Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae

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Abstract

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

Original languageEnglish
JournalInternational Journal Of Medical Microbiology
Volume309
Issue number3-4
DOIs
Publication statusPublished - 2019

Fingerprint

Biofilms
Streptococcus
Proteins
Growth
Electron Scanning Microscopy
Glass
Dairying
Bovine Mastitis
Endopeptidase K
Structural Models
Polystyrenes
Microbiology
Protein Binding
Confocal Microscopy
Trypsin
Extracellular Matrix

Keywords

  • Biofilm
  • Biofilm regulatory protein
  • BrpA inhibitors
  • Molecular docking
  • Streptococcus dysgalactiae subsp. dysgalactiae

Cite this

@article{b191999d401a4b8896be98cb4fd74754,
title = "Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae",
abstract = "Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.",
keywords = "Biofilm, Biofilm regulatory protein, BrpA inhibitors, Molecular docking, Streptococcus dysgalactiae subsp. dysgalactiae",
author = "Cinthia Alves-Barroco and Catarina Roma-Rodrigues and Natesan Balasubramanian and Guimar{\~a}es, {Marcia Aparecida} and Ferreira-Carvalho, {Bernadete T.} and Jayaraman Muthukumaran and Daniela Nunes and Elvira Fortunato and Rodrigo Martins and Teresa Santos-Silva and Figueiredo, {Agnes M. S.} and Fernandes, {Alexandra R.} and Ilda Santos-Sanches",
note = "info:eu-repo/grantAgreement/FCT/5876/147258/PT# info:eu-repo/grantAgreement/FCT/3599-PPCDT/131609/PT# PTDC/CVT-EPI/6685/2014. FCT-MEC is also acknowledged the grant SFRH/BD/118350/2016 to CAB and SFRH/BPD/97719/2013 to JM.",
year = "2019",
doi = "10.1016/j.ijmm.2019.02.001",
language = "English",
volume = "309",
journal = "International Journal Of Medical Microbiology",
issn = "1438-4221",
publisher = "Elsevier Science B.V., Amsterdam.",
number = "3-4",

}

TY - JOUR

T1 - Biofilm development and computational screening for new putative inhibitors of a homolog of the regulatory protein BrpA in Streptococcus dysgalactiae subsp. dysgalactiae

AU - Alves-Barroco, Cinthia

AU - Roma-Rodrigues, Catarina

AU - Balasubramanian, Natesan

AU - Guimarães, Marcia Aparecida

AU - Ferreira-Carvalho, Bernadete T.

AU - Muthukumaran, Jayaraman

AU - Nunes, Daniela

AU - Fortunato, Elvira

AU - Martins, Rodrigo

AU - Santos-Silva, Teresa

AU - Figueiredo, Agnes M. S.

AU - Fernandes, Alexandra R.

AU - Santos-Sanches, Ilda

N1 - info:eu-repo/grantAgreement/FCT/5876/147258/PT# info:eu-repo/grantAgreement/FCT/3599-PPCDT/131609/PT# PTDC/CVT-EPI/6685/2014. FCT-MEC is also acknowledged the grant SFRH/BD/118350/2016 to CAB and SFRH/BPD/97719/2013 to JM.

PY - 2019

Y1 - 2019

N2 - Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

AB - Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

KW - Biofilm

KW - Biofilm regulatory protein

KW - BrpA inhibitors

KW - Molecular docking

KW - Streptococcus dysgalactiae subsp. dysgalactiae

UR - http://www.scopus.com/inward/record.url?scp=85061783871&partnerID=8YFLogxK

U2 - 10.1016/j.ijmm.2019.02.001

DO - 10.1016/j.ijmm.2019.02.001

M3 - Article

VL - 309

JO - International Journal Of Medical Microbiology

JF - International Journal Of Medical Microbiology

SN - 1438-4221

IS - 3-4

ER -