Plasmid vector(s) in Bacillus thuringiensis harbor genes for insect pest control and for neglected infectious diseases in humans

Plasmids (circular DNA molecules) represent an ingenious strategy for horizontal gene transfer in prokaryotes and eukaryotic cells. Plasmids harbored in bacteria are responsible for the spread of traits such as antibiotic resistance, virulence factors, and the machinery for the horizontal gene transfer e.g., type IV secretion systems. Remarkably, Bacillus thuringiensis (Bt) cryptic plasmids encode and carry genes that, under the host environment, replicate and concomitate with sporulation, producing parasporal crystalline proteins of two major types, crystalline (Cry) and cytolytic (Cyt), the former toxic against different orders of insects such as Lepidopterans, Coleopterans, and Dipterans (Cry proteins, MW 50 – 130 KDa); Cyt proteins, produced by B. thuringiensis subspecies israelensis ( Bti )(MW 27-kDa) are toxic against Dipterans, i.e., mosquitoes and black ﬂ ies. The X-Ray tridimensional structure for both types of toxins, formed by three domains, mostly of beta sheets antiparallel (Domain II and Domain III) linked through loops of different lengths. Domain I is a bundle of alpha helices. This structure is characterized by ﬁ ve conserved blocks, implying a conservation in the mode of action. Cyt proteins possess two alpha helices and some beta sheets with a structure similar to the antimicrobial peptides. Indeed, the mode of action proposed is mediated by the toxin-lipid interaction that hypothetically could result in transmembrane ionic channel formation. Several pieces of evidence support the action of both toxins in insects and mammals. The question is to what extent these Bt / Bti plasmid-encoded Cry or Cyt genes can be applied as bioinsecticides individually or in combination with Lysinibacillus sphaericus . The feasibility of being considered a promising and safe biological strategy for crop pests and vector-borne neglected infectious diseases is an issue pinpointed in the present review.


Introduction
The genetic microbial strategy for DNA exchange is conjugation, a process shaping microbial communities (1,2).The genetic process driving and leading to diversity is the horizontal transfer of mobile genetic elements or plasmids (3).This process allows the acquisition of exogenous DNA and recombinant bacteria with novel and potential properties or new phenotypes.It is paramount that Bacillus thuringiensis (Bt) has cry genes encoded in plasmids, and these genes are transferred to other bacteria of the same genus or different, expressed for the production of crystalline proteins (1,4).Understanding the hidden lifestyle of the plasmid vectors in the genus Bacillus family (B cereus, B. thuringiensis, and B. anthracis) unveils the dynamic bacterial communication and genetic transfer of diverse traits such as antibiotic resistance, virulence factors, and secretory system components.The spreading resistance in the microbial communities of the genus Bacillus is genetic information that allows survival, diversity, adaptation, and evolution.Indeed, data from 5, support the fact that there is an active process of genetic exchange of plasmids (128 and 100 kb in size) (5)(6)(7) leading to the dissemination and spread of traits (3,5,6,8).Therefore, the understanding and identification of all the extrachromosomal elements in the different strains of Bt might provide genetic tools for improvement in the control strategies against mosquitoes (9)(10)(11).At this point, pest vector management and control are of utmost importance for worldwide health as it is threatened by the chemical insecticides used to overcome them.It becomes an urgent need and paramount priority search for novel technological strategies to control pests in crops of agronomical and economic importance.
One of the most successful strategies to counteract it is the entomopathogen Bacillus thuringiensis.This Gram-positive soil bacteria discovered in the flour moth larvae in the province of Thüringia in 1911 (12)(13)(14) caused toxicity to different orders of insects.Despite the emergence of insect resistance populations to Bt and its products, they are used as bioinsecticides in proper formulations and efforts are being made to improve their range of action as well as their application (15)(16)(17).Another alternative that has been approached and developed with promising results is the transfer of plasmids encoding Crystalline (Cry) and the cytolitic (Cyt) proteins, which have been transformed with a cocktail of genes for a more diverse plant resistance response to insect attacks (18-20).But still, this strategy raised concerns about the endemicity of crops.
The most recent advances in the biological control of crop pests are the genetic and molecular techniques, in particular, the genetic engineering that has played a role in the design of biopesticides that can properly aid in vector control (mosquitoes) of disease transmission (e.g., dengue, malaria) (2,9,10,21).By harnessing the synergy of the Bt products, Cry and Cyt toxins, as recently reviewed by Silva-Miranda et al. (11), are among the different strategies for the control of mosquitoes; the most feasible and safe is based on Bacillus thuringiensis subsp israelensis because the low persistence of the latter and the toxicity of both in a short period (11,21).Gaining insight into this might represent a frontier to focus on.

