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The relevance of L. sphaericus as a mosquito-control agent

The relevance of L. sphaericus as a mosquito-control agent

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94



Maria Helena Neves Lobo Silva Filha et al.



Table 3.1 Examples of Lysinibacillus sphaericus strains and their larvicidal properties to

mosquito larvae

Genes encoding

mosquitocidal proteinsb

Strain



Origin



Flagellar

serotype



DNA

group



Larvicidal

activitya



Crystal Mtxs



Cry48/

Cry49



Kellen

K



USA



1a



IIA



Low



À



1, 2, 3



Nd



SSII-1



India



2a2b



IIA



Medium



À



1, 2, 3



Nd



Medium



+



1, (2 and 3 +

Nd)



c



IAB881 Ghana



2a2b



Nd



LP1-G China



3



IIA



Medium



+



1, (2 and 3 +

Nd)



1593M Indonesia 5a5b



IIA



High



+



1, 2 (3 Nd) À



2362



Nigeria



5a5b



IIA



High



+



1, 2, 3



À



1691



El

Salvador



5a5b



IIA



High



+



1, 2, 3



Nd



IAB59



Ghana



6



Nd



High



+



1, 2, 3



+



2297



Sri Lanka 25



IIA



High



+



1, 2, 3



À



IAB872 Ghana



48



Nd



High



+



1, (2 and 3 Nd

Nd)



C3-41



5a5b



IIA



High



+



1 (pseudo) À

2, 3, 4



China



a



Based on criteria defined by Charles et al. (1996).

+ Presence, À absence.

Not determined.

Modified and extended from Charles et al. (1996)



b

c



The experiences of Ls larvicide utilisation provided a solid background

for its adoption in mosquito-control programmes worldwide (Section 4).

These larvicides were first introduced for Cx. pipiens control in many areas

of France as early as 1987 to reduce the nuisance caused by this species

(Thiery et al., 1996). An initiative by the World Health Organisation

(WHO) created a multi-centre study for the evaluation of the field effectiveness of Ls to control Cx. quinquefasciatus larvae in urban areas of tropical

countries, with particular attention to the role of this mosquito as the vector

of Wuchereria bancrofti the causative agent of filariasis (WHO, 1993). These

pioneer trials, along with others, supported the broad utilisation of these



Microbial Toxins for Mosquito Control



95



larvicides. The control of Culex spp. has gained more importance recently

in regard to their role as vectors of emergent arboviruses, such as the West

Nile Virus that has provoked important epidemics in human populations

(Kramer et al., 2008; Petersen and Fischer, 2012). The control of anophelines is a challenge, and previous studies have shown that some relevant species involved in Plasmodium transmission are susceptible to Ls. The control

of Anopheles (An.) stephensi and An. sinensis in India and China, respectively,

showed the operational viability of Ls to control this group of mosquitoes

(Kumar et al., 1994; Thiery et al., 1996; Yuan et al., 2000). In addition to its

application for vector control in urban areas, the selective activity and biocompatibility of Ls is of great utility when the target species breed in environmentally sensitive areas. The utilisation of Ls also raises concerns about

the selection of resistance. High levels of resistance achieved due to its

utilisation have been reported, and the major findings on this issue are presented in Section 5 along with the strategies that can be introduced for the

management of resistance in Section 6. These strategies can ensure the

effectiveness of Ls when used in the scope of integrated control programmes

and can overcome the potential onset of resistance. Different aspects of Ls

and its properties as an entomopathogenic bacterium have been covered by

previous reviews and book chapters that can provide additional information

(Baumann et al., 1991; Becker, 2000; Berry, 2012; Charles and NielsenLeRoux, 2000; Charles et al., 1996, 2010; Delecluse et al., 2000; Lacey,

2007; Porter et al., 1993; Regis and Nielsen-LeRoux, 2000).



