Lantibiotics produced by Actinobacteria and their potential applications

13 Pages·2017·2.14 MB·English

REVIEW
Gomesetal.,Microbiology2017;163:109–121
DOI10.1099/mic.0.000397
Actinobacteria
Lantibiotics produced by and their potential
applications (a review)
KarenMachadoGomes,1,*RafaelSilvaDuarte1andMariadoCarmodeFreireBastos2
Abstract
The phylum Actinobacteria, which comprises a great variety of Gram-positive bacteria with a high G+C content in their
genomes, is known for its large production of bioactive compounds, including those with antimicrobial activity. Among the
antimicrobials,bacteriocins,ribosomallysynthesizedpeptides,representanimportantarsenalofpotentialnewdrugstoface
the increasing prevalence of resistance to antibiotics among microbial pathogens. The actinobacterial bacteriocins form a
heterogeneous group of substances that is difficult to adapt to most proposed classification schemes. However, recent
updates have accommodated efficiently the diversity of bacteriocins produced by this phylum. Among the bacteriocins, the
lantibiotics representasourceofnewantimicrobialstocontrolinfectionscausedmainly byGram-positivebacteriaandwith
a low propensity for resistance development. Moreover, some of these compounds have additional biological properties,
exhibiting activity against viruses and tumour cells and having also potential to be used in blood pressure or inflammation
control and in pain relief. Thus, lantibiotics already described in Actinobacteria exhibit potential practical applications in
medicalsettings,foodindustryandagriculture,withexamplesatdifferentstagesofpre-clinicalandclinicaltrials.
INTRODUCTION naturalorsemisyntheticderivativesofactinobacterialandfun-
galproducts[6].Thesedatahighlighttheimportanceandthe
The phylum Actinobacteria is one of the major phyla of the
potential practical application of the actinobacterial anti-
domainBacteriaandcontainsGram-positivefilamentousbac-
microbial substances, notably to control pathogenic micro-
teriawithahighG+CDNAcontentanddifferentmorpholog-
organisms.
ical, physiological and metabolic characteristics [1–3]. Most
bacteriabelongingtothisphylumarefree-livingmicro-organ- The actinobacterial antimicrobials belong to many different
ismsthatareubiquitouslyfoundinbothaquaticandterrestrial groups, including conventional antibiotics and bacteriocins
ecosystems and include the following: (i) several pathogens, [1]. In this review, we will focus on lantibiotics produced by
suchasspeciesofCorynebacterium,Mycobacterium,Nocardia, these micro-organisms as members of this bacteriocin class
PropionibacteriumandTropheryma;(ii)soilinhabitants,such represent new alternatives to fight against emerging multi-
as Micromonospora spp. and Streptomyces spp.; (iii) plant drug-resistantpathogens.Wewillalsoupdatetheclassification
commensals, such as Frankia spp.; and (iv) gastrointestinal scheme of the bacteriocins already described in this phylum,
commensals,suchasBifidobacteriumspp.[1].Theirdiversity based on the proposal of Alvarez-Sieiro and coworkers [7],
ensures one of the main features of this group: versatility in and discuss the best characterized actinobacteriallantibiotics,
the production of biologically active compounds, including highlightingtheirpotentialbiotechnologicalapplications.
antimicrobialsubstances[4].Until2010,about34000micro-
bialbioactivecompoundshadalreadybeenreportedandclose
BACTERIOCINS PRODUCED BY
to 40% of them are produced by Actinobacteria, especially
ACTINOBACTERIA
Streptomyces spp., which produce nearly 80% of the actino-
bacterial compounds. Approximately 10000 actinobacterial Bacteriocinsareribosomallysynthesizedantimicrobialpepti-
substances exhibit antimicrobial and/or antitumour activity des or proteins produced by prokaryotes [8]. They are usu-
[5]. Moreover, most of the antimicrobials already used in ally small peptides (<10 kDa) which are cationic and
human and veterinary medicine or agriculture are either amphipathic [9–12]. Actinobacterialbacteriocins inhibit the
Received9October2016;Accepted17November2016
Authoraffiliations:1DepartamentodeMicrobiologiaM(cid:19)edica,InstitutodeMicrobiologiaPaulodeGóes,UFRJ,RiodeJaneiro,Brazil;2Departamentode
MicrobiologiaGeral,InstitutodeMicrobiologiaPaulodeGóes,UFRJ,RiodeJaneiro,Brazil.
*Correspondence:KarenMachadoGomes,[email protected]
Keywords:bacteriocin;antimicrobialpeptides;lantibiotics;Actinobacteria;biotechnologicalapplication.
Abbreviations:CDI,Clostridiumdifficileinfections;Dha,didehydroalanine;Dhb,didehydrobutyrine;HIV,humanimmunodeficiencyvirus;HSV,herpes
simplex virus; Lan, lanthionine; Lab, labionin; MeLan, b-methyl-lanthionine; PLA, phospholipase A; DPH, 3,3-dimethylamino-1-propylamide hydro-
2 2
chloride;PE,phosphatidylethanolamine.
