the risk, and they did not produce the needed ground-based investigations on mechanisms. The 2009 selection addresses collective changes of organisms within the human microbiome, and the 2014 selection addresses viral reactivation. The selected studies will provide information applicable to the gaps Micro-101 to better understand the potential impact of microgravity on microbial virulence and Micro-201 to better understand the contribution of these changes on adverse health events. Completion of the proposed work will provide clear evidence as to the operational applicability of these original microbial virulence data to a variety of microorganisms and will include measurements of host immune responses to microbial challenge.

Access to Previous Crew Data: This proposed study will leverage previous microbiology operational and research data as well as previously published immunology research data to provide a better understanding of impacts of microbial changes to the host and to determine the need for countermeasure evaluation as outlined in our PRR.

Salmonella Enteritidis: • Growth kinetics at Arizona State University (ASU) were repeated and validated at Johnson Space Center (JSC) in support of astronaut blood immunological analysis and preparation for animal infection experiments.

Pseudomonas aeruginosa: • Obtained new PA14 strain due to atypical phenotypic variations in the original strain and repeated and validated growth kinetics for this organism at ASU and JSC multiple times for statistical relevance • Growth kinetics at ASU were repeated and validated at JSC in support of astronaut blood immunological analysis and preparation for animal infection experiments

Burkholderia cepacia: • Stress assay testing on this microorganism continued • Currently preparing for 3-D tissue culture infection studies

Streptococcus pneumoniae: • Obtained a new S. pneumoniae strain due to limited survival characteristics of the original strain • We have been working closely with the Consultants to optimize growth conditions for subsequent studies

Enterohemorrhagic E. coli: • ASU worked closely with JSC and Consultants to refine the appropriate time point for phenotypic analysis to avoid variability associated with autolysin production by this strain • Growth kinetics completed multiple times for statistical relevance by both JSC and ASU • Using the updated time point, in vitro stress assays are underway

The Institutional Animal Care and Use Committee (IACUC) proposal has been revised in accordance with JSC IACUC recommendations and has been resubmitted for review.

ASU continues to provide technical support to JSC for growth conditions in support of the Immunological component of this study.

In parallel to the observations regarding microgravity exposure and bacterial virulence, multiple studies onboard ISS have documented and characterized the dysregulation of the human immune system associated with spaceflight. The phenomenon is generally characterized by altered leukocyte reductions in T and Natural Killer (NK) cell function, altered plasma cytokine profiles, and the reactivation of latent herpesviruses. Most of these studies were generally performed by returning astronaut biosamples for evaluation. Akin to microbial studies, spaceflight analogue cell culture is also well established as a terrestrial analogue that mimics key aspects of microgravity on immune cell activation. For example, both T and NK cells exhibit inhibited activation during spaceflight analogue cell culture.

Unknown, and almost completely uninvestigated, is the possible synergistic effect between increased microbial virulence and reductions in immune cell function during microgravity/spaceflight conditions. Should virulence increase for multiple pathogens during crewed deep space missions, synergy with diminished immunity could increase crew health risk for infectious diseases during pending missions of exploration. The NASA Human Research Program created a specific ‘Knowledge Gap’ in their Integrated Research Plan regarding this issue, but to date, no study has provided an integrated systematic approach to address this Gap.

Using spaceflight analogue technology, the proposed studies will incorporate microbial and mammalian cell culture, animal studies, and ISS crew immune cell studies in an integrated systematic approach to better understand how these systems shape the dynamics of the host-pathogen interaction that lead to infectious disease in microgravity conditions. Our hypothesis is that the incidence of higher virulence observed in both spaceflight and spaceflight analogue culture for the foodborne pathogen S. Typhimurium is not limited to this organism, and that multiple bacteria will exhibit similar increases in virulence when cultured under spaceflight analogue conditions. We further hypothesize that spaceflight-induced alterations in crew immune cell function will lead to compromised defenses against pathogen infection, which when combined with alterations in microbial virulence, will lead to a synergistic response that will reflect greater risk to crew health.

Accordingly, the goal of this study is to gain an understanding of medically important microorganisms relevant to crew health that exhibit altered virulence and pathogenesis-related properties and the impact of those changes on the crew immune cell response using spaceflight analogue culture conditions. This study will incorporate relevant ISS bacterial pathogens that have either been identified from operational microbial monitoring activities or that have a clear route of infection for the crew, including potential foodborne pathogens applicable to future development of bioregenerative food systems.

