All posts filed under “Antibiotics

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Biosensors to detect macrolide antibiotics in high throughput screening

Wiki Source, DNA binding motif of TetR

  Antibiotics are often natural products discovered in other organisms, including but not limited to bacteria, fungi, plant, frogs and humans. Although they may have different functions in their natural setting, they are exploited by medicine for their ability to kill bacteria.  Their discovery often relies on traditional testing for antimicrobial activity in a crude extract, followed by the purification of the active ingredient. We have progressed the search for new antibiotics and employ elegant new approaches to produce and detect antibiotic activity, combined with high throughput screening methods that permit us to find the’ needle in a haystack’ of tens of thousands of compounds.

Erythromycin,  wiki.  

  Macrolides are a broad-spectrum, bacteriostatic class of antimicrobials that bind to the large 50S subunit of the ribosome and block the translocation step during protein synthesis. Erythromycin was discovered by an Eli Lilly researcher in the late 1940s, who discovered this antibiotic from a Streptomyces isolate recovered from a soil sample.  This polyketide class of antimicrobials also has other important biological activities, including antifungal, antiprotozoal and even anticancer activity. Macrolides have complex structures and are synthesized by type I polyketide syntheses in a modular fashion. Rationally designing these enzymes for novel biosynthetic projects has proved challenging. Rather than engineer these complex modular enzymes to produce new macrolides, the work described here uses a biosensor strategy to detect macrolides with a tailored substrate specificity and increased sensitivity.

  The MphR protein is a TetR repressor protein that represses the expression of a macrolide phosphotransferase resistance protein. In general, the TetR family are transcriptional repressors that bind and repress target promoters, but in the presence of a chemical effector, allow for activation of the target genes. This is a common regulatory mechanism used by bacteria to sense changing environmental conditions and then express appropriate genes to cope with the change.

  In the presence of the macrolide erythromycin, MphR binds the antibiotic, causing MphR to fall off the target promoter and induce expression of the resistance gene. In this system, MphR is the only known bacterial protein that binds to and therefore ‘senses’ the presence of macrolide antibiotics in the cytoplasm. MphR is “de-repressed” in the presence of several natural and semi-synthetic macrolides, including erythromycin and others. Note, resistance to macrolides can also occur through other resistance mechanisms, including efflux pumps or by mutations or modification in the small ribosome subunit.

  Rather than engineer and adjust the macrolide biosynthesis enzymes and pathways to create new novel macrolides, the authors attempted to engineer MphR so that it recognized a different range of macrolides, or better differentiated natural and synthetic compounds, and increased its sensitivity in order to create more effective biosensors.

  The biosensor system was previously described and uses plasmid-based system in E. coli. The plasmid encodes both MphR (biosensor) and the gfp reporter under the control of an MphR- regulated promoter. In the presence of erythromycin, the E. coli biosensor now produces green fluorescence from turning on the gfp reporter. As a proof of concept, error prone PCR and multi-site saturation mutagenesis were performed on MphR, which led to variants of MphR that were up to 10-fold more sensitive to its’ natural ligand, erythromycin. Mutations were recovered in the coding sequence, as well as in the non-coding ribosome binding site (RBS). Utilizing the 3D structure of MphR, mutations were targeted to key amino acids in the repressor that are involved in recognizing macrolides.

 It would be ideal to have an MphR biosensor that recognizes a specific macrolide structure within a complex mixture. Given that MphR has a relatively broad substrate specificity, the goal was to mutagenize MphR and select for variants with a narrowed specificity. The first attempt was to try and find mutants that could differentiate natural from semi-symthetic macrolide structures. After the error prone PCR libraries were constructed and introduced back into E. coli, FACS sorting was  used to screen for constitutive gfp clones, which were excluded, and then erythromycin-responsive gfp expressing clones, followed by determining the kinetics and sensitivity levels. Randomly generated mutants were reconstructed with targeted mutations, which confirmed that MphR variants could now respond to a narrow range of macrolides. The amino acid mutations were mapped to the protein structure, but it wasn’t immediately obvious some of these changes led to a narrow specificity.

The next aim was to engineer MphR variants that could now recognize macrolides that the wild type does not, despite its broad substrate specificity. Several macrolides are produced by Streptomyces venezuelae, such as pikromcyin. The error prone PCR-generated libraries of MphR variants, were screened for those that could now detect pikromcyin. Resulting from this FACS-based screen, a biosensor strain  grown in the presence of pikromycin that expressed a single amino acid variant of MphR, was 123 times more sensitive than the wild type protein.

