Fibronectin
Keratin
Fibrinogen
Results
The development of a whole cell high-throughput assay to detect surface adhered S. aureus
- Nichol K.A.
- Adam H.J.
- Roscoe D.L.
- Golding G.R.
- Lagace-Wiens P.R.S.
- Hoban D.J.
- Zhanel G.G.
- Zhanel G.G.
- Hoban D.J.
- Adam H.J.
- Karlowsky J.A.
- Baxter M.R.
- Nichol K.A.
- Lagace-Wiens P.R.S.
- Walkty A.
- on behalf of the Canadian Antimicrobial Resistance Alliance (CARA)
- Al-Rawahi G.N.
- Reynolds S.
- Porter S.D.
- Forrester L.
- Kishi L.
- Chong T.
- Bowie W.R.
- Doyle P.W.


Validation of the high-throughput whole cell S. aureus adhesion assay


Construction of a S. aureus genetic adhesion network highlights the anti-adhesive target space
Gene | Locus | Keratin (0.125 μg/well) % adhesion | Keratin p value | Fibronectin (0.125 μg/well) % adhesion | Fibronectin p value | Fibrinogen (0.25 μg/well) % adhesion | Fibrinogen p value |
---|---|---|---|---|---|---|---|
agrC | SAUSA300_1991 | 106.8 | 0.57 | 102.4 | 0.75 | 77.0 | 0.040* |
arlR | SAUSA300_1308 | 107.4 | 0.33 | 111.5 | 0.25 | 57.9 | 0.00096* |
atl | SAUSA300_0955 | 51.6 | 0.026* | 23.7 | 0.0015* | 50.7 | 0.0044* |
clfA | SAUSA300_0772 | 104.0 | 0.72 | 130.1 | 0.20 | 44.4 | 0.00050* |
clfB | SAUSA300_2565 | 42.2 | 0.0055* | 81.4 | 0.45 | 101.9 | 0.26 |
codY | SAUSA300_1148 | 68.3 | 0.038* | 49.4 | 0.012* | 97.2 | 0.80 |
fmtA | SAUSA300_0959 | 60.5 | 0.028* | 73.2 | 0.12 | 76.7 | 0.049* |
fmtC | SAUSA300_1255 | 96.1 | 0.44 | 82.7 | 0.13 | 54.5 | 0.00093* |
hypo | SAUSA300_0602 | 100.4 | 0.95 | 100.4 | 0.97 | 65.1 | 0.026* |
mazE | SAUSA300_2026 | 98.9 | 0.92 | 68.9 | 0.0058* | 96.3 | 0.75 |
recD2 | SAUSA300_1576 | 41.8 | 0.00029* | 38.4 | 0.0019* | 20.6 | 0.000082* |
rpiR | SAUSA300_2264 | 116.6 | 0.050 | 67.1 | 0.0056* | 136.9 | 0.020 |
rpoF | SAUSA300_2022 | 18.6 | 0.0013* | 25.7 | 0.0012* | 35.2 | 0.0018* |
rsbU | SAUSA300_2025 | 16.5 | 0.0012* | 17.0 | 0.00082* | 35.3 | 0.0016* |
saeR | SAUSA300_0691 | 108.1 | 0.43 | 79.6 | 0.030* | 121.7 | 0.056 |
saeS | SAUSA300_0690 | 104.9 | 0.44 | 86.0 | 0.037* | 117.5 | 0.016 |
sarA | SAUSA300_0605 | 26.0 | 0.00016* | 29.6 | 0.000051* | 17.7 | 0.000085* |
sarS | SAUSA300_0114 | 104.3 | 0.55 | 70.4 | 0.014* | 116.1 | 0.14 |
srtA | SAUSA300_2467 | 20.9 | 0.0015* | 21.4 | 0.00083* | 34.4 | 0.0016* |
ydiL | SAUSA300_1984 | 102.8 | 0.86 | 115.7 | 0.068 | 69.0 | 0.015* |

Assessing the proteolytic activity of adhesion attenuated mutants

Discussion
Materials and methods
Bacterial strains, plasmids, and growth conditions
Primers | Sequence (5′-3′) | Transposon primer used | Source |
---|---|---|---|
Confirmation of bursa aurealis transposon insertion site | |||
Upstream | CTCGATTCTATTAACAAGGG | Bae et al. (2008) | |
Buster | GCTTTTTCTAAATGTTTTTTAAGTAAATCAAGTAC | Bae et al. (2008) | |
New_Upstream | CTTCAAACTTGACTTCAGC | This study | |
New_Buster | CCAGTCTGGATCCAGTTG | This study | |
accC_Tn_check | TTCGTTGTTTAATTGCG | Buster | This study |
arlR_Tn_check | TTGATTACGGTGCAGA | Upstream | This study |
atl_Tn_check | GTTGCATTAACGCTTGTAG | Upstream | This study |
clfA_Tn_check | AACACGCAATTCGGAA | Buster | This study |
clfB_Tn_check | TTGAAAAAAAGAATTGATTATTTGTC | Buster | This study |
codY_Tn_check | ACGAGAGAGTTAAACACG | Upstream | This study |
fmtA_Tn_check | GGTTGCGCCGTCTAAAC | fmtA_r versus_check | This study |
fmtA_rversus_check | CACCCTTCGTATTGTAAGG | fmtA_Tn_check | This study |
fmtC_Tn_check | GGCATCGCTTGTTATTC | Upstream | This study |
fnbA_Tn_check | CTTAGGTACGGCATTAG | Buster | This study |
fnbB_Tn_check | GCAATCTTAGATACGGC | Buster | This study |
GNAT4_Tn_check | GAGACTTGTTTCGACAG | Upstream | This study |
graR_Tn_check | TGGGTGATATGGATGC | Upstream | This study |
hypo_rversus_check | GCAAGTGGCAACTCTATTG | New_Buster | This study |
ilvE_new_check | GCAATCACCATGTCACAAG | ilvE_rversus_check | This study |
ilvE_rversus_check | GTACAACGACTCTCCAAC | ilvE_new_check | This study |
mazE_Tn_check | TGATTAGACGAGGAGATG | Upstream | This study |
PutLipo_Tn_check | GACACTGGGATGTTTAC | Upstream | This study |
RecD2_Tn_check | TGTCAGACCCTACACT | Upstream | This study |
rot_Tn_check | GCATAAGTTAGCACATACAA | Buster | This study |
rpiR_Tn_check | CGCGTTAAACAACAATAGC | New_Upstream | This study |
rpoF_Tn_check | GCGAAAGAGTCGAAATC | Buster | This study |
rsbU_Tn_check | GCCTGAAGACATTGTCG | Upstream | This study |
rsbW_rversus_check | TTATCGAAATGCGCG | Buster | This study |
sarA_Tn_check | GAGTTGTTATCAATGGTCAC | New_Upstream | This study |
sarS_Tn_check | AGTGCATATACAAGGAGA | NaT_fwd_check | This study |
sdcS_NaT_Tn_check | GTGCGGGACAACTTATTG | NaT_fwd_check | This study |
NaT_fwd_check | GCACTGATTAAGTTTACCC | SdcS_NaT_Tn_check | This study |
spa_rversus_check | CTAGGTGTAGGTATTGCATC | New_Buster | This study |
srtA_Tn_check | TTATTTGACTTCTGTAGCTACAAAGATTTTACG | Upstream | This study |
srtB_Tn_check | TTAACTTACCTTAATTATTTTTGCGAC | Upstream | This study |
vraG_Tn_check | TGGCGTTAATTATGACC | Upstream | This study |
xerC_Tn_check | GAATCATATTCAAGATGCGT | Upstream | This study |
yabJ_rversus_check | CACAACAAGATTACCGG | Upstream | This study |
YdiL_Tn_check | GGGCATCATTGCTAACTG | New_Buster | This study |
YfeH_Tn_check | ATGTTGCGACATTAGG | Upstream | This study |
Antibodies and reagents
The ELISA-based S. aureus adhesion assay
Crystal violet adhesion assay
Profiling the NTML to identify adhesion attenuated mutants
Assessing mutant proteolytic activity
Data availability
Acknowledgments
Supplementary Material
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This article contains supporting information.
Author contributions—L. E. P., A. C. L., and G. C. data curation; L. E. P., A. C. L., and G. C. formal analysis; L. E. P., A. C. L., and J. M. validation; L. E. P., A. C. L., and J. M. investigation; L. E. P. and A. C. L. visualization; L. E. P. and G. C. methodology; A. C. L. and G. C. writing-review and editing; G. C. conceptualization; G. C. resources; G. C. supervision; G. C. funding acquisition; G. C. writing-original draft; G. C. project administration.
Funding and additional information—This work was supported by New Frontiers in Research Fund-Exploration Grant NFRFE-2018-01058, a Medical Research Grant from the J. P. Bickell Foundation, and the Canada Foundation for Innovation Grant JELF 37730 (to G. C.).
Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.
Abbreviations—The abbreviations used are: CWA
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