Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. DNA Manipulations
2.3. Construction of the Lipoprotein Maturation Isogenic Mutants
2.4. Complementation of the Mutants
2.5. Growth Analysis
2.6. Bacterial Surface Hydrophobicity Assay
2.7. Bacterial Adhesion and Invasion Assays Using Porcine Brain Microvascular Endothelial and Tracheal Epithelial Cells
2.8. Biofilm Assay
2.9. Generation of Bone-Marrow-Derived Dendritic Cells (bmDCs)
2.10. Preparation of Heat-Killed S. suis
2.11. Preparation of Bacterial Supernatant
2.12. S. suis Activation of bmDCs
2.13. S. suis Virulence Mouse Model of Systemic Infection
2.14. Measurement of Plasma (Systemic) Pro-Inflammatory Mediators
2.15. Statistical Analyses
3. Results
3.1. Characteristics of the Δlgt and Δlsp Mutants Derived from the ST25 89-1591 Strain
3.2. Lack of Lipoprotein Maturation Enzymes Does Not Impair Adhesion to and Invasion of Respiratory Epithelial and Brain Microvascular Endothelial Swine Cells Regardless of the Sequence Type of the Strain
3.3. Lgt and Lsp Enzymes Are Important for S. suis Biofilm Formation Regardless of the Sequence Type of the Strain
3.4. The Diacyl Motif and the Peptide Signal Cleavage Are Important for the Recognition by Innate Immune Cells of Periplasmic and/or Secreted Lipoproteins of S. suis Serotype 2 ST25 Strain
3.5. The Absence of Lgt Enzyme, but Not Lsp, Significantly Affects the Virulence of S. suis Serotype 2 ST25
3.6. Absence of the Lgt or Lsp Enzymes Significantly Reduces the In Vivo Inflammatory Response of Mice Infected with Either the Wild Type S. suis Serotype 2 Strain ST25 or Its Respective Δlgt or Δlsp Mutant
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain or Plasmid | Characteristics | References |
---|---|---|
Streptococcus suis | ||
P1/7 | Virulent serotype 2 ST1 strain isolated from a case of pig meningitis in the United Kingdom | [21] |
P1/7 Δlgt | Isogenic mutant derived from P1/7; in frame deletion of lgt gene | [8] |
P1/7 Δlsp | Isogenic mutant derived from P1/7; in frame deletion of lsp gene | [8] |
P1/7 comp Δlgt | Mutant Δlgt complemented with pMX1-lgt complementation vector | [8] |
P1/7 comp Δlsp | Mutant Δlsp complemented with pMX1-lsp complementation vector | [8] |
P1/7 ΔcpsF | Isogenic mutant derived from P1/7; in frame deletion of cpsF | [22] |
89-1591 | Virulent North American ST25 strain isolated from a case of pig sepsis in Canada | [23] |
89-1591 Δlgt | Isogenic mutant derived from 89-1591; in frame deletion of lgt gene | This study |
89-1591 Δlsp | Isogenic mutant derived from 89-1591; in frame deletion of lsp gene | This study |
89-1591 comp Δlgt | Mutant Δlgt complemented with pMX1-lgt complementation vector | This study |
89-1591 comp Δlsp | Mutant Δlsp complemented with pMX1-lsp complementation vector | This study |
89-1591 ΔcpsF | Isogenic mutant derived from 89-1591; in frame deletion of cpsF | [16] |
Escherichia coli | ||