 Screening for new macrolide structures using conventional approaches and mass spectrometry is very complex, time consuming and costly. The ultimate goal of this project would be to use the relatively simple and high throughput MphR-based biosensor to detect a new macrolide structure.  As a proof of concept, the culture supernatants from a bacterial strain that only produces erythromycin A and not the intermediate B and C products, were recovered and added to the macrolide biosensors. The engineered MphR biosensor was 5 times more sensitive to detect erythromycin A directly from bacterial supernatants that required no purification or concentration.

While the research described here 1 highlights the potential to engineer macrolide biosensors for detecting new substrates with greater sensitivity, others have used a similar approach with the wild type MphR biosensor to detect macrolides from Streptomyces supernatants 2. It is impressive that single or double amino acid mutants can be recovered that are capable of detecting very subtle differences between macrolide structures.

These kind of projects laid the groundwork for future macrolide discovery, relying on the elegant macrolide sensing mechanisms of transcriptional repressor proteins. Biosensors could be used to detect new antibiotics in large collections of semi-synthetic drug libraries or natural products. They are also useful in screening the complex soup of compounds within culture supernatants from other macrolide producing strains, possibly with engineered macrolide biosynthesis pathways, or by screening the culture supernatants from metagenomic libraries. In the latter approach, environmental DNA from any soil sample is cloned and expressed in a lab friendly bacterial host. A library of 1000s of isolates can effectively express all the DNA in a given soil sample, which overcomes our current ability to grow the vast majority of microbes in soil and in most environments. It therefore allows us to access and screen the diverse biosynthetic potential from microbes that are uncultivable. Many new antibiotics will undoubtedly be discovered from this “microbial dark matter“.

References.

1.  Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology. Kasey CM, Zerrad M, Li Y, Cropp TA, Williams GJ ACS Synth Biol. 2017 Oct 12. doi: 10.1021/acssynbio.7b00287. [Epub ahead of print] PMID:28950701

2.  Biosensor-guided screening for macrolides. Möhrle V, Stadler M, Eberz G. Anal Bioanal Chem. 2007 Jul;388(5-6):1117-25. Epub 2007 May 12. PMID: 17497142

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Synthetic biology reporter of bacterial growth rate during an infection

Graphical abstract, Open Access

  The hallmark of chronic infections is the difficulty in treating and curing infection with antibiotics. Biofilms are often the cause of chronic infections,  due to their increased antibiotic tolerance and immune protective, aggregate structure. The antibiotic tolerance of biofilms is attributed mainly to the inability of the antibiotic to penetrate the biofilm, or the slow growth rate of bacteria in a biofilm. A recent study using a synthetic biology approach to examine the bacterial growth rate during an infection has provided some interesting results that challenge the current thinking regarding bacterial growth rates during infections.

  In a brief report published in Cell, Host and Microbe, the team employs an engineered E. coli strain as a reporter for the bacterial growth rate during an infection model that mimics an orthopaedic device implanted into a bone that becomes infected. It is common during various medical procedures for the implant device or catheter to become colonized, and due to the inability to treat this infection, the device needs to be removed and reinserted.

Toggle switch = trigger and reporter elements

  A toggle switch is an example of a synthetic biological circuit engineered in a cell to provide a reproducible and tuneable mechanism to turn on and off specific behaviours or reporters.  In this case, the toggle switch is derepression of the TetR repressor upon exposure to the anhydrotetracycline (ATH) inducer, leading to induction of the Cro repressor. The Cro repressor now represses the cl regulator, both derived from the lambda cl/cro system, which ultimately leads to induction of the lacZ reporter. In the baseline state,  lacZ is repressed, and the colony is white in the classic blue/white screen. During active growth in log phase, after 4 hrs of inducer treatment, all colonies switch to the lacZ expression state, producing blue colonies. The reporter works well in recapitulating the active growth state of log phase cultures, and the frequency of lacZ positive colonies decreases in non-growing or slow growth rates, such as the entry into stationary phase. Even with repression of the cl regulator, active cell division is still needed to dilute the active cl protein at the time of adding the tetracycline inducer.  Therefore,  the lacZ positive phenotype only occurs in actively dividing cells, but slow-growing cells will remain in the lacZ negative state.

  The antibiotic levofloxacin, which kills actively dividing cells best, caused a decrease in cell number, and a decrease in the lacZ positive phenotype. Conversely, it does not kill stationary phase cells very well, and these cells had the lowest  lacZ expression, all of which demonstrates further that the toggle switch is a reliable indicator of growth states.

Are bacteria in a slow growth, antibiotic tolerant state during an infection?

