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Hindawi Publishing CorporationClinical and Developmental ImmunologyVolume , Article ID ,pageshttp://dx.doi.org/.//

Review ArticleRole of Pore-Forming Toxins in Neonatal Sepsis

Andreas F.-P. Sonnen and Philipp Henneke

Center of Chronic Immunodeciency, Medical Center, University of Freiburg, Breisacher Strae , Freiburg, Germany

Correspondence should be addressed to Andreas F.-P. Sonnen; [email protected]

Received February ; Accepted March

Academic Editor: Robert Bortolussi

Copyright A. F.-P. Sonnen and P. Henneke. Tis is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Protein toxins are important virulence actors contributing to neonatal sepsis. Te major pathogens o neonatal sepsis, group BStreptococci,Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus, secrete toxins o differentmolecular nature, whichare key or dening the disease. Amongst these toxins are pore-orming exotoxins that are expressed as soluble monomers prior toengagement o the target cell membrane with subsequent ormation o an aqueous membrane pore. Membrane pore ormation isnot only a means or immediate lysis o the targeted cell but also a general mechanism that contributes to penetration o epithelialbarriers and evasion o the immune system, thus creating survival niches or the pathogens. Pore-orming toxins, however, can alsocontribute to the induction o inammation and hence to the maniestation o sepsis. Clearly, pore-orming toxins are not the soleactors that drive sepsis progression, but they ofen act in concert with other bacterial effectors, especially in the initial stages oneonatal sepsis maniestation.

1. Introduction

Te birth canal, that is, the area between the ully dilateduterus andthe outside o the vagina, harbours a polymicrobialcommunity that may ull the denition o a biolm []. Nextto apathogenic species, such asLactobacillusspp., potentiallypathogenic bacteria including group B streptococci (GBS),Escherichia coli(E. coli),Listeria monocytogenes(L. monocy-togenes), andStaphylococcus aureus(S. aureus) are ound inthe vagina o up to % o women. During birth, the etus

needs to pass rom the sterile uterus through these bacteria.Accordingly, aspiration o bacteria during birth is regardedas a major cause o neonatal sepsis in the rst three to sevendays o lie (early-onset sepsis). In line with this model,early-onset sepsis is predominantly caused by GBS,E. coli,L.monocytogenes, and S. aureus. Yet most inants successullycontrol the bacteria at the mucocutaneous suraces.

Subsequent to aspiration, bacteria like GBS can prolierateto striking densities in the neonatal lung, as shown in

newborn primates with neonatal GBS pneumonia (911

colony-orming units (CFUs)/g lung tissue, []). Te antimi-crobial quality o the local pulmonary environment, orexample, the concentration o suractant, may be important

or the metabolic activity in the bacterial community andthereore orthe expression o bacterial virulence actors suchas bacterial toxins [].

Sepsis imposes a major threat to newborn inants. Itis estimated that sepsis causes over hal a million neona-tal deaths annually, thereby accounting or about % oall neonatal deaths worldwide []. Whereas sepsis causesapproximately .% o inant deathsin developed countries, itis responsible or up to % o neonatal deaths in developingcountries[, ]. Moreover, neonatal sepsis ofen occurs as

meningoencephalitis, which leaves almost % o affectedpatients with lielong disabilities []. On the other hand,GBS, E. coli, and S. aureus are normal components o themucocutaneous microbiome, and it is impossible to predictthe risk to an individual baby.

2. Bacterial Membrane-Damaging Toxins

Te rst membrane-damaging bacterial toxin was describedby Paul Ehrlich in [], who ound that Clostridiumtetani extracts lyse erythrocytes. oday, three different mech-anisms o membrane damage by proteinaceous agents canbe delineated. First, toxins can solubilise target membranes
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Clinical and Developmental Immunology

Detergent-like action Pore formation Lipid hydrolysis

F : Ways to damage a lipid membrane. Tere are various mechanisms o membrane damage by protein toxins. Amphiphilic toxinscan integrate into the membrane and essentially solubilise the lipid membrane like a detergent (structure:S. aureus-toxin, PDB ID KAM).Similarly, the membrane lipids can be hydrolysed by phospholipases also resulting in the destructiono the membrane (structure:Clostridium,perfringens -toxin, PDB ID KHO, []). By ar the largest class o membrane damaging toxins is that o the pore-orming toxins (structure:-toxin rom S. aureus, PDB ID AHL,[]). Tese toxins integrate as stable channels into the lipid bilayer, thus creating an aqueousconnection between the cytosol and the extracellular space o the target cell.

1

2 3

F : Pore ormation is a dynamic process with structurally and unctionally distinct states. Distinct molecular states exist on the pathto membrane pore ormation by PFs. Te toxin is secreted by the bacterial pathogen into the extracellular medium in a water-soluble orm,usually as a monomer. Upon engagement o the membrane via binding to a receptor (step ), or example, a membrane lipid or protein, themonomers assemble to a prepore oligomer (step ). Te membrane beneath the prepore oligomer remains intact and is only punctured once

the preporereoldsto the membrane-inserted pore oligomer(step ). Tis stepusually goesalong with considerablestructural rearrangements.

acting essentially as amphiphilic suractants.-oxins romvarious staphylococcal species [,] and the cyclolipopep-tides romBacillus subtilisare prominent examples[] (seeFigure ). Second, toxins can act as phospholipases anddamage membranes by enzymatic hydrolysis o phospholipidester bonds. -Hemolysin rom S. aureus, or instance, isa sphingomyelin-specic phospholipase, which cleaves sph-ingomyelin to ceramide and phosphorylcholine. However,the large majority o membrane damaging proteins belong

to the class o pore-orming proteins/toxins (PFs). PFs,which make up approximately % o all protein toxins inpathogenic bacteria [], have evolved in all domains o lie.Tey are secreted as water-soluble proteins and subsequentlyintegrate into oreign membranes.

