What is not a reason for the use of bacteria in the initial use of genetic engineering?

Genetically Engineered Microorganisms

Prophylactic and therapeutic applications for genetically modified bacteria are not a new idea. In the early 1990s such proposals were widely debated, even though CRISP/Cas9 (clustered, regularly interspaced, short palindromic repeats), zinc-finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs), and other gene engineering technologies were not available [91].

Much earlier, in the 1940s, viruses with affinity for solid tumors [92] and subsequently bacteria with similar properties such as Salmonella typhimurium were already receiving attention. The hypothesis of genetic manipulation of such microbes to convert them into vehicles for anticancer agents and other drugs was subsequently envisaged.

Biotechnological drug manufacturing, relying on genetically engineered organisms such as Escherichia coli and the yeasts Saccharomyces cerevisiae and Pichia pastoris is nowadays an industry standard. The gut microbiome could provide the long-term personal platform to manufacture as well as release low doses of the required molecule, especially if ordinary commensals were selected for genetic manipulation. Such tailored treatment would be an elegant complement to precision medicine.

Salmonella typhimurium can colonize the human gut. As a tumor-seeking organism, a manipulated strain expressing flagellin B (FlaB) suppressed tumors and metastasis and assured prolonged survival through a TLR-5–dependent mechanism in a mouse model [93].

A modified strain of the same species has been experimentally endowed with antimelanoma properties by means of expression of interferon gamma [94].

Genetically engineered E. coli Nissle 1917 strains for the treatment of urea cycle disorder and phenylketonuria are advancing. Inflammation-sensing bacteria able to both diagnose and orchestrate a therapeutic response in patients with IBD are also being pursued [95]. At least one molecular basis for such antiinflammatory result, namely α-MSH, has already been elucidated, as previously alluded to in the study by Qiang et al. [70].

Attenuated Salmonella strains

Facultative anaerobic bacteria are attractive cancer therapeutics because of their selective growth in hypoxic regions of solid tumors. Genetically modified bacteria from several strains of Salmonella, including S. typhimurium and S. choleraesuius, can be specifically targeted to tumors following systemic administration. The bacteria then replicate only within tumor tissue, allowing efficient delivery of genes and other proteins to tumor tissue.

Besides their utility for targeted drug delivery, attenuated Salmonella directly inhibit tumor growth. In a mouse melanoma model, an attenuated Salmonella typhimurium (VNP20009) selectively targeted both primary tumors and metastatic lesions, significantly slowing tumor growth.63 Antitumor effects are thought to occur by several different mechanisms, including production of inflammatory cytokines, such as TNF-α, and toxic proteins that induce tumor apoptosis. The bacteria stimulate innate immunity through production of an attenuated lipopolysaccharide (LPS), a potent stimulus for enhanced DC function.64,65

Thamm et al. recently completed a phase I clinical evaluation of systemic VNP2009 administration in dogs with a variety of malignant tumors.66 In this study, 41 dogs received either weekly or biweekly intravenous infusions of the modified Salmonella product and were monitored for acute toxicity and tumor response. The dose-limiting toxicities were fever and vomiting, occurring at the high end of the dose interval in this dose-escalating trial. Colonization of the bacteria within tumor tissue was detected in about 40% of the dogs, with significant clinical responses (either complete remission or partial remission) occurring in 15% of patients. Overall, 37% of dogs experienced either disease stabilization or a transient response to treatment. Given the advanced stage of disease in the dogs of this study, the low incidence of toxicity, and the high overall response rate, further evaluation of VNP2009 is warranted.

