Staphylococcus aureus

Streptococcus pneumoniae

Virulence of S. pneumoniae

All over the world, Streptococcus pneumoniae causes the life threating invasive diseases pneumonia, sepsis and meningitis. In early childhood the diplococcus colonizes the epithelium of the upper respiratory tract as a commensal bacterium. Under certain circumstances, it gets a threat by tissue invasion. Still up to date, not much is known regarding the mechanism behind the shifting from a colonizer organism to an invader or how S. pneumoniae is able to cross the blood-brain barrier to cause meningitis.[http://www.ncbi.nlm.nih.gov/pubmed/19880020] To cross the line from an opportunist to a pathogen, S. pneumoniae needs to express specific virulence factors in a coordinated way. Apart from virulence factors like the polysaccharide capsule, the pore forming toxin pneumolysin or surface proteins like PspA and PspC, also biofilm production plays an important role for persistence in the human nasopharynx and contributes to pneumonia and meningitis [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618759/].

One aspect of its infectiousness is the ability to perform natural transformation. Thus, it can take up extracellular DNA containing the information for resistance against diverse antibiotics and permanently incorporate the DNA into its genome via recombination. The main problem [1] of treating S. pneumoniae infections is the increasing spread of ß-lactam resistance. Luckily, the molecular mechanism behind natural transformation in S. pneumoniae is the focus of current research.

Natural Transformation in S. pneumoniae

In S. pneumoniae, biofilm production and competence for natural genetic transformation are triggered by a two component regulatory system, responsive to environmental stimuli. There, a peptide pheromone called competence-stimulating-peptide (CSP) binds to and consequently activates the membrane-embedded sensor kinase ComD by changing the conformation of the polytopic kinase. ComD autophosphorylates and subsequently transphosphorylates the cognate response regulator ComE. ComE acts as transcription activator via binding to the -10 promotor regions of early and late competence genes. These ComE binding sites (CEbs) consist of a 9 bp direct repeat seperated by a stretch of 12 nucleotides.

A positive feedback loop ensures the availability of the two component system by activating the expression of the comCDE operon, where comDE encodes for the two component system ComDE and comC for the precursor peptide pre-CSP. Pre-CSP is cleaved to the mature peptide pheromone and transported to the extraplasmic room by the ABC transporter ComAB, whose expression is also activated by ComE. [http://www.ncbi.nlm.nih.gov/pubmed/?term=regulation+of+natural+genetic+transformation+and+acquisition+of+transforming+DNA]. In addition, ComE~P is able to trigger the expression of comX and comW , encoding for the alternative σ factor X, and its stability factor ComW. This alternative σ factor is required for the transcription of the so called late competence genes involved in the process of DNA uptake and recombination.

We are working with the genomic DNA of the nonencapsulated apathogenic strain R6 of S. pneumoniae, whose genome is completely sequenced [http://www.ncbi.nlm.nih.gov/pubmed/11544234]. CSP is a 17 amino acid long linear protein with the following amino acid sequence: Glu-Met-Arg-Leu-Ser-Lys-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH [http://www.microbesonline.org/cgi-bin/fetchLocus.cgi?locus=134925&disp=4]with a charged N-terminal residue and a positively charged C-terminal tail [http://www.ncbi.nlm.nih.gov/pubmed/16484185].

Sources

QS Two Component System

agrAC for S. aureus and XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX

The operon encoding for the histidine sensor kinase ComD and the response regulator ComE comDE for S. pneumoniae sensing was designed in two different ways: On one hand, the native comDE operon with its native ribosome binding site (RBS) ([http://parts.igem.org/Part:BBa_K1351015 BBa_K1351015]), on the other hand thecomDE operon with the B. subtilis adapted RBS (TAAGGAGG) in the BioBrick standard rcf25 ([http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]). Unfortunately, the operon contains two EcoRI and one SpeI restriction site. The attempts to mutagenize these restriction sites were not complettely successfull as one recognition site remained. Thus, the comDE operon could not been sent to the registry - but this will be accomplished as soon as possible.