2. TOXINS AND MODE OF ACTION

2.1. Spectrum of action

Ls strains in DNA group IIA may produce a range of toxins as detailed in this

section, including those with activity against mosquitoes, which are the Bin,

the Mtx and the Cry48Aa/49Aa toxins. The profile of toxins produced by

individual strains is varied and contributes to the designation of bacteria

as either high- or low-toxicity strains with respect to their activity against

mosquito larvae. The strains with the highest activity are characterised by

the presence of the Bin protoxin that is produced as parasporal crystals during

sporulation (de Barjac et al., 1980; Payne and Davidson, 1984; Yousten,

1984a). Strains lacking Bin crystals display low toxicity and some of them

produce Mtx toxins, however, the latter undergo degradation during their

production in the vegetative phase and they do not contribute to provide a

high activity to the strains. The performance of strain 1593 that produces Bin



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Maria Helena Neves Lobo Silva Filha et al.



and Mtx toxins to larvae is, for instance, 3000-fold superior compared to the

SSII-1 strain that produces Mtx toxins only (Myers et al., 1979). Additional

studies on different strains have shown that Bin accounts for most activity

recorded for the sporulated cultures and this is the main active ingredient

of biolarvicides based on Ls, as reviewed by Charles et al. (1996). According

to the Insect Resistance Action Committee (www.irac-online.org), the

insecticidal toxins from Ls are classified into the mode of action group

11 (Moa11), along with Bti, and those agents are defined as “bacterial

disruptors of insect midgut membranes”. The midgut of mosquito larvae

is the central site for the action of these toxins, since they act following ingestion, are processed under specific conditions in this environment and they

act on specific receptors located on the epithelium, to cause mortality of larvae. More details of insect midgut are provided in Chapter 1.

Mosquitoes are the principal targets of the Ls toxins and this is reflected in

the activity spectrum of the individual toxins. However, Ls toxicity to

Phlebotomus sandflies has been reported for high-toxicity strains 1593 and

2362 that may result in larval death and reduced fecundity of surviving

insects (Penner and Wilamowski, 1996; Robert et al., 1997, 1998;

Wahba, 2000). Strain 2362 also showed low toxicity against Lutzomyia

sandflies (Wermelinger et al., 2000). In addition, larvicidal effects of Ls

extracts against the nematode Trichostrongylus colubriformis have also been

reported (Bone and Tinelli, 1987) and some toxicity was seen against the

crustacean Palaemonetes pugio (Key and Scott, 1992).

Within the mosquitoes, there is differential toxicity to the species studied.

The most susceptible are Culex spp, in particular, those from the Cx. pipiens

complex, but one exception in this genus is Cx. cinereus larvae (Nicolas and

Dossou-Yovo, 1987). Anophelines including species of medical importance such as An. gambiae, An. stephensi, An. albimanus, An. quadrimaculatus,

An. darlingi and An. nuneztovari are also susceptible to the Bin toxin

(Arredondo-Jimenez et al., 1990; Davidson, 1989; Karch et al., 1992; Lacey

et al., 1988b; Rodrigues et al., 1998, 1999; Young et al., 1990). Aedes or

Ochlerotatus show a variable scenario including susceptible species such as

Oc. atropalpus, Ae. vexans and Oc. trivittatus, as well as Ae. aegypti larvae that

are refractory to Bin toxin (Berry et al., 1993; Delecluse et al., 2000;

Nielsen-Leroux and Charles, 1992). The lethal concentration (LC) of Ls

for these larvae is between 100- and 1000-fold higher than the respective

LC for Cx. pipiens larvae (Thiery and de Barjac, 1989). The screening of Ls

activity has also demonstrated susceptible larvae from Psorophora and Mansonia

species. On the other hand, Simulium larvae that are susceptible to Bti cannot be



97



Microbial Toxins for Mosquito Control



targeted by the Bin toxin. Table 3.2 presents a non-exhaustive list of mosquito

susceptibilities to Ls strains. The most common species targeted by Ls in fieldcontrol trials or programmes are described in Section 4.



