1H, 13C and 15N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae

doi:10.21203/rs.3.rs-4391209/v1

Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein's N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. We succeed to assign the ¹³C-¹⁵N- self-complemented MrkA monomer, with carbon atoms resonances accounting for 81%, backbone nitrogen atoms resonances for 92%, and proton resonances for 92%. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 175-180. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies.

Neonatal sepsis is a major cause of death across low- and middle-income countries (LMICs) [1]. These infections, occurring in newborns, are acquired both in communities and in health-care facilities [2]. Klebsiella pneumoniae (Kp) has been identified by different surveillance networks as a leading cause of neonatal sepsis [3]. Kp is a gram-negative, encapsulated bacterium, belonging to the Enterobacteriaceae family, often found in a variety of environmental niches [4]. Kp produces several biomolecules that are essential for virulence, including fimbriae that aid in the initial colonization of the host and capsular polysaccharides that protect the organism from phagocytosis, complement and inhibit macrophage differentiation [5]. Fimbriae are typically extracellular appendages with 0.5–10 µm length and 2–8 nm width, which are encoded by the mrk gene cluster (mrkABCDF) that is comprised of five genes encoding the structural and assembly components of the appendages [6]. Two major adhesive fimbriae structures are responsible for adherence of Kp to eukaryotic epithelial cells: the mannose-sensitive type 1 fimbriae composed of a major fimbrial FimA subunit and a minor tip adhesin FimH; and the mannose-resistant type 3 fimbriae, composed of the major fimbrial subunit MrkA and the minor tip adhesin MrkD [7–9]. The type 3 fimbriae are believed to be assembled using the chaperone/usher pathway used by a variety of fimbrial systems. In fact, exploring other fimbrial gene clusters MrkB and MrkC are recognized to belong to the family of periplasmic chaperones and scaffolding proteins implicated in fimbrial assembly [10–12]. In this assembly pathway, fimbrial subunits are transported via the general secretory pathway to the periplasm where a chaperone, in the case of type 3 fimbriae encoded by mrkB, forms a complex with the fimbrial subunit proteins. This complex is directed to the scaffolding protein MrkC, located at the outer membrane. Fimbrial assembly is accomplished by addition of MrkA subunits to the growing appendage and MrkD as its tip [10, 11]. Previous studies have indicated that MrkF may be randomly incorporated into the growing fimbrial appendage to confer stability or may serve as an adaptor protein for MrkD and MrkA; its precise location in the fimbriae is unknown [6, 13]. MrkA, a 20 KDa protein with a high conserved amino acid sequence among the Enterobacteriaceae strains analyzed so far [14], has been recognized as the common protein antigen expressed by the majority of Kp strains with the function of biofilm formation and establishment of infection [15–17]. To date, its 3D molecular structure is not known. Here we take the challenge to assign the NMR signals of this protein, as first step toward its more in-depth structural characterization. Such studies are essential to investigate this protein as a potential antigen and to look into its mechanism of action. We assign the recombinant form of the protein, by generating a self-complemented variant of MrkA, which is extended at the C-terminus by a hexaglycine linker followed by a second copy of the MrkA donor strand (residues 1–20 in wild-type (wt) MrkA). The donor strand is a key element for fimbrial proteins [18]: it is reported that the elongation to a fimbriae is due to the interaction via donor strand complementation among the subunits, where the incomplete, immunoglobulin-like fold of each subunit is complemented by an N-terminal donor strand of the subsequent subunit [19, 20].

Design, expression and purification of the self-complemented MrkA monomer

The donor strand displacement strategy is applied to MrkA of Kp in order to obtain a self-complemented monomer not able to elongate to a fimbria Fig. 1. Specifically, the donor strand (first 20 aa in the mature protein after leader sequence cleavage) present at the N-terminus is moved at the C-terminus and a hexaglycine linker is added to let the donor strand assuming an antiparallel orientation within the beta sheet as it has been already observed for inter-molecular donor strand complementation in FimA polymers [20].

