Keywords

Ever since their discovery in 1966–1967, research on OMVs has progressed steadily and many more investigators have contributed significantly towards revealing the structure, both physical and chemical, and functions of the OMVs derived from numerous Gram-negative bacteria. But the search for knowledge is an unending process and accordingly, there are many more features of the OMVs that have remained unexplored or unanswered until now. In this context, the present authors have taken the opportunity or liberty to do some wild thinking, which is hoped to be relevant, about the past, present and future of the OMVs and their applications towards the welfare of mankind. Some attempts have also been made to offer questions, right or wrong, that may stimulate the minds of future investigators.While considering the different facets of these nanoparticles, this monograph has dealt with (1) bacterial growth conditions, (2) pathogenicity, (3) impact of LPS structure, (4) antibiotic treatment, (5) stress response, and so on as conditions affecting the production of OMVs by Gram-negative bacteria in general. But a detailed study of the genetics of the organisms controlling the OMV production under any situation still remains to be worked out, although some isolated studies have already been reported. Again not enough information is available on the genes, in particular, exerting positive or negative regulation on the production of OMVs. Why the OMVs are produced under such diverse conditions and what are the driving forces working under those conditions are some of the disturbing questions that remain to be answered.

The production of OMVs has been interpreted as a novel secretion mechanism of Gram-negative bacteria. Although there are several different secretion mechanisms operating in Gram-negative bacteria, the reason or the signal for evolving this additional secretion mechanism, production of OMVs, has remained unknown. Also the details of the energetics of this mechanism remain to be worked out.It has now been recognized that different virulence factors, proteins, toxins, antibiotics, genetic elements, and the like are packaged into OMVs for transfer and delivery into different prokaryotic and eukaryotic cells as hosts. There are enough reasons to believe that such transfer through OMVs ensures that the packaged materials are not damaged or destroyed by the hostile environment in the extra-cellular space. It is implied that some signaling mechanism works between the bacterial cell and the outer medium so as to ensure that the materials to be secreted do not face any hostile environment outside. But what this signaling mechanism and, if present at all, how it works are matters that deserve the attention of future investigators. It is known that a signaling molecule, PQS, works in Pseudomonas aeruginosa to control in some way or other the production of OMVs, but, here again, the exact molecular mechanism involved and particularly its genetic basis remains to be elucidated.

Investigations have revealed that not all the proteins of the outer membrane (OM) or of the periplasm (PPM) are transferred and packaged into OMVs in equal proportions and that a selection process works and controls this affair. There are reasons to believe that a sorting process works at this stage. But what is the nature of this sorting mechanism and what is its genetic basis are not known at present.

Formation of biofilm is a defense mechanism of bacteria and particularly the pathogenic ones. Enough evidence has been presented about the release of OMVs by the bacteria in the biofilm state and plenty of OMVs have been found in association with the biofilm. Whether the OMVs contribute towards formation of the biofilm or they simply help the survival of the parent bacteria in the biofilm or both is an intriguing question.

Recent proteomic analyses of the Gram-negative bacterial OMVs using mass spectrometry, ultracentrifugation, gel electrophoresis, and the like have opened a new chapter in OMV research, but have already produced results that are debated. The presence of cytoplasmic and inner membrane proteins in the OMVs is difficult to explain. This demands a very stringent method of isolation and purification of OMVs. Only a synchronized cell culture can ensure to a great extent the absence of some bacteria undergoing lysis during the logarithmic phase of growth. Such lytic bacteria may release the cytoplasmic and inner membrane proteins in the extra-cellular medium to be picked up by the OMVs. Also a very stringent method of isolation and purification of OMVs is required to ensure the absence of any contamination from the culture filtrate. Nevertheless, the analyses of proteomic profiles of OMVs derived from different Gram-negative bacteria are very important not only for elucidating the functional aspects of the different vesicular proteins but also for designing the proper mode of delivery of antigens and toxins for production of effective vaccines against Gram-negative pathogens.

Many biological molecules, proteins, toxins, antibiotics, and so on cannot directly enter different eukaryotic cells, but have found access into these cells through the OMVs. This is an important and biologically useful function of the OMVs. However, the packaging of all such molecules into the OMVs from the parent bacterial cells may not be easy or at all possible in as much as all of them cannot simply pass through or get translocated across the inner membrane to get into the PPM. In this respect, the autolysin molecule, ClyA, has the unique property of easily getting translocated across the inner membrane to reach the PPM and then to the OMVs. Not only that, ClyA can combine with different molecules and this combination can also easily get translocated across the inner membrane and entrapped into the OMVs. This gives a unique scope for producing the so-called engineered recombinant OMVs and to broaden thereby the functional spectrum of the OMVs. These engineered recombinant OMVs will, in future, play a great role in modulating the immunogenicity of the eukaryotic systems and in producing effective vaccines against many Gram-negative bacterial pathogens.

The OMV vaccines are acellular and regarded as safe for use in humans and should thus be preferred to conventional live or heat-killed or virulence-attenuated vaccines. The advantages of OMVs over subunit vaccines such as purified proteins are (1) purification of OMVs need simple ultracentifugation, effectively eliminating the requirement of costly infrastructure; (2) OMVs are effective as vehicles for vaccine delivery so that adjuvants are not required and (3) improvement of efficacy and safety can be done by addition of effective antigens and/or detoxification of LPS through simple genetic engineering of bacterial strains used for OMV production. The development of OMV-vaccines against different bacterial pathogens thus has a bright future.

The concept of “OMV vaccines” was developed during the 1980s when it was tried against group B meningococcal diseases. These vaccines have a long-standing safety and effectiveness record as available through a number of clinical trials. The collaborative study of the New Zealand ministry of health (NIPH) and the private firm, Chiron/Novartis, revealed that OMV vaccines can be upscaled and the quality, consistency and cost-effectiveness can be maintained. This gives the scope and direction in which further studies can be made towards improving the efficacy and quality of the vaccine.

Recent developments including sequencing of complete genomes and the reverse vaccinology approach have enabled us to predict a number of protective antigens. Technologies are at present available for determining the cross-protective and universal antigens to be included in the future OMV vaccine. Thus in the coming few years, a global OMV-based vaccine strategy incorporating suitable antigens is expected to be developed and might offer protection to susceptible individuals against diseases caused by the meningococci of all serogroups.

After the first report of the OMV vaccines against the serogoup B meningococcal disease, OMVs from various other organisms including Vibrio cholerae, Salmonella typhimurium, P. aeruginosa, Bordetella pertussis, and others were shown to exhibit immunogenic properties and were reported to have vaccine potential. The future will thus see a lot of research and clinical efforts towards development and use of effective immunogens and/or vaccines against many Gram-negative organisms, in addition to N.meningitidis.Thus, the OMVs have already crossed the boundaries of the research laboratory and reached the open field, where the researchers and the clinicians will have a lot of copes, in future, to interact with each other for the welfare of the living world.