Manganese Depletion Leads to Multisystem Changes in the Transcriptome of the Opportunistic Pathogen Streptococcus sanguinis

Abstract

Streptococcus sanguinis is a primary colonizer of teeth and is typically considered beneficial due to its antagonistic relationship with the cariogenic pathogen Streptococcus mutans. However, S. sanguinis can also act as an opportunistic pathogen should it enter the bloodstream and colonize a damaged heart valve, leading to infective endocarditis. Studies have implicated manganese acquisition as an important virulence determinant in streptococcal endocarditis. A knockout mutant lacking the primary manganese import system in S. sanguinis, SsaACB, is severely attenuated for virulence in an in vivo rabbit model. Manganese is a known cofactor for several important enzymes in S. sanguinis, including superoxide dismutase, SodA, and the aerobic ribonucleotide reductase, NrdEF. To determine the effect of manganese depletion on S. sanguinis, we performed transcriptomic analysis on a ssaACB mutant grown in aerobic fermentor conditions after the addition of the metal chelator EDTA. Despite the broad specificity of EDTA, analysis of cellular metal content revealed a decrease in manganese, but not in other metals, that coincided with a drop in growth rate. Subsequent supplementation with manganese, but not iron, zinc, or magnesium, restored growth in the fermentor post-EDTA. Reduced activity of Mn-dependent SodA and NrdEF likely contributed to the decreased growth rate post-EDTA, but did not appear entirely responsible. With the exception of the Dps-like peroxide resistance gene, dpr, manganese depletion did not induce stress response systems. By comparing the transcriptome of ssaACB cells pre-and post-EDTA, we determined that manganese deprivation led to altered expression of diverse systems. Manganese depletion also led to an apparent induction of carbon catabolite repression in a glucose-independent manner. The combined results suggest that manganese limitation produces effects in S. sanguinis that are diverse and complex, with no single protein or system appearing entirely responsible for the observed growth rate decrease. This study provides further evidence for the importance of this trace element in streptococcal biology. Future studies will focus on determining mechanisms for regulation, as the multitude of changes observed in this study indicate that multiple regulators may respond to manganese levels.

Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic and nucleic acid metabolites

“…As we are unaware of the existence of a strong, specific chelator of Mn, we elected to use a Mn-transporter mutant of S. sanguinis in combination with the nonspecific metal chelator, EDTA. As described in Puccio et al ( 2020 ), EDTA treatment of Δ ssaACB aerobic fermentor-grown cells results in the depletion of Mn but no other biologically relevant metals, such as Fe, Zn, or Mg, as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) (Fig. 1 ).…”

“…Extraction, derivatization, and annotation were completed by Metabolon, Inc. ICP-OES, inductively coupled plasma optical emission spectroscopy; UPLC-MS/MS, ultra performance liquid chromatography with tandem mass spectrometry. Fermentor sample collection, metal analysis, and H 2 O 2 analysis charts adapted from Puccio et al ( 2020 ) …”

“…3 b, d). We hypothesized that the hexose diphosphate is primarily FBP and its accumulation results from the reduced activity of two potentially Mn-cofactored FBP-consuming enzymes in glycolysis/gluconeogenesis: fructose-bisphosphate aldolase (Fba) and fructose-1,6-bisphosphatase (Fbp) (Puccio et al, 2020 ).…”

“…S. sanguinis strain SK36 was isolated from human dental plaque (Kilian et al, 1989 ; Xu et al, 2007 ). The Δ ssaACB strain (JFP169) was generated from SK36 previously by replacement of the ssaACB genes with the aphA-3 gene encoding kanamycin resistance (Baker et al, 2019 ; Puccio et al, 2020 ). Overnight pre-cultures were created by inoculation of Brain Heart Infusion (BHI) broth (BD) with single-use aliquots of cryopreserved cells by 1000-fold dilution.…”

“…Aerobic fermentor growth of Δ ssaACB cell culture was achieved using a BIOSTAT® B bioreactor (Sartorius Stedim) and samples were collected as described in Puccio and Kitten ( 2020 ) and Puccio et al ( 2020 ). Briefly, a 40-mL overnight pre-culture of S. sanguinis was grown as described above and centrifuged for 10 min at 3740× g at 4 °C (Beckman-Coulter).…”