6.09.4.6 Bioreporters

The bioavailability, though not the bioaccessibility, of contaminants can be addressed most directly by applying so-called whole-cell living bioreporters. These are usually genetically modified bacteria, which respond to the presence of specific chemicals with easily detectable signals. In most cases, promoters responsible for the cellular reaction on the availability of substrates or the presence of toxicants are coupled to genes coding for luciferases or fluorescent proteins. Exposure of these bioreporter bacteria to the target chemicals leads to dose-dependent signals. Light emission has the advantage that it permits remote sensing, by, for example, luminometry or fluorescent microscopy. Noninvasive observation and quantification of bioavailability is thus possible. Bioreporters can be designed in ways that they act as sensor–degrader organisms, meaning that they do not only sense but also degrade the target chemical, which leads to a dynamic bioavailability situation. This makes them appropriate substitutes for metabolically active bacteria used for bioremediation. Other bioreporters come as pure sensors. Due to the lack of a catabolic machinery for the target chemical, they do not influence bioavailable concentrations and may be considered as substitutes of metabolically inactive biological targets of chemical toxicity. A potential advantage of bioreporter bacteria, which, however, requires awareness, is the fact that they report the overall suitability of the prevailing environmental conditions for biodegradation. As bioreporter signals typically integrate over chemical bioavailability and toxicity and also account for other aspects of the living conditions in the environment they are applied to, signal interpretation has to be done with care and often requires experimental controls such as spiking of target chemical for the control of bioreporter functioning.

6 Probiotics as Drug Delivery Agents for Experimental Colitis

A potentially exciting niche for probiotics may be as drug delivery agents for novel therapeutic agents. This therapeutic approach has been examined in several rodent colitis studies. One novel pharmacological approach used genetically modified bacteria to deliver a regulatory cytokine to the colon. Specifically, investigators utilized a Lactococcus lactis secreting IL-10 strain to successfully treat chronic DSS-induced colitis in mice, as well as to prevent colitis in IL-10 deficient mice [71]. This specific approach is currently being utilized in an ongoing clinical trial for CD [72]. Moreover, the same investigators utilized a related approach to deliver cytoprotective and mucosa repair-promoting peptides (trefoil factors) to the murine colon. This localized peptide delivery resulted in prevention and healing of DSS-induced colitis [72]. Furthermore, this novel pharmacological approach also was successful in improving established chronic colitis in IL-10–/– mice [72].

In addition, Carroll and colleagues tested the efficacy of Lactobacillus gasseri expressing manganese superoxide for improving colitis in IL-10 deficient mice. This innovative colonic delivery of an antioxidant reduced the severity of murine colitis [73]. It is apparent from these studies that novel drug delivery approaches may find a niche for the treatment of intestinal inflammation in humans. Relevant probiotic carrier organisms may include the aforementioned Lactobacillus strains, as well as E. coli Nissle 1917 [74].

2.1 Asilomar Conference

The need to regulate GM technology was mooted by scientists themselves. A number of scientists expressed concerns over the potential risks of developing new strains of bacteria as a result of transferring genetic material from viruses and other bacteria. The GM bacteria were feared to compete and outdo the natural microbial population or transmit viral genes that might cause cancer. These concerns resulted in two historic conferences among scientists in the mid-1970s. The first conference took place in January 1973, spearheaded by the National Science Foundation's Human Cell Biology Steering Committee and the National Cancer Institute (NCI) of the National Institutes of Health (NIH). The consensus of that meeting was that researchers should proceed cautiously, that they should attempt to quantify the potential risks of working with such genes, and that additional efforts should be expended to determine what safety precautions should be taken to avoid spreading a potential carcinogenic risk through the environment (Pew Initiative on Food and Biotechnology, 2001).

This conference was followed by the renowned 1975 Asilomar Conference which discussed the overall safety issues related to GM technology. Although most of the participants believed that the technology neither posed significant health risks nor created new hazards, they agreed to abide by a set of research guidelines for the safe use of the technology. Chief among these was the agreement to limit work to disabled bacteria that were not able to grow outside a laboratory environment. Thus, one of the first recognized risk management decisions applied to the technology was the adoption of voluntary controls by an otherwise unregulated community of scientists, primarily in academic laboratories (Pew Initiative on Food and Biotechnology, 2001).