Plasmids encode genes for horizontal gene transfer
Plasmids are small circular DNA in bacteria defined as mobile genetic elements that promote the spread of several traits and geographical distribution worldwide (22)(23)(24).Moreover, plasmids favor bacterial fitness adaptation and evolution.Plasmids favor horizontal gene transfer and gene recombination.Both genetic mechanisms allow bacteria to transfer genetic information to related and unrelated bacteria.The transfer of genetic determinants, i.e., carrying utmost antibiotic resistance genes and virulence factors (biofilm formation) proteins of the type IV secretion system (22)(23)(24), is present in Gram-positive and Gramnegative bacteria.The plasmids of the family Enterobacteriaceae comprise more than 200 different F-like plasmids.F-plasmids or Ffactor, described in 1940, is a large 100 Kb circular conjugative plasmid of Escherichia coli described as a vector for horizontal gene transfer and recombination (Figure 1).
Since then, these F-plasmids and F-related F-like plasmids serve for bacterial conjugation purposes (25).Around 200 different F-like plasmids participate with highly related DNA transfer genes, including those of utmost relevance for the assemblage of a type IV secretion system.An outstanding feature of the F-plasmids in enterobacterial hosts is that they are isolated from clinical and environmental samples from different geographical regions.In the F-plasmid family (pOX38, ColB2, and R100-1), members recognize outer membrane proteins at the surface of the recipient cell (OmpA, OmpF, OmpK36, OmpW) (25-27) (Figures 2I-II).At this point, there was further investigation of defined mutations in the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in vitro (liquid media) by the F-plasmid members.The transfer rate of these members is affected differentially by mutations in the rfa (LPS) locus (27, 28).Indeed, the F-plasmids showed increased sensitivity to mutations that affected rfaP gene expression, which encodes the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS.Furthermore, ompA mutation significantly affected the mating efficiency of an F-plasmid carrying a mutation in the mating-pair stabilization protein TraN (25,27,28).During the transfer, adhesion present in the F-pilus tip could be playing a role in the specific recognition of LPS, OmPA, or the TraT (29-32) (Figures 2I-II).
On the other hand, Bacillus cereus (B.cereus) and its relatives harbor a plethora of plasmids, including conjugative plasmids, representing the heart of the group species differentiation and specification (2,33).Over the years more than 20 plasmids from B. cereus have been found to be conjugative.The large plasmid can potentially "circulate" among members of the Bacillus cereus group (2).In contrast, XO16's known natural distribution is limited to B. thuringiensis var.israelensis.At this point, it is noteworthy to pinpoint that Bacillus thuringiensis, a Gram-positive entomopathogenic soil microorganism, harbors large plasmids encoding the insecticidal crystalline proteins, with bioinsecticide and virulence factor properties (34,35).
The plasmids replicate with bacteria, allowing Cry genes to express and produce the crystalline proteins.These extrachromosomal elements play a pivotal role in spreading along with antibiotic resistance and other traits, transferring these genes to another member of the genus Bacillus and even to other non-related bacteria (36,37).These plasmids that carry genetic traits pivotal to bacterial survival adaptation and evolution participate in the horizontal gene transfer (HGT) (36,(38)(39)(40).
One of them is an integrative or conjugative plasmid with a similar structure to the modular backbone of the F-plasmids (37,41).The conjugative plasmids transfer themselves between most bacteria, thus being one of the main causal agents of the spread of antibiotic resistance among pathogenic bacteria.How the plasmids accomplish this task is as follows.The modular structure or backbone of the integrative and conjugative plasmids (ICPs) consists of three modules: -Module for maintenance -Module for dissemination -Module for regulation.Moreover, additional module structures are possible through insertion sequences, transposons, and specific recombinases.Integrative and conjugative elements (ICEs) in the plasmid vectors transfer of the antibiotic resistance genes (40,41), but now it is evident that ICEs can mediate the transfer of a very diverse set of functions.The advantages of ICEs are that they shape bacterial genomes, promote variability between strains of the same species, and distribute genes between unrelated bacterial genera using conserved integration sites (42,43).ICEs allow bacteria to adapt to new environmental conditions and to colonize new niches (44).
Like phages and conjugative plasmids (45), ICEs can mediate the transfer of virulence determinants and may promote the mobilization of genomic islands (45).
The integrative and conjugative F-plasmids of t60 kbp can be placed into four major genetic modules: -Module for plasmid replication (DNA replication and DNAbinding proteins) -Module for stable maintenance (plasmid stability influenced by plasmid growth and horizontal transfer rates), -Module for DNA transfer which occupies the most conserved part.-Module for accessory cargo genes (within transposons or integrons).