000397ã2017TheAuthors
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Gomesetal.,Microbiology2017;163:109–121
growthofspeciesrelatedtotheproducer,suchasmichiganin lanthionine (Lan), b-methyl-lanthionine (MeLan), didehy-
A[13],althoughmanyofthempossessabroadspectrumof droalanine (Dha) and didehydrobutyrine (Dhb), among
inhibitoryactivitythatincludesnon-relatedbacteria(micro- others [30]. Lan and MeLan possess internal rings formed
bisporicin),protozoans(thiostrepton),yeastsandfungi(cin- by thioether bridges formed between Dha or Dhb, respec-
namycin)andviruses(labyrinthopeptinA1andduramycin) tively, and a cysteine (Cys) residue [8, 10, 34]. Therefore,
[14–18]. In addition to antimicrobial activity, some actino- lanthipeptide modifications depend on the occurrence of
bacterialbacteriocinsandtheirsemisyntheticderivativesalso twomainreactions:(i)dehydrationofserine(Ser)orthreo-
exhibitanti-inflammatory[19–21],anti-allergic[20],antitu- nine (Thr) residues to form Dha or Dhb, respectively, and
mour [22–24] and antinociceptive activities [25, 26], and (ii)cyclizationforformationofLanorMeLan,respectively.
may also act in blood pressure regulation [19, 27]. Some of The lanthipeptides that exhibit antimicrobial activity are
thesepeptideshavealreadyfoundclinicalapplications,such calledlantibioticsandtheirantibacterialactivityisrelatedto
asthiostrepton,athiopeptideusedindermatologicointment theinhibitionofcellwallbiosynthesisbybindingtolipidII
forveterinaryuse[28],andothersareinstagesofpre-clinical (to its pyrophosphate-sugar moiety), thus preventing the
andclinicaltrials,suchasNAI-107,NAI-112[29]andlanco- incorporation of the cell wall precursor units into the
vutide/duramycin (https://clinicaltrials.gov/ct2/show/study/ nascentpeptidoglycan,and/ortoporeformation,interfering
NCT00671736?term=Lancovutide&rank=1; accessed Sep-
with cellular membrane functions [8, 34, 35]. Of medical
tember 17, 2016), which reinforces the great potential of
importanceisthefactthatbindingof lantibioticstolipidII
practical application of bacteriocins produced by
is not antagonized by vancomycin, a drug commonly used
Actinobacteria.
inthecontrolofinfectionscausedbyGram-positivepatho-
The increasingnumber of new bacteriocins with distinctive gens [36]. Since their mechanisms of action are different
features being continuously described makes difficult the from those of other antimicrobials already used in clinical
establishment of a classification system for these com- settings, lantibiotics have been considered important alter-
pounds. There are several publications that discuss and natives for treatment of infections caused by resistant bac-
update the bacteriocin classification [8, 9, 11, 30–33]. teria carrying prevailing resistance mechanisms [8, 10, 34].
Alvarez-Sieiroandcoworkers[7]proposedabroaderbacte- Actinobacteriallantibiotics generallytargetlipidII.Aslipid
riocin classification scheme, which will be adopted in this IIisnottheproductofasinglegenebutderivedfrommulti-
review since it allows us to best accommodate the diversity ple enzyme-catalysed reactions, such lantibiotics have a
of actinobacterial bacteriocins. This classification scheme reducedpropensity forresistancedevelopment[37]. There-
divides the bacteriocins into only three classes. Class I fore, their antimicrobial activity combined with their low
includes peptides smaller than 10 kDa that undergo enzy- tendency to generate resistance makes these lantibiotics
matic post-translational modifications of some amino acid highlyattractiveformedicalapplications[38].
residues, known asribosomally synthesized and post-trans-
Lantibiotics may exhibit either linear or globular structures
lationally modified peptides. Class II bacteriocins are
andaredividedinto threetypesorsubclassesbasedontheir
unmodified peptides smaller than 10 kDa and class III is
biosyntheticmachinery[8].ThetypeIlantibioticsaremodi-
formed by unmodified bacteriocins larger than 10 kDa.
fiedbythedehydrataseLanBandthecyclaseLanC;thetype
Actinobacteria produce bacteriocins belonging to all three
II is modified by a bifunctional enzyme, LanM, with both
classesandmostsubclasses,exceptforsubclassesIIb(whose
activities, and the type III is modified by a trifunctional
antimicrobialactivitydependsontwounmodifiedpeptides),
enzyme, LanKC (with lyase, kinase and cyclase activities)
IIc (which comprises single-peptide, unmodified bacterio-
(Table2).
cins that are synthesized without leader peptide sequences)
and IId (which comprises single-peptide and unmodified Lantibioticsaregenerallysynthesizedintheformofinactive
bacteriocinsthatarenotpediocin-likebacteriocins).Table1 precursor peptides. Their production depends on the pres-
summarizesthebacteriocinclassificationschemeadoptedin ence of a gene cluster, organized in one or more operons,
this review, describing the main features of each class and
locatedeitheronmobilegeneticelements,suchasplasmids,
subclassandgivingexamplesofactinobacterialbacteriocins.
or on the bacterial chromosome of the producer strain [10,
ForclassI,onlytheribosomallysynthesizedandpost-trans-
11, 34]. The number of genes found in these clusters varies
lationally modified peptide subclasses already described in
(generally from 6 to 15) depending on the lantibiotic pro-
Actinobacteria are listed, such as lanthipeptides, thiopep-
duced. Besides the lantibiotic structural gene, these genes
tides, lasso peptides, linaridins, bottromycins and linear
encodeatleastfunctionsrelatedtopost-translationalmodi-
azole-containingpeptides.
fications of amino acids, proteolytic processing of the pre-
cursor peptide for production of the mature and bioactive
ACTINOBACTERIAL LANTIBIOTICS AND THEIR peptide,bacteriocin transport andimmunity. Some clusters
alsocarrygenesinvolvedinregulationoflantibioticproduc-
POTENTIAL BIOTECHNOLOGICAL
tion.Thesegeneclusterswillnotbedescribedinthepresent
APPLICATIONS
review as detailed information about them can be found in
Lanthipeptides are peptides that undergo post-translational the numerous reviews on lantibiotics that can be found in
modifications which give rise to rare amino acids, such as theliterature[10,11,34,35,39,40].