QS dependent Promoters

In S. aureus, P2 and P3 xxxxxxXXXXXXXXXXXXXX

For S. pneumoniae sensing, the two ComE dependent promoters P_comC [http://parts.igem.org/Part:BBa_K1351024 BBa_K1351024] and P_comAB [http://parts.igem.org/Part:BBa_K1351025 BBa_K1351025] and additional promoters containing putative ComE binding sites (CEbs) P_mreA, P_natAB) were taken for evaluation. (Figure XX)

Fig1: ComE dependent promoters containing bases matching (blue) and differing (red) from the CEbs consensus sequence. (figure modified from Martin et al. 2013)

To test the functionality of our model, first, the two component system was cloned into the integrative vector [http://parts.igem.org/wiki/index.php?title=Part:BBa_K823027 pBS2E] from the Bacillus BioBrick Box under the control of P_xyl, an inducible promoter that responds to extracellular xylose (see fig. A). The corresponding QS-dependent promotor was cloned in the vector [http://parts.igem.org/wiki/index.php?title=Part:BBa_K823025 pBS3C-lux], thus fused to luxABCDE. If QS-molecules are detected from the two component system, P_QS will trigger the expression of luxABCDE, resulting in luminescence which was measured by a multi-mode plate reader (18 hrs, 37 °C, shaking).

In the distant future, the quorum sensing two component system, under the regulation of an inducible promoter, will be combined with an quorum sensing promoter expressing specific killing factors (see fig. B).

We optimized the BioBrick <bbpart>BBa_K316018</bbpart> encoding for the comC promoter [http://parts.igem.org/wiki/index.php/Part:BBa_K316018] by sending it to the registry. For the previous version, no DNA was available.

Surface display

Bacterial cell surfaces are covered with a wide range of proteins functioning as transporters, interacting with molecules and cells or fulfilling a variety of other functions. As they are naturally displayed on the cell’s surface, they can also be used for presenting heterologous proteins or peptides.

One class of proteins that have been used for surface display are B. subtilis autolysins, in particular LytC and LytE [1, 2]. Autolysins hydrolase peptidoglycan and thus play a role in cell wall turnover, vegetative growth and cell division. They consist of an N-terminal cell wall binding (CWB) domain with a signal peptide directing export via the Sec pathway and of a C-terminal catalytic domain [3, 4]. Fusions of the CWB domains of LytC and LytE to heterologous proteins have been shown to successfully facilitate surface display of their passenger protein in B. subtilis [1, 2]. Another promising system is based on the B. subtilis sortase YhcS and its substrate YhcR. Sortases represent a class of membrane-anchored proteins in gram-positive bacteria, which recognize their substrates via a conserved C-terminal pentapeptide sequence. They catalyze covalent binding of their substrates to lipid II, an intermediate of peptidoglycan synthesis [5]. Two putative sortase-substrate pairs have been identified in B. subtilis, one of them being YhcS/YhcR, which has already been used for successful surface display of recombinant proteins [5, 6].

Pathogen-binding peptides

Bacterial adhesins mediate binding of the cells to all kind of surfaces, thereby preventing removal by physical influences like sheer forces. Attachment of pathogens to their host is often crucial for infection, which is why many adhesins are listed as virulence factors [7]. Binding partners of adhesins are often components of the extracellular matrix, which would be difficult to express on the BaKillus surface. In contrast, proteins of human cells that get bound by adhesins are optimal candidates for translational fusions to surface display systems. BaKillus mimics human cells by expressing parts of the proteins on its surface, leading to binding of the pathogens to BaKillus. Barbu et. al. 2010 [8] and Muchnik et. al. 2013 [9] used phage display to identify the binding peptide interaction partners of Staphylococcus aureus virulence factor SdrC and Streptococcus pneumoniae adhesin NOX, respectively. Those peptides are the specific binding sequences of SdrC and NOX within surface proteins of human cells. Thus, expression of the peptides on the BaKillus surface should enable it to adhere to the targeted pathogens.

Sources

1. Chen, C.L., et al., Development of a LytE-based high-density surface display system in Bacillus subtilis. Microb Biotechnol, 2008. 1(2): p. 177-90.

2. Kobayashi, G., et al., Accumulation of a recombinant Aspergillus oryzae lipase artificially localized on the Bacillus subtilis cell surface. J Biosci Bioeng, 2000. 90(4): p. 422-5.