Insufficient time for solicitation: Continued delays in initiating the proposed study will continue to impact the schedule and decrease our likelihood of gaining the knowledge needed to close the risk. Note that the delay in this work may impact the Path to Risk Reduction (PRR) color change from yellow to green and put the studies outside of the window for use of the International Space Station (ISS). Two prior solicitations have been released (in 2009 and 2014) for ground-based proposals to understand microbial responses to simulated microgravity. Even though the prior solicitations were written clearly, the selected studies did not focus on identifying the microbial alterations that would gain the understanding needed to inform the risk, and they did not produce the needed ground-based investigations on mechanisms. The 2009 selection addresses collective changes of organisms within the human microbiome, and the 2014 selection addresses viral reactivation. The selected studies will provide information applicable to the gaps Micro-101 to better understand the potential impact of microgravity on microbial virulence and Micro-201 to better understand the contribution of these changes on adverse health events. Completion of the proposed work will provide clear evidence as to the operational applicability of these original microbial virulence data to a variety of microorganisms and will include measurements of host immune responses to microbial challenge.

Access to Previous Crew Data: This proposed study will leverage previous microbiology operational and research data as well as previously published immunology research data to provide a better understanding of impacts of microbial changes to the host and to determine the need for countermeasure evaluation as outlined in our PRR.

Salmonella Enteritidis:

• Characterization of spaceflight analogue culture on growth kinetics, stress responses, and in vitro infection of 3-D biomimetic intestinal cell culture models, and fixation of samples for RNA has been completed.

Pseudomonas aeruginosa:

• Growth kinetics completed for this organism multiple times for statistical relevance; • Initiating RNA isolation

Burkholderia cepacia:

• Growth kinetics completed multiple times for statistical relevance ; • Several in vitro stress assays performed multiple times for statistical relevance.

Streptococcus pneumoniae:

• Optimization of growth conditions and media requirements for Streptococcus pneumoniae.

Enterohemorrhagic E. coli:

• Growth kinetics completed multiple times for statistical relevance ; • Several in vitro stress assays performed multiple times for statistical relevance.

ASU Institutional Animal Care and Use Committee (IACUC) approval has been obtained for all animal infection studies using respiratory pathogens.

Participation of NASA Johnson Space Center (JSC) in animal infection studies using intestinal pathogens has been discussed with JSC IACUC.

JSC IACUC panel review of animal studies occurred in summer 2021.

ASU supplied four test organisms to NASA JSC in support of the Immunological component of this study.

ASU provided technical support to JSC for growth conditions in support of the Immunological component of this study.

Specific Aims:

Aim 1: Characterize the effect of spaceflight analogue culture on microbial pathogenesis related stress responses and in vitro host-pathogen interactions. Analysis will include microbial stress responses as well as colonization and viability following pathogen challenge of three-dimensional (3-D) tissue co-culture models containing immune cells.

Aim 2: Characterize the effect of spaceflight analogue culture on the virulence potential of pathogenic microorganisms. Changes in virulence will be assessed using a mouse model of infection. To reduce and refine the use of animals for virulence studies, the selection of microorganisms for Aim 2 will be based on a combination of microbial responses from Aim 1 and previously reported spaceflight and spaceflight analogue experimental data of similar microorganisms. This will be the first study to apply an integrated systematic approach to understand the relationship between spaceflight, immune cell function, and infectious disease risk for the crew. The results from this study will enhance the current infectious disease risk assessment for the crew, elucidate the relationship to clinical disease, and support future development and application of effective countermeasures for treatment and prevention.

Insufficient time for solicitation: Continued delays in initiating the proposed study will continue to impact the schedule and decrease our likelihood of gaining the knowledge needed to close the risk. Note that the delay in this work may impact the Path to Risk Reduction (PRR) color change from yellow to green and put the studies outside of the window for use of the International Space Station (ISS). Two prior solicitations have been released (in 2009 and 2014) for ground-based proposals to understand microbial responses to simulated microgravity. Even though the prior solicitations were written clearly, the selected studies did not focus on identifying the microbial alterations that would gain the understanding needed to inform the risk, and they did not produce the needed ground-based investigations on mechanisms. The 2009 selection addresses collective changes of organisms within the human microbiome, and the 2014 selection addresses viral reactivation. The selected studies will provide information applicable to the gaps Micro-101 to better understand the potential impact of microgravity on microbial virulence and Micro-201 to better understand the contribution of these changes on adverse health events. Completion of the proposed work will provide clear evidence as to the operational applicability of these original microbial virulence data to a variety of microorganisms and will include measurements of host immune responses to microbial challenge.

Access to Previous Crew Data: This proposed study will leverage previous microbiology operational and research data as well as previously published immunology research data to provide a better understanding of impacts of microbial changes to the host and to determine the need for countermeasure evaluation as outlined in our PRR.