2.2. Binary toxin

The Bin toxin, comprising the BinA and BinB proteins, is the best

characterised of the toxins from Ls. It is produced during early sporulation

by highly mosquitocidal strains (El-Bendary et al., 2005; Kalfon et al., 1984)

and is deposited as a parasporal crystalline inclusion within the exosporium

(Davidson and Myers, 1981; Kalfon et al., 1984; Yousten and Davidson,

1982). In these strains, Bin contributes to the majority of the toxicity and

this fact, in turn, is a factor in the relative ease with which mosquitoes

can develop resistance to Ls (see Section 5). Bin toxin acts in the midgut

and the major steps of its mode of action in culicid larvae are: ingestion

of crystals; dissolution of the crystal matrix under the alkaline pH conditions

and release of Bin protoxin in the midgut lumen; processing of the Bin

protoxin into active toxin; binding of the active toxin to specific receptors

available on the midgut epithelium; occurrence of cytopathological effects

on the midgut are followed by larval death, which is provoked by

Table 3.2 Sensitivity of mosquito species to Lysinibacillus sphaericus

Susceptibility (strain

Species

tested)

References



Culex pipiens



High (1593, 2013-4);

moderate (SSII-1)



Cx.

High (1593); moderate

quinquefasciatus (SSII-1)



Thiery and de Barjac (1989), Wraight

et al. (1987)

Cheong and Yap (1985), Mulligan

et al. (1978), Wraight et al. (1987)



Cx. nigripalpus High (1593, 1404, SSII-1) Ramoska et al. (1977)

Cx. salinarius



High (1593)



Wraight et al. (1987)



Cx. restuans



High (2013-4)



Wraight et al. (1987)



Cx. tarsalis



High (1593)



Mulligan et al. (1978)



Cx. cinereus



Refractory (2362)



Nicolas and Dossou-Yovo (1987)



Culiseta

melanura



High (1593)



Wraight et al. (1987)



Mansonia

uniformis



High



Cheong and Yap (1985)

Continued



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Maria Helena Neves Lobo Silva Filha et al.



Table 3.2 Sensitivity of mosquito species to Lysinibacillus sphaericus—cont'd

Susceptibility (strain

Species

tested)

References



Psorophora

columbiae



High (1593); moderate

(SSII-1)



Ramoska et al. (1977)



Anopheles

stephensi



High (multiple strains)



Davidson (1989), Thiery and de Barjac

(1989)



An. gambiae



High (2362)



Davidson (1989), Nicolas et al. (1987)



An. albimanus



High (2362)



Davidson (1989)



An.

High (2362)

quadrimaculatus



Davidson (1989), Young et al. (1990)



An. darlingi



High (2362)



Rodrigues et al. (1998, 1999)



An.

nuneztovari



High (2362)



Rodrigues et al. (1998, 1999)



An. braziliensis High (2362)



Rodrigues et al. (1998, 1999)



An.

balabacensis



Moderate



Cheong and Yap (1985)



Ochlerotatus

nigromaculis



High



Mulligan et al. (1978)



Oc. atropalpus



High



Berry et al. (1993)



Oc. intrudens



High (1593)



Wraight et al. (1987)



Oc. triseriatus



Moderate (SSII-1); low

(1593)



Wraight et al. (1987)



Oc. canadensis



Moderate (SSII-1)



Wraight et al. (1987)



Oc. fitchii



Moderate (1593)



Wraight et al. (1987)



Oc. stimulans



Moderate (1593); low

(SSII-1)



Wraight et al. (1987)



Oc.

taeniorhynchus



Moderate (1593, 1404,

SSII-1)



Ramoska et al. (1977)



Aedes vexans



Moderate



Wraight et al. (1987)



Ae. aegypti



Refractory (1593); low

(SSII-1 vegetative cells)



Thiery and de Barjac (1989), Wraight

et al. (1987)



Toxorhynchites Refractory (2362, 2297)

rutilus



Lacey et al. (1988b)



Microbial Toxins for Mosquito Control



99



mechanisms that are still under investigation. Recently, activity of Bin toxins

against human cancer cells has also been reported (Luo et al., 2014).

