Part:BBa_K4645003

Fusion protein formed by CsgA and Hsa with (GGGGS)4 as linker.

The NR2 domain of Hsa is the sialic-binding core of Hsa protein, and CsgA is the extracellular fiber component of Escherichia coli biofilm formation. The two proteins were connected with a flexible linker to enable them to colonize on cat teeth and form a stable biofilm, creating conditions for the realization of subsequent neutralization.A 6x His tag was added to the C-terminus of the protein for purification or specificity testing in experiments.

Usage and Biology

Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit. CsgA proteins can self-assemble into amyloid nanofibers, characterized by their hierarchical structures across multiple length scales, outstanding strength and their structural robustness under harsh environments.The extracellular stacking of this protein forms the scaffold of the E. coli biofilm. Many streptococci adhere to protein-attached carbohydrates expressed on cell surfaces using Siglec-like binding regions (SLBRs). SLBRs are usually found within the context of serine-rich repeat proteins, which form fibrils extending from the bacterial surface. The Siglec domain contains a ΦTRX sequence motif15 that recognizes Neu5Acα2-3Gal in the context of larger glycans[1].

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 37
  Illegal PstI site found at 861
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 37
  Illegal PstI site found at 861
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 37
  Illegal PstI site found at 861
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 37
  Illegal PstI site found at 861
  
• 1000
  COMPATIBLE WITH RFC[1000]

Gene Design in Circuits

The circuit of the adhesion part is as follows(Figure 1)

Figure 1. Gene circuit of adhesion part.

The Molecular Structure Of the Protein

Before the experiment, we predicted the protein structure by Alpha Fold 2 and made its 3D structure simulation map （Figure 2）.

Figure 2. Molecular structure prediction of CsgA-Hsa based on Alpha Fold 2.

Validation Of Function Trials

To obtain the CsgA-Hsa protein, we constructed pET-28a(+)- csgA-linker-hsa and transferred it to E. coli BL21(DE3) (Figure 3). The E. coli strain was cultured to OD₆₀₀=0.6, induced with 1 mM IPTG, and alllowed the bacterial to grow 16 hours at 16℃, 160 rpm. CsgA-Hsa's molecular weight is about 43 kDa. The fusion protein CsgA-Hsa was confirmed to be expressed by SDS-PAGE analysis (SDS-PAGE Preparation kit , Sangon Biotech (Shanghai) Co., Ltd), although the linker of the fusion protein may have been broken during sonication.

Figure 3. Colony PCR assay of the csgA-hsa fragment.
Figure 4. SDS-PAGE analysis of CsgA-Hsa expression.

Since more protein fractures were found in purified protein samples after ultrasonic treatment, we had to redesign the verification scheme. To verify the adhesion effect of the CsgA-Hsa fusion protein, we cultured BL21 (DE3) strain without plasmid (WT), BL21 (DE3) strain with plasmids induced by gradients of 0.1 mM, 1 mM, and 5 mM IPTG (IPTG+).Subsequently, the cultured bacterial solution was sonicated and centrifuged at 5000rpm at 4℃ to obtain the supernatant as the total bacterial protein solution for use. The total protein solution in each group was controlled at 3mg/mL by dilution with PBS buffer.

In our experiments to verify adhesion, we referred to a method called "Tooth Blot" used in the 2020 HZAU Tooth Fairy project, Designed our "Adhesion tests based on detection of chemiluminescence" to vertify the existence and function of CsgA-Hsa.
The cat's teeth, a gift from Pet-King Pet Hospital, were physically cut into five similar-sized pieces, and the 5 tooth fragments were incubated in the same tube of cat saliva at 37℃ and 200rpm for 14 hours. Then The cat teeth incubated with saliva were removed, and each tooth fragment was placed on a petri dish with a diameter of 10cm and washed twice with 2 mL PBS for 2-3 min each time. After that, The cleaned tooth fragments were placed in the previous bacterial crushing supernatant and incubated at 37℃ and 200 rpm for 20 hours to bind Hsa to sialic acid；The tooth fragments in the cell crushing solution were removed and placed in a petri dish with the same specifications as before. The unbound proteins were washed with PBS, and washed with 2 mL PBS for 2-3min each time, washing twice.
Use 5% skim milk with 1: 5000 Dilute Anti-6xHis rabbit polyclonal antibody (Sangon Biotech (Shanghai) Co.,Ltd.), add 2 mL diluent of antibody to each plate, and shake in a shaker for 2 h at room temperature. Then the uncombined primary antibody was cleaned with 1x TBST, and the washing dosage of each plate was 3 mL, the washing time was 10 min, and washing three times. After washing, 2 mL HRP-conjugated Goat Anti-Rabbit IgG (Sangon Biotech (Shanghai) Co.,Ltd.) diluted 1:5000 with 5% nonfat milk was added to each plate and shaken for 2 h at room temperature on a shaker (Figure 5). After incubation, 1x TBST was added to rinse the unbound goat anti-rabbit antibody. Wash each plate with 3 mL TBST for 10 min each time, washing three times. Finally, the teeth were transferred to a 96-well plate and 100μL Ultrasensitive ECL Chemiluminescence Kit (Sangon Biotech (Shanghai) Co.,Ltd.) was added to each well to react for 10 min. The luminescence intensity of each well was measured using a microplate reader as a reference for the amount of adhered Hsa (Figure 6).
Although we could not carry out more repeated tests due to the lack of cat tooth material, we could see that the chemiluminescence intensity of the total protein solution expressing the fusion protein was greater than that of the non-transfected plasmid, which means that the fusion protein could bind to the sialic acid-coated teeth.