  During chronic infections, it is proposed that treatment fails because it cannot accumulate to high enough concentrations in biofilms, or that bacteria are not actively growing, and therefore tolerant to antibiotic exposure. To test the latter of these two hypotheses, the E. coli reporter was used in this device-associated infection model, rather than Staphylococcus, the more typical cause of orthopaedic implant-related infections. The E. coli infection was maintained for 6 weeks, and after 4 days, about half of the bacterial cells in the infection are actively dividing, and half are not dividing. This was one of the first interesting results, which suggests that cells causing this infection do not mimic a stationary phase culture,  a uniformly non-growing population. Then levofloxacin was used to treat the infection, and although bacterial load decreases compared to untreated  infections (~1 log), it was predicted that the population of actively dividing cells would decrease. However, the opposite was shown, where the infection was enriched for actively dividing cells, which directly contrasts the killing data from in vitro experiments.

  Were bacteria in the infection developing resistance? A few hundred isolates were recovered from the infection and none demonstrated any in vitro levofloxacin resistance. The authors concluded that the presence of non-dividing bacteria at the infection site does not lead to an antibiotic-tolerant infection. This conclusion directly contrasts what we know about the development of resistance under laboratory conditions, and highlights the utility of this kind of growth rate reporter during an actual infection.  When the amount of antibiotic was increased, the infection could be cured, indicating that the non-dividing bacteria either begin to divide at some point, or an increase in concentration is enough to kill. The authors argue that either interpretation supports the unexpected observation that during infection, there are mixed growth rates of actively dividing and non-growing states, and that antibiotic resistance does not necessarily arise from the non-growing bacteria.

  While the authors did not test the other tolerance hypothesis, that bacterial biofilms are the cause of chronic infections, it is assumed that biofilms formed on the inoculated pins. However, no studies were performed to confirm biofilm growth. Even in the presence of biofilms, these infections were cured with higher dose treatments, arguing perhaps that this was not necessarily a biofilm infection. This growth rate reporter would be interesting to examine under biofilm forming conditions, to test some of the underlying assumptions about heterogeneous growth rates in biofilms.

  This straightforward, synthetic biology approach to design a reporter of the bacterial growth rate during an infection will be a useful tool in future antibiotic discovery research. Examining the bacterial response to antibiotics in a host system is ultimately more valuable than studying bacteria growing in a tube of broth.

 

 

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CRISPR Antimicrobials: A bacterial defense strategy turned on itself

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Bacteria are innovators. Life as a bacterium means lethal and frequent chemical assaults from antibiotic exposure and physical assault from the most abundant “biological entities” living on earth, bacteriophages. The myriad of resistance mechanisms used in response to these threats illustrates the creativity of bacterial survival. The CRISPR-Cas system is one shining example of how microbes resist killing by viruses. Bacteria recognize foreign DNA injected from viruses and degrade it, along with keep a record of all viral DNA sequences from previous infections, as a kind of ‘memory’ for the bacterial immune system.

 Components of a CRISPR system.

Encoded on the bacterial chromosome of numerous species, the CRISPR immune system has 3 main components:

  • An enzyme that cuts double stranded DNA (Cas gene)
  • A short RNA guide (tracRNA)
  • CRISPR array of “clustered regularly interspaced short palindromic repeat” sequences separated with “spacer” regions that contain the target DNA

The original function of CRISPR/Cas system is to degrade incoming plasmid or viral DNA, but it has been developed as a powerful genome engineering tool. It is easy and cheap to employ this pioneering system towards cutting any desired target DNA, which explains its widespread and rapid usage in many academic research labs. The CRISPR technology permits DNA cutting, editing, introducing mutations, deletions or insertions, with unprecedented precision. There can be “off-target”mutations that arise and this is an area where the technology needs refining to limit the creation of potentially harmful DNA changes.

Game changing applications of CRISPR and the patent race to own it.

The list of potential applications is growing and underscores its transformative potential. CRISPR may give the field of gene therapy a boost, with the ability to correct numerous human genetic diseases including blindness, Duchenne muscular dystrophy, Cystic Fibrosis, sickle cell anemia and others. CRISPR gene drives are being developed to engineer sterile mosquitoes to help eliminate vector-borne diseases like malaria, dengue fever, West Nile and Zika. Plants could be edited for resistance to drought and pathogens, which can proceed without the addition of any foreign genes, but rather editing existing genes. This approach to not introduce foreign genes offers a solution to the problems associated with GM organisms. The other huge opportunity is to engineer T cells to combat HIV infection or to treat cancer. Since HIV infects T cells, it is proposed to engineer T cells to either resist HIV infection, or to redirect HIV immune responses. As part of the oversimplified “cancer moonshot”, CRISPR is also being considered for use in treating cancer.