The analysis of Bin proteins isolated from parasporal crystals was reported

in the mid-1980s (Baumann et al., 1985; Narasu and Gopinathan, 1986) and

initially it appeared that they were derived from a larger precursor protein

(Broadwell and Baumann, 1986). Subsequent cloning of the gene encoding

BinA (Berry and Hindley, 1987; Hindley and Berry, 1987) and BinB

(Baumann et al., 1987, 1988) showed that, in fact, the two components were

produced from a single operon as independent proteins of approximately

42 and 51 kDa, respectively. The proteins are produced in approximately

equimolar amounts and form a co-crystal in sporulating Ls whereas the individual components expressed in recombinant Ls did not form crystals

(Charles et al., 1993). The combination of BinA + BinB forms crystals in

Ls and Bt strains but not in recombinant B. subtilis (Baumann and

Baumann, 1991; Broadwell et al., 1990a; Charles et al., 1993; Yuan

et al., 1999) suggesting that the former, insect pathogenic bacteria, encode

a factor that facilitates crystallisation and that is absent from B. subtilis. Bin

protein synthesis is enhanced by recombinant co-expression of the P20 protein from Bt (Park et al., 2007) but a region downstream of the bin operon in

Ls strain 2297 reduces Bin synthesis (Park et al., 2009). The activity of the

Bin toxin appears to be synergistic with the Cyt1Aa protein from Bt when

the two are co-expressed in acrystaliferous Bt (Li et al., 2000) but expression

of Cyt1Ab in Ls did not show synergy although it did help to overcome Bin

resistance in Culex larvae (Thiery et al., 1998). Other studies have shown

synergy of Bt Cyt and Cry toxins with Ls against wild-type or Bin-resistant

Culex, which may indicate synergy with Bin toxins although the use of Ls

cells in these assays may also indicate synergy with other toxins that they produce (see below) (Wirth et al., 2000a,c, 2001a, 2004).

Circular dichroism analysis has suggested that BinA and BinB are predominantly composed of beta sheet (Hire et al., 2009; Kale et al., 2013;

Srisucharitpanit et al., 2012) although BinB in wild-type and truncated forms

has also been reported to contain considerable alpha helix (Tangsongcharoen

et al., 2011). Crystallization of BinB protein (Chiou et al., 1999;

Srisucharitpanit et al., 2013) and BinA/BinB co-crystals (Smith et al., 2004)

have been described and the structure of BinB has recently been published

(Srisucharitpanit et al., 2014). This protein has an N-terminal domain with

a beta-trefoil architecture found in lectins and a C-terminal region rich in

extended beta sheets that shows structural similarity to aerolysin beta pore forming toxins. These results may suggest a role for the N-terminal region in



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Maria Helena Neves Lobo Silva Filha et al.



receptor binding and roles for the C-terminal region and the related BinA

structure in formation of a beta pore. Association of the two proteins with

each other and the membrane may result in conformational changes to the

structures (Boonserm et al., 2006; Kale et al., 2013).

As described, the ingestion of the Bin proteins by mosquito larvae results

in their solubilisation in the alkaline environment of the gut (Charles, 1987)

and activation of the protoxin forms by proteolytic cleavage mediated by gut

proteinases (Aly et al., 1989; Broadwell and Baumann, 1987; Brownbridge

and Margalit, 1987; Davidson et al., 1987, 1990). These will activate both

subunits BinA (51 kDa) and BinB (42 kDa) into smaller polypeptides of

43 and 39 kDa, respectively. After proteolysis, the 43 kDa BinB derivative

results from the removal of 21 and 53 residues from the N- and C-termini,

respectively (Clark and Baumann, 1990). For the BinA active fragment of

39 kDa, cleavage of 10 and 17 amino acids occur in these respective positions (Broadwell et al., 1990c). The correct processing of the Bin subunits

and their presence in equimolar amounts are essential conditions that assure

the optimal activity of this Bin toxin (Broadwell et al., 1990b; Davidson

et al., 1990; Nicolas et al., 1993; Oei et al., 1990). Similar patterns of

protoxin cleavage occur on exposure to digestive enzymes from nonsusceptible larvae, indicating that the protoxin processing is not the origin

of insect specificity (Nicolas et al., 1990). In solution, NaOH solubilised

BinA/BinB crystal proteins may associate into a BinA2BinB2 heterotetramer

but this association may be lost on trypsin activation (Smith et al., 2005) with

activated proteins showing weak interactions between the two proteins

(Kale et al., 2013), although formation of oligomeric complexes between

activated toxins has also been suggested (Srisucharitpanit et al., 2012).