The organization of the different functions in the modules could be related to the host-specific plasmid evolution that might explain the spread of clinical antibiotic-resistance plasmids.In referring specifically to the plasmid pXO16, which is a large conjugative plasmid from Bacillus thuringiensis var.israelensis (Bti), it has several properties (34,42,46,47): self-transfer with high efficiency (48); -mobilize the small pUB110 plasmid from Btithermotolerant Bacillus cytotoxicus at high frequencies (3.3 × 10-3 and 5.2 × 10-4 transconjugants per donor (T/D), respectively (49,50).-Retro-mobilize (the capture of DNA from a recipient by a donor cell) non-conjugative plasmids, including non-transfer plasmids (2,4).-Promote their transfer as well as that of coresident plasmids; transfer chromosomal loci; -displays a remarkable aggregation phenotype associated with conjugation under liquid conditions, [(Natural liquid foods (cow milk, soy milk, and rice milk)], where conjugation, mobilization, and retromobilization were shown to occur at frequencies of 8.0 x 10- Plasmids as circular extrachromosomal DNA elements functioning as vectors of genetic information that can be transferred to other related bacteria and non-related bacteria.The size of plasmids can vary, but in general are usually large plasmids such as those of genus Bacillus, encoding different traits, such as antibiotic, heavy metal resistant genes, virulence factors, and other proteins.In addition, in these plasmids are encoded the nonstructural and structural proteins that are necessary for the horizontal genetic transfer (HGT).

Mechanism for plasmid horizontal genetic transfer: the case of conjugation.
Bacterial communities establish communication through molecular crosstalk to share genetic information that influences their physiology and their lifestyle outside of their host (51-54) and is essential for them for survival, adaptation, and evolution.One genetic process by which bacteria can do this is through bacterial conjugation, a mechanism of mobile genetic elements (55-57) that uses the F-pilus that serves as a conduit for DNA transfer (26,27,29,32,40,51,56).DNA transfers through a conjugation mechanism into eukaryotic host cells.In this process, plasmids function as mobile genetic elements conformed in a conjugation module with the relaxosome complex targeting the type IV secretion system or to the sexual piles for conjugation or mating (41,42,46).Microorganisms respond and adapt to environmental conditions by acquiring genetic material in large amounts (58).Conjugation of plasmids contributes to this process, allowing lateral Conjugation as a mechanism.Horizontal gene transfer (HGT) is described initially for Gram-negative bacteria (I) and through evolution to the Gram-positive (II) and eukaryotic cells.Recently, this process was proposed as a kit tool for conjugation and, therefore, for gene transfer of encoded toxins, accessory proteins, antibiotics and enzymes.Different adhesins in Gram-negative bacteria recognize donor cells at the cellular surface membrane.Plasmids through this conjugosome are endowed with the capacity of HGT because these mobile genetic elements encode the proteins and the accessory factors for DNA replication (relaxase) and DNA transfer (VirB1, VirD4) as well as some non-structural proteins promote the conjugation process.The stabilization mating by TraN and OMPA in the recipient contribute significantly also to the transfer of ssDNA and conjugation efficiency.
gene flow in prokaryotes, carried out through ICE self-transmissible mobile genetic elements that contribute to shaping genomes and lateral gene flow in prokaryotes through the conjugative process.

Two hallmarks of the plasmid bacterial conjugation 2.1.1.1 What participates
-The type IV secretion systems (T4SSs) encoded in the Fplasmid, e.g., the pED208 encoded the T4SS (TrapRD/208).E. coli and other Gram-negative and -positive bacteria employ a type IV secretion system (T4SS) (62)(63)(64) to translocate DNA and protein substrates, generally by a contact-dependent mechanism, into other cells.In Enterobacteriaceae family (including E. coli), T4SS identified to date function exclusively in conjugative DNA transfer.In these species of bacteria, in the plasmid-encoded systems classified as P, F, and I types, Ancestral P-, F-, and I-systems adapted throughout evolution to yield the extant effector translocators, highlighting the adaptive and mosaic nature of these highly versatile machines (Figures 2I-II).
The T4SS has two main subfamilies: conjugation systems and effector translocators.The first ones are for inter-bacterial transfer of antibiotic resistance genes, virulence factors, and genes encoding other traits of potential benefit to the bacterial host.The effector translocators used by many Gram-negative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection (62-64) (Figures 2I-II).
