Staphylococcus aureus

Virulence of S. aureus

S. aureus is living as a commensal in humans with a prevalence of up to 30% of the population in Europe [http://www.nivel.nl/sites/default/files/ECCMID%202013%20-%20Resistance%20results_0.pdf (1)]. It is also responsible for a variety of diseases ranging from skin infections to life threatening toxinosis and is quickly adapting to the use of common antibiotics by becoming resistant. This dual lifestyle and the expression of a multitude of virulence factors is tightly regulated by a process termed quorum sensing [http://jb.asm.org/content/193/21/6020 (2)].

Quorum sensing in S. aureus

Quorum Sensing is a mechanism of bacterial cell-cell communication, granting bacteria the ability to measure the density of their surrounding cell population, and react to this by upregulation of specific genes. In S. aureus this process is one of the major regulator of its virulence, making sure that energy-costly virulence factors are only produced when a large enough number of pathogens is present to overcome host-defence mechanisms.

In this mechanism a certain peptide termed autoinducing peptide (AIP) is produced from a pro-peptide called AgrD and further processed and exported by a membrane protein AgrB to create a cyclic AIP. When a certain threshold concentration of AIP in the environment is reached, AIPs activate the receptor-histidine kinase AgrC. Upon induction AgrC phosphorylates the intracellular response regulator AgrA with its cytosolic histidine kinase domain [http://www.annualreviews.org/doi/abs/10.1146/annurev.genet.42.110807.091640 (3)]. Phosphorylated AgrA then binds to distinct sites in the P2 and P3 promoter-region and activates these promoters. [http://jb.asm.org/content/193/21/6020 (2)] The P2 promoter activates transcription of the agrBDCA-Operon and thereby represents a positive feedback-loop. The P3 promoter activates transcription of the RNA-III transcript, involved in virulence gene expression [http://jb.asm.org/content/182/22/6517.full (4)]).

Fig.1: Quorum sensing in Staphylococcus aureus. Taken from Novick and Geisinger, "Quorum Sensing in Staphylococci", Annual Review of Genetics, Vol. 42: 541-564 (2008)

Interestingly, accumulated mutations in the hypervariable region of the agrBDCA-operon have led to four different pherotypes of S. aureus, using different AgrC-types, and producing different AIPs (AIPI, AIPII, AIPIII, AIPIV), which cross-inhibit each other and might correlate with certain diseases associated with S. aureus [http://www.sciencemag.org/content/287/5452/391.full.html (5)].

For this project we used genomic DNA derived from the strain Staphylococcus aureus N315, which is a MRSA strain, belonging to the AIP-II group.

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 (6)]. 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 (7)].

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 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 promoter 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.

Fig. 2: Competence regulation in Streptococcus pneumoniae. See text for more details. (Jonsborg et al. 2009)

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 (8)]. 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 (9)]. CSP is a 17 amino acid long linear protein with the following amino acid sequence: Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH [http://www.microbesonline.org/cgi-bin/fetchLocus.cgi?locus=134925&disp=4 (10)] with a charged N-terminal residue and a positively charged C-terminal tail [http://www.ncbi.nlm.nih.gov/pubmed/16484185 (11)].

Sources

[1] den Heijer, C. D., van Bijnen, E. M., Paget, W. J., Pringle, M., Goossens, H., Bruggeman, C. A., . . . Team, A. S. (2013). Prevalence and resistance of commensal Staphylococcus aureus, including meticillin-resistant S aureus, in nine European countries: a cross-sectional study. Lancet Infect Dis, 13(5), 409-415. doi: 10.1016/S1473-3099(13)70036-7

[2] Reyes, D., Andrey, D. O., Monod, A., Kelley, W. L., Zhang, G., & Cheung, A. L. (2011). Coordinated regulation by AgrA, SarA, and SarR to control agr expression in Staphylococcus aureus. J Bacteriol, 193(21), 6020-6031. doi: 10.1128/JB.05436-11

[3] Novick, R. P., & Geisinger, E. (2008). Quorum sensing in staphylococci. Annu Rev Genet, 42, 541-564. doi: 10.1146/annurev.genet.42.110807.091640

[4] Jarraud, S., Lyon, G. J., Figueiredo, A. M., Lina, G., Vandenesch, F., Etienne, J., . . . Novick, R. P. (2000). Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus. J Bacteriol, 182(22), 6517-6522.