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Gomesetal.,Microbiology2017;163:109–121
References [45] [57] [93] [14,96,97][98] [99] [100,101] [102] [103,104] [23] [105] [106] [107]
al
Activity (cid:0)ntibacterial(G+andG) ntibacterial(G+) ntibacterial(G+),antiviral,antinociceptiventibacterial(G+),antiprotozoal,anticancer(cid:0)ntibacterial(G+andG) ntibacterial(Mycobacterium) (cid:0)ntibacterial(G+andG)andantivir ntibacterial(Streptomyces) ntibacterial(G+andMycoplasma) ntibacterial(Micrococcusluteus),anticancer ntibacterial(G+) ntibacterial(Streptomycesipomoeae) ntifungal
A A A A A A A A A A A A A
ns
Examples Planosporicin Actagardine Labyrinthopepti Thiostrepton GE2270A Lariatins Siamycin Goadsporin BottromycinA2 Cypemycin BifidocinB ND ND ND Ipomicin AFP-1
d
e
at
yl
h
et
m
cles bon-
ocy car
heter and
as ole
well hiaz
t
ngas minal eria.
hemeforbacteriocinsproducedbyActinobacteria Distinctivefeatures kDa,modifiedpeptides) bContainsulfur--carbonlinkages Containacentralpyridine,adihydropyridineorpiperidineri Possessalassostructure Possessheterocyclicringsofthiazoleand(methyl)oxazole Containamacrocyclicamidine,adecarboxylatedcarboxy-teraminoacidsPossessalinearstructurewithdehydratedaminoacids kDa,unmodifiedpeptides) Pediocin-likepeptides Two-peptide Leaderlesspeptides Non-pediocin-like,single-peptide >10kDaproteins) Bacteriolysins Non-lytic (cid:0)bacteria;G+,Gram-positivebacteria;G,Gram-negativebact
Classificationscable1. ClassI(heat-stable,<10 Lanthipeptides Thiopeptides Lassopeptides Linearazole-containingpeptideBottromycins Linaridins ClassII(heat-stable,<10 IIa IIb IIc IId ClassIII(thermo-labile, IIIa IIIb ,NotdetectedinActinoD
T N
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Gomesetal.,Microbiology2017;163:109–121
Table2.ClassificationschemeoflantibioticsproducedbyActinobacteria
Type Distinctivefeatures Example ProducerActinobacteria Reference
I ProcessedbyLanBandLanC MicrobisporicinA1andA2 Microbisporacorallina [15]
Planosporicin Planomonosporaalba [45]
II ProcessedbyabifunctionalLanM Variacin Kocuriavarians [52]
Actagardine ActinoplanesgarbadinensisandActinoplanesliguriae [54]
DeoxyactagardineB Actinop.liguriae [68]
MichiganinA Clavibactermichiganensissubsp.michiganensis [13]
Cinnamycin Streptomycescinnamoneus [17]
Duramycin Strep.cinnamoneus [81]
Ancovenin Streptomycesspp. [27]
III Containlabionin;processedbyLanKC LabyrinthopeptinsA1,A2andA3 Actinomaduranamibiensis [93]
NAI-112 Actinoplanesspp. [25]
AlthoughlantibioticsaremainlyproducedbyspeciesofFir- and anaerobic Gram-positive pathogens. Its spectrum of
micutes, potent lantibiotics havebeen detected in species of activityincludesdrug-resistantbacterialstrainssuchasmetic-
Actinobacteria [41], as described below. However, only the illin-resistant Staphylococcus aureus, vancomycin-resistant
best characterized lanthipeptides that exhibit antimicrobial EnterococcusandStreptococcuspneumoniaeresistanttopeni-
activitywillbecoveredbythisreview. cillin,presentingefficacycomparableandsometimessuperior
tothoseobservedforvancomycinandteicoplanin.Thislanti-
biotic also exhibits activity against Clostridium spp. and Pro-
TYPE I LANTIBIOTICS
pionibacterium spp., and some Gram-negative bacteria as
Microbisporicin or NAI-107 well,suchasNeisseriaspp.,HaemophilusinfluenzaeandMor-
axella catarrhalis [15]. Microbisporicinproved to be effective
MicrobisporicinorNAI-107(Fig.1a)isproducedasacom-
in treating different types of infections (sepsis, endocarditis
plex of two structurally related lantibiotics containing 24
and granuloma pouch) caused by Gram-positive multiple
amino acids (aa), five thioether bridges (three Lan, one
drug-resistant pathogens in vivo [47]. Pharmacokinetic and
MeLan and one AviCys; the latter amino acid results from
pharmacodynamic studies using a neutropenic murine thigh
theoxidativedecarboxylationofaterminalCysresidue)and
infection model showed a dose-dependent bactericidal effect
amino acid modifications that have never been reported in
of microbisporicin/NAI-107 and a prolonged suppression of
the literature, such as a 5-chloro-tryptophan (Trp) and a
bacterial growth (>35h). In addition, the in vivo NAI-107
proline (Pro) hydroxylation [15, 42]. Microbisporicin/NAI-
effectiveness was similar to or evenbetter than that observed
107 has been detected in strains of Microbispora corallina
for drugs already used to treat meticillin-resistant Staph.