-The non-structural genes are closely linked.These genes are not part of the T4SSs for conjugative transfer, such as the membrane pore, the relaxosome, and replication machinery.Moreover, the non-structural genes assist in core conjugative functions and mitigate the cellular burden on the host.During conjugation, they modulate dormancy, transfer, and establish a commensal relationship with the host, allowing manipulation of the host for efficient T4SS ensemble and assist in conjugative evasion of recipient cell immune functions.These genes take in a broad ecological context and ensure proper propagation of the conjugation system in a natural environment.Proteins with functions associated with plasmid maintenance, e.g.PsiB and SSB, suppress the mating-induced SOS response.This property establishes a novel biological function for conjugative protein translocation and suggests the potential for diverse outcomes from interbacterial protein translocation, influencing bacterial communication, physiology, and evolution.Conjugative protein translocation machinery through the TrapRD/208 requires the engagement of the pED208 relaxosome with the TraD substrate receptor or coupling proteins for activating the signal for protein translocation (Figures 2I-II).This provides evidence of novel biological functions for conjugative protein translocation in mitigating the potentially negative consequences to plasmids and genome integrity resulting from SOS-induced recombination and mutation events.The set of known substrates of conjugation systems includes proteins with functions associated with plasmid maintenance.Of relevance is the fact that the first report of a conjugation-like event between strains of B. cereus sensu lato (s-l-) was 40 years ago.Many have studied the potential of plasmid transfer across the group, especially for plasmids encoding toxins, e.g.Bt Cry toxins.B. cereus s.l.(sensu lacto), in diverse environmental niches, mimics laboratory conditions to study the conjugation-related mechanism.
On referring to the conjugation system in Bt subsp.israelensis encoded on the large plasmid pXO16 (350 kb), the system is characterized by the formation of macroscopic aggregates (Agr+) in social exponential growth in liquid cultures.The recipients Agr-has been identified in the genus Bacillus, specifically those belonging to the Bacillus cereus group, such as Bacillus subtilis, Bacillus megaterium, Bacillus sphaericus, and 24 subspecies of B. thuringiensis (Bt) (1,2,4,68).The transfer of the plasmid pXO16 to Bti Agr-strains (n= 14) was 100% effective and all recipients had acquired the aggregation-plasmid pXO16 and converted to the Agr+ phenotype.The genetic basis for this remarkable conjugative transfer system is not known, since no type IV secretion system homologs have been found.However in a more recent study it was reported a novel novel T4SS-mediated DNA transfer used by the Bti pXO16 plasmid.It was identified a 'transfer-Bti-plasmid' (tip) region encoding FtsK/SpOIIIE ATPase for an unrelated conjugative system T4SSs necessary for conjugative transfer, and distantly to the other Gram-positive bacteria.Furthermore, in the study it was observed up to 791 kb Bti chromosomal regions mobilization (2,4,68).
At this point, the question of what are the recipient genetic requirements for the conjugative transfer?Several studies have addressed this (69, 70).One of them using the IncI2 -(self transferable plasmids of the incompatibility group P-1 considered important carriers of genes for antibiotic resistance) plasmid TP114 which was recently shown to transfer at high rates in the gut microbiota (71,72).In transfer experiments in vitro, 4000 singlegenes deletion mutants of E. coli in a solid medium impaired transfer rates not associated with a specific cellular function.In contrast, broth medium were largely dependent on the lipopolysaccharide biosynthesis pathway.The specific structures used as recipient cells surface receptors by PilV adhesins associated with the type IVb accessory pilus F TP114 (71,72).Moreover, using live-cell microscopy, while most transfer events occur between cells in direct contact, the F pilus serves as a conduit for the DNA during transfer between physically distant cells.Therefore, the F pilus function aids in understanding how is accomplished the dissemination of drug resistance and virulence genes within complex bacterial communities (60, 73-77).Thus, the genetic requirements for the recipients to participate in bacterial conjugation vary between different plasmids, among them: 1) the receptor molecules recognized by the conjugative pilus or other accessory pili involved in mating pair formation (MPF) (25, 78-80).

Plasmids encode genes for insect control and for infectious diseases
Large plasmids are present in the different strains of Bacilus thuringiensis (Bt).These extrachromosomal elements have the particular and outstanding feature of harboring the encoded genes.The product of these genes are proteins with endowed properties that allow for broad application as bioinsecticides, but also potential use as immunogens, carriers, or adjuvants (84-89).At the same time, insects are vectors of pathogens, such as viruses, fungi, and even other bacteria, and transmit to animals and plants.
In this way, plasmids become a fundamental vehicle for horizontal genetic transfer and play a role in spreading virulence factors (4,68,90).B. thuringiensis harbor these plasmids which express the delta-endotoxins that are toxic against a wide range of insect orders (17-20, 91, 92).Some outstanding features of the plasmids gene encoded Cry and Cyt proteins are the structure, composition and mode of action (12,(91)(92)(93).Remarkably these proteins are produced during the sporulation phase as inactive parasporal bodies (12, 91-93).