The solubilised and active toxin binds regionally to the larval midgut in

the gastric caecum and posterior midgut (Davidson, 1988, 1989; Mulla et al.,

1984a; Oei et al., 1992) leading to toxicity. In Culex larvae, the BinB component of the toxin is responsible for receptor binding and the BinA component subsequently binds to BinB or the BinB/receptor complex (Charles

et al., 1997; Oei et al., 1992). The situation in An. gambiae appears a little

more complex with a possible role for BinA in binding as well (Charles

et al., 1997). The receptor for Bin binding has been identified in Cx. pipiens

as a midgut-bound α-glucosidase (Silva-Filha et al., 1999). Orthologs of this

protein have been identified in other mosquito species including An. gambiae

(Opota et al., 2008) and the refractory Ae. aegypti (Ferreira et al., 2010) and

differences in this receptor are believed to be the crucial factor in determining sensitivity (Section 3). Changes in the receptor are also known to cause

resistance to the Bin toxin (Section 5).



Microbial Toxins for Mosquito Control



101



Toxin binding to the midgut receptors is essential, and soon after the

ingestion of Bin crystals, cytopathological alterations can be observed.

The pathogenesis associated with Ls treatment was first described based

on the study on the action of strain SSII-1, which produces only Mtx toxins,

on Cx. quinquefasciatus larvae (Davidson, 1979). Subsequently, studies

showed the cytopathological alterations in midgut cells of larvae treated with

different strains all of which produced the Bin toxin as their major toxin

(Charles, 1987; de Melo et al., 2008; Silva Filha and Peixoto, 2003;

Singh and Gill, 1988). For Cx. pipiens, the major alterations observed in

the midgut cells are the intense disruption of microvilli, intense cytoplasmic

vacuolisations (or cytolysosomes) with broken membranes, pronounced

swelling of mitochondria and break-down of the endoplasmatic reticulum.

Ultra-structural effects investigated using an in vitro processed form of Bin

toxin to treat Cx. pipiens-cultured cells, showed similar effects as those seen

in midgut cells from larvae (Davidson and Titus, 1987). The study of Singh

and Gill (1988) also recorded damage in neural and muscles tissues that were

detected later than the major effects that were primarily observed in the midgut cells. Treatment of resistant Cx. pipiens larvae that lacked the midgut

receptors with high concentration of Bin toxin showed minor alterations

that were comparable with those observed for Ae. aegypti, a refractory species

that does not have functional receptors in their midgut (Charles, 1987; de

Melo et al., 2008). Physiological studies of the Bin action are not available

except for one that shows an inhibition of the oxygen uptake of mitochondria and in the activity of the enzyme choline acetyl transferase in larvae

treated with the Bin toxin (Narasu and Gopinathan, 1988).

The mode of action of the Bin toxin, following receptor binding, remains

somewhat unclear. Many reports have suggested that to exhibit toxicity, both

BinA and BinB components are absolutely required (Broadwell et al., 1990b;

Charles et al., 1993; Nicolas et al., 1993; Oei et al., 1990), with optimal activity

reported when components are present in approximately equimolar amounts

(Davidson et al., 1990). Nevertheless, toxicity of BinA in purified form (Hire

et al., 2009) or produced in B. subtilis (de la Torre et al., 1989) or in Bt (Nicolas

et al., 1993) has been reported by some authors. When the Cx. pipiens Bin

receptor Cpm1 was expressed in Madin Darby canine kidney cells, patchclamp experiments showed that toxin binding is followed by the induction

of currents that are likely to be due to the opening of pores (Pauchet et al.,

2005). Experiments with Culex cells in culture (Cokmus et al., 1997) and artificial membranes (Schwartz et al., 2001), also suggest that the toxin may be able

to form pores and indicated that BinA was better able to form pores than BinB,

consistent with the model whereby BinB is the receptor-binding component



102



Maria Helena Neves Lobo Silva Filha et al.



and BinA forms a pore. In contrast, a separate report described the ability of

BinB to interact with artificial membranes and form pores in the absence of

BinA (Boonserm et al., 2006). This pore formation was proposed to be

through membrane insertion of beta sheet rather than alpha helical structures.

