Media ion composition controls regulatory and virulence response of Salmonella in spaceflight

Abstract

The spaceflight environment is relevant to conditions encountered by pathogens during the course of infection and induces novel changes in microbial pathogenesis not observed using conventional methods. It is unclear how microbial cells sense spaceflight-associated changes to their growth environment and orchestrate corresponding changes in molecular and physiological phenotypes relevant to the infection process. Here we report that spaceflight-induced increases in Salmonella virulence are regulated by media ion composition, and that phosphate ion is sufficient to alter related pathogenesis responses in a spaceflight analogue model. Using whole genome microarray and proteomic analyses from two independent Space Shuttle missions, we identified evolutionarily conserved molecular pathways in Salmonella that respond to spaceflight under all media compositions tested. Identification of conserved regulatory paradigms opens new avenues to control microbial responses during the infection process and holds promise to provide an improved understanding of human health and disease on Earth.

Figure 1. S. typhimurium virulence in LB, M9 and LB-M9 spaceflight cultures.

A) Ratio of LD₅₀ values of S. typhimurium spaceflight and ground cultures grown in LB (Lennox Broth), M9, or LB-M9 salts media. Female Balb/c mice were perorally infected with a range of bacterial doses from either spaceflight or ground cultures and monitored over a 30-day period for survival. B) Time-to-death curves of mice infected with spaceflight and ground cultures from STS-115 (infectious dosage: 10⁷ bacteria for both media). C) Time-to-death curves of mice infected with spaceflight and ground cultures from STS-123 (infectious dosage: 10⁶ bacteria for LB and 10⁷ bacteria for M9 and LB-M9 salts). Infectious dosages were selected such that the rates in time-to-death facilitated normalized comparisons across the different media.

Figure 2. qRT-PCR analysis confirms S. typhimurium gene expression altered during spaceflight in LB and M9 media.

Total RNA harvested from spaceflight and ground cultures in the indicated media was converted to single-stranded cDNA and used as a template in qRT-PCR analysis with primers hybridizing to the indicated genes. PCR product levels were normalized to the 16S rRNA product and a ratio of each gene level in flight and ground cultures was calculated. All differences in expression between spaceflight and ground cultures were found to be statistically significant using student's t-test (p-value<0.05). The error bars represent the standard deviation for three to nine independent technical replicate experiments.

Figure 3. Increased phosphate ion concentration prevents altered S. typhimurium acid tolerance in ground-based spaceflight analog culture.

Cultures of S. typhimurium grown in the indicated medium in the rotating wall vessel in the low-shear modeled microgravity (LSMMG) or control orientation were subjected to acid stress (pH 3.5) immediately upon removal from the apparatus. A ratio of percent survival of the bacteria cultured at each orientation in each media is presented. The error bars represent the standard deviation for two to five independent experimental trials each plated in triplicate. All differences in survival ratios were found to be statistically significant at p-value<0.05.