As is discussed in Chapter 5, finding new ways to detect pathogenic bacteria, either in the environment or in a living organism, is vital to reducing the amount of antibiotics we use and arresting the development of antimicrobial resistance. In 2012, Pacheco-Gómez et al. published their work towards developing an assay that detects the difference in theLDsignal of an M13 bacteriophage-antibody complex in the presence or absence of pathogenic bacteria [107]. This assay was patented by Prof Timothy Dafforn and Dr Matthew Hicks prior to this publication [108]. Figure 4.14 illustrates the principle behind the detection system designed by Dafforn and Hicks.
Figure 4.14: Illustration of the LD assay to detect pathogens in solution. A: In the
absence of any pathogen target, the M13 bacteriophage aligns very well, and hence gives a strong LD signal. B: Where a target bacterium is present (textured red),
the M13 complexes bind to it and lose much of their freedom to align in the flow direction. Therefore, presence of a pathogen will be seen as a loss in LD signal. Figure 4.14 shows the outer pVIII proteins (green) and the end pIII proteins (yellow) endogenous to the M13 bacteriophage. Attached to the pIII proteins is an antibody
and secondary antibody complex (both grouped in solid red), which are selected to be specific to a pathogenic target. One may wish to use fluorophores attached to the pVIII proteins (orange) although these are not strictly necessary for the experiment, since aligned M13 has its own LD signal. Clearly, the assay is heavily reliant on the ability to detect an oriented M13 bacteriophage LD signal, and its sensitivity to relatively small concentrations of pathogens in solution is highly dependent on the ability of the detection system to measure small losses in M13 alignment. An example LD spectrum of 100 µg/mL of M13 bacteriophage, oriented in Couette flow, is given in Figure 4.15.
180 200 220 240 260 280 -0 .0 2 0.00 0.02 0.04 Wavelength / nm L D 100 µg/mL
Figure 4.15: LD spectrum of a 100µg/mL sample of M13 bacteriophage, oriented in
micro-volume Couette flow. The dashed grey line shows where LD is equal to zero.
This spectrum was recorded using the instrument parameters given in Table 4.2.
The largest peak in the LD spectrum of M13 bacteriophage, given in Figure 4.15, is in the far-UV region and comes from the ⇡⇤ ⇡ and ⇡⇤ n transitions of the
peptide backbone [15]. In the near-UV region of the spectrum, the broad, shallow peak is from the absorbance of the aromatic amino acids, tryptophan and tyrosine. Like many biological samples, M13 bacteriophage is produced and purified in buffers that contain salts [107], which absorb light in the far-UV and can distort signals from the chromophore of interest. The sample used to record the LD spectrum in
Figure 4.15 was diluted down to 100 µg/mL from a 18 mg/mL stock solution with water, and so the effect of the salts in the buffer are negligible. However, when using high concentration buffers it would generally be preferable to use the signals in the near-UV to track changes in alignment.
As has been demonstrated in the spectra of the DNA binding fluorescent probes above, F DLD has the potential to be used to detect a fluorophore at much lower concentrations than can be achieved when using absorbance methods. M13 bacteriophage contains one tryptophan and two tyrosines per PVIII protein, and each bacteriophage contains approximately 2,700 copies of this protein along its shaft [109]. As both of these amino acids are fluorescent we predicted we should be able to detect M13 bacteriophage alignment using F DLD. This is indeed the case, as illustrated in the F DLD spectra given in Figure 4.16.
Figure 4.16 shows the F DLD spectra of a range of concentrations of M13 bacteriophage. It is clear from the figure that it is possible to detect down to a concentration of 50 µg/mL, with a very good signal to noise ratio. This is despite using the same experimental setup as was used for the F DLD measurements of the DNA-dye complexes. The reduced noise in this example may be due to the choice of long-pass filter collecting more of the emitted photons. Comparing the 100 µg/mL sample with the LD spectrum, given in Figure 4.15, we see that 17.5 times the signal magnitude in the aromatic region is obtained when using F DLD
(LD280 = 0.00184646, F DLD280 = 0.0323457). This shows that F DLD could add
a great deal to the efficiency of the detection of pathogenic bacteria assay described above.
180 200 220 240 260 280 0.00 0.02 0.04 0.06 0.08 Wavelength / nm F D L D 50 µg/mL 100 µg/mL 200 µg/mL 300 µg/mL 400 µg/mL
Figure 4.16: F DLD spectra of a range of concentrations of M13 bacteriophage,
oriented in micro-volume Couette flow. All spectra were recorded using the parameters given in Table 4.2, with a variable HT voltage. A Semrock 300 nm long-pass edge filter was used to block transmitted incident light in each measurement.
It is also interesting to see the high resolution of the signature 1L
a and 1Lb bands of the tryptophan’s indole chromophore around the 280 nm region of the spectra in Figure 4.16 [110]. Similar to the F DLD spectra of small molecules oriented on stretched PEOX, given in Chapter 3, this is a further example of the increased ability of F DLD to resolve the individual contributions to an absorption band.
We know that each of the M13 bacteriophage particles possesses two fluorophores, tryptophan and tyrosine, however, we do not know the degree to which each was contributing to theF DLD spectra. To try and gain insight into this, we measured the intensity of fluorescence emissions over a range of excitation and emission wavelengths. The result is illustrated in Figure 4.17.