3. Tjalsma, H., et al., Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev, 2000. 64(3): p. 515-47.

4. Smith, T.J., S.A. Blackman, and S.J. Foster, Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology, 2000. 146(2): p. 249-262.

5. Nguyen, H.D., T.T. Phan, and W. Schumann, Analysis and application of Bacillus subtilis sortases to anchor recombinant proteins on the cell wall. AMB Express, 2011. 1(1): p. 22.

6. Liew, P.X., C.L. Wang, and S.L. Wong, Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display. J Bacteriol, 2012. 194(1): p. 161-75.

7. Coutte, L., et al., Role of Adhesin Release for Mucosal Colonization by a Bacterial Pathogen. The Journal of Experimental Medicine, 2003. 197(6): p. 735-742.

8. Barbu, E.M., et al., beta-Neurexin is a ligand for the Staphylococcus aureus MSCRAMM SdrC. PLoS Pathog, 2010. 6(1): p. e1000726.

9. Muchnik, L., et al., NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice. PLoS One, 2013. 8(4): p. e61128.

Surface Display

Constructs for non-covalent and covalent surface display were cloned similar to those described by Chen et. al., 2008 [1], and Liew et. al., 2012 [6], respectively.

As depicted in figure 1A, cell-wall binding domains (CWB) of LytC or LytE ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1351006 BBa_K1351006] or [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1351007 BBa_K1351007]) were translationally fused to a protein of interest for non-covalent anchoring of the latter to the cell wall. These CWB domains also included the native signal peptide (SP) of the respective proteins to direct export via the Sec pathway. For covalent anchoring, signal peptide ([http://parts.igem.org/Part:BBa_K1351008 BBa_K1351008]) and cell wall anchoring domain (CWAD, [http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) of the sortase substrate YhcR and a 57 amino acid linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) were assembled with the protein of interest as depicted in figure 1B to generate a YhcR-like fusion.

To evaluate the functionality of the constructs, the latter were cloned into the replicative B. subtilis vector pBS0K under the control of the inducible promoter P_spac. Two different proteins were used to test for successful display: Firstly, a superfolder GFP variant (sfGFP) was used for fluorescence microscopy to check if the constructs locate to the cell wall as shown in figure 2. This variant of GFP had been shown previously to fold in the periplasm of gram-negative bacteria [10] and could thus be expected to be fuctional also in the extracellular environment, in contrast to normal GFP that does not fold under reducing conditions. As controls for the sfGFP constructs, only the signal peptides without the respective cell wall binding or anchoring domains were fused to sfGFP, which should lead to secretion of the fusion into the growth medium.

Secondly, fusions to alkaline phosphatase (PhoA) were used to detect surface accessibility of the presented protein, as PhoA is only active when exported out of the cytoplasm [11]. Agar plates containing the PhoA substrate XP (5-bromo-4-chloro-3-indolyl-phosphate) indicate enzyme activity by turning blue where the substrate is hydrolyzed and further oxidized to an indigo dye [12]. PhoA was also fused to intracellular GFP as a negative control and to an extracellular domain of LiaI as a positive control. The PhoA-deficient B. subtilis strain MH3402 was used for expression of all PhoA constructs.

Pathogen binding

Binding of BaKillus to pathogens in general, and for this project to S. aureus and S. pneumoniae in particular, should be mediated by specific peptides that had been identified via phage display. Unfortunately, evaluation of the S. aureus-specific peptide Nrx1b ([http://parts.igem.org/Part:BBa_K1351000 BBa_K1351000]) was not possible due to the lack of an S2 laboratory, as the Nrx1b-binding partner SdrC is a virulence factor of S. aureus.

Evaluation of peptides binding to S. pneumoniae was done by a biochemical pull-down assay with their binding partner NADH oxidase (NOX). As shown in figure 3, NOX was bound to streptavidin-coupled beads via an N-terminal fused Strep-Tag II, for which the pASK-IBA expression vector system was used. S. pneumoniae-specific peptides C4P, CSP and L5P ([http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003], respectively) were fused C-terminally to a maltose-binding protein (MBP) and a 6xHis-tag. The MBP prevents degradation of the short peptides by proteases and was used for detection of the peptide fusions on an SDS gel. To increase specificity of the detection, the 6xHis-tag was used for Western Blot analysis of the pulled down proteins. Additionally, S. pneumoniae itself was used as cellular beads for a pull down of the same peptide-MBP-His constructs, again followed by analysis with SDS gel and Western Blot.