7.6 Future trends

In addition to antimicrobial treatments using microbes, antimicrobial peptides that are not of microbial origin could be exploited by genetic manipulation to enable their production in microbes. Furthermore, the use of bacteriophage-derived enzymes (lysins), produced by genetically modified bacteria, may also be possible, but technically challenging. A successful example of this is the production of murein hydrolase, an endolysin from bacteriophage ϕ3626 that attacks Clostridium perfringens. Cl. perfringens produces an enterotoxin that can cause food-borne disease and is responsible for severe economic losses in chicken production, as is the aetiological agent responsible for necrotic enteritis. The ϕ3626 endolysin was expressed in E. coli and shown to be active against 48 different strains of Cl. perfringens (Zimmer et al., 2002). The structures and actions of phage enzymes may provide data allowing the development of synthetic therapeutics (Bernhardt et al., 2002), and phages may also be modified to deliver specific toxins to infecting bacteria (Westwater et al., 2003). Genetic modification of strains to produce bacteriocin is one area where preliminary reports are encouraging. The inhibition of S. Typhimurium in the chicken intestinal tract by a transformed avirulent avian E. coli, with a plasmid coding for the production of microcin 24, was demonstrated by Wooley et al. (1999). Similarly, it has been proposed to engineer avirulent bacteria to produce the antimicrobial peptides produced by many eukaryotic organisms, called defensins. However, it is becoming apparent that the role of defensins is not restricted to antibacterial activity. These proteins have wider antimicrobial properties and can interact with immune regulatory components. Another possibility is the bioengineering of bacteriocins, particularly the Lantibiotic group, to generate enhanced forms of these peptides (Piper et al., 2009).

The development of new microbial treatments for poultry is beginning to result in feasible alternatives to conventional antimicrobials. The use of biotechnological tools may accelerate their development, but the public desire for more ‘natural’ food should not be ignored. However, both bacteriophage and bacteriocins provide the possibility of novel, acceptable solutions to the problems of microbiological safety in the poultry industry.

6.6 Future directions

Due to their impacts on mosquito growth and development, reproduction, and vector competence, members of the gut microbiota of mosquitoes are emerging as novel targets for the control of a number of mosquito-borne pathogens, and paratransgenic approaches using genetically modified bacteria have already shown promise in Anopheles mosquitoes for the control of malaria (Wang and Jacobs-Lorena, 2013). Efforts to identify and disseminate unmodified bacteria that naturally inhibit pathogen infection or transmission are also ongoing. However, despite recent progress in our understanding of mosquito-microbiota interactions, a number of important basic questions remain underexplored:

How do various microorganisms, including non-bacterial members of the gut microbiota, affect the vectorial capacity of mosquitoes? Numerous types of microorganisms, including bacteria, algae, protists, fungi, rotifers, and viruses, have been identified from the mosquito gut and have the potential to produce anti-pathogen molecules and/or alter host immunity or fitness (Belda et al., 2017; Bolling et al., 2015; Bozic et al., 2017; Chandler et al., 2015; Demaio et al., 1996; Hinman, 1930; Merritt et al., 1990; Muturi et al., 2016a; Roundy et al., 2017; Steyn et al., 2016; Tawidian et al., 2019; Thongsripong et al., 2018; Walker et al., 1988). However, studies to date have almost exclusively focused on bacteria and few have stepped beyond taxonomic characterization to provide insights into the mechanisms underlying how gut microbiota impact mosquito survival, reproduction, and/or growth and development. Future experimentation using axenic (microbe-free) and gnotobiotic mosquitoes colonized by a defined gut microbiota will be necessary to identify specific taxa with functions of interest (e.g. the ability to antagonize human pathogens).