[5] R. P. Novick, H.F. Ross, A. M. S. Figueiredo, G. Abramochkin (2000) Activation and Inhibition of the Staphylococcal AGR System. Science 21 Vol. 287 no. 5452 p. 391

[6] van der Poll, T., & Opal, S. M. (2009). Pathogenesis, treatment, and prevention of pneumococcal pneumonia. Lancet, 374(9700), 1543-1556. doi: 10.1016/S0140-6736(09)61114-4

[7] Oggioni, M. R., Trappetti, C., Kadioglu, A., Cassone, M., Iannelli, F., Ricci, S., . . . Pozzi, G. (2006). Switch from planktonic to sessile life: a major event in pneumococcal pathogenesis. Mol Microbiol, 61(5), 1196-1210. doi: 10.1111/j.1365-2958.2006.05310.x

[8] Johnsborg, O., & Havarstein, L. S. (2009). Regulation of natural genetic transformation and acquisition of transforming DNA in Streptococcus pneumoniae. FEMS Microbiol Rev, 33(3), 627-642.

[9] Hoskins, J., Alborn, W. E., Jr., Arnold, J., Blaszczak, L. C., Burgett, S., DeHoff, B. S., . . . Glass, J. I. (2001). Genome of the bacterium Streptococcus pneumoniae strain R6. J Bacteriol, 183(19), 5709-5717. doi: 10.1128/JB.183.19.5709-5717.2001

[10] Sequence obtained from http://www.microbesonline.org

[11] Johnsborg, O., Kristiansen, P. E., Blomqvist, T., & Havarstein, L. S. (2006). A hydrophobic patch in the competence-stimulating Peptide, a pneumococcal competence pheromone, is essential for specificity and biological activity. J Bacteriol, 188(5), 1744-1749. doi: 10.1128/JB.188.5.1744-1749.2006

QS Two Component System

The two component system [http://parts.igem.org/Part:BBa_K1351036 agrCII-A] for S. aureus has been designed as a composite part derived from Staphylococcus aureus N315 gDNA. The two intermediated parts agrC-II and agrA were amplified by PCR using primers with modified RFC25 prefix and suffix. And then fused by using the restriction sites EcoRI and SpeI for agrC-II, as well as XbaI and PstI for agrA, thus creating the composite part [http://parts.igem.org/Part:BBa_K1351036 agrCII-A].

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 RFC25 ([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 completed successfully in time 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, the AgrA-sensitive promoters P2 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037] and P3 [http://parts.igem.org/Part:BBa_K1351038 BBa_K1351038] with four AgrA binding sites downstream of the -35 signals were used for evaluation. The intergenic region with suspected regulator binding sites is shown in Fig. 3

Fig. 3: AgrA dependent promoters P2 and P3 with the intergenic region containing several regulator binding sites for AgrA (arrows) and SarA/R (dashed lines) (figure taken from Reyes et al. 2000)

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 (see figure 4).

Fig. 4: 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).

S. aureus Sensing

We constructed the biobrick <bbpart>BBa_K316036</bbpart> encoding for a composite part containing the AIP-II sensing two component system AgrC-IIA [http://parts.igem.org/wiki/index.php/Part:BBa_K316036] And the AIP-II sensing dependant promoters P2 and P3 <bbpart>BBa_K316037</bbpart>, <bbpart>BBa_K316038</bbpart> P2 [http://parts.igem.org/wiki/index.php/Part:BBa_K316037] and P3 [http://parts.igem.org/wiki/index.php/Part:BBa_K316038]

As a first step we recorded the promoter backgrounds of the P2 and P3 promoters with a luminescence reporterassay, using the strategy outlined in design. (Fig.3) As expected according to the strength of the -35 and -10 signals, P2 is the stronger promoter showing a higher background, compared to P3.

In a next step we induced the expression of PxylA-agrCII-A by adding 0%, 0.02%, 0.2% Xylose at an OD₆₀₀ of about 0.1. The result is depicted in Figure 5. This result is in accordance with our expactations since strong overexpression of the two-component system should lead to unspecific induction of the P2 and P3 promoters, and thereby can be used as a method to measure relative expression of agrC-IIA.

