Biology subjects

Puccio, T.

Publications and source records attributed to Puccio, T..

4 recordsLinked to original sources

bioRxiv2021

Contribution of metal transporters of the ABC, ZIP, and NRAMP families to manganese uptake and infective endocarditis virulence in Streptococcus sanguinis

Streptococcus sanguinis is an important cause of infective endocarditis. In strain SK36, the ABC-family manganese transporter, SsaACB, is essential for virulence. We have now identified a ZIP-family protein, TmpA, as a secondary manganese transporter. A tmpA mutant had no phenotype, but a {Delta}ssaACB {Delta}tmpA mutant was far more attenuated for serum growth and somewhat more attenuated for virulence in a rabbit model than its {Delta}ssaACB parent. The growth of both mutants was restored by supplemental manganese, but the {Delta}ssaACB {Delta}tmpA mutant required twenty-fold more and accumulated less. Although ZIP-family proteins are known for zinc and iron transport, TmpA-mediated transport of either metal was minimal. In contrast to ssaACB and tmpA, which appear ubiquitous in S. sanguinis, a mntH gene encoding an NRAMP-family transporter has been identified in relatively few strains, including VMC66. As in SK36, deletion of ssaACB greatly diminished VMC66 endocarditis virulence and serum growth, and deletion of tmpA from this mutant diminished virulence further. Virulence was not significantly altered by deletion of mntH from either VMC66 or its {Delta}ssaACB mutant. This and the accompanying paper together suggest that SsaACB is of primary importance for endocarditis virulence while secondary transporters TmpA and MntH contribute to growth under differing conditions.

microbiology↗

bioRxiv2021

Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a {Delta}ssaACB mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and {Delta}ssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its {Delta}ssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis and point to strategies for enhancing the safety of oral probiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/446192v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a5afa7org.highwire.dtl.DTLVardef@1af3cdforg.highwire.dtl.DTLVardef@1c58693org.highwire.dtl.DTLVardef@1063980_HPS_FORMAT_FIGEXP M_FIG Depiction of methods. Streptococcal strains used are depicted at the top. The four methods illustrated are: 1. S. sanguinis cells were grown in media at pH 7.3 and pH 6.2 and quantified by plating. 2. S. sanguinis cells were grown in a fermentor vessel for RNA-sequencing and metal analysis. 3. S. sanguinis and S. mutans cells were grown in 12-well plates singly or in competition, then plated and the pH of the media measured. 4. S. sanguinis and S. mutans cells were inoculated into the mouths of mice. Oral swabs and dental biofilms from molars were assayed for bacterial composition by qPCR. (Biorender) C_FIG

microbiology↗

bioRxiv2020

Time-course analysis of Streptococcus sanguinis after manganese depletion reveals changes in glycolytic, nucleotide, and redox metabolites

IntroductionManganese is important for the endocarditis pathogen, Streptococcus sanguinis. Little is known about why manganese is required for virulence or how it impacts the metabolome of streptococci. ObjectivesWe applied untargeted metabolomics to cells and media to understand temporal changes resulting from manganese depletion. MethodsEDTA was added to a S. sanguinis manganese-transporter mutant in aerobic fermentor conditions. Cell and media samples were collected pre- and post-EDTA treatment. Metabolomics data were generated using positive and negative modes of data acquisition on an LC-MS/MS system. Data were subjected to statistical processing using MetaboAnalyst and time-course analysis using Short Time series Expression Miner (STEM). ResultsWe observed quantitative changes in 534 and 422 metabolites in cells and media, respectively, after EDTA addition. The 173 cellular metabolites identified as significantly different indicated enrichment of purine and pyrimidine metabolism. Further multivariate analysis revealed that the top 15 cellular metabolites belonged primarily to lipids and redox metabolites. The STEM analysis revealed global changes in cells and media in comparable metabolic pathways. Products of glycolysis such as pyruvate and fructose-1,6-bisphosphate increased, suggesting that enzymes that act on them may require manganese for activity or expression. Nucleosides accumulated, possibly due to a blockage in conversion to nucleobases. Simultaneous accumulation of ortho-tyrosine and reduced glutathione suggests that cells were unable to utilize glutathione as a reductant. ConclusionDifferential analysis of metabolites revealed the activation of a number of metabolic pathways in response to manganese depletion, many of which may be connected to carbon catabolite repression.

microbiology↗

bioRxiv2020

Manganese depletion leads to multisystem changes in the transcriptome of the opportunistic pathogen Streptococcus sanguinis

1Streptococcus sanguinis is a primary tooth colonizer 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 {Delta}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 {Delta}ssaACB cells pre- and post-EDTA, we determined that manganese deprivation led to altered expression of diverse systems, including ethanolamine utilization, CRISPR/Cas, and a type IV pilus. 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.

microbiology↗