Polyamines are required for virulence in Salmonella enterica serovar Typhimurium

Abstract

Sensing and responding to environmental cues is a fundamental characteristic of bacterial physiology and virulence. Here we identify polyamines as novel environmental signals essential for virulence of Salmonella enterica serovar Typhimurium, a major intracellular pathogen and a model organism for studying typhoid fever. Central to its virulence are two major virulence loci Salmonella Pathogenicity Island 1 and 2 (SPI1 and SPI2). SPI1 promotes invasion of epithelial cells, whereas SPI2 enables S. Typhimurium to survive and proliferate within specialized compartments inside host cells. In this study, we show that an S. Typhimurium polyamine mutant is defective for invasion, intracellular survival, killing of the nematode Caenorhabditis elegans and systemic infection of the mouse model of typhoid fever. Virulence of the mutant could be restored by genetic complementation, and invasion and intracellular survival could, as well, be complemented by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection. Interestingly, intracellular survival of the polyamine mutant was significantly enhanced above the wild type level by the addition of exogenous putrescine and spermidine to the bacterial cultures prior to infection, indicating that these polyamines function as an environmental signal that primes S. Typhimurium for intracellular survival. Accordingly, experiments addressed at elucidating the roles of these polyamines in infection revealed that expression of genes from both of the major virulence loci SPI1 and SPI2 responded to exogenous polyamines and was reduced in the polyamine mutant. Together our data demonstrate that putrescine and spermidine play a critical role in controlling virulence in S. Typhimurium most likely through stimulation of expression of essential virulence loci. Moreover, our data implicate these polyamines as key signals in S. Typhimurium virulence.

Figure 1. Gene organization and graphic representation of putrescine/spermidine transporters and biosynthesis pathways.

(A) The putrescine and spermidine transporters are localized at three distinct loci; potABCD interrupted by sifA, potFGHI, and potE. The genotype of the transporter mutant (potCD;I;E) is indicated by an asterix above deleted genes. Below is shown a graphic presentation of the transporters with their substrate affinity indicated by p for putrescine and s for spermidine. Expression during infection of cell-cultures is indicated above genes , . (B) The putrescine and spermidine biosynthesis genes are localized at five distinct genetic loci; speA, speB, speC, speDE, and speF. The genotype of the biosynthesis mutant (speB;C;E;F) is indicated by an asterix above deleted genes. Below is shown a graphic presentation of the biosynthesis pathways present in bacteria, reviewed in . SAM: S-adenosylmethionine.

Figure 2. Growth in vitro.

Indicated bacterial strains were grown at 37°C, 200 rpm for 16 hrs in either LB or M9 before sub-culturing into fresh media at a 40 fold dilution (LB or M9), and growth was followed every 15 min for 18 hrs using a Bioscreen C. Where indicated, the M9 was supplemented with either 100 µg ml⁻¹ of putrescine or spermidine, respectively, during the growth experiment.

Figure 3. Invasion of epithelial cells.

Int-407 cells were infected with exponential phase M9 cultures (blue bars) or overnight M9 cultures (red bars) of the indicated strains. Non-adherent bacteria were removed and adherent bacteria were enumerated by plating (not shown). For determination of invasion extracellular bacteria were killed by gentamicin and intracellular bacteria were enumerated by plating. The strains tested are: wt; S. Typhimurium 4/74, spe (biosynthesis), pot (transport), invH; SPI1 invasion mutant, spe/pACYC177; spe-mutant with blank complementation plasmid, spe/pLOJ318; spe-mutant complemented with speB (putrescine biosynthesis). pot/pLOJ326; pot-mutant complemented with potCD (spermidine/putrescine uptake). +p and +s denotes that the bacterial cultures have been supplemented with 100 µg ml⁻¹ of putrescine or spermidine, respectively, prior to invasion. The experiments were repeated at least 4 times with similar results and shown is an average of these. Errorbars indicate standard deviations. Significant differences between the wt and the mutants are indicated with aterixs (* P<0.05; ** P<0.001). The P-values were calculated by a one-way ANOVA using Bonferronís post-test.

Figure 4. Expression of SPI1 genes.

Relative expression levels of selected SPI1 genes in S. Typhimurium wt, spe-mutant and the complemented spe-mutant (spe/pLOJ318). RNA was extracted from overnight M9 cultures. +p and +s denotes that the bacterial cultures have been supplemented with 100µg ml⁻¹ of putrescine or spermidine, respectively, prior to RNA extraction. A value of 1 indicates no detectable difference in expression between wt and mutant, values <1 indicate lower expression in the mutant and values >1 indicate higher expression in the mutant. Dotted lines mark the cut-off of two-fold regulation compared to wt. Results from 6 independent experiments are plotted for each strain. Outliers were removed using the Grubbs' test. Bar indicate average value of these for each strain. Significant differences between mutant and wt grown in M9 are indicated as follows: *** = P<0.0001; ** = P<0.001; * = P<0.05. The P-values were calculated by a one-way ANOVA using Dunnets post-test.

Figure 5. Intracellular survival/replication.

Int-407 cells were infected with exponential M9 or LB cultures of the indicated strains of S. Typhimurium for 15 min at 37°. At this time point non-adherent bacteria were removed and extracellular bacteria were killed by gentamicin. To determine intracellular survival/replication intracellular bacteria were enumerated by plating after 2 hrs and 8 hrs, respectively. The strains tested are: wt; S. Typhimurium 4/74, spe (biosynthesis), pot (transport), spe/pACYC177; spe-mutant with blank complementation plasmid, spe/pLOJ318; spe-mutant complemented with speB (putrescine biosynthesis). pot/pLOJ326; pot-mutant complemented with potCD (spermidine/putrescine uptake). +p and +s denotes that the bacterial cultures have been supplemented with 100 µg ml⁻¹ of putrescine or spermidine, respectively, prior to invasion. The experiments were repeated at least 4 times with similar results and shown is an average of these. Errorbars indicate standard deviation. Significant differences between wt grown in M9 vs other strains/growth conditions are indicated as follows: ** = P<0.001; * = P<0.05. The P-values were calculated by a one-way ANOVA using Dunnets post-test.

Figure 6. Expression of SPI2 genes.

Relative expression levels of selected SPI2 genes in S. Typhimurium wt, spe-mutant and the complemented spe-mutant (spe/pLOJ318). RNA was extracted from overnight M9 cultures. +p and +s denotes that the bacterial cultures have been supplemented with 100 µg ml⁻¹ of putrescine or spermidine, respectively. A value of 1 indicates no detectable difference in expression between wt and mutant, values <1 indicate lower expression in the mutant and values >1 indicate higher expression in the mutant. Dotted lines mark the cut-off of two-fold regulation compared to wt. Results from 6 independent experiments are plotted for each strain. Outliers were removed using the Grubbs' test. Bars indicate average value of these for each strain. Significant differences between mutants and wt grown in M9 are indicated as follows: *** = P<0.0001; ** = P<0.001. The P-values were calculated by a one-way ANOVA using Dunnets post-test.

Figure 7. C. elegans killing assay.

C. elegans pha-1(e2123ts) strain worms were seeded onto bacterial lawns on M9 plates of the wt (squares), the spe-mutant (diamonds) and the complemented spe-mutant (triangles) of S. Typhimurium. The plates were scored for live and dead worms every 24 hours. Three independent trials were performed for each strain and shown is an average of these experiments. Survival of the wt vs the spe-mutant was determined to be significantly different using the log-rank test for survival, p<0.0001.