Structure and composition
The three-dimensional structure of the Cry proteins determined to 1.5 A of the resolution, highly conserved in structure and function (14,(92)(93)(94)(95). 3D Cry toxins show a dual role, either as bioinsecticides for crop pests of agronomical importance endowed with the ability to colonize the insect world or as potential candidate adjuvants.Furthermore, and on referring to the Cyt toxins (cytolitic toxins), the three-dimensional X-ray structure of the Cyt2Aa1 was determined by Li et al., (96), comprised of two outer alpha helix hairpins (A-B and C-D)(rev (97, 98) and a core of mixed beta sheets (1 to 7).The structure of Cyt1Aa1 was similar.The alignment of amino acid sequences of six Cyt proteins from different B. thuringiensis subspecies revealed high scores and high statistical significance in the four blocks that comprise helix A, the loop after helix D plus beta strand four, the strands five and six, and finally the strand six-a and the following loop (94,96,97,(99)(100)(101).

Mechanism of action
The mode of action of the Bt Cry proteins has been proposed is a multistep process, starting with a) ingestion of mix spore+crystal by the insect(s) (12, 91-93); b) solubilized under the alkaline midgut insect conditions; c) activated after proteolytic digestion; d) Sequential binding to midgut receptors like-proteins; e) conformational change favouring pore formation; f) Osmotic swelling of the cell, and g) insect death (13,15,16,20), In the case of the mode of action of the Cyt1A toxins is not well understood.However, structural studies have revealed that these toxins have an affinity for unsaturated fatty acids in the lipid portion of the microvillar membrane (21,97,98,101,108,109).Cyt1A toxins insert into the microvillar membrane and then aggregate with each other in small clusters of lytic pores (21,98,108,110).However, more recent evidence favors a membrane perturbing, detergent-like mode of action in which faults are created in the lipid bilayer of the membrane, disrupting and causing cell lysis and cell death (21, 96-101, 103, 107, 111).In vivo, the effects of the Cyt1A toxins, the soluble crystal delta-endotoxin proteins caused hemolysis in rats, mice, sheep, horse, and human erythrocytes (105, 110, 112).Furthermore, Cyt toxins can function as Cry receptors, a type of synergy favoring the biocidal action of the Cry proteins (113-115).

The Cyt and Cry proteins for the insect biological control 3.2.1 Pest control management
Crop pests (rodents, nematodes, mites, and insects), plant pathogens (bacteria, fungi, and viruses), and weeds impact the world's agriculture and livestock and cause hundreds of millions of disease cases every year due to the transmission of pathogens and parasites (116-118).Moreover, there has been an increase in the undernourished population from 777 million in 2015 to 815 million in 2016 (119,120).The risk of insufficient food for the global population is higher in subtropical and tropical geographical regions where there is no control of crop pests and vectors that transmit pathogens of humans (malaria, dengue, paludism, filariasis, and chagas) (117,118,(121)(122)(123)(124).This is an issue that demands urgent priority (125,126).
Indeed, insect crop pests cause 10% to 30% loss of crop yields annually worldwide, and vector-borne pathogens have become a cause of emerging diseases of crop plants and of neglected infectious diseases (118,123,124).In the last decades, crop pest control has relied on pesticides, such as insecticides, fungicides, bactericides, and herbicides, which provide the mainstay of crop protection (119,120).These chemical compounds affect animal health and humans (119,120,127,128).In arthropods (insects), chemicals have an effect at the level of the nervous system by inhibiting acetylcholinesterase, voltage-gated sodium channels, and GABA receptors, and therefore interrupting synapse and immune regulation (127,128).
Furthermore, the action of the pesticides is through targeting several metabolic, physiological and biochemical pathways, the nerve receptors of pests, and even microbial organisms (122,129).While insecticides are part of and a strategy in integrated pest management, they represent barriers to effective biological control (130)(131)(132)(133) and can increase the development of resistance to principal substance classes worldwide and in many different species.
Of relevance is the observation that HGT favour is the insect vectors' ability to transmit between different organisms, like a spatial bridge, and thus increase the infectious opportunities.Furthermore, a pathogen can adapt to an insect vector transmission rate and pathogen dispersal (117,134,135).For instance, HGT might represent under this natural setting a drawback for the control of pathogen-vectored insects.At this point, combined strategies might aid in the decrease of the rate of transmission of crop pests and vector-borne diseases (21, 104, 110, 135-140) (Figure 3).The combined system strategies include the delivery of novel and specific chemical insecticides, the development of vaccines (which only exist for yellow fever), bacterial larvicides, and transgenic mosquitoes for reducing pathogen transmission (11,105,117,(141)(142)(143)(144).
The integrated planning might include insecticides and natural enemies (145, 146), combined tactics between selective insecticides and selective genetically modified crops or chemical tools and genetically modified (GM) crops (147) or broad-spectrum organophosphate with biological control conservation (148).The environmental impacts of chemical pesticides and resistance among pests have stimulated the development and utilization of microbial agents to improve human well-being and agricultural productivity (149).