Despite the many hurdles faced to obtain these diverse goals, it is no surprise that there is a mad rush to patent the CRISPR technology and researchers at the Broad Institute (MIT) (Feng Zhang) and the University of California Berkeley (Dr Jennifer Doudna – along with Emmanuelle Charpentier) are engaged in a combative patent dispute. The value of the technology is difficult to estimate but the impact will be comparable to the invention of PCR by Kary Mullis, and will also likely lead to a Nobel prize. Professor, open science advocate, and co-founder of the Public Library of Science (PLOS) journals, Michael Eiesen, has been refreshingly honest and critical of the tactics used in the CRISPR patent debate. He even went so far as to propose that neither group should be able to patent a technology that was government funded research, and therefore ultimately owned by the tax-paying citizens.

Novel CRISPR antimicrobial treatment that does not involve antibiotics.

Widespread antibiotic resistance is an increasing, global problem due to the excessive use of antibiotics in hospitals and more importantly, in agriculture. The arms race between microbes and antibiotics is a losing battle for antibiotic discoverers. As soon as a new antibiotic is discovered and employed, there is often the immediate discovery of clinical resistant isolates in response to treatment. It has also been shown than antibiotic resistance even precedes the clinical usage of antibiotics, as bacteria need to defend against antimicrobial attack our in the real world. Resistance is out there.

As an alternative to continuing our search for new antibiotics (which is ongoing in new and exciting ways), scientists are developing new approaches to kill microbes and treat bacterial infections. The CRISPR-based antimicrobial approach described here is one such alternative approach to killing microbes without antibiotics. A bacterial defense strategy against viruses is being turned on itself.

Early work showed that the CRISPR-cas system can be directed to cleave DNA sequences in the bacterial genome, which led to irreparable damage and bacterial death (1). Recent studies have moved this concept further by developing a system to deliver cytotoxic, CRISPR-Cas9 systems to microbes. Phagemids are plasmids derived from phage genomes, viruses that specifically target bacteria. The phagemids have been engineered to express all the components of a CRISPR system, in addition to the information required to package the phagemid DNA into a phage particle. Bacteriophages are experts at landing on the outer bacterial surface, injecting its DNA into the bacterium, taking over the bacterial machinery and producing hundreds of viruses. The factory ultimately bursts, leading to death and release of an army of viruses to maintain the attack on neighbouring cells.

CRISPR Antimicrobials.

In back-to-back papers in Nature Biotechnology, two groups have published remarkably similar reports on the use of the Streptococcus pyogenes type II Cas 9 protein as a killing enzyme (2,3). The basic concept was to develop a CRISPR-mediated approach that can cause specific bacterial death. The element of specificity is particularly interesting in light of our increasing awareness of the dangers of broad-spectrum killing of healthy and helpful commensal, microbiome members. Drugs that cause little collateral damage is a new principal of antimicrobial design, which is a large shift from conventional thinking that broad-spectrum is best. Also, these papers explored the use of CRISPR to limit the transfer of antibiotic resistance genes.

Both approaches used phage-derived vectors to encode the CRISPR components of the the Cas9 enzyme, the short RNA guide (tracRNA) and a CRISPR array containing the desired cut sites within the spacer regions. When directing cleavage of kanamycin or ampicillin resistance genes, both groups could successfully show a loss of antibiotic resistance in the target organism, after delivery of the phagemid-encoded CRISPR systems. The same principal was applied to monitoring the efficiency of transformation, by measuring the number of viable cells in strains containing the target genes compared to strains that lacked the target. There was a 1000-fold decrease viability (and in the efficiency of conjugation) in hosts that contained the targeted cleavage site, indicating that successful DNA disruption and killing. Interestingly, there was never a complete killing of the target population in these experimental conditions.

To further examine the feasibility of this approach, various infection models were used to determine the capacity of CRISPR killing when applied to pathogens during an infection. In both a skin infection and the Galleria larval infection models, the pathogen numbers could be reduced by CRISPR-Cas antimicrobials, which permitted skin healing and survival of the larvae. The final experiments were to test the system when exposed to simple, mixed communities, in order to show specific pathogen targeting and non-targeting of commensals.

Although phages are being revisited for use as an alternative to antibiotics, engineered phages with CRISPR capacity have perhaps the added value of narrow spectrum killing. The biggest obstacle to improving these transmissable, programmed antimicrobials is to improve the method of delivery. It is not unreasonable to image a scenario where due to the low costs of DNA synthesis, custom, specific CRISPR-Cas-phage particles could be synthesized within a few days, and used as needed to treat any possible infectious disease.

References

1 . CRISPR design for next-generation antimicrobials. Beisel CL, Gomaa AA, Barrangou R. Genome Biol. 2014 Nov 8;15(11):516. doi: 10.1186/s13059-014-0516-x.

2.  Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA.                  Nat Biotechnol. 2014 Nov;32(11):1146-50. doi: 10.1038/nbt.3043..

3. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.Citorik RJ, Mimee M, Lu TK. Nat Biotechnol. 2014 Nov;32(11):1141-5. doi: 10.1038/nbt.3011.