[15,42,43]andActinoallomurusspp.[43,44].Thislantibi-
aureus, such as ceftaroline, daptomycin, oxazolidinones and
oticwasfirstisolatedinaprojectthataimedtoidentifynew
vancomycin [48]. Sustained concentrations of microbispori-
compounds that act as cell wall synthesis inhibitors from
cin/NAI-107wereobservedinplasma,whichmayexplainthe
screening a collection of about 40000 rare actinomycetes
compound efficacy in different animal models of infection,
[15, 45]. The most abundant congenersofmicrobisporicin/
such as the acute lethal infection model in mice, the granu-
NAI-107 are named A1 (2246 Da) and A2 (2230 Da), and
lomapouchmodelinrats,ratendocarditis[47]andtheneu-
theydifferonlyinposition14,witha3,4-dihydroxy-proline
tropenicmurinethighinfectionmodel[48].Microbisporicin/
in microbisporicin A1 and a 4-hydroxy-proline in variant
NAI-107isatthefinalstagesofpre-clinicaldevelopment[29]
A2. Both congeners exhibit the same mechanism of action
proving to be a potent lantibiotic with promising chances of
andsimilarinhibitionspectrum,provingtobepotentlanti-
beingusedtotreatinfectionscausedbyavarietyofGram-pos-
biotics that act by blocking cell wall synthesis [15]. This
itivebacteriaofmedicalimportance.
blockingoccursbythemicrobisporicin/NAI-107bindingto
lipid II as well as to other bactoprenol-bound precursors, A brominated variant of microbisporicin/NAI-107, known
impairingmembranefunctionsandresultinginarapidbac- as NAI-108, carrying a 5-Br-tryptophan, was detected in
tericidal activity [46]. Other minor congeners have also cultures of Microbispora spp. and Actinoallomurus spp.
been described in strains of Microbispora spp., differing by
when potassium bromide (KBr; at 8.6 mM) was added to
thepresenceoftwo,oneorzerohydroxylgroupsatPro-14,
thegrowthmedium.NAI-108wasproducedbysubstitution
by the presence of a chlorine at Trp-4 and/or by the pres-
of chlorine found in NAI-107 structure (in Trp-4) with
enceofasulfoxideonthethioetherlinkage of thefirst Lan.
bromine and it represents the first example of a bromine-
However, these minor congeners showed some variation in
containing lantibiotic. This variation was responsible for
antimicrobial activity, being less effective than A1 and A2,
the generation of a more potent lantibiotic, with an inhibi-
particularlyagainststaphylococciandenterococci[42].
tory spectrum similar to that of microbisporicin/NAI-107
Microbisporicin/NAI-107 is considered one of the most but effective at lower concentrations than those required
potentlantibiotics,exhibitingastrongactivityagainstaerobic for inhibition by the parental peptide. NAI-108 turned out
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Gomesetal.,Microbiology2017;163:109–121
(a) (b)
NH
1
OH 16
S
Cl 8
4
S
S
4 S S
1 NH
12 S
S
8 16
20 S OH 12
Microbisporicin A2 Duramycin
(c) (d) 4
16
4
S 1 S
S
S 24
1 12
12
S S=o
S
8 8
16
20 S
Planosporicin Actagardine S
(e) (f)
4
S S
S CH2 16
S
1 1 8
4 16
8 12 S
S
CH2 12
S 20
Labyrinthopeptin A2 Michiganin A
Fig. 1.Representativeschemeofthemainactinobacteriallantibiotics.
to be about twice as active as microbisporicin/NAI-107 Planosporicin or lantibiotic 97518
against important pathogens such as Staphylococcus spp.,
Planosporicin (Fig. 1c), a cell wall biosynthesis inhibitor of
Streptococcusspp.andEnterococcusspp.At0.25µgml(cid:0)1,it
24 aa (2194 Da), is produced by Planomonospora sp. DSM
also rapidly killed the target strain Staph. aureus ATCC 14920 and it was detected in the same project that
29213, leading to a 5-log10 reduction in viable cells within described microbisporicin [45]. Planosporicin and micro-
30min.Alowerinhibitionwasobservedwithmicrobispori- bisporicin/NAI-117 exhibit a similar primary structure,
cin/NAI-107 that caused only a 3-log10 reduction in viable with over 70% sequence similarity (differing in only six
cells, when used at the same concentration. For both, amino acids, occupying the positions 1, 4, 6, 14, 19 and
microbisporicin/NAI-107 and NAI-108, the target strain 22), and five thioether linkages. Planosporicin possesses
growth rate in the presence of the antimicrobial was com- fourLanresiduesandoneMeLan residue asthislantibiotic
parable to that observed in the control with no antimicro- presents a Lan residue instead of an AviCys residue at the
bial added. Therefore, NAI-108 was consistently more C-terminal region [49, 50]. Planosporicin production,
activethanmicrobisporicin/NAI-107.AlthoughNAI-108is which is encoded by a cluster of 15 genes, is confined to
still at an early study phase, it is another attractive option the stationary phase of growth in broth and to the onset of
for treatment of infections caused by multidrug-resistant morphological differentiation when Planomonospora spp.
Gram-positivebacteria[43]. aregrownonsolidmedium[51].
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Gomesetal.,Microbiology2017;163:109–121
Despite sharing remarkable structural and antibacterial variacinat1%offermentedingredientinhibitedthegrowth
spectrum similarities, inhibiting important Gram-positive of B. cereus in all products and models to which it was
pathogens, planosporicin exhibited a less potent antimicro- applied. At 12(cid:14)C, the addition of 3% fermented ingredient
bial activity than microbisporicin/NAI-117 against all was required to inhibit outgrowth of the spores [53]. How-
tested micro-organisms, including staphylococci, strepto- ever,furtherstudies,suchas(i)testsofthevariacincytotox-
cocci and enterococci, among others [15]. The difference icity to eukaryotic cells at the concentrations used in the
in the effectiveness of these two lantibiotics seems to be study of O’Mahony and coworkers, (ii) the effectiveness of
related to subtle structural differences in the region thebacteriocininotherfoodmatricesand(iii)otherbacteri-
between amino acids 12 and 18. In this region, microbi- ocinproperties(suchasshelf-lifeandsensitivitytogastroin-
sporicin is more flexible. Moreover, planosporicin presents testinal conditions), should be encouraged so the feasibility
a negative charge due to the presence of a glutamic acid ofusingthislantibioticasanaturalantimicrobialtocontrol
residue (Glu-14) in this region, not found in microbispori- undesirablemicro-organismsinfoodproductsisconfirmed.
cin. These two features seem to influence the interaction
of both lantibiotics with the membrane and, therefore,
Actagardine and derivatives
their antibacterial activity [50].