The corresponding gene of self-complemented MrkA monomer (preceded by a methionine and a 10-histidine tag) is inserted into a pET29b (+) Twist Bioscience plasmid, resulting in a construct of 201 residues. The plasmid is used to transform E. coli BL21 (DE3) competent cells by ThermoFisher Scientific. Cell growth is performed in ¹⁵N and ¹³C-¹⁵N ISOGRO medium by Sigma-Aldrich (5g/L; addition of 100 g/L K₂HPO₄, 50 g/L KH₂PO₄, 50 g/L MgSO₄ and 37 g/L CaCl₂) at 30°C in order to obtain both mono-labeled and double-labeled MrkA monomer. When the culture reaches an OD₆₀₀ of 0.8–1, 1 mM IPTG is added to induce protein expression, and the cells are incubated at 20°C overnight. Cells are harvested and lysed using CelLytic Reagent by Sigma-Aldrich, following the manufacturer’s instructions. After incubation, the lysate is centrifuged and the supernatant containing the soluble protein fraction is diluted with 50 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole pH 7.4, filtered using a 0.22 µm filter and then loaded in a HisTrap FF affinity chromatography column by Cytiva. The column is then washed with an imidazole gradient and MrkA protein eluted with 500 mM imidazole, pH 7.4. A size exclusion chromatography step is finally performed to ensure the removal of aggregates from the final protein sample. A Superdex 75 Increase prepacked column by Cytiva has been chosen with an isocratic elution in 50 mM sodium phosphate, 100 mM NaCl pH 7.0. Peak fractions are pooled together and checked by SDS-PAGE gel analysis to confirm the monomeric form of the proteins and their purity (Fig. 2).

NMR spectroscopy

All NMR experiments used for resonances assignment of MrkA are recorded on a Bruker AVANCE 950 MHz spectrometer on ¹³C-¹⁵N-labeled sample. Heteronuclear relaxation measurements, ¹⁵N- R₁, ¹⁵N-R₂ and ¹H-¹⁵N NOE are recorded on a Bruker AVANCE 500 MHz spectrometer equipped with a triple resonance cryoprobe TXI on a ¹⁵N-MrkA. For ¹H-¹⁵N NOE measurements, delays of 5s are used between repetitions of the pulse sequence. For ¹⁵N- R₁ and ¹⁵N -R₂ 3s of delay is used. Amide resonances are integrated using CARA software (Keller 2006) and ¹⁵N- R₁ and ¹⁵N-R₂ values are obtained by fitting peak intensities using single exponential decay:

I_(t) = I₀ exp (-t/T_1,2)

where I_(t) is the peak intensity, t is the time, and I₀ is the intensity at time 0 using ORIGIN software (Origin (Pro), Version 2023 OriginLab Corporation, Northampton, MA, USA). The analysis of the uncertainties of the ¹⁵N- R₁ and ¹⁵N-R₂ values is carried out by comparing the peak heights on duplicate spectra at 10 ms (shortest value of relaxation delay). The heteronuclear steady-state and ¹H-¹⁵N NOE values are obtained from the ratios of peak intensities in the saturated spectrum to those in the unsaturated spectrum. The radio frequency pulses, carrier frequencies, acquisition and processing parameters of all the NMR experiments needed for the backbone and side-chain resonances assignment are reported in Table 1.

The NMR samples has a protein concentration of about 400 µM for ¹³C-¹⁵N-MrkA and 450 µM for ¹⁵N-MrkA in 50 mM sodium phosphate, 100 mM NaCl pH 7.0 and 10% (v/v) D₂O. All NMR spectra for resonances assignment are collected at 298 K, processed using the standard Bruker software Topspin (version 4.3) and analyzed through the CARA program (Keller 2006).

Extent of assignments and data deposition

The ¹H- ¹⁵N HSQC spectra of MrkA show well-dispersed resonances indicative of an essentially folded protein (Fig. 3). The backbone resonance assignment is obtained from the analysis of the triple resonance spectra. 181 out of the expected one 196 ¹⁵N backbone amide resonances are assigned. The amide resonances are missing for residues Met 1, Gly 2, Ser 3, His 4- His 13, Gly 179 and Gly 180. The assignment of the aliphatic side chain resonances is performed through the analysis of 3D CC(CO)NH and (H)CCH-TOCSY spectra, together with ¹⁵N-NOESY-HSQC and ¹³C-NOESY-HSQC spectra. The assignment of the aromatic spin systems is performed with 2D NOESY and TOCSY maps and a 3D ¹³C-NOESY-HSQC spectrum with the carrier centered in the aromatic region at 130 ppm. In total, the resonances of 81% of carbon atoms, 92% of backbone nitrogen atoms, and 92% of protons are assigned, leaving only Met 1, Gly 2, Ser 3, His 4–13 and Gly 179 completely unassigned.

We determine the amino acid specific secondary structure properties of MrkA from the assigned backbone chemical shifts (HN, Cα, Cβ, CO, N); using TALOS-N program (Shen & Bax 2013) we reveal that the secondary structure of MrkA comprises three small α-helices and eight β-strands (Fig. 4).

