A SNP in the Cache 1 Signaling Domain of Diguanylate Cyclase STM1987 Leads to Increased In Vivo Fitness of Invasive Salmonella Strains

Abstract

Nontyphoidal Salmonella (NTS) strains are associated with gastroenteritis worldwide but are also the leading cause of bacterial bloodstream infections in sub-Saharan Africa. The invasive NTS (iNTS) strains that cause bloodstream infections differ from standard gastroenteritis-causing strains by >700 single-nucleotide polymorphisms (SNPs). These SNPs are known to alter metabolic pathways and biofilm formation and to contribute to serum resistance and are thought to signify iNTS strains becoming human adapted, similar to typhoid fever-causing Salmonella strains. Identifying SNPs that contribute to invasion or increased virulence has been more elusive. In this study, we identified a SNP in the cache 1 signaling domain of diguanylate cyclase STM1987 in the invasive Salmonella enterica serovar Typhimurium type strain D23580. This SNP was conserved in 118 other iNTS strains analyzed and was comparatively absent in global S Typhimurium isolates associated with gastroenteritis. STM1987 catalyzes the formation of bis-(3',5')-cyclic dimeric GMP (c-di-GMP) and is proposed to stimulate production of cellulose independent of the master biofilm regulator CsgD. We show that the amino acid change in STM1987 leads to a 10-fold drop in cellulose production and increased fitness in a mouse model of acute infection. Reduced cellulose production due to the SNP led to enhanced survival in both murine and human macrophage cell lines. In contrast, loss of CsgD-dependent cellulose production did not lead to any measurable change in in vivo fitness. We hypothesize that the SNP in stm1987 represents a pathoadaptive mutation for iNTS strains.

Keywords: CsgD independent; STM1987; cellulose; cyclic-di-GMP; diguanylate cyclase; invasive Salmonella; macrophage; virulence.

FIG 1

Schematic of stm1987 allele in S. Typhimurium 14028 and D23580. The 570-amino acid STM1987 protein is represented by the black rectangle and is shown to scale, with transmembrane (TM), cache 1 signaling, and GGDEF-DGC domains highlighted. The magnified region shows nucleotides between position 553 and position 579, with nucleotide 566 in bold (C in 14028 and G in D23580). Single-letter amino acids corresponding to each codon are shown above for S. Typhimurium 14028 and below for strain D23580. Amino acid residue 189 is shown in bold red (T for 14028 and R for D23580).

FIG 2

Phenotypic comparison of stm1987 alleles from S. Typhimurium 14028 and D23580. S. Typhimurium 14028 Δstm1987 was transformed with pBR322 (control) or pBR322 harboring the stm1987^ST14028 or stm1987^D23580 alleles. (A) Cells from overnight cultures were grown on tryptone agar supplemented with calcofluor white, and colonies were visualized under UV light after 12 h of growth at 28°C. (B) Strains were grown in LB for 24 h at 37°C, and tubes were examined for the presence of surface-attached biomass at the air-liquid interface. (C) The amount of attached biomass was quantified using CV staining, with the absorbance of the resulting solution being measured at 590 nm (A₅₉₀). Columns represent the average values and error bars represent the standard deviations from five biological replicates. ****, P < 0.0001.

FIG 3

Conservation of stm1987 SNP in invasive S. Typhimurium lineages. The maximum likelihood phylogenetic tree was constructed from sequenced S. Typhimurium isolates (10) as reported previously (40). The isolates analyzed were divided into S. Typhimurium groups associated with gastroenteritis or invasive disease (lineages I and II) (10). The presence or absence of the STM1987 SNP is noted for each group; the red star shows the position of strain D23580 in the tree.

FIG 4

Analysis of the role of STM1987 and cellulose production in S. Typhimurium intramacrophage survival. Human THP-1 and murine RAW 264.7 macrophages were infected with S. Typhimurium D23580 or D23580 (stm1987^ST10428) (A), S. Typhimurium 14028 or 14028 (stm1987^D23580) (B), S. Typhimurium 14028 Δstm1987 strains transformed with pBR322, pBR322/stm1987^ST14028, or pBR322/stm1987^D23580 (C), or S. Typhimurium Δstm1987 ΔbcsA strains transformed with the same plasmids (D). Percent survival was determined as the proportion of intracellular CFU remaining at 18 h postinfection, compared to the initial CFU values measured after 30 min. Statistical significance between groups was noted. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant, P > 0.05.

FIG 5

Transcription of stm1987 is not CsgD dependent in S. Typhimurium. Expression curves for stm1987 (A) or adrA (B) promoter-lux fusions are shown for S. Typhimurium 14028 (black), 14028 ΔcsgD (red), or S. Typhimurium D23580 (gray) grown at 28°C in 1% tryptone medium. For each fusion, the luminescence values (counts per second [CPS]) were plotted as a function of time, with measurements recorded every 30 min. Each expression curve corresponds to an individual biological replicate culture.

FIG 6

Role of the CsgD-independent and CsgD-dependent cellulose production pathways in S. Typhimurium virulence. CI infections were performed with S. Typhimurium 14028 and 14028 (stm1987^D23580) strains (A and B) or S. Typhimurium 14028 and 14028 (PcsgD^D7795) strains (C and D). C57BL/6 mice were challenged orally with 10⁷ CFU (A and C) or i.p. with 10⁴ CFU (B and D); each inoculum consisted of a 1:1 ratio of each pair of strains. At 4 to 7 days after oral infection or 72 h after i.p. infection, bacteria were enumerated from the spleen, liver, MLN, cecum, and kidney. CI values were calculated for each organ as follows: (CFU 14028_mutant/14028_wild-type)output/(CFU 14028_mutant/14028_wild-type)input. A CI value of 1, which represents a situation in which strains are equally virulent, is represented by the horizontal dotted line on each graph. Red circles represent CI values at which the 14028 strains containing the stm1987^D23580 or PcsgD^D7795 mutations were recovered in greater proportions than the wild-type strain. Statistical differences between groups of mice were noted. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant, P > 0.05.