Actagardine (Figs 1d and 2) was first described in 1976
underthenamegardimycin.Thislantibioticisproducedby
TYPE II LANTIBIOTICS Actinoplanes garbadinensis and Actinoplanes liguriae [54–
Variacin 56].Itiscomposedof19aa(1890Da)andfourintramolec-
ular rings, one Lan and three MeLan, with the C-terminal
VariacinwasdetectedintwostrainsofKocuriavarians(for- MeLanoxidizedtosulfoxide[57–59].Thissulfoxidebondis
merlyreferredtoasMicrococcusvarians)isolatedfromItal-
notcommonamonglantibiotics.Theoxidation ofthethio-
ian-type raw salami as part of the natural meat microbiota.
etherlinkagebetweenresidues14and19ofthematurepep-
Thematurelantibioticiscomposedof25aa(2658Da),con-
tide for formation of the sulfoxide bond is catalysed by the
tainingoneLanandoneMeLanrings,andexhibitsasignifi-
product of the garO gene, which encodes a luciferase-like
cant similarity (92%) to lacticin 481, a type II lantibiotic
monooxygenase.DeletionofgarOresultedinproductionof
produced by Lactococcus lactis. Variacin inhibits a wide
deoxyactagardine,withaslightlyloweractivitythanitscog-
range of Gram-positive food spoilage bacteria; important
nate, suggesting a certain influence of the sulfoxide group
food-borne pathogens, such as Enterococcus spp., Listeria
on antimicrobial activity [60]. This result, however, con-
monocytogenesandStaph.aureus;andvegetativeformsand
trasts with a study performed with nisin, a lantibiotic pro-
spores of Clostridium spp. and Bacillus spp. In addition to
duced by Lc. lactis, in which the oxidation of its Lan
its broad inhibitoryspectrum, which suggestsa potentialof
residuesledtonisininactivation[61].
this lantibiotic as a food biopreservative, variacin is also
thermostableandresistanttoawidepHrange(pH2to10), Actagardine acts by inhibiting cell wall synthesis by block-
desirable features for this application [52]. When variacin ingthetransglycosylationreaction[36,62,63].Thislantibi-
wasaddedintheformofaspray-driedfermentedingredient otic exhibits a potent activity against Gram-positive
(at1or3%oftotalweight)inchilleddairyproducts,itwas pathogens, particularly Streptococcus spp. and Clostridium
effectiveincontrollingacocktailofpsychotrophicstrainsof spp. [54–56]. In vivo tests using mice experimentally
the spore-forming food-borne pathogen Bacillus cereus, infectedwithStrep.pneumoniaeorStreptococcushaemolyti-
even under conditions of temperature abuse. At 8(cid:14)C, cus (formerly referred to as Diplococcus haemolyticus)
Actagardine
Ala(0)-actagardine Cl
Actagardine B
NVB333 NH
Cl
NVB302 NH NH
2
NAI-802
Ala(0)-NAI-802
Fig. 2.Peptidesequencesofactagardineanditsderivatives.Thethioetherbondsthataresharedbyallthesepeptidesareshownas
blacklinesontheactagardinesequence.Aminoacidsthatdifferfromactagardinesequencearemarkedinred.
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Gomesetal.,Microbiology2017;163:109–121
showed that actagardine has an antimicrobial activity com- diarrhoea caused by Clost. difficile [71, 72], NVB302 effec-
parabletothoseofampicillinandcephaloridine[54]. tiveness was compared to that of vancomycin. In vitro tests
were then performed using a human gut model inoculated
Upon detection of actagardine, several natural and semisyn-
withafaecalemulsionpreparedfromClost.difficile-negative
theticderivativeshavebeendescribed(Fig.2).Theselantibiot-
faecesofhealthvolunteers.Ondays14and21,spores(~107
icsshareintheirstructuresatleastthreethioetherbridgesand
c.f.u.)ofavirulentClost.difficilestrainwereinoculatedinto
14ofthe19aaoftheirchains[64].TheDmetabolite,acciden-
the system, followed by instillation of either NVB302
tally generated during actagardine purification, differs from
(125 mg l(cid:0)1) or vancomycin (125 mg l(cid:0)1) on day 30. Each
actagardine by only the absence of a MeLan residue, and in
antimicrobialwasinstilledfourtimesdailyfor7days.From
vitroandinvivotestsshowedsignificantlybetterantimicrobial
day 37 until day 50, gut microbiota populations and the
activity of this compound against Streptococcus spp. and
Clost. difficile cytotoxin titres were monitored. NVB302
Staph.aureus[65].Severalbasiccarboxamidesofactagardine
exhibited an activity comparable to that of vancomycin as
have already been synthesized. The monocarboxamides were
both antimicrobial instillations effectively eliminated vege-
showntobetwicetofourfoldmoreeffectivethanactagardine
tative Clost. difficile from the gut model, leading also to
in a murine model of Streptococcus pyogenes septicaemia.