TOP10 | F− mrcA Δ(mrr-hsdRMS-mcrBC) φ80 lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu) 7697 galU galK rpsL (Strr) endA1 nupG | Invitrogen |
MC1061 | F− Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ-e14-mcrA0 relA1 rpsL150(StrR) spoT1 mcrB1 hsdR2(r-m+)Host for pMX1 derivatives | [24] |
Plasmids | ||
pCR2.1 | Apr, Kmr, pUC ori, lacZΔM15 | Invitrogen |
pSET4s | Spcr, pUC ori, thermosensitive pG+host3 ori, lacZΔM15 | [25] |
pMX1 | Replication functions of pSSU1, MCS pUC19 lacZ Spcr, malX promoter of S. suis, derivative of pSET2 | [25,26] |
p4Δlgt | pSET-4s carrying the construct for lgt allelic replacement | This study |
p4Δlsp | pSET-4s carrying the construct for lsp allelic replacement | This study |
pMX1-lgt (P1/7) | pMX1 carrying intact lgt gene | [8] |
pMX1-lsp (P1/7) | pMX1 carrying intact lsp gene | [8] |
pMX1-lgt (89-1591) | pMX1 carrying intact lgt gene | This study |
pMX1-lsp (89-1591) | pMX1 carrying intact lsp gene | This study |
Name | Sequence (5′–3′) | Construct |
---|---|---|
lgt-ID1 | GGAACGCTATGGAACAGGTC | p4Δlgt |
lgt-ID2 | CACTCCATGAAAAGGCGACG | p4Δlgt |
lgt-ID3 | CGTAGACGGCCAAAATTCC | p4Δlgt |
lgt-ID4 | CGCTTATCTGCTGGATTCTCC | p4Δlgt |
lgt-ID5 | GCCAATCGTCTGCATCAAGG | p4Δlgt |
lgt-ID6 | GGGTTGATAGAATGGGATTGCATACCAACG | p4Δlgt |
lgt-ID7 | CGTTGGTATGCAATCCCATTCTATCAACCC | p4Δlgt |
lgt-ID8 | GACCGACTTGCTGGTCAAAC | p4Δlgt |
lsp-ID1 | TGAGAAAACTGTTGTGGGTA | p4Δlsp |
lsp-ID2 | AGAGCACCAGCAATCATCAA | p4Δlsp |
lsp-ID3 | TTGATGATTGCTGGTGCTCT | p4Δlsp |
lsp-ID4 | TAGACAGCGAACAGAGATAC | p4Δlsp |
lsp-ID5 | TACGCTACGTTGTAGCCATTGC | p4Δlsp |
lsp-ID6 | ACCTACACCAACTGTTAATACTACCATCAA | p4Δlsp |
lsp-ID7 | TTGATGGTAGTATTAACAGTTGGTGTAGGT | p4Δlsp |
lsp-ID8 | CGCGCTGCAGCCAAAGTGTAGTCACCAAAA | p4Δlsp |
pMX1-lgt-F | CCGCCATGGACAGATGGGGTTTGATGCAAC | pMX1-lgt |
pMX1-lgt-R | CGCGAATTCGGACAAGGCAATAATCAAGAC | pMX1-lgt |
pMX1-lsp-F | GTGCCATGGACTTTATTGAAACCATGCAGG | pMX1-lsp |
pMX1-lsp-R | ATCGAATTCAATACCACCAACCTCAACTCT | pMX1-lsp |
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Payen, S.; Roy, D.; Okura, M.; Segura, M.; Gottschalk, M. Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain. Pathogens 2023, 12, 1325. https://doi.org/10.3390/pathogens12111325
Payen S, Roy D, Okura M, Segura M, Gottschalk M. Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain. Pathogens. 2023; 12(11):1325. https://doi.org/10.3390/pathogens12111325
Chicago/Turabian StylePayen, Servane, David Roy, Masatoshi Okura, Mariela Segura, and Marcelo Gottschalk. 2023. "Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain" Pathogens 12, no. 11: 1325. https://doi.org/10.3390/pathogens12111325
APA StylePayen, S., Roy, D., Okura, M., Segura, M., & Gottschalk, M. (2023). Study of the Role of Lipoprotein Maturation Enzymes in the Pathogenesis of the Infection Caused by the Streptococcus suis Serotype 2 Sequence Type 25 North American Prototype Strain. Pathogens, 12(11), 1325. https://doi.org/10.3390/pathogens12111325