Fig.5: Luminescence Reporterassay of the strain W168 lacA::PxylA-agrCA; sacA::P2/P3-luxABCDE

Using the strategy outlined in design, we tried to induce the AgrC-IIA two-component system, with synthetic AIP-II. The synthetic peptids have been dissolved in 100% DMSO to 1 mM concentration and further diluted in dH₂0. The constructs have then been induced with a range of different concentrations of AIP-II at an OD₆₀₀ of 0.1. The result is shown in Figure 6. Unfortunatly we were not able to show induction of the system by addition of AIP-II. Several other approaches like the addition of a protease inhibitor cocktail to the growth media, several washing steps, using LB instead of MCSE or inducing with 50 µM AIP-II did not change this observation.

Fig.6: Luminescence Reporterassay of the strain W168 lacA::PxylA-agrCA; sacA::P2/P3-luxABCDE induced with AIP-II

There are several possible reasons why the induction didn't work as expected. One idea is that the extracellular proteases produced by Bacillus subtilis destroy the synthetic AIPs. So we tried adding protease inhibitor cocktails to the growth media and using the protease deficient strain Bacillus subtilis WB700. We successfully transformed the WB700 strain with the reporter construct pBS4S-PxylA-agrCA. However the reporter constructs pBS3C-P2/P3-luxABCDE refused transformation. All in all this set of constructs needs several further protocol optimisations and testing.

S. pneumoniae Sensing

First, 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 our BioBrick <bbpart>BBa_K1351024</bbpart> to the registry. For the previous version, no DNA was available.

The BIG result we obtained in the night of the wiki freeze performing a luminescence assay with different constructs in B. subtilis P_xyl - comDE P_QS - lux based on [http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]. Three different P_QS were tested: P_comC, P_comAB, P_mreA. The comDE operon expression was induced with 0 %, 0,02 % and 0,2 % xylose. Adding 0 mg/ml CSP, 100 mg/ml CSP and 1000 mg/ml CSP luxABCDE expression is only activated if B. subtilis was able to sense CSP due to the comDE operon. For P_mreA, no significant luminescence output could be measured concerning differing CSP concentrations. Nevertheless, at a xylose concentration of 0,02 % and induction of 1000 mg/ml CSP P_comC and P_comAB showed a 3 fold increased luminescence output compared with an induction of 0 mg/ml and 100 mg/ml CSP.

These results were obtained with cultures growing in MCSE medium two hours after induction with CSP. A concentration of 0.02 % xylose was optimal to measure CSP dependent luminescence.

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]) should be 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 should be cloned into the replicative B. subtilis vector pBS0K under the control of the inducible promoter P_spac. Two different proteins should be for successful display: First, 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) should be 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 will be used for expression of all PhoA constructs.

Unfortunately, the status of these constructs is still "cloning".

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 into the pKLD66 vector. This was achieved by digestion of the BioBricks and pKLD66 with NotI and HindIII and ligation of the products. 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 for 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 the supernatant (SN) from the pellet (P). 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 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 3:‎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. 3) shows strong bands for the lysates at 40kDa, indicating a high amount of the His-MBP tagged peptides. The empty vector shows a relatively low expression of 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. 3, black arrows) the amount of protein is clearly less. To determine whether the band represents the His-MBP tagged peptides, a western blot was performed (Figure 4).

Figure 4: 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 antibodies.

The blot shows a clear signal for the applied lysates and all the pellet fractions of the cells incubated with the peptides, indicating their adhesion to S.pneumoniae. The band for the His-MBP of empty vector is missing (or very weak), which might be due to the lower expression, but one can clearly detect the bands corresponding to the His-MBP in the SDS-PAGE (Fig. 4). Our results strongly indicate that the previously described adhesion of the peptides to S. pneumoniae works. Thus, our BioBricks [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] should serve as a tool for adhesion to S. pneumoniae.

Fig. 1. Gene organization for the sdpABC sdpRI operons. The hairpin symbolizes thetranscriptional terminators. [http://www.sciencemag.org/content/301/5632/510.full [1] ]

In vegetative cells, both operons are repressed by the unstable AbrB regulator. However, during early stages of sporulation AbrB itself is repressed by the master regulator of sporulation Spo0A, making sdpABC and sdpRI accessible for RNA polymerase. [http://www.sciencemag.org/content/301/5632/510.full [1]]