cytotoxin reduction to undetectable levels 5–7 days after
Among the monocarboxamides, the 3,3-dimethylamino-1-
drug instillation. Moreover, NVB302 exhibited a reduced
propylamide hydrochloride (DPH) was chosen for further
effect on the intestinal microbiota. However, neither
analysesand,likeactagardine,displayedabactericidalactivity,
NVB302 nor vancomycin displayed activity against Clost.
although at lower concentrations. The actagardine and DPH
difficile spores. Taken together, these results showed that
killedatleast99.9%oftheStrep.pyogenesviablecellsexposed
NVB302 succeeded in treating simulated CDI in a human
to a concentration of 10 times the MIC, the MIC being
DPH gut model [70]. Animal model tests have also shown that
equal to 0.4 µg ml(cid:0)1 and the MIC equal to 1.6 µg
Actagardine NVB302 is effective in treating CDI, reaching high concen-
ml(cid:0)1)[66].
tration levels in the colon and without significant toxic
TheAla(0)-actagardine(1961Da;Fig.2),producedbyActi- effects.NVB302wasalsoresistanttopHandenzymespres-
nop.liguriaeATCC31048,differsfromactagardinebyonly entinthegastricfluidanditwasnotabsorbedsystemically,
an additional alanine (Ala) residue at its N-terminus whichminimizestheappearanceofcollateraleffects[73].
(Fig. 2), and it displays a slightly higher activity than acta-
NVB333 (Fig. 2) was synthesized from binding 3,5-
gardine, especially against strains of Staphylococcus spp.
dichlorobenzylaminetotheC-terminalregionofdeoxyacta-
However, regarding streptococci and enterococci, Ala(0)-
gardine B. This semisynthetic lantibiotic presents activity
actagardineexhibitedaninhibitoryactivitysimilartothatof
against clinically relevant Gram-positive pathogens such as
actagardine. The semisynthetic derivatives Lys(0)-actagar-
Staph. aureus resistant to meticillin, vancomycin, linezolid
dine and Ile(0)-actagardine displayed an activity either bet-
and daptomycin; vancomycin-resistant Enterococcus; and
ter(fromtwofoldtofourfoldlowerMICs)orsimilartothat
penicillin-resistant Strep. pneumoniae. Although the
ofAla(0)-actagardineagainstthestrainstested[67].
NVB333MICsagainstmoststrainstestedwereeithertwice
Actinop.liguriaeNCIMB41362producesanotherlantibiotic or fourfold higher than those determined for vancomycin,
relatedtoactagardine,thedeoxyactagardineB(Fig.2).This this peptide showed excellent results in in vivo tests using
peptide has also 19 aa and differs from actagardine in resi- disseminated infection, thigh infection and lung infection
dues 15 and 16 [actagardine contains valine (Val) and iso- models, with results comparable to and even better than
leucine (Ile) and deoxyactagardine, leucine (Leu) and Val, those observed for vancomycin. Additionally, NVB333 was
respectively]andbytheabsenceofthesulfoxidebondinthe welltoleratedatthedoselevelsusedandtherewerenosigns
MeLan bridge between residues 14 and 19 [68]. Semisyn- of any drug-related adverse effects. These results coupled
thetic derivatives of deoxyactagardine B, NVB302 and with a low resistance rate to NVB333 make this compound
NVB333, display antimicrobial activity against important a novel promising candidate for the treatment of systemic
bacterialpathogensandarealreadyinclinicaltrials. infectionscausedbyGram-positivepathogens[37].
NVB302 (aminoheptylamido-deoxyactagardine; Fig. 2) More recently, two other actagardine-related lantibiotics
completed part of the phase I of clinical trials for the treat- have been described, NAI-802 (2188 Da; Fig. 2), produced
ment of Clostridium difficileinfections(CDI) (www.evalua- byActinoplanessp.ID104802andID104771,andasemisyn-
tegroup.com/Universal/View.aspx?type=Story&id=344702; thetic derivative, obtained following amidation of NAI-802
accessed17September2016).Clost.difficileisanimportant with benzylamine, which resulted in the diamidated deriva-
pathogenic micro-organism implicated in 20–30% of cases tive3.NAI-802hastwoadditionalaminoacids,anN-termi-
ofantibiotic-associateddiarrhoeaand90%ofcasesofpseu- nalAlaandaC-terminalarginine(Arg),whencomparedto
domembranous colitis [69]. One advantage of using actagardine,theseadditionsbeingtheonlydifferencesfound
NVB302inthetreatmentofCDIisthefactthatthisantimi- between both lantibiotics. The NAI-802 producer strains
crobial exhibits selective invitro activity against Clost. diffi- also produce minor amounts of its Ala(0)-congener 2
cile compared with other intestinal bacteria [70]. As (Fig.2).TheaminoacidadditionsfoundinNAI-802seemto
vancomycin is used to treat severe cases of colitis and have improved its activity against Staphylococcus spp. and
115
Gomesetal.,Microbiology2017;163:109–121
Streptococcus spp., with MICs ranging from 0.5 to 32 µg Cinnamycingroup
ml(cid:0)1forNAI-802,whereastheMICsforactagardineranged
Cinnamycin (2042 Da), duramycin (2014 Da; Fig. 1b) and
from 2 to 64 µg ml(cid:0)1. In relation to enterococci, the MICs
ancovenin (1959 Da) form a group of natural variants that
observedforNAI-802((cid:21)128µgml(cid:0)1)weresimilartothose
presentotherbiologicalactivitiesinadditiontoaweakanti-
for actagardine [64]. The improved activity of NAI-802 is
microbial action. Lantibiotics belonging to the cinnamycin
probablyrelatedtotheadditionalpositivechargeduetothe
group exhibit a similar structure as globular 19 aa peptides
presenceofaC-terminalArg.Positivelychargedresiduesare
with one Lan, two MeLan and an unusual lysinoalanine
believedtoplayanimportantroleintheinteractionoflanti-
bridge between Lys-19 and Ser-6 (Fig. 3). They also exhibit
biotics with the negatively charged bacterial membranes
a modification in position 15, an aspartate hydroxylation
[74]. Such importance is confirmed by the antimicrobial
yielding the erythro-3-hydroxy-aspartic acid. Among the
activity of the diamidated derivative 3, which carries
cinnamycin group, ancovenin is the most different variant,
oneadditionalpositivechargethroughconversionoftheC-
neither presenting the aspartate 15 modification nor the
terminal carboxylate into the corresponding basic amide. lysine–alaninebridge(Fig.3)[20].
