Roles of rel(Spn) in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39

Abstract

RelA/SpoT homologue (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of Streptococcus pneumoniae, Rel(Spn), can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 Deltarel(Spn) mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel(+)(Spn) and Deltarel(Spn) strains treated with mupirocin revealed rel(Spn)-independent (translation stress), rel(Spn)-dependent (stringent response) and Deltarel(Spn)-dependent changes, suggesting that rel(Spn) and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the rel(Spn)-dependent response included significant upregulation of the ply operon encoding pneumolysin toxin, whereas the Deltarel(Spn)-dependent response affected expression linked to the VicRK and CiaRH two-component systems. Finally, a D39 Deltarel(Spn) mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel(+)(Spn).

Fig. 1

Organization of the rel_Spn gene region and design of constructs used in this study at the native rel_Spn locus (A) or in the ectopic bgaA locus (B). Reading frames are indicated by arrows; genes of interest are indicated in white, antibiotic resistance genes are in black, others are in gray. Gene names are indicated above the arrows. Promoters are indicated by arrows: solid arrows represent endogenous promoters, broken arrows represent either the synthetic constitutive P_c promoter (Claverys et al., 1995) or the L-fucose-inducible P_fcsK promoter (Chan et al., 2003). The P_rel-rel_Spn construct used for complementation studies contains the rel_Spn gene and 147 bp of upstream sequence. Lollipop symbols indicate putative terminator sequences. Regions are drawn to scale. CBA= BOX repetitive element; dtd encodes D-tyrosyl tRNA^Tyr deacylase.

Fig. 2

(p)ppGpp production by D39 rel⁺_Spn, Δrel_Spn, and complemented Δrel_Spn bgaA∷P_rel-rel⁺_Spn strains in the absence (A) and presence (B) of 100 ng mupirocin per mL. Cells were non-uniformly ³²P_i labeled (150 μCi per mL) in MOPS-CDM (- Ile) and extracted with formic acid, and extracts were analyzed by PEI TLC as described in Experimental procedures. The strains assayed were: IU1912 (D39 luxABCDE rel⁺_Spn), IU1943 (D39 luxABCDE Δrel_Spn), and IU2025 (D39 luxABCDE Δrel_Spn bgaA∷P_rel-rel⁺_Spn). IU2025 contained an amino acid replacement (Glu227Gly, GAG to GGG) that matches the sequence of E. coli SpoT at that position, and a silent change at Cys607 (TGT to TGC). Labeled nucleotides were identified by comparison with standards synthesized by strain TX2737 (Table S1) expressing the constitutive E. coli RelA′ enzyme from an IPTG-inducible promoter (Svitil et al., 1993).

Fig. 3

Growth of D39 rel⁺_Spn (IU1690) and D39 Δrel_Spn (IU1921) strains in BHI broth (A) and outgrowth in BHI following mupirocin treatment (B). In (A), mupirocin was added to 100 ng per mL (open symbols) and arrows indicate when samples were harvested for RNA preparation for microarray and QPCR analyses. In (B), strains were grown in BHI and treated with 100 ng mupirocin per mL. Samples of treated cultures were removed at the indicated times and diluted into BHI containing no drug as described in Experimental procedures. Growths of untreated cultures are shown for comparison.

Fig. 4

Growth of the D39 rel⁺_Spn (IU1690) and D39 Δrel_Spn (IU1921) strains in CDM (A) and in CDM supplemented with BHI or CuSO₄, MnSO₄, and ZnSO₄ (B). Strains were grown in BHI overnight, collected by centrifugation, washed and resuspended in CDM, and diluted into the indicated media as described in Experimental procedures. In (A), D39 Δrel_Spn bgaA∷P_fcsK-rel_Spn complementation strains IU2024 and IU2560 contained a silent change at Asn475 (AAT to AAC) or an amino acid replacement (Lys319Glu, AAA to GAA), respectively, in Rel_Spn. In (B), standard CDM contained 2.8 mM MgSO₄, 26.4 μM MnSO₄ (1× Mn), 9.8 μM FeSO₄, and 1.5 μM Fe(NO₃)₃. Where indicated, CuSO₄ was added to 1.56 μM (6× Cu), MnSO₄ was added to 158.4 μM (6× Mn), and ZnSO₄ was added to 9.8 μM (1× Zn). See the text for additional details.

Fig. 5

Predicted amino acid biosynthesis pathways in S. pneumoniae D39 based on genomic sequences (Hoskins et al., 2001; Lanie et al., 2007). Arrows are not scaled to reflect lengths of pathways. “X” indicates incomplete or missing pathway. “?” indicates a complete pathway that appears nonfunctional under tested conditions. D39 did not grow in CDM drop-out media lacking boxed amino acids. Relative transcript amounts for some biosynthesis genes increased (amino acids indicated with asterisks) or decreased (pound signs) upon mupirocin treatment (see text and Table 1, lines 1, 2, 9).

Fig. 6

Correspondence of relative expression ratios obtained by QPCR and microarray analyses. Relative transcript amounts were determined by QPCR and normalized to 16S rRNA, rpoB or gyrA as described (Experimental procedures). Expression ratios obtained by QPCR and by microarray analyses were log2-transformed and plotted against each other. (A) Comparison of transformed expression ratios obtained for the D39 rel⁺_Spn strain treated with mupirocin. Relative transcript levels were determined for rpoB (RNA polymerase subunit β), ply (pneumolysin), gyrA (DNA gyrase subunit), and eno (enolase). Correlation values for QPCR data normalized to the different RNAs: 16S rRNA (r²=0.99); rpoB mRNA (r²=0.99); gyrA mRNA (r²=0.99). (B) Comparison of transformed expression ratios obtained for the D39 Δrel_Spn strain treated with mupirocin. Relative transcript levels were determined for rpoB, ply, rpsJ (ribosomal protein S10), gyrA, spxB (pyruvate oxidase), eno and ileS (isoleucyl-tRNA synthetase). Correlation values for QPCR data normalized to the different RNAs: 16S rRNA (r²=0.98); rpoB mRNA (r²=0.98); gyrA mRNA (r²=0.97). Normalization to 16S rRNA resulted in a line that did not pass through the plot origin and was not appropriate for validation of the D39 Δrel_Spn data (see text).

Fig. 7

Disease progression and survival of mice infected with D39 luxABCDE rel⁺_Spn (IU1912), D39 luxABCDE Δrel_Spn (IU1943), and complemented D39 luxABCDE Δrel_Spn bgaA∷P_rel-rel⁺_Spn (IU2025) strains. Mice were inoculated intranasally with 6 × 10⁶ CFU and disease progression was followed in real time by survival curve analysis (A) and biophotonic imaging (B) as described in Experimental procedures. Ten animals were infected with each strain in two independent experiments, and a representative survival curve and biophotonic images are shown. Survival curves were analyzed by Kaplan-Meier statistics and log-rank tests, and P values relative to the D39 rel⁺_Spn lux strain are given. In (B), the time at which each image was captured is indicated along with the relative color bar scale extending from blue (fewer bacteria) to red (more bacteria). All pictured mice are from the same experiment. Luminescence visible on the tail and lower foot of the rel⁺_Spn-infected mouse (Fig. 8B, top) is light reflected from an adjacent animal photographed at the same time (not shown). Two representative Δrel_Spn-infected mice are shown at 93 h and 115 h after inoculation to illustrate altered disease progression (Fig. 8B, bottom). Additional details are in the text.