The sdpABC Operon – Production and Secretion of the Cannibalism Toxin SDP

The production of the Cannibalism Toxin SDP is a multi-step process. The sdpC sequence encodes the Pro-SdpC1-203,,which is translated by the ribosome.It is a precursor peptide which needs to be processed by a signal peptidases and the two membrane proteins SdpA and SdpB to become functional. This active form of SDP is a 42-amino-acid antimicrobial peptide (AMP) containing a disulfide bond between two cysteine residues located at the N-terminus.(Fig. 2). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697648/ [2]]

Fig. 2. SDP production requires multiple steps. In the cytosol, the full length SdpC (pro-SdpC1-203) is secreted via the Sec pathway. Following secretion, the signal peptidases SipS and SipT cleave the N-terminal signal peptide sequence of SdpC. Disulfide bond formation occurs independently of SdpAB. Finally, posttranslational cleavage of SdpC occurs via SdpAB to produce a 42-amino-acid SDP that will be secreted extracellulary as the active SDP peptide. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697648/ [2] ]

SDP has been shown to be a very effective AMP against a variety of Gram-positive bacteria in the Phylum of the Firmicutes (Fig. 3). It rapidly collapses the proton motive force (PMF), thus inducing autolysis. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839633/ [3]]

Fig. 3. SDP inhibition curves for pathogenic microbes. Relative growth of the strains named abovewith the presence of increasing concentrations of SDP is shown in the curve. As a negative control the gram-negative bacteria K. pneumoniae and P. aeruginosa are depicted, which are unaffected by the toxin SDP, as it specifically targets gram-positive bacteria. B. subtilis is a gram-positive bacteria, but expresses the immunity protein SdpI and is therefore relatively resistent to the toxin SDP. the Stapylococcus species (also MRSA) though are quite drastically reduced in the presence of the SDP. [http://www.pnas.org/content/107/37/16286.full [4] ]

The sdpIR Operon – Production and Regulation of the Immunity Protein SdpI

In the absence of SDP the gene coding for the immunity protein SdpI is repressed by the autorepressor SdpR. If extracellular SDP binds the transmembrane protein SdpI it causes a conformational change. As a consequence SdpR is sequestered at the membrane, forming a complex with SDP and SdpI. Since the repression by SdpR is relieved the RNA polymerase can bind the sdpRI promotor region and start transcription (Fig. 4). [http://www.sciencemag.org/content/301/5632/510.full [1]]

Fig. 4. Model of how SDP induces expression of the sdpRI immunity operon. The signal SDP interacts with the immunity protein SdpI to induce a conformational change that allows SdpI to bind the repressor SdpR, which is sequestered at the membrane. [http://www.sciencemag.org/content/301/5632/510.full [1] ]

ECF41: An alternative sigma factor for building an orthogonal Delay Switch

The alternative σ-factor ecf41_{bli aa 1-204}, which derives from B. licheniformis is a truncated version and binds to the target promoter P_ydfG. It is an orthogonal σ-factor since there are no cross-reactions with other native promoters. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ [5]] The introduction of this loop leads to a translational delay.

FsrA: a small RNA Represses Expression of Succinate Dehydrogenase

Regulation of bacterial iron homeostasis is often controlled by the iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur protein acts as an iron-dependent repressor for siderophore biosynthesis and iron transport proteins. Furthermore it also coordinates an iron-sparing response that acts to repress the expression of iron-rich proteins (e.g. succinate dehydrogenase) when iron is limited. It has been shown that the downregulation of succinate dehydrogenase (SDH) is most likely caused by complementary binding of a small RNA, named fsrA, to the leader region of the sdhCAB mRNA, namely SdhC' (Fig. 5). In this project this principle is exploited to downregulate the translation of the immunity protein SdpI in a delayed response to the detection of the QS-molecules of S. aureus or S. pneumoniae. [http://www.pnas.org/content/105/33/11927.full.pdf [6]]

Fig. 5. Predicted pairing between fsrA and the 5’-leader region of sdhC, the first gene of the sdhCAB operon. [http://www.pnas.org/content/105/33/11927.full.pdf [6] ]

Sources

[http://www.sciencemag.org/content/301/5632/510.full [1]] Gonzalez-Pastor, J. E. (2011). "Cannibalism: a social behavior in sporulating Bacillus subtilis." FEMS Microbiol Rev 35(3): 415-424.

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697648/ [2]] Perez Morales, T. G., et al. (2013). "Production of the cannibalism toxin SDP is a multistep process that requires SdpA and SdpB." J Bacteriol 195(14): 3244-3251.