The diamidated derivative 3 exhibited an improved antimi-
crobialactivitywhencomparedtoNAI-802,exhibitingfrom Cinnamycin is produced exclusively by Streptomyces spp.
a fourfold to an eightfold reduction in MIC values, even [79]. Despite this lantibiotic having also been described in
againstenterococci.NAI-802andderivative3showedalsoa Streptoverticilliumspp.,thisgenuswasunifiedwithStreptomy-
significantactivityagainstGram-positiveanaerobicbacteria, cesin1990[80].ThislantibioticinhibitsthegrowthofBacillus
such as Clost. difficile, Clostridium butyricum, Clostridium subtilis, anaerobic bacteria, fungi and yeasts (although less
perfringens and Peptostreptococcus asaccharolyticus with intensely). Additionally, it also inhibits the proliferation of
MIC ranges of 0.25–2 µg ml(cid:0)1 and <0.125–8 µg ml(cid:0)1, for herpes simplex virus (HSV) type1[17].Duramycin wasiso-
NAI-802 and derivative 3, respectively. These MIC values lated from Streptomyces cinnamoneus and initially studied
werecomparabletothosedeterminedforvancomycin(0.25– because of its inhibitory action against Rhodococcus fascians
1µgml(cid:0)1).Hence,NAI-802anditsdiamidatedderivative3 [81],aphytopathogenthatinfectsawiderangeofplantscaus-
arepotentialnovelantimicrobialagentsforthetreatmentof ingaleafygallformation[82].Somenaturalvariantsofdura-
infections caused by Gram-positive pathogens, including mycin, duramycins B (1951 Da) and C (2008 Da), have
resistantstrains[64]. alreadybeendescribed[34].
Michiganin A Ancoveninandcinnamycininhibittheactivityoftheangio-
tensin-converting enzyme, presenting a potential applica-
Clavibacter michiganensis subsp. michiganensis is known
tion in blood pressure regulation [19, 27]. Cinnamycin and
notonlyastheagentofbacterialwiltandcankeroftomato
duramycin act as phospholipase A (PLA ) inhibitors [20,
but also for producing many antimicrobial substances [75, 2 2
21].Infact,itisanindirectinhibitionbecausethelantibiot-
76], including michiganin A [13]. Michiganin A is a 21 aa
ics bind at a 1:1 ratio and with high affinity and exclusive
heat-stable lantibiotic (2145 Da; Fig. 1f) related to actagar-
specificity to the substrate of PLA , the phosphatidyletha-
dine and, like other actagardine-related bacteriocins, 2
nolamine(PE)[21,83].Thisbindingalterstheoperationof
presentstworingsofMeLanandoneofLan.MichiganinA ionchannels[84–86],afeaturethatisexploitedbythephar-
differs from actagardine by the absence of the sulfoxide
maceutical industry for cystic fibrosis treatment, as dis-
bond and by only four residues, the amino acids added to
cussed below [87, 88]. Furthermore, as PLA provides
the N- and C-termini and the residues 5 and 15, which are 2
arachidonicacid,byreleasingthisfattyacidfromphospho-
Val and Val in actagardine, and Leu and Ile in michiganin
lipidsinthecellmembranestotheenzymesresponsible for
A,respectively[13,60].Holtsmarkandcoworkers[13]sug-
the synthesis of eicosanoids, which are associated with
gestedthatmichiganinAactsbyinhibitingcellwallsynthe-
inflammation, such lantibiotics can also be used in regula-
sis due to its high similarity to mersacidin and actagardine,
tion of inflammatory processes [19, 89]. The inhibition of
including the presence of a conserved Glu residue, known
PLA has also been explored in treatment of diseases such
to be important for interaction with lipid II. However, 2
asatherosclerosis,diabetesandcancer[89].
michiganinAhasarathernarrowspectrumoftargetorgan-
isms. This lantibiotic inhibits, at nanomolar concentrations Invitrostudiesusingairwayepitheliumshowedthatduramy-
(in the10–100 nMrange),the growth of theclosely related cin enhances the chlorine secretion (via alternative chloride
bacteriumClavibactermichiganensissubsp.sepedonicus[13, channel activation) due to the intracellular calcium increase
77], an important phytopathogen that causes potato ring [85].Becauseofthisactivity,duramycin,alsocalledMoli1901
rot, responsible for major economic losses worldwide [78]. orlancovutide,isinphaseIIofclinicaltrialstobeusedinthe
Despitetherequirementofmorestudies,michiganinAisa treatmentofcysticfibrosis.Cysticfibrosisisageneticdisorder
potential new antimicrobial for plant disease control. thatcausesanexocrineorgandysfunction.Thelungdiseaseis
Although its spectrum of activity is quite narrow, such characterized by inhibition of chlorine secretion into airway
afeature offers anadvantage, contributingto coveragricul- surface liquid, creating an ion imbalance which results in a
ture’s need for more sustainable and effective strategies for thick mucuslayerwhichinturn hinderstheciliaryfunctions
control of specific phytopathogens, without affecting plant- and airway clearance, forming a favourable environment for
beneficialmicro-organisms[77]. multiplicationofmicro-organisms[90].Thenduramycinacts
116
Gomesetal.,Microbiology2017;163:109–121
OH
Cinnamycin
OH
Duramycin
H
Ancovenin
Fig. 3.Comparisonofthesequencesofthepeptidesbelongingtothecinnamycingroup.Thethioetherbondsthataresharedbyall
peptidesareshownasblacklinesontheactagardinesequence.Thedashedlinerepresentsthelysine–alaninebridge,whichisnot
presentinancovenin.