Sources

1. Chen, C.L., et al., Development of a LytE-based high-density surface display system in Bacillus subtilis. Microb Biotechnol, 2008. 1(2): p. 177-90.

6. Liew, P.X., C.L. Wang, and S.L. Wong, Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display. J Bacteriol, 2012. 194(1): p. 161-75.

11. Manoil, C. and J. Beckwith, A genetic approach to analyzing membrane protein topology. Science, 1986. 233(4771): p. 1403-8.

12. Brickman, E. and J. Beckwith, Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and φ80 transducing phages. Journal of Molecular Biology, 1975. 96(2): p. 307-316.

Results

Overexpression of tagged NOX and peptides

Figure 1:SDS-PAGE of the overexpression of NOX compared to the empty vector. Different fractions of two clones (C1, C2) carrying the NOX-insert were compared to a clone carrying the empty vector. Cultures were lysed and centrifuged to separate a supernatant (SN) from the pellet (P) and samples of the cells were taken at different time points (PI = pre-induction, AI = 2h after induction). 20 μl of fractions were loaded.

The protein composition of the two clones show no difference in contrast to the strain carrying the empty vector. A band at around 55 kDa would have been expected for the NOX. Thus, the expression of NOX was not successful.

Figure 2:SDS-PAGE of induced (I) and uninduced (UI) strains carrying the peptides C4P, CSP or A5P fused to MBP. Strains carrying the plasmids coding for the His-MBP tagged peptides were induced, lysed and centrifuged to separate the supernatant (SN) from the pellet (P). Samples were taken at different time points (PI = pre-induction, AI= 2h after induction) and uninduced strains serve as an control for expression. The lanes were loaded with 15μl.

The overexpression of MBP-tagged peptides was successful. The supernatant fractions showed a thick band at approximately 40kDa, corresponding to the size of MBP, which was not present in the uninduced samples. A smaller amount of the protein was also present in the lane of the pellet, but the major amount is cytosolic. Since the overexpression of NOX did not work, the planned pull-down of NOX with beads could not be performed. Instead the binding of peptides was tested by applying the His-MBP tagged peptides to S. pneumoniae cells.

Adhesion of peptides to S.pneumoniae – Pulldown with cells

Figure 4:‎SDS-PAGE of the lysates and the fractions of the incubated S. pneumoniae cells. E. coli cells carrying the plasmids coding for C4P, CSP and A5P were lysed. S. pneumoniae cells were resuspended in the lysate and incubated for 30 minutes, followed by pelleting to separate the supernatant (SP) from the cell pellet (P). 3 μl of sample were applied per lane.

The gel of the SDS-PAGE (Fig. 4) shows strong bands for the lysates at 40kDa, indicating a high amount of the His-MBP tagged peptides. The empty vector shows a relative low expression of the His-MBP. The pellet fractions of the S. pneumoniae cells incubated with the tagged C4P and CSP peptides show light bands at the same height indicating adhesion of the peptides to the cells (Fig. 4, white arrows). Although the cells incubated in buffer alone also show a band at the same height indicating a native protein of that size ((Fig. 4, black arrows), the amount of protein is less. To determine whether the band represents the His-MBP tagged peptides, a western blot was performed (Figure 5).

Figure 5: Western-Blots of the lysates and the fractions of the incubated S. pneumoniae cells. Duplicates of the gel from Figure 4 (A) and another gel loaded with 10 μl/ lane (B) were processed by western blot with Anti-His antibodes.

The blot shows a clear signal for all the pellet fractions of the cells incubated with the peptides, with the strongest signal for the C4P peptide, and also for the applied lysates. Only the band for the empty vector is missing, which might be due to the lower expression of the His-MBP. Our results strongly indicate that the previously described adhesion of the peptides to S. pneumoniae works. Thus, our BioBricks BBa_K1351001, BBa_K1351002 and BBa_K1351003 should serve as a tool for adherence to S. pneumoniae.