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Uncovering Roles of Streptococcus gordonii SrtA-Processed Proteins in the Biofilm Lifestyle.

ABSTRACT: Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent.IMPORTANCE S. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.

SUBMITTER: Nairn BL

PROVIDER: S-EPMC7950405 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pioneer oral bacteria, including Streptococcus gordonii, initiate the formation of oral biofilms on tooth surfaces, which requires differential expression of genes that recognize unique environmental cues. An S. gordonii::Tn917-lac biofilm-defective mutant was isolated by using an in vitro biofilm formation assay. Subsequent inverse PCR and sequence analyses identified the transposon insertion to be near the 3' end of an open reading frame (ORF) encoding a protein homologous to a Streptococcus pneumoniae repressor, AdcR. The S. gordonii adc operon, consisting of the four ORFs adcR, adcC, adcB, and adcA, is homologous to the adc operon of S. pneumoniae, which plays a role in zinc and/or manganese transport and genetic competence in S. pneumoniae. AdcR is a metal-dependent repressor protein containing a putative metal-binding site, AdcC contains a consensus-binding site for ATP, AdcB is a hydrophobic protein with seven hydrophobic membrane-spanning regions, and AdcA is a lipoprotein permease with a putative metal-binding site. The three proteins (AdcC through -A) are similar to those of the binding-lipoprotein-dependent transport system of gram-positive bacteria. Reverse transcriptase PCR confirmed that adcRCBA are cotranscribed as an operon in S. gordonii and that the transposon insertion in S. gordonii adcR::Tn917-lac had resulted in a polar mutation. Expression of adcR, measured by the beta-galactosidase activity of the adcR::Tn917-lac mutant, was growth phase dependent and increased when the mutant was grown in media with high levels of manganese (>1 mM) and to a lesser extent in media with zinc, indicating that AdcR may be a regulator at high levels of extracellular manganese. A nonpolar inactivation of adcR generated by allelic replacement resulted in a biofilm- and competence-defective phenotype. The biofilm-defective phenotype observed suggests that AdcR is an active repressor when synthesized and acts at a distant site(s) on the chromosome. Thus, the adc operon is involved in manganese acquisition in S. gordonii and manganese homeostasis and appears to modulate sessile growth in this bacterium.

Project description:Polymicrobial biofilms play important roles in oral and systemic infections. The oral plaque bacterium Streptococcus gordonii is known to attach to the hyphal cell wall of the fungus Candida albicans to form corn-cob like structures in biofilms. However, the role of C. albicans in formation of polymicrobial biofilms is not completely understood. The objective of this study was to determine the role of C. albicans transcription factors in regulation of polymicrobial biofilms and antibiotic tolerance of S. gordonii. The proteins secreted by C. albicans and S. gordonii in mixed planktonic cultures were determined using mass spectrometry. Antibiotic tolerance of S. gordonii to ampicillin and erythromycin was determined in mixed cultures and mixed biofilms with C. albicans. Additionally, biofilm formation of S. gordonii with C. albicans knock-out mutants of 45 transcription factors that affect cell wall integrity, filamentous growth and biofilm formation was determined. Furthermore, these mutants were also screened for antibiotic tolerance in mixed biofilms with S. gordonii. Analysis of secreted proteomes resulted in the identification of proteins being secreted exclusively in mixed cultures. Antibiotic testing showed that S. gordonii had significantly increased survival in mixed planktonic cultures with antibiotics as compared to single cultures. C. albicans mutants of transcription factors Sfl2, Brg1, Leu3, Cas5, Cta4, Tec1, Tup1, Rim101 and Efg1 were significantly affected in mixed biofilm formation. Also mixed biofilms of S. gordonii with mutants of C. albicans transcription factors, Tec1 and Sfl2, had significantly reduced antibiotic tolerance as compared to control cultures. Our data indicates that C. albicans may have an important role in mixed biofilm formation as well as antibiotic tolerance of S. gordonii in polymicrobial biofilms. C. albicans may play a facilitating role than being just an innocent bystander in oral biofilms and infections.

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Uncovering Roles of Streptococcus gordonii SrtA-Processed Proteins in the Biofilm Lifestyle.

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