Genetic engineering has proved to be one of the most promising technologies to improve crop resistance to disease and pests through the generation of lethal genes (OxitecO) to interrupt vector pathogen compatibility (150-152) (Figures 3, 4).
However, one alternative explored and applied is the release of sterile insects and insect recombinants.However, there is concern about the ecological threat and risks because nontargeted and indigenous species could harm and alter the wild and natural interaction host-pathogen (11, 116) (Figures 3, 4).
Another approach, is the theoretical models, which take into account all the factors that could have a role in the host-pathogen interaction (134,139,(153)(154)(155)(156).Modeling can be helpful to understand how the environmental conditions and abiotic and biotic factors could influence the underlying mechanism of how to limit the spread of the vectored insects.Several other potential strategies are mixed strategies such as chemical and biological The mode of action of the B. thuringiensis subsp israelensis(Bti) is a complex of proteins, Cry4A, Cry11A Cyt1A, and Cyt2A, toxic against dipterans, mosquitoes, and flies.Cry and Cyt possess the structural information to act at the level of the cellular membrane of insect midgut to cause cell lysis and death but also to bind to proteins in the cellular membrane of the vertebrate (mammals), resulting in signalization of the pathways involved in the induction of immune responses, or pathways involved in cell death, caspases, or apoptosis programs.The frontier in the knowledge of the pest crops and utmost of Neglected Infectious Diseases is to unravel the biology of the interaction host-pathogen.Basic knowledge research is pivotal to approach the control specifically of the mosquitoes (please refer to the review by Silva-Miranda et al., (11) for further details on the different strategies used until that year after the pandemic.Herein, we aimed to pinpoint some aspects of the control of mosquitoes.
Despite several alternatives for the control of dipterans, mosquitoes and flies, main pest crops, and Neglected diseases in developed and developed countries, the synergism between the toxins produced by B. thuringiensis subsp israelensis (Bti) and Lysinibacillus sphaericus remains as the most promising and safe strategy for the biological control of vectored dipterans.Among other reasons is that the combined toxicity and low persistence make these two entomopathogenic bacteria with a lower risk for insect resistance development (11,21), which continues to be a hot spot in South American countries, e.,g.Brazil country is endemic to dengue and malaria vectorized by mosquitoes.
Several studies have shown that Bt is the most widely used biological insecticide (16-21, 92, 110, 139).Bt strains produce a variety of toxic proteins used in insecticidal formulations and transgenic crops against caterpillars, beetles, and flies (16,18,163).In particular, the specific toxicity of Bti against larval mosquitoes and black flies was discovered by Goldberg and Margalit in 1977 (164), leading to the development and registration for use in aquatic environments (164,165).
Referring to the use of Bti in a proper dosage (166), several studies have revealed direct effects on non-target diptera, particularly chironomids (167)(168)(169)(170), but also on other insect taxa such as lepidoptera (171) and coleoptera (172) Bti is recommended as a means of control.Beside the direct effects and their consequences on insect species (173), concerns have been raised about its persistence in the ecosystem, specially attributed to the spore stage (162,(174)(175)(176).
Bacillus thuringiensis subsp isralensis serotype H14 discovered in Israel ( 177) highly toxic at an LC50 of 13-20 ng/ml against the fourth instar of most Aedes, Ochlerotatus and Culex species (21, 103, 105, 106, 177-184) (Figure 3).Thus, Bt subsp Israelis extend its mosquito spectrum and overcome insect resistance (110,111,(185)(186)(187)(188).Indeed, when combined with Bacillus sphaericus (Bs) (189) (Lysinibacillus sphaericus) (21,190,191) at the same ratio, Cyt1A improved significantly the toxicity of Bs to Aedes.aegypti, a species considered insensitive to this bacterium (192)(193)(194) Lastly, selection studies have shown that resistance to Bti Cry proteins develops much more slowly when Cyt1A is present in the toxin mixture used for selection (Figure 3).The Cyt1A's unique capacity to avoid, delay, or overcome resistance and extend its spectrum of activity is due to its unique mode of action, specifically its affinity for the lipid portion of the microvillar membrane (21,98,184,190,191).This property enables mosquitocidal endotoxins to bind to the midgut The control of mosquito vectors of pathogens that cause neglected infectious diseases (e.g., malaria, paludism, filiarasis) through integral control management (ICM) has had some success.Genetic Engineering and Molecular techniques such as gene driver, CRISP-Cas9, and Transgenesis have proved to be one of the most promising technologies to improve crop resistance to disease and pests for the generation of lethal genes (OxitecO) to interrupt vector pathogen compatibility.Furthermore, sterile technique of the insects and insect