3. Mechanism of Membrane Pore Formationby Pore-Forming Toxins

Common structural themes o protein/membrane associa-tion are insertion o transmembrane -helices or -sheetbarrel arrangements, anchoring by prosthetic glycolipids, or

direct linkage to hydrophobic lipid tails, such as myristic orpalmitic acid. Most pores or channels allowing or commu-nication across biological membranes are ormed by integralmembrane proteins spanning the lipid bilayer. However, PFsorm pores by acting initially extraneously o the lipid bilayer.Tey start out as soluble molecules and then turn themselvesinto integral membrane proteins, with a membrane-spanningregion that denes the pore. Pore ormation is a dynamicprocess with structurally and unctionally distinct states (see

Figure ). Initial binding to the membrane, or example, to amembrane lipid or protein receptor, is ollowed by homotypicoligomerisation to a prepore state on the membrane surace(Figure , arrows and ). In this state, the protomer congu-ration resembles that o the soluble monomer, and the wholeoligomer still stands prone to the membrane with an intactlipid bilayer beneath the assembled ring. InFigure (a), thisis depicted or pneumolysin romStreptococcus pneumoniae,a close homologue o listeriolysin rom L. monocytogenesand, interestingly, also o perorin secreted by cytotoxic cells [], and o the complement membrane attack complex[, ], which indicates that bacterial attack and immunedeence employ the same mechanisms. Tis prepore state


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(a1)

(a2)

(b1) (b2)

(c1)

(c2)

(c3)

F : Structures o PFs that are important or neonatal sepsis. Panel (a) shows available structures o cholesterol-dependent cytolysinsto illustrate listeriolysins mechanism o pore ormation. (a) displays the crystal structure o the soluble, monomeric orm o perringolysinrom Clostridium perfringens (lef, PDB ID PFO, []). Te cryo-electron microscopy (cryo-EM) reconstruction o the prepore (EMdatabank: ) o the listeriolysin homologue pneumolysin romStreptococcus pneumoniae displayed on the right revealed that the protomerconguration in the prepore resembles that o the soluble monomer []. Lipid membrane is coloured yellow. Molecular modeling othe protomer tted into the cryo-EM pore structure below (EM databank: ) revealed the considerable structural rearrangements thataccompany membrane pore ormation. Te-helices that reold into-sheets are coloured in red. Panel (b) shows the different structuresavailable or ClyA romE. coli, (b) the soluble state (PDDid QOY, []) monomer and (b) a protomer rom the dodecameric pore state,which is shown as side and top view on the right (PDB ID WCD, []). Pore-lining-helices are in red and the -tongue in yellow. Panel(c) shows the PFs romS. aureus. (c) shows rom top to bottom LukF (PDB ID LKF, []), LukF-PV (PDB ID PVL,[]), and LukS-PV(PDB ID R, []). (c) shows the octameric pore structure o-hemolysin (PDB ID B,[]), protomer on the lef, side and top viewson the right. (c) displays the heptameric pore structure o the AF pore (PDB ID AHL, [ ]), individual protomer, side and tops views.Te-stem that unolds into the membrane lining, extended-hairpin is shown in red.

then undergoes drastic conormational rearrangements to beinserted as a stable pore into the membrane (see Figure ,arrow ). Tis rearrangement can even involve the reold-ing o -helices in the soluble state to -sheets in themembrane-inserted orm []. While this general mechanismo pore ormation can be proposed or nearly all PFs, thestructural changes, which the individual proteins undergo,remain largely elusive. Detailed mechanistic models on howhydrophilic proteins can suddenly change their solubility andintegrate into biological membranes are available only ora ew PFs involved in neonatal sepsis (see Figure ). Notsurprising when considering that one not only needs to be

able to study the solution state in sufficient structural detail,orexample, via X-ray crystallography, butthe structureo themembrane state also needs to be resolved. PFs are classicallydivided into two main groups based on the structural motisthat orm the pore[]. Pores can be ormed by-helices,-PFs or by-sheets, -PFs. For certain pore-ormingproteins, it was recently proposed that lipids might play adirect role in pore ormation, but our understanding o howthis can be achieved is limited by the available structural datato date[]. Te structures, where known, o the solubleand membrane states o the PFs discussed in this review aredisplayed inFigure .However, not all membrane pores areequal. Te pore diameter, or instance, o the -hemolysin

membrane heptamer is considerably smaller than that olisteriolysin with up to protomers (c. Figures (a) and(c)). Clearly, the size o the membrane pore has importantconsequences or the targeted cell, as a large pore diameteris not selective or what it can conduct across a membrane,potentially mediating diffusion o larger molecules, such asAP or even small proteins.

Interestingly, clearing o toxin pores rom host mem-branes, a mechanism that is o marked importance whenconsidering the amounts o toxin that are produced dur-ing sepsis, seems also to be size dependent. Cholesterol-

dependent cytolysins, such as listeriolysin, can induce Ca2+-

dependent resealing o membrane pores by induction oendocytosis[] but-hemolysin romS. aureuscannot[]. Tis is counterintuitive, as van der Goot and coworkersnicely state[] that small pores are harder to repair thanlarger ones.

4. Bacterial Pore-Forming Toxins inPathogens Causing Neonatal Sepsis

In the major pathogens isolated rom newborn inantswith sepsis, PFs are key virulence actors. Tey initiate amultitude o events ranging rom direct necrotic cell deaths


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to the induction o signalling cascades, or instