Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells
Abstract
:1. Introduction
2. Results
2.1. Infection of Human HCT116 Cancer Cells Results in Reduced M. fermentans MF-I1 Replication, Increased Expression of dnaK DNA Copy Number and Detection of Intracellular DnaK
2.2. Analysis of MF-I1 dnaK Transcripts Exposes the Emergence of Bacteria with Several Mutations in Important Regulatory Regions
2.3. M. fermentans MF-I1 Strain dnaK mRNA Length Analysis Reveals Presence of Several Types of Transcripts
2.4. Bacteria Grown in Different Conditions Show Differences in dnaK mRNA and Its Internal Promoter
3. Materials and Methods
3.1. Culture of Bacteria
3.2. Colony Forming Unit Assay
3.3. Cells and MF-I1 Infection
3.4. Accession Numbers
3.5. DNA and RNA Extraction
3.6. Analysis of Subcellular Fractions by SDS-PAGE and Immunoblot
3.7. Cloning of MF-I1 dnaK
3.8. Quantitative PCR (qPCR) for MF-I1 and Host Cell Genome Copy Numbers
3.9. Circularized RT-PCR (cRT-PCR) for Analysis of 5′ and 3′ Ends of MF-I1 Isolate mRNA
3.10. Sequence Analyses
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations
MF-I1 | Mycoplasma fermentans-IHV-substrain 1 |
PCR | Polymerase Chain Reaction |
PARP1 | Poly (ADP-Ribose) Polymerase-1 |
USP10 | Ubiquitin carboxyl-terminal hydrolase 10 |
CFU | Colony Forming Units |
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Time Point | Internalized MF-I1 (CFU/106 HCT116 Cells) | MF-I1 in Supernatant from HCT116 Culture (CFU/mL) | MF-I1 in McCoy’s 5A Medium Axenic Culture (CFU/mL) |
---|---|---|---|
Day 1 | 8242 ± 2224 | 8193 ± 3461 | 905 ± 75 |
Day 3 | 62,647 ± 3512 | 33,303 ± 3461 | 3568 ± 1275 |
Day 6 | 26,371 ± 596 | 336,665 ± 42,757 | 7874 ± 903 |
dnaK Promoters | −35 Box | −10 Box |
---|---|---|
Pr. 1 | (−173) TTGATT | (−153) ATTTAATTT |
Pr. 2 | (+197) TTGCAT | (+219) GGGTACAAA |
Pr. 3 | (+617) ATGAAA | (+638) TGCTAAAAT |
Pr.4 | (+1205) TTGAAA | (+1224) TGTTACAAT |
CLONE | POINT MUTATIONS | INSERTIONS | DELETIONS | |
---|---|---|---|---|
Nucleotide substitution | Amino acid substitution | |||
H5 | A1190G | Glu397Gly | ||
H6 | C1578G | Asn526Lys | ||
H8 | A756G | Leu255Leu | ||
H9 | A1190G | Glu397Gly | ||
H10 | A1190G | Glu397Gly | ||
H11 | A1190G | Glu397Gly | ||
H12 | G373T, T600C, A1236G | Ala125Ser, Gly200Gly, Lys412Lys | ||
H16 | A1488G | Glu483Gly | ||
H19 | A1512G | Ala504Ala | ||
H21 | A1348 (Lys450) | |||
H24 | A1299G | Thr433Thr | ||
H28 | T1304C, G1357A | Ile435Thr, Ala453Thr | ||
H32 | G1321A | Gly441Arg | ||
H33 | A1714G | Ser572Gly | ||
H35 | G1471A, A1484G | Glu491Lys, Asn495Ser | ||
H39 | A1211G | Asn404Thr | ||
H41 | A1200G | Thr400Thr | ||
C2 | A1368G, T1564C | Ile456Met, Val522Ala | ||
C9 | 790G (His264) | |||
C10 | T648C | Ile216Ile | ||
C11 | G4A, C1309T | Pro2Ser, Ala437Thr | ||
C13 | C528G | Phe176Leu | ||
C15 | T1263C | Ala421Ala | ||
C16 | G1715A | Ser572Asn | ||
C20 | C732T | Asp244Asp | ||
C24 | A1075G, T1076C | Ile359Ala | ||
C26 | A11G, T57C | Glu4Gly, Ala19Ala | ||
C34 | A1503C | Lys501Asn | ||
C35 | C761T, A1396G | Ser254Leu, Thr466Ala | ||
C37 | A37G | Thr13Gly | ||
C40 | C710T | Ala237Val | ||
M25 | T1062G | Val354Val | ||
M26 | T1070C | Arg390Arg | ||
M30 | G632T | Trp211Leu | ||
M34 | T764G, G765T | Leu259Leu, Val260Phe | ||
M35 | C1298T | Thr433Ile |
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Curreli, S.; Tettelin, H.; Benedetti, F.; Krishnan, S.; Cocchi, F.; Reitz, M.; Gallo, R.C.; Zella, D. Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells. Int. J. Mol. Sci. 2021, 22, 3885. https://doi.org/10.3390/ijms22083885
Curreli S, Tettelin H, Benedetti F, Krishnan S, Cocchi F, Reitz M, Gallo RC, Zella D. Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells. International Journal of Molecular Sciences. 2021; 22(8):3885. https://doi.org/10.3390/ijms22083885
Chicago/Turabian StyleCurreli, Sabrina, Hervé Tettelin, Francesca Benedetti, Selvi Krishnan, Fiorenza Cocchi, Marvin Reitz, Robert C. Gallo, and Davide Zella. 2021. "Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells" International Journal of Molecular Sciences 22, no. 8: 3885. https://doi.org/10.3390/ijms22083885
APA StyleCurreli, S., Tettelin, H., Benedetti, F., Krishnan, S., Cocchi, F., Reitz, M., Gallo, R. C., & Zella, D. (2021). Analysis of DnaK Expression from a Strain of Mycoplasma fermentans in Infected HCT116 Human Colon Carcinoma Cells. International Journal of Molecular Sciences, 22(8), 3885. https://doi.org/10.3390/ijms22083885