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NASA NTRS2005

Analysis of Pdeudomonas aeruginosa Growth and Virulence in Modelled Microgravity

Stress, radiation and microgravity cause astronauts to experience secondary immunosuppression. Spaceflight conditions enhance bacterial growth and alter antimicrobial susceptibility. Clinostats are used to model microgravity effects at lxg. In controls rotated on the vertical axis, the g-vector acts on cells as in static cultures. Salmonella enterica serovar Typhimurium virulence genes are up-regulated in modelled microgravity (MMG); a MMG regulon has been postulated. We hypothesize that the virulence of P. aeruginosa (PA) may be affected similarly by microgravity, which could be observed in MMG. This study focused on regulation of the ETA protein by PA during growth in MMG. PA103 was grown in an ETA production medium at 37 C. One series of media was inoculated with frozen cultures and grown using horizontal (MMG) or static incubation. Another series inoculated with refrigerated cultures included vertical rotating controls. Analyses included optical density (OD), agar plate counts (PC) on R2A, ETA ELISA, and protein expression by 2-D gel analyses. Growth and ETA results differed depending on inoculum, with minor effects of MMG. Proteomic analysis of 2-D gels indicate differences in protein expression with MMG. Growth and ETA results show that consistent methodology is critical when studying environmental effects. This study provides information on the relationships between environmental changes and virulence regulation, especially for flight experiments, when ground experiments are used to predict potential spaceflight effects.

Guadarrama, Seratna↗

NASA NTRS2002

Isolation and characterization of a novel calmodulin-binding protein from potato

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.

NASA Discipline Plant Biology↗

NASA NTRS2000

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

NASA Discipline Plant Biology↗

NASA NTRS2010

Response of Staphylococcus Aureus to a Spaceflight Analogue

The decreased gravity of the spaceflight environment creates quiescent, low fluid shear conditions. This environment can impart considerable effects on the physiology of microorganisms as well as their interactions with potential hosts. Using the rotating wall vessel (RWV), as a spaceflight analogue, the consequence of low fluid shear culture on microbial pathogenesis has provided a better understanding of the risks to the astronaut crew from infectious microorganisms. While the outcome of low fluid shear culture has been investigated for several bacterial pathogens, little has been done to understand how this environmental factor affects Staphylococcus aureus. S. aureus is an opportunistic human pathogen which presents a high level of infection risk to the crew, as it has been isolated from both the space shuttle and International Space Station. Given that approximately forty percent of the population are carriers of the bacteria, eradication of this organism from in flight environments is impractical. These reasons have lead to us to assess the response of S. aureus to a reduced fluid shear environment. Culture in the RWV demonstrated that S. aureus grown under the low-shear condition had lower cell concentrations after 10 hours when compared to the control culture. Furthermore, the low-shear cultured bacteria displayed a reduction in carotenoid production, pigments responsible for their yellow/gold coloration. When exposed to various environmental stressors, post low-shear culture, a decrease in the ability to survive oxidative assault was observed compared to control cultures. The low fluid shear environment also resulted in a decrease in hemolysin secretion, a staphylococcal toxin responsible for red blood cell lysis. When challenged by the immune components present in human whole blood, low-shear cultured S. aureus demonstrated significantly reduced survival rates as compared to the control culture. Assays to determine the duration of these alterations demonstrated that the low-shear response could be lost in as few as 2.5 hours. These changes in phenotypic properties prompted investigation into variations occurring at the genetic level. Microarray analysis of low-shear cultured S. aureus revealed the differential regulation of genes involved in metabolism, stress response, and phosphate transfer. Additional genetic analysis with quantitative real-time PCR revealed alterations in the expression of Hfq, the conserved RNA chaperone protein involved in global gene regulation. Hfq has been connected to the regulation of a spaceflight microgravity response in S. typhimurium. These findings in S. aureus suggest an evolutionary conserved response to spaceflight conditions among structurally-diverse microorganisms. Furthermore, the reduction in pigmentation, hemolysin secretion, and survival against oxidative stress and immunologically active whole blood demonstrate an overall decrease in the virulence factors of S. aureus in response to spaceflight-like conditions.

Castro, S. L.↗