Urinary tract infections, Uro-pathogens and their associated virulence factors

Urinary tract infections (UTIs) are one of the most common pathological conditions in both community and hospital settings. It has been estimated that about 150 million people worldwide develop UTI each year. Among the common uropathogens associated with UTIs, UroPathogenic Escherichia coli (UPEC) is the primary cause. UPEC strains possess a plethora of both structural (as fimbriae, pili, curli, flagella) and secreted (toxins, iron-acquisition systems) virulence factors that contribute to their capacity to cause disease, although the ability to adhere to host epithelial cells in the urinary tract represents the most important determinant of pathogenicity.⁷ Among the large variety of bacterial adhering factors (adhesins) that promote adhesion and internalization into host cells, type 1 fimbriae are remarkably versatile virulence factors. Therefore our project is primarily focused on the Type 1 fimbriae, since it is commonly found in most of the UPECs and other uropathogens.⁸

Type 1 fimbriae: the virulence factor

Type 1 fimbriae, specifically helps the uropathogens to adhere to a spectrum of alpha-D-mannosides found in mammalian tissue surface. It has a 7nm wide and 1 μm long heteropolymer structure, out of which approximately 1000 copies are of the major subunit, FimA,that are polymerised into a right-handed helical structure. Additionally it also contains a few percent of the minor components FimF, FimG and FimH. ¹

The functions of the components of Type 1 fimbriae are:

1. FimH acts as a receptor- recognition element of type 1 fimbriae . It is located at the tip of the organelle as an integral part of a short fibrillum.
2. FimF and FimG proteins act as adapters for integration of adhesins into the fimbrial organelle.
3. FimC and FimD are the proteins used to assemble fimbriae on the surface of the cell.¹

Since FimH is the main protein interacting with the alpha D mannoside receptors of the host cell , our project aims to use FimH as a bait to detect Uropathogens using a combination of aptamers that show high affinity towards FimH.

FimH - the target protein

FimH is the mannose-binding adhesin found at the tip of the type 1 pilus produced by UPEC. It is a two-domain protein in which the N-terminal domain contains the receptor-binding site and the C-terminal domain is required for organelle integration.¹ The interaction between these two domains, determines the conformational state of the FimH adhesin, and therefore influencing the level of affinity of FimH with the related molecule/receptor/ligand. The conformation of FimH is highly dynamic, and interdomain interactions can be influenced by different factors, including the shear stress.⁹ Besides UPEC, many other uropathogens also express FimH as their virulence factor.⁸

The figure shows the 3 dimensional cartoon representation of FimH structure containing the two primary domains -The N-terminal domain or lectin domain (depicted in cyan) that is required for receptor-binding and the C-terminal domain or the pillin domain (depicted in blue) which is required for organelle integration. An elongated linker (in orange) connects the pilin and the lectin domain of FimH. The protein is bound to alpha D mannose molecule that is depicted as atom colored Vanderwaal spheres.(Adapted from EvaMaria Krammer et al., Molecules 2018)¹⁰

Basic Parts

These are the parts of our cloned constructs that will be generated in our project Apt4UTI:

Serial	Description	Part Number	Type	Length(bp)
1	Expression of FimH1 gene	BBa_K4449000	Coding	540
2	Expression of FimH2 gene	BBa_K4449001	Coding	534
3	Expression of Staphylococcus epidermidis Bap like Bhp gene	BBa_K4449002	Coding	6909
4	pET28b FimH1 F	BBa_K4449003	Primer	32
5	pET28b FimH1 R	BBa_K4449004	Primer	30
6	pET15b FimH2 F	BBa_K4449005	Primer	29
7	pET15b FimH2 R	BBa_K4449006	Primer	29
8	pET28b Bhp F	BBa_K4449007	Primer	28
9	pET28b Bhp R	BBa_K4449008	Primer	28
10	Aptamer Sequences	BBa_K4449012	DNA	80

FimH1 and FimH2

Both FimH1 and FimH2 primarily contains the coding sequence of the FimH gene retrieved from the plasmid pBAD - FimH -9X His that is deposited in the Addgene repository (Addgene # 97305) by Per Svenningsen Lab.

But, in order to clone the FimH gene in two different vectors some modifications had been done in the sequences to maintain the reading frame and hence we have two sequences FimH1 and FimH2, without affecting their functionality.

Here FimH1(BBa_K4449000) is cloned in the pET28b+ vector between Nco1f and Xho 1 sites to generate the construct Apt4UTI C1 (BBa_K4449009). Again, the FimH2 (BBa_K4449001) gene was cloned between the BamH1 and Blp1 sites of pET15b vector to generate the construct Apt4UTI C2 (BBa_K4449010).

Aptamer Sequences

We have generated our own aptamer library which will be utilized in the subsequent rounds of the ligand based SELEX , to select for the best aptamer candidates that will show highest affinity towards FimH1 and FimH2 . This library is characterized by the presence of 40 random nucleotides in between flanked by a common known 5’ and 3’ sequences on both sides which will act as the primer binding sites . These known sequences were added so that the selected aptamer sequences can be subjected to PCR amplification in the positive selection rounds of SELEX technique.

