Heterologous expression of Streptococcus mutans cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOIn S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors811829FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO2014/07231-0; 2013/25080-7308644/2011-

Transcriptional and phenotypic characterization of novel Spx-regulated genes in Streptococcus mutans

CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOIn oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Delta spx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress.In oral biofilms, two of the major environmental challenges encountered by the dental pathogen Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the S. mutans transcriptional regulators SpxA1 and SpxA2 (formerly SpxA and SpxB, respectively) are involved in stress survival by activating the expression of classic oxidative stress genes such as dpr, nox, sodA and tpx. We reasoned that some of the uncharacterized genes under SpxA1/A2 control are potentially involved in oxidative stress management. Therefore, the goal of this study was to use Spx-regulated genes as a tool to identify novel oxidative stress genes in S. mutans. Quantitative real-time PCR was used to evaluate the responses of ten Spx-regulated genes during H2O2 stress in the parent and Delta spx strains. Transcription activation of the H2O2-induced genes (8 out of 10) was strongly dependent on SpxA1 and, to a lesser extent, SpxA2. In vitro transcription assays revealed that one or both Spx proteins directly regulate three of these genes. The gene encoding the FeoB ferrous permease was slightly repressed by H2O2 but constitutively induced in strains lacking SpxA1. Nine genes were selected for downstream mutational analysis but inactivation of smu127, encoding a subunit of the acetoin dehydrogenase was apparently lethal. In vitro and in vivo characterization of the viable mutants indicated that, in addition to the transcriptional activation of reducing and antioxidant pathways, Spx performs an important role in iron homeostasis by regulating the intracellular availability of free iron. In particular, inactivation of the genes encoding the Fe-S biogenesis SUF system and the previously characterized iron-binding protein Dpr resulted in impaired growth under different oxidative stress conditions, increased sensitivity to iron and lower infectivity in rats. These results serve as an entryway into the characterization of novel genes and pathways that allow S. mutans to cope with oxidative stress104CAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIORFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES [6849-12-1]FAPESP [2012/032278-3, 2014/03816-4]6849-12-12012/032278-3; 2014/03816-

Characterization of Streptococcus mutans Strains Deficient in EIIAB(Man) of the Sugar Phosphotransferase System

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression

Different Roles of EIIAB(Man) and EII(Glc) in Regulation of Energy Metabolism, Biofilm Development, and Competence in Streptococcus mutans

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major carbohydrate transport system in oral streptococci. The mannose-PTS of Streptococcus mutans, which transports mannose and glucose, is involved in carbon catabolite repression (CCR) and regulates the expression of known virulence genes. In this study, we investigated the role of EII(Glc) and EIIAB(Man) in sugar metabolism, gene regulation, biofilm formation, and competence. The results demonstrate that the inactivation of ptsG, encoding a putative EII(Glc), did not lead to major changes in sugar metabolism or affect the phenotypes of interest. However, the loss of EII(Glc) was shown to have a significant impact on the proteome and to affect the expression of a known virulence factor, fructan hydrolase (fruA). JAM1, a mutant strain lacking EIIAB(Man), had an impaired capacity to form biofilms in the presence of glucose and displayed a decreased ability to be transformed with exogenous DNA. Also, the lactose- and cellobiose-PTSs were positively and negatively regulated by EIIAB(Man), respectively. Microarrays were used to investigate the profound phenotypic changes displayed by JAM1, revealing that EIIAB(Man) of S. mutans has a key regulatory role in energy metabolism, possibly by sensing the energy levels of the cells or the carbohydrate availability and, in response, regulating the activity of transcription factors and carbohydrate transporters