¹⁵ N-relaxation experiments

Reliable ¹⁵N R₁, R₂, and ¹H-¹⁵N NOE values, which provide information on internal mobility, are obtained for 181 of the 196 assigned backbone NH resonances. Peaks are integrated using CARA software and the relaxation rates are calculated using EXCEL/ORIGIN software. R₁, R₂, and ¹H-¹⁵N NOE average values of MrkA are 1.41 ± 0.1 s^− 1, 14.5 ± 0.73 s^− 1, and 0.77 ± 0.06, respectively. The relaxation parameters are essentially homogeneous along the entire polypeptide sequence with exception of glycine stretch located in loop 175–180 (Fig. 5). The correlation time for molecule reorientation (τ_m), estimated from the R₂/R₁ ratio, is 10.2 ± 0.7 ns, as expected for a protein of this size in a monomeric state (Khashami, 2024).

The complete assignment of the bacterial protein antigen is a key step in the full characterization of MrkA. Thanks to this preliminary work performed with solution NMR spectroscopy, we set the basis for solving the NMR solution structure of this antigen. In perspective, structural studies are essential to characterize and better design the protein as antigen in vaccinology.

Data availability

The chemical shift values for ¹H ¹⁵N and ¹³C resonances of MrkA are deposited BioMagResBank BMRB, under access number 52205.

Acknowledgements

The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work was supported by the project PAN-HUB 2021-T4-AN-07-CUP B93C22000920001, “Hub multidisciplinare e interregionale di ricerca e sperimentazione clinica per il contrasto alle pandemie ed all'antibiotico resistenza”.

VM V. Monaci is a PhD student at the University of Florence and participates in a post graduate studentship program at GSK.

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Competing interest

The authors declare that they have no competing conflict of interest

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Table 1. Acquisition parameters for NMR experiments performed on MrkA.

Experiments	Time domain data size			Spectral width			T (K)	ns	D1	Acquisition time			Magnetic Field
	(points)			(ppm)			T (K)	ns	(s)	(ms)			(MHz)
	t₁	t₂	t₃	F1	F2	F3				F1	F2	F3
HNCA	96	48	2048	30 (¹H)	38 (¹⁵N)	14 (¹³C)	298	8	0.25	4.8	6.1	92	950
HNCO	88	48	2048	16 (¹³C)	38 (¹⁵N)	14 (¹H)	298	4	0.25	9.9	6.3	92	950
HNCOCA	96	48	2048	30 (¹H)	38 (¹⁵N)	14 (¹³C)	298	16	0.25	4.8	6	92	950
HNCACO	88	48	2048	16 (¹H)	38 (¹⁵N)	14 (¹³C)	298	24	0.25	9	6	90	950
15N-separated NOESY	256	68	2048	14 (¹H)	32 (¹³C)	14 (¹H)	298	8	1	7.6	1.4	61	950
13C-separated NOESY	256	68	2048	14 (¹H)	32 (¹³C)	14 (¹H)	298	4	1	7.6	1.4	61	950
HNCACB	96	48	2048	80 (¹³C)	38 (¹⁵N)	14 (¹H)	298	24	1	2.5	6.2	77	950
CBCACONH	96	48	2048	80 (¹³C)	38 (¹⁵N)	14 (¹H)	298	16	1	2.5	6.2	77	950
HCCH TOCSY	1	64	2048	80 (¹³C)	80 (¹³C)	14 (¹H)	298	16	1.2	7.6	1.4	61	950
¹⁵N-HSQC	1024	128	-	16 (¹H)	38 (¹⁵N)	-	298	8	1.2	64	16	-	500-950
¹⁵N -R₁ ¹⁵N-HSQC*	1024	128	-	16 (¹H)	38 (¹⁵N)	-	298	16				-	500
¹⁵N-R₂-HSQC*	1024	128	-	16 (¹H)	38 (¹⁵N)	-	298	16				-	500
* A series of twelve ¹⁵N -R₁ ¹⁵N-HSQC experiments are recorded using period of 0.010 s, 0.04 s, 0.08 s, 0.125 s, 0.200 s, 0.370 s, 0.500 s, 0.675 s, 0.800 s, 1 s, 1.55 s and 2.5 s. A series of eleven ¹⁵N-R₂-HSQC experiments are recorded using period of 8.48 ms, 16.96 ms, 33.92 ms, 50.88 ms, 67.84 ms, 101.76 ms, 135.64 ms, 152.64 ms, 186.56 ms, 203.52 ms and 220.48 ms.

No competing interests reported.

¹H, ¹³C and ¹⁵N assignment of self-complemented MrkA protein antigen from Klebsiella pneumoniae

Status:

Journal Publication

Version 1

Abstract

Figures

Biological context

Methods and experiments

Design, expression and purification of the self-complemented MrkA monomer

NMR spectroscopy

Extent of assignments and data deposition

Conclusion

Declarations

References

Table

Additional Declarations

Status:

Journal Publication

Version 1