How does the gut environment shape the acquisition and maintenance of key microbial species? Successful implementation of microbe-based control strategies will rely on our ability to effectively disseminate candidate microbes into mosquito populations in the field, via attractive sugar baits to target adult mosquitoes (Bilgo et al., 2018; Mancini et al., 2016) or introduction into larval habitats. Thus, a better understanding of how microbes persist in environments where mosquitoes feed and the mechanisms underlying how they colonize and persist in mosquito hosts is essential.

How do microbial interactions impact the stability and function of mosquito gut microbial communities? Finally, heterogeneity in gut microbiota composition between individuals and across mosquito populations could have unpredictable effects on introduced microbes due to competition or other interactions (Bahia et al., 2014; Coon et al., 2014; Hegde et al., 2018; Terenius et al., 2012). As previously noted, the gut microbiota may also influence the efficacy of other mosquito and pathogen control agents, including entomopathogens and Wolbachia. The ability of Wolbachia to spread rapidly through mosquito populations makes characterizing the interactions between stable Wolbachia infection and gut microbiota a particularly high priority for future research.

Conclusion

Progress in the study of mosquito–microbiota and mosquito–pathogen interactions has allowed paratransgenesis research to advance in earnest. Promising results in proof-of-principle experiments have demonstrated that pathogen transmission can be reduced through genetic manipulation of the mosquito microbiota. Furthermore, bacteria have already been introduced into mosquitoes that were subsequently released into the environment [88,89]. Nevertheless, there may be challenges in utilizing genetically modified bacteria to reduce the prevalence of mosquito-borne disease. For instance, the ability of target pathogens to develop resistance to chosen effector mechanisms should be explored in greater detail. To date, no obligate symbiont of mosquitoes has been identified. An obligate mosquito symbiont is desired by researchers wishing to employ a paratransgenic strategy similar to genetically modified R. rhodnii in triatomes [47]. Genetically modified bacteria have to be introduced into the mosquito, and more studies are required to determine the optimal route of introduction. Similarly, more research is necessary to determine if genetically modified bacteria will be competitive against wild-type bacteria. Despite the challenges, bacterial paratransgenesis is a promising approach in the war against mosquito-borne disease and addressing these challenges in the laboratory will refine the methodology behind field deployment of paratransgenic organisms.

Microorganisms

Several GM microorganisms have been approved for food use, the yeast Saccharomyces cerevisiae being the first GM organism to be approved for use in food production. GM yeasts are an important source of enzymes used in food production (e.g., cheese-making) and in improving the quality of beer and wine. GM bacteria are used in the production of enzymes such as milk-clotting enzymes for cheese production and food/feed additives such as aspartame and l-lysine. An important feature relating to the safety of these approved GM microorganisms is that they have been modified by utilizing genetic material from within the host species and from microorganisms with GRAS (Generally Regarded As Safe) status (in Europe QPS—Qualified Presumption of Safety).

The safety of the approved GM microorganisms as components of food and livestock feed is attested to by the fact that no authenticated reports of allergenicity or toxicity have been published in any peer-reviewed journals.

3 Conclusion

Nanoparticles play a vital role and are crucial to many sectors. The process of synthesizing a nanoparticle via a biological method is environmentally friendly, reliable, and less costly than chemical and physical ways where toxic chemicals and high temperatures are involved. Various metallic nanoparticles can be synthesized using plants, algae, bacteria, fungi, and actinomycetes. Genetically modified bacteria can synthesis a high rate of nanoparticles as they will be more resistant to metal toxicity and they can grow more rapidly in the media and can reduce the metals to its oxides and further to nanoparticles even in industrial scales. Fungi generally have a better surface area, and the yield of nanoparticle will be more since the conversion of ions to metallic nanoparticles is easier. To scale up to industry level is also possible using fungi because of the ease of bio-separation technique. Although multiple methods are used to synthesize metallic nanoparticles, extensive studies have to be carried out to determine the best mechanism and the best route, which is harmless to the environment and society.