indirectly, rehydrating airway surface liquid of patients with preventing cell entry of HIV and HSV, and cell-to-cell viral
cystic fibrosis. The finished phase IIb of clinical trials transmissionofHIV.Itsanti-HSVactivitywascomparableto
approved intrapulmonary administration by inhalation of thoseshownbyantiviralcompounds(acyclovirandcidofovir)
nebulized duramycin in the treatment of cystic fibrosis andthis lantibiotickept its anti-herpeticactivityeven against
patients, proving to be safe and with improvement in lung drug-resistantstrains.Additiveorsynergisticeffectswerealso
function[87,88]. demonstrated between LabA1 and antiviral drugs already
used in the clinic (tenofovir, acyclovir, saquinavir, raltegravir
Recent studies have also shown inhibitory activity of dura-
andenfuvirtide).Therefore,LabA1presentsfavourableprop-
mycin on the multiplication of viruses and tumour cells.
ertiesthatqualifyitsapplicationinpreventionandcontrolof
Both pancreas tumour cells and viruses of the Flaviviridae
transmissionofsexualdiseasescausedbyHIVandHSV[16].
and Filoviridae families have PE on their surfaces. In
Ontheotherhand,althoughLabA2showedonlyamoderate
tumour cells, duramycin binds to PE, increasing apoptosis
anti-HSVactivityandnoanti-HIVactivity[16,26],itpresents
and necrosis and reducing cell proliferation in a dose-
apotentantipainactivity[26],asalsoobservedforNAI-112.
dependentmanner(between0.125and12.5µmoll(cid:0)1)[91].
For the West Nile, dengue and ebola viruses, duramycin NAI-112
inhibitedtheviralentryprocesses.Therefore,duramycin,as
NAI-112 was another lantibiotic that was detected during a
other lantibiotics that bind to PE, has potential to inhibit
screening program that aimed to search for new cell wall
theactivityoftumourcellsandviruses[18,91].
inhibitors. NAI-112 is a Lab-containing lantibiotic produced
byActinoplanessp.DSM24059.Itisa22aaneutrallycharged
TYPE III LANTIBIOTICS peptide presenting, besides Lab residues, a methyl-labionin
(MeLab)initsC-terminalregion.Inaddition,NAI-112carries
Labyrinthopeptins a 6-deoxyhexose moiety N-linked to Trp-13. Thus, NAI-112
The labyrinthopeptins are lanthipeptides that do not possess was the first lantibiotic in which the presence of MeLab and
Lan andMeLanresidues.Instead,theselantibioticscontaina N-glycosylation was reported. Despite its modest antimicro-
Lan variant known as labionin (Lab), which is a carbacyclic bialactivityobservedagainstStaphylococcusspp.andStrepto-
ring which requires two Dha and one Cys for its formation. coccus spp., which contrasts with the properties of most
LabisthusformedbyaLanresiduecovalentlyboundtoDha lantibiotics,NAI-112displayedantinociceptiveactivityinani-
by a methylene bridge [26, 92]. Labyrinthopeptins were ini- malmodels,reducingpainsymptomsinmiceinboththefor-
tially detected in Actinomadura namibiensis DSM 6313 malin and the chronic constriction injury tests, without
cultureandthisbacteriumproducesthreetypesoflabyrintho- toxicitysigns.Therefore,likeLabA2,NAI-112haspotentialto
peptinscalledLabA1,LabA2andLabA3.Thelabyrinthopep- beusedforpainreliefapplications.However,themechanism
tins are composed of 18–21 aa and, in addition to Lab, they of action of NAI-112 as antinociceptive agent has not been
also possess a disulfide bond between two Cys residues. The establishedyet[25].
variants LabA1 and LabA3 differ only by addition of an
aspartic acid (Asp) in the N-terminal region of LabA3, and CONCLUDING REMARKS
LabA2(Fig.1e)isthesmallestvariantwith18aa[26,93].
The phylum Actinobacteria comprises important human
LabA1exhibitsaveryeffectiveantiviralactivity,actingagainst pathogens, such as Mycobacterium tuberculosis, Mycobac-
both human immunodeficiency virus (HIV) and HSV, terium leprae and Corynebacterium diphtheriae, among
117
Gomesetal.,Microbiology2017;163:109–121
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K.M.G. was a recipient of a scholarship from CAPES/Brazil. The 837–845.
research conducted in our laboratory is supported by grants from
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CNPq (470.443/2012-0) and FAPERJ (E26.102.336/2013) to M.C.F.B.
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infection by Ebola, dengue, and West Nile viruses. Proc Natl
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Conflictsofinterest
improve therapeutic potential. Curr Protein Pept Sci 2005;6:
Theauthorsdeclarethattherearenoconflictsofinterest. 61–75.
20. Fredenhagen A, Fendrich G, Ma€rki F, M€arki W, Gruner J
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