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839633/ [3] Lamsa, A., et al. (2012). "The Bacillus subtilis cannibalism toxin SDP collapses the proton motive force and induces autolysis." Mol Microbiol 84(3): 486-500.

[http://www.pnas.org/content/107/37/16286.full [4]] Liu, W. T., et al. (2010). "Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis." Proc Natl Acad Sci U S A

[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426412/ [5]] Wecke, T., et al. (2012). "Extracytoplasmic function sigma factors of the widely distributed group ECF41 contain a fused regulatory domain." Microbiologyopen 1(2): 194-213.

[http://www.pnas.org/content/105/33/11927.full.pdf [6]] Gaballa, A., et al. (2008). "The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins." Proc Natl Acad Sci U S A 105(33): 11927-11932.

The suicide switch module requires a high level of combinability regarding to its components. We therefore designed every new part in RFC10 or RFC25 standard.

Production of the toxin SDP and the immunity protein SdpI

By the activation of the QS-dependent promoter P_QS, BaKillus produces the Cannibalism Toxin SDP (Fig. 1A). For evaluation purposes we cloned the sdpABC operon under the control of inducable xylose promoter P_xyl (Fig. 1B) into a ∆sdpAB and a ∆sdpC mutant strain of B. subtilis W168. The construct was then tested by spot-on-lawn assays on B. subtilis W168 and B. subtilis W168 ∆sdpI mutant lawns.

To ensure a co-regulated production of the immunity protein SdpI we used the native promoter of the sdpRI operon P_sdpRI which is accesible for RNA polymerase in the presence of extracellular SDP. Between the promoter and the immunity sequence sdpI we inserted the binding site of fsrA namely sdhC’ (Fig. 2A). By expressing the SdpI immunity protein under the control of the P_xyl promoter (Fig. 2B) we were able to evaluate the functionality of SdpI by growth curves measurements.

Delayed loss of immunity

A time delayed loss of the immunity protein SdpI in BaKillus can be achieved by interposing the alternative σ-factor ECF41_{bli aa 1-204} [http://parts.igem.org/Part:BBa_K823043 BBa_K823043] which is also under the control of the quorum sensing promoter P_QS (Fig.3A). When expressed, ECF41_{bli aa 1-204} binds to the target promoter P_ydfG [http://parts.igem.org/Part:BBa_K823041 BBa_K823041] leading either to the expression of fsrA, a small RNA, or transcription of sdpI-antisense, a RNA, a complementary RNA to the sdpI mRNA (Fig. 3B). To veri- and quantify the time delay due to the interposition of Ecf41_{bli aa 1-204} we replaced the fsrA coding sequence with the lux-cassette luxABCDE allowing lumi assay measurements (Fig. 3C).

For the loss of immunity we implemented two strategies, both causing translational inhibition of the sdpI mRNA. The first strategy is based on an artificial synthesized RNA called sdpI-antisense RNA, which is complementary to the sdpI mRNA sequence. The sdpI-antisense sequence is regulated by P_ydfG (Fig. 4A) which is activated by the binding of Ecf41_{bli aa 1-204}. As a consequence the sdpI-antisense RNA and the sdpI mRNA form a double-stranded duplex which is inaccessible for the ribosome making the cell SDP-sensitive. We tested the sdpI-antisense RNA in B. subtilis W168 with the xylose-inducable promoter P_xyl. (Fig. 4B)

The second strategy involves fsrA, a small RNA regulated by P_ydfG which binds to its binding site sdhC' (the 5'-UTR of sdhC mRNA) which is cloned between P_sdpRI and sdpI (Fig. 5A). Binding of fsrA to the sdhC' site on the transcript inhibits the ribosomal translation process of the sdpI-mRNA. To evaluate this small RNA and its binding site in B. subtilis we designed two strains. One ∆abrB ∆sdpI mutant strain where we replaced the P_ydfG promoter with the P_xyl promoter (Fig. 5B). The second construct containing fsrA under control of P_xyl and replaced P_sdpRI-sdhC'sdpI with the P_lepA-sdhC'gfp, where GFP is consecutively expressed, (Fig. 5C) was transformed into B. subtilis W168.

The suicide switch as well as the delay loop are part of our modelling work:

Mathematical model of suicide switch dynamics
Mathematical model with additional sigma factor for delay