Thus, an alternative model for Bin toxicity may involve receptor binding and

pore formation by BinB coupled with an unknown role for BinA or by a

BinA/BinB complex and this may be supported by the crystal structure of

BinB (Srisucharitpanit et al., 2014).

In addition to the possibility of pore formation, a further effect is characteristic of Bin intoxication. The vacuolisation of target cells is seem

(Charles, 1987; Davidson, 1988; Pauchet et al., 2005) accompanied by

the uptake of labelled toxins into vesicles (Davidson, 1988), a phenomenon

that only occurs when both BinA and BinB components are present

together (Oei et al., 1992). A detailed study of this phenomenon was carried

out using Madin Darby cells expressing the Bin receptor Cpm1 (Opota

et al., 2011). This investigation showed the opening of cationic pores in

the membrane and demonstrated that the large vacuoles formed in target

cells were autophagic. These structures were transient but, having disappeared from the cells, these vacuoles then reappeared following cell division: a novel phenomenon termed post-mitotic vacuolation. The uptake of

Bin into the cells, along with their receptor, was shown to be via recycling

endosomes; structures that are distinct from the large transient autophagic

vesicles. Thus, Bin intoxication induces autophagy, while Bin uptake in separate structures protects it from degradation by targeting to recycling pathways. The overall significance of these events for toxicity remains to be

clarified but Bin trafficking may allow it access to tissues beyond the midgut.

The BinA and BinB proteins are related to each other and to a family of

Bin-like proteins including Cry49 from Ls (see below), Cry35 and Cry36 from

Bt, and sequences of unknown function from B. cereus group strains (e.g. accession number ZP_17404242) and Chlorobium phaeobacteroides (accession number Y_911930) (Baumann et al., 1988; Jones et al., 2007). Cry36 acts alone to

cause insect mortality, whereas Cry35 requires the 14 kDa Cry34 protein for

toxicity and Cry49 requires the three-domain family toxin Cry48 for its function. The various interactions that this family of proteins may require for

toxicity, further complicates our understanding of their modes of action.

The Bin toxin proteins themselves are highly conserved. Strains isolated

from around the world produce Bin toxins (Priest et al., 1997) but only six

variants of BinA (in which nine amino acids are altered) and four variants of

BinB (in which six amino acids are altered) have been described (Hire et al.,

2009; Humphreys and Berry, 1998; Priest et al., 1997). Two variants, Bin1



Microbial Toxins for Mosquito Control



103



and Bin2 have been shown to share the same receptor (Silva-Filha et al.,

2004) and cross-resistance between variants is seen (Yuan et al., 2003). Nevertheless, Bin toxin variants can show differential activity against mosquito

targets. BinA variants were shown to alter the activity against the marginal

target Ae. aegypti and to alter the progression of growth and mortality for Cx.

quinquefasciatus and these effects were localised to amino acids 99 and 104 in

this protein (Berry et al., 1993). Reciprocal exchange of the amino acid at

position 93 of the BinA protein between the BinA2 variant, which is highly

active against Cx. pipiens larvae and the BinA4 variant, which is non-toxic to

this insect, showed that this residue was also a key determinant of activity

(Yuan et al., 2001). Deletion experiments have defined the essential core

regions of the Bin toxins. BinA can be truncated by 17 residues at both

the N- and C-termini without loss of toxicity. BinB can be truncated by

34 residues at the N-terminus and 53 residues at the C-terminus without

loss of toxicity (Broadwell et al., 1990c; Clark and Baumann, 1990, 1991;

Limpanawat et al., 2009; Oei et al., 1990; Sebo et al., 1990). Analysis of

the binding of non-toxic variants suggested that the N-terminal region of

BinB may have a role in interaction with the receptor and its

C-terminus, along with both the N- and C-termini of BinA may be

involved in the interaction of the two proteins (Oei et al., 1992). Predictions

of structural disorder within the BinA and BinB proteins have suggested that

the N- and C-termini may be flexible, consistent with a role in protein–

protein interactions (Kale et al., 2013). More detailed analysis of the

N-terminal region of BinB and receptor binding, confirmed the importance

of residues 33–158 in this interaction and, particularly, the sequences IleArg-Phe (residues 85–87) and Phe-Gln-Phe (residues 147–149) (Roma˜o

et al., 2011). Mutagenesis studies on BinB indicated that individual substitution of Pro35, Glu36, Phe41 and Tyr42, resulted in reduced activity but all

were able to interact with BinA (Singkhamanan et al., 2013). Pro35 and

Phe41 to alanine substitutions could bind to the larval midgut at a comparable level to the wild-type BinB but binding of the Tyr42 to alanine mutant

was reduced and the Pro35Ala replacement decreased penetration of the

membrane. Block mutation of BinB from residues 113–150 showed the

protein to have some tolerance to mutation in this region (Singkhamanan

et al., 2010). Alanine replacement of individual amino acids Phe149 and

Tyr150 resulted in loss of toxicity and loss of midgut binding for the latter

mutant. Toxicity could be rescued by replacement of these two residues

with other aromatic residues. In BinB, the substitution of Cys67 or

Cys161 reduced BinB interaction with BinA and eliminated toxicity while

replacement of Cys241 had no effect (Boonyos et al., 2010). In similar



104



Maria Helena Neves Lobo Silva Filha et al.



experiments with BinA, alanine or serine replacement drastically reduces

(Cys195) or abolishes (Cys31, Cys47) activity (Promdonkoy et al., 2008).

These three cysteine residues in each protein were shown not to be involved

in disulphide bonding and the nature of their roles is not clear at present.

Substitution of charged residues with alanine in BinA produced a loss

(Arg97) or reduction in activity (Glu98, Arg101, Glu114) but no change

in the ability of BinA to interact with BinB (Sanitt et al., 2008). Substitution

of Arg312 has also been shown to eliminate toxicity (Elangovan et al., 2000).

Replacement of tryptophan residues at positions 222 and 226 in BinA produces proteins that are still able to permeabilise liposomes but which have

lost biological activity (Kunthic et al., 2011). It has been shown that alanine

mutants in both BinA and BinB can eliminate toxicity and that, for each protein, a non-toxic variant mutated close to the N-terminus and a non-toxic

variant mutated close to the C-terminus, can complement each other to produce a toxic combination (Shanmugavelu et al., 1998).



2.3. Cry48/Cry49

While the Bin toxin is the major sporulation-associated toxin in most Ls

strains, the ability of the spores of a small number of strains to overcome

Bin resistance in mosquitoes, indicated the presence of another toxin in

these strains and an approximately 49 kDa protein was identified as a candidate (Pei et al., 2002; Shi et al., 2001). The genes encoding the Cry49 protein and a second crystal protein (Cry48) related to the three-domain toxins

of Bt has been cloned and expressed ( Jones et al., 2007) and show a very

narrow target range, so far active only against Culex mosquitoes ( Jones

et al., 2008). It is of interest that the two proteins form a novel type of

Bin toxin since both components are necessary for activity and no other

three-domain protein has ever been shown to have a requirement for

another protein for its activity ( Jones et al., 2007). Consistent with the presence of a Bin-type toxin and a three-domain toxin, the cytopathology of

Culex cells exposed to the Cry48/Cry49 toxin shows features of both toxin

types, including the vacuolation observed on Bin intoxication (de Melo

et al., 2009) (Fig. 3.2). Similar effects were seen when Bin susceptible larvae

were treated with the synergistic combination of Bin and Cry11Aa toxins.



2.4. Mosquitocidal toxin 1

The mosquitocidal toxin 1 (Mtx1) was first discovered in Ls strain SSII-1, a

strain that shows low toxicity and lacks the Bin toxin (Thanabalu et al.,



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