The fluorescence properties of the two fluorescent amino acids are well known — tryptophan possesses excitation and emission maxima at 280 nm and 350 nm, respectively [111], and tyrosine’s excitation and emission maxima are at 275 nm and 303 nm [112]. Although tryptophan’s emission wavelength is highly dependent on its surroundings, and in a hydrophobic environment can be 330 nm or lower [113].
From Figure 4.17 we can see that at both the excitation maxima in the phageF DLD spectrum, the emission maxima are around 330 nm, which suggests the signal is predominately coming from the tryptophan residues, in hydrophobic environments.
Figure 4.17: 3D plot showing the change in fluorescence emission intensity with excitation wavelength. This measurement was made on a Jasco FP-6500 spectrofluorimeter.
A further confirmation of the fact that the signals present in the M13 bacteriophage F DLD spectra originate from tryptophan, is that tyrosine fluorescence is reduced significantly by the presence of tryptophan and the peptide bonds in proteins, and so tyrosine will most likely have a lower quantum yield in the environment of our measurement than tryptophan [114]. Tyrosine fluorescence is also significantly effected by pH [115]. These factors are important in our discussion of the bacterial actin homologue MreB (Chapter 5), which contains five tyrosine and zero tryptophan residues.
4.4 Conclusions
In this section we have shown the utility of LD for determining the binding mode that small molecules adopt when bound to DNA. On observing anyLD signal from the molecules we know that they are bound, as they are too small to orient in Couette flow on their own, and from the sign of the signal we can determine whether they are intercalated between the base pairs of the DNA (giving a negativeLDsignal) or bound to the major or minor groove of the molecule (giving a positive LDsignal). We have also shown that in cases where the bound molecule is a fluorophore, it is possible to record a micro-volume Couette flowF DLD spectrum of the sample. Due to the distance of the oriented sample from the detector in the current Couette flow cell, these spectra have a decreased signal to noise ratio than theLD spectra of the same samples. However, they give signals of much higher magnitude in the examples given above, due to the inherent increased sensitivity of fluorescence detection, and a new design of Couette flow cell where the sample chamber is closer to the detector would increase the signal to noise ratio.
A further advantage of fluorescence detection is in the selectivity of the measurement when dealing with samples of complex mixtures, which is also clearly demonstrated in the F DLD spectra given above. In the examples of the intercalating dyes ethidium bromide and propidium iodide, it is possible to see the the sign of the bands overlapping with the DNA’s absorbance from their LD spectra, however, F DLD reveals the full dye spectral shape, absorption maxima and the relative magnitude of peaks throughout this region.
The F DLD spectra of the minor-groove binding dyes DAPI and Hoechst 33258 bound to DNA show a large increase in signal magnitude for their near-UV transitions, when compared to their LD spectra. Additionally, in both cases the transitions in the middle-UV region can also be observed in their F DLD spectra — transitions that are completely obscured by the DNA’s absorbance signal in theLD measurements. The negative sign of these peaks provides additional insight into the orientation of the bound dye.
oriented by Couette flow. TheF DLDspectra showed a large enhancement of signal in the aromatic region of the spectra, which we argue would add significantly to the ability of a previously devised LD assay used to detect pathogenic bacteria in solution [107]. The F DLD spectra also show highly resolved bands of the tryptophan residues, which further demonstates the additional information that can be obtained when using the technique.
CHAPTER 5
Expression and characterisation of the
E.
5.1 Introduction
It is well known that antimicrobial resistance (AMR) is a serious threat to global public health. There have been numerous reports from the World Health Organisation that have emphasised the current — and not just future — danger of AMR [116]. The first World Antibiotic Awareness Week begun on the 16th November 2015, and the UK government has recently published its own five year strategy to tackle the problem [117]. The problem of AMR increased with the use of antimicrobial drugs [118]. These drugs, whilst creating an environment too hostile for most bacteria to survive, promotes the survival of pathogenic bacteria which have a mutation that makes them drug resistant. In the absence of any antibiotic it may be that mutant populations are less fit for survival, and so one strategy to help reduce AMR is to use fewer antibiotics [1].
Reducing the amount of antibiotics we use is critical to combatting AMR, and there is a global drive to increase awareness of the risks associated with the overuse of antibiotics. One example here in the UK is the Treat Yourself Better Campaign (www.treatyourselfbetter.co.uk). The need to use fewer antibiotics, however, will only serve as an afterthought to a dairy farmer whose livelihoodmay be jeopardised through mastitis in his herd, or a mother whose childmay have a bacterial infection. In cases such as these, the real work must be done in the development of better diagnostics, so as to eliminate any doubt about whether or not the use of antibiotics is required. One way in which fluorescence detected linear dichroism may help achieve this goal is discussed in Chapter 4.3.3.
A second solution to the problem of AMR is to discover new drug targets and develop new antibiotics that function in a novel way [1]. With a large body a drugs that all kill pathogenic bacteria by a unique mechanism, it would be possible to cycle their use so that bacteria are less likely to evolve to become resistant to any one. The focus of this chapter is the expression, purification, and structural analysis of one such potential target: the bacterial actin homologue MreB (MreB). What follows is an introduction to this protein, our methods for obtaining it, and the results and conclusions of this work.