Figure 5. Primary and secondary antibodies were incubated.

Figure 6. Chemiluminescence detection results.
Control 1 is a blank ECL chromogenic solution, Control 2 is the WT

In order to verify the effect of CsgA in the fusion protein on the biofilm formation of engineered bacteria, we determined the OD value after crystal violet staining to determine the effect of CsgA on the biofilm formation[2]. After BL21 (DE3) was induced with 0.1 mM IPTG, 10 uL of BL21 (DE3) was taken and added into 96-well plates, while uninduced BL21 (DE3) was set as a negative control group and a blank control group with only medium, and 9 replicate experiments were done in each group. After 1% crystal violet staining and elution, the OD₆₀₀ detection was performed by enzyme marker and the results of Figure 7. were obtained. We can determine that the biofilm formation ability of the engineered bacteria containing the target gene is very significantly different from the two control groups at an inducible concentration of 0.1 mM IPTG, which confirms that the csgA-hsa gene indeed enhances the formation of bacterial biofilm.

Figure 7.The results of induction with IPTG after 10 hours.
Because the difference between the experimental group and the two control groups was too large, the Y-axis is expressed in logarithmic format.

In order to obtain more direct experimental results of biofilm formation and adhesion, we designed a cover slip with saliva treatment in a large cup-shaped dish. The engineered bacterial solution was added to the cup-shaped dish, and the OD₆₀₀ was between 0.6-0.8. The group without bacterial solution was used as the control(CT).
First, Escherichia coli was inoculated in LB plates overnight (20h), and single colonies were picked and shaken in 10 ml LB medium for about 20 hours. Six coverslips (soaked in 2% hydrochloric acid overnight in advance and 75% ethanol for later use) were immersed in saliva extract for 1 h before placing the coverslips in the bottom of a 6-well plate. 6 ml of LB culture medium was added to each well. The bacterial solution was diluted with normal saline to an OD₆₀₀ value of 0.8-0.13, and 200 ul of bacterial solution was added to each well. The 6-well plates were incubated at 37 ℃ in a constant temperature incubator. The OD value was measured during the culture, and IPTG was added when the appropriate OD value was reached. After induction of IPTG at 16℃ overnight, the slides were removed on the second day and washed repeatedly with PBS to remove non-adherent bacteria. The excess water was blotted by absorbent paper and fixed with 2.5% glutaraldehyde for 1.5 h. After development in PBS, the slides were stained with PI at 4 ° C in the dark for 15 minutes. Excess water was blotted out after rinsing with PBS and blocked in 40% glycerol on clean slides. Finally, the slides were placed under a fluorescence microscope and excited with green light, and the biofilm formation and adhesion were observed under a microscopic imaging system[3] (Figure 8).

Figure 8. The fluorescence of Escherichia coli stained with propidium iodide was observed by microscopic imaging system. The magnification was 10x10.
A, B, C.Saliva-treated coverslips were incubated with 0.1 mM, 0.5 mM, and 1 mM IPTG-induced bacterial solutions.
D.The bacterial broth was not induced with IPTG. E.Cover slips treated with saliva, but without bacterial solution. F.Image of sample.

References

[1]Bensing BA, Stubbs HE, Agarwal R, Yamakawa I, Luong K, Solakyildirim K, Yu H, Hadadianpour A, Castro MA, Fialkowski KP, Morrison KM, Wawrzak Z, Chen X, Lebrilla CB, Baudry J, Smith JC, Sullam PM, Iverson TM. Origins of glycan selectivity in streptococcal Siglec-like adhesins suggest mechanisms of receptor adaptation. Nat Commun. 2022 May 18;13(1):2753.
[2]CoffeyBM, Anderson GG. Biofilm formation in the 96-well microtiter plate. MethodsMol Biol. 2014;1149:631-641.
[3]David J.Lynch, Tracey L. Fountain, Joseph E. Mazurkiewicz, Jeffrey A. Banas,Glucan-binding proteins are essential for shaping Streptococcus mutans biofilm architecture, FEMS Microbiology Letters, Volume 268, Issue 2, March 2007, Pages 158–165