The aptamer sequences were generated using python with the help of Jupyter Notebook.²



Here are the list of generated Aptamer Sequences: Click Here

The given script was used to generate the output:

list_1 = [ ]

for y in range(0,500):

s = "AAAAAAAAAAAACCCCCCCCCCCCGGGGGGGGTTTTTTTT"

t = ''.join(random.sample(s,len(s)))"

list_1.append(t)

print(list_1)

Composite parts

These are the proposed parts that will be generated in our project Apt4UTI:

Serial number	Description	Part Number	Components	Stratergy	Type	Length(bp)
1	Apt4UTI C1	BBa_K4449009	BBa_K3830009 BBa_K389005 BBa_K4103004 BBa_K4047028 BBa_K2259010 BBa_K082004 BBa_K3842018 BBa_I719005 BBa_K4284000 BBa_K4159001 BBa_K4449000 BBa_K1362468 BBa_K731721	Cloning the gene encoding the FimH1(BBa_K4449000) obtained from modifying the FimH gene from plasmid pBAD-FimH-9XHis (Addgene plasmid #97305) into pET28b+ (EMD Biosciences) expression vector between the Nco1 and Xho1 restriction sites.	Composite	5773
2	Apt4UTI C2	BBa_K4449010	BBa_K3982027 BBa_K4284007 BBa_K4103004 BBa_K2259010 BBa_K082004 BBa_K3842018 BBa_I719005 BBa_K4284000 BBa_K4159001 BBa_K1362468 BBa_K1362456 BBa_K4449001 BBa_K731721	Cloning the gene FimH2(BBa_K4449001) (gene sequence obtained from modifying the FimH gene from plasmid pBAD-FimH-9XHis Addgene plasmid #97305) into pET15b (Novagen) expression vector between the Bam H1 and Blp1 restriction sites.	Composite	6197
3	Apt4UTI C3	BBa_K4449011	BBa_K3830009 BBa_K389005 BBa_K4103004 BBa_K4047028 BBa_K2259010 BBa_K082004 BBa_K3842018 BBa_I719005 BBa_K4284000 BBa_K2685025 BBa_K4449002 BBa_K1362468 BBa_K731721	Cloning the gene encoding Staphylococcus epidermidis Bap like Bhp (BBa_K4449002)( coding sequence obtained from European Nucleotide Archive browser) in pET28b+ expression vector (EMD Biosciences) between the Nco1 and Xho1 restriction sites.	Composite	12136

Apt4UTI C1

The DNA insert, FimH1 (BBa_K4449000) in this construct Apt4UTI C1(BBa_K4449001) contains the coding sequence of the E.coli UTI89 FimH signal peptide and lectin domain which forms a subunit of Type 1 fimbriae, located at the tip of the organelle as an integral part of short fimbriae. This construct will enable the expression of a C terminal -6 X His tagged FimH1 protein which will be purified by a Ni-NTA affinity column and will subsequently be used for conducting experiments like SDS PAGE, Receptor Blots and ELISA Based binding assays. The expressed protein, FimH1-6XHis (target) will be also tested with our aptamer library during the ligand based SELEX technique to find out the aptamer showing highest affinity towards the target. The construct map was created by using SnapGene.

To know more related to testing of this part visit Contribution Page



Apt4UTI C2

The DNA insert FimH2 in this construct contains the coding sequence of the E.coli UTI89 FimH signal peptide and lectin domain which forms a subunit of type 1 fimbriae, located at the tip of the organelle as an integral part of short fimbriae. This construct will enable the expression of a N terminal -6 X His tagged FimH2 protein with a thrombin site which will be purified by a Ni-NTA affinity column and will subsequently be used for conducting experiments like SDS PAGE, Receptor Blots and ELISA Based binding assays. The thrombin site will be used to remove the 6X His tag by using the thrombin enzyme which cleaves between the Arginine and Glycine residue. This composite part will enable us to validate if our candidate aptamers are truly binding to the purified FimH2 protein and are showing no interaction with the His Tag. By designing this construct we will be setting up a platform to purify tag free FimH protein. The expressed protein, FimH2 (target) will be also tested with our generated aptamer library during the ligand based SELEX technique to find out the aptamer showing highest affinity. The construct map was created by using SnapGene.

To know more related to testing of this part visit Contribution Page



Apt4UTI C3

The DNA insert (Bap like Bhp) in this construct Apt4UTI C3 encodes a virulence factor of S. epidermidis that helps in biofilm accumulation is Bap (biofilm associated protein) which is known as a surface adhesion protein that is commonly found in S. epidermidis strains.³ This construct will enable the expression of Bhp protein with a C terminal 6X His tag in E Coli strain BL21DE3. The expressed fusion protein will subsequently be purified by using the Ni-NTA affinity column due to the presence of the His tag. Bhp will be used for the negative selection round of ligand based SELEX and will serve as a negative control in our ELISA based binding assays. We have chosen this part from Staphylococcus epidermis because Staphylococcus epidermidis comprises a minority of the microflora of the lower part of the Urethra , and can be expected to be present as a contaminant in the urine cultures.⁴ experiments like SDS PAGE, Receptor blots and ELISA based binding assays will be conducted for testing this part. The construct map was created by using SnapGene.



Existing parts

The following existing parts will be used in our project:

Biobrick	Part Number
f1 ori	BBa_K3830009
KanR	BBa_K389005
ori	BBa_K4103004
bom	BBa_K4047028
rop	BBa_K2259010
lacI	BBa_K082004
lacI promotor	BBa_K3842018
T7 promoter	BBa_I719005
lac operator	BBa_K4284000
RBS	BBa_K2685025
6x His	BBa_K1362468
T7 terminator	BBa_K731721
Thrombin site	BBa_K1362456
AmpR promoter	BBa_K3982027
Amp R	BBa_K4284007