recombinants, alternatives explored and applied with potential future.Recent work on the toxicity and synergism of the toxins produced by Lysinibacillus sphaericus and B. thuringiensis subsp.israelensis supports the biotechnological development of both in proper formulation and dosage since there is a balance between toxicity, persistence in the environment, and avoiding insect resistance development compared to chemical insecticides and transgenic insect release.microvillar membrane independent of receptors (195)(196)(197).In a recent review related to the mechanisms of action and resistance of the Cyt proteins produced by Bti and Lysinibacillus sphaericus (21,98,184,190,191) highlights the relationship structure and function and the importance of using combined or a cocktail of toxins with different specificity that allow overcoming insect resistance (18, 21,185,186).The strain 2362 of Bs toxic against the fourth instar of Culex mosquitoes (196) produced the binary toxin two composed of two proteins, a 41.9 kDa toxin domain (BinA) and a 51.4 Kda binding domain (BinB) that co-crystallize into a single small parasporal body.Strain 2297 of Bs possesses a larger parasporal body and lower toxicity than strain 2362 (190,197,198).After ingestion of the inactive binary toxin by mosquito larvae, the 51.4 and 41.9 kDa proteins are cleaved by proteases yielding peptides of 43 and 39 kDa, respectively, that form the active toxin (196) (Figure 3).These associated toxins bind to receptors (e.g.alpha-glucosidase) (199) on the brush border insect midgut and cause cell lysis after internalization (200)(201)(202).Many strains of Lysinibacillus sphaericus produce other mosquitocidal toxins referred to as Mtx toxins.Two of these have been well-studied.Mtx (100 kDa) and Mtx (30.8 kDa), but they are not as toxic as the binary toxin (186,190).Lysinibacillus sphaericus targeting dipterans (111,179,184,186,(203)(204)(205)(206) (Figure 3).The Mosquitoes (Diptera: Culicidae) of the Aedes species (Ae spp) are main vectors in tropical and subtropical of different serotypes of several viruses, such as dengue, the yellow fever virus (203,(207)(208)(209)(210)(211) the Zika virus (ZKV) (138,(212)(213)(214)(215), and the virus Chikungunya (CHIKV) (157-161, 216-219).Therefore, mosquitoes, as vectored of pathogens, represent a threat because of their resistance and require frequent control (124,133,139,159,211,220,221).Neglected vector diseases cause morbidity and mortality (121, 124-126, 133, 137, 139) with an estimated 2 billion people worldwide living in areas where these are endemic (125,211,222,223).One of the alternatives that have risen hopes to control insect vectors of pathogens causing human diseases (mosquitoes) is the formulations based on Cry-Cyt toxins, one of the most optimal and safe biological controls.Interestingly while Cry toxins are of low persistence (110,111,186), Cyt toxins have high persistence (21,185).Therefore, a combination of these capabilities between Bti and LBs (138,185,188,(224)(225)(226), or a combination of Wolbachia and Leptolegnia chapmanii (facultative bacteria pathogen of diverse species of mosquitoes spp of the genus Aedes, Culex and Anopheles) can be highly effective to control to the insect vectored of mosquitoes (21,(223)(224)(225).
Interestingly, the potential for pest biological control of these insects depends on several factors, among them the levels of specificity to the different larvae stages of mosquito spp (227,228).Interestingly, crop lines expressing active insecticidal cry genes from Bt have to achieve an efficient control of insect pests.Recently, identification of a mutation in the Bt toxin receptor recognizing Bt molecular patterns.Mutation genes of the toxin receptors lead to changes in the immune system response of the insect (129, 169, 170, 228-231) (Figures 2, 3), compromising the defensive systems of the mosquito (proteases for the peritrophic matrix) or exploiting receptors used by the virus to deliver toxins via the midgut to mosquito larvae Insight knowledge and understanding of the biology and the mechanism of interaction of insect pests and Bti, LBs, and their products (11,18,110,231) represent a frontier for the advances in the development of improved or novel mosquito control strategies (Figures 3, 4).

Bt Cry proteins as carriers of immune dominant antigens of insect vectors pathogens causing human neglected infectious diseases
The mechanism of action for the bacterial toxins is dependent on receptors present in the epithelial cells, such as ganglioside (GM1) distributed in the human body, and the activation of G proteins and adenylate cyclase (232-239).Interestingly, in the case of the pCry1Ac and the 3D-Cry toxins, studies pointed out a specific interaction with brush border membranes in mice intestines (240).
More recent reports have found that pCry1Ac, as an antigen, interacts with the TLR present on the antigen-presenting cells, triggering signalization pathways such as mitogen-activated protein kinase (MAPK), specifically the extracellular signal-regulated kinase (ERK), a subclass of the MAPK pathway (240, 241), activated by a wide variety of receptors involved in growth and differentiation including receptor tyrosine kinases (RTKs), integrins, and ion channels.
This leads to the regulation of targets in the cytosol and further translocation to the nucleus, where it can phosphorylate several transcription factors to regulate gene expression (241).In another setting in the mouse model, the immunogenic properties of pCry1Ac were administered by different routes, with significant induction of IgG antibodies (84, 85, 242).Furthermore, there was a structural implication of the N-terminal region in the induction of antibodies IgG and IgA after systemic and intranasal route immunization by 3D-Cry1A toxins (Cry1Aa, Cry1Ab, and Cry1Ac) (243).Moreover, pCry1Ac and the 3D-Cry toxins have been carriers of and properties of important clinical epitopes or antigens of clinical importance (diphtheria toxin, HIV) (82,(86)(87)(88).

Highlights and perspectives
It is clear that plasmids and the natural conjugation mating systems play a role in spreading virulence factors but also endow the bacteria with an ecological niche.To harness the biological control of crop pests and vectored insects of pathogens that affect animal health integrated tools to achieve meaningful results, unraveling and identifying the abiotic and biotic complex interaction factors among insect pests, humans, and entomopathogens remains a hope for the advancement and development of novel strategies to protect humans without affecting biodiversity and environmental health.The interaction of pests and pathogens favored host defenses by secreting virulence factors injected into their targets.
What about the limitations and challenges of the plasmidencoded cry or cyt genes producing bioinsecticide proteins?The limitations are the insect response and the climatic change that plays a role.Another limitation and challenge is the development of resistance to the cry and cyt gene products.The challenge is biological control of the crop pests without affecting wild species.The effort is toward a proper and safe formulation and dosage of combined Cry or Cry + Cyt toxins.The challenge is to combine several strategies.The insect response to the Cry and Cyt toxins could provide targets to keep balance with the synergy between the Cry and Cyt proteins' capabilities-the genetic engineering of crops.However, the fitness cost is too high for the ecosystem.An alternative is the biological control of mosquitoes using an endosymbiotic bacteria, Wolbachia to block virus entry.Thus, transgenic mosquitoes with this bacteria could potentially lead to a relatively biological control of mosquitoes.The challenge in either of these alternatives for biological control is the genetic diversity of the insects and the pathogen.The diversity could even lead to unexpected results.We expect no variation, even though the genetic background plays a role.In either case, the combinations of different Cry and different Cyt proteins against different insect orders, the genetics of the host, and the pathogen play a role.The cry or cyt genes can lead to a more feasible horizontal genetic transfer between insect orders.In addition, the remarkable properties of these bacterial toxins could perfectly match biotechnological and pharmacological issues.
The perspective is not just to use bioinsecticides for biological control but to harness the insect immune response (REPAT) for biomarker identification that allows further targeting.Indeed, it is evident that despite the recent advancements in molecular techniques such as driver gene, CRISPR-Cas9 (transgenic insects), and RNAi, the toxins from B. thuringiensis subsp israelensis and Lysinibacillus sphaericus, alone or in combination, are the most feasible to continue the development of a formulation that does not compromise the biodiversity and the health in the ecosystems.
Recent technology called biomimetic lure-and-kill exploits biomimetic principles of biocompatible/biodegradable biopolymers (e.g., natural hydrogel) to develop new substrates that selectively attract insects by reproducing specific natural environmental conditions (biomimetic lure) and kill them by hosting and delivering a natural biopesticide or through mechanical action.
Biomimetic lure-and-kill-designed substrates point to provide a new attractive system to develop/improve and make more costcompetitive new and conventional devices (e.,g.traps).The tiger mosquito Aedes albopictus has been proposed as a model to gain insight into this novel technology.Finally, the clinical perspective of the Cry and Cyt proteins in infectious disease as adjuvants of clinical antigens and in non-infectious disease (for example in cancer) is an issue that remains latent and to continue investigation of their structures allows them to interact with epithelial surfaces and with the host immune system.

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What is needed for the transfer a) Transfer of mobile genetic elements (MGES) and plasmids together with their cargoes of antibiotic resistance and virulence genes.T4SSs translocate MGEs as single-stranded DNA intermediates (T-strands), which triggers the SOS response in recipient cells.b).Protein transfer (e.g., single-stranded DNA-binding proteins (SSB) such as ParA, ParB1, PaarB2, PsiB, and PsiA)(set of shared proteins) (59-61).The genesis or ssb encodes the SOS inhibitor protein PsiB and single-stranded DNA-binding protein SSB, eliciting a significantly stronger SOS response (59-61).Interestingly, translocation of PsiB or SSB, but not PsiA, through the TrapRD/208T4SS suppressed the mating-induced SOS response being triggered in recipient cells upon the acquisition of the single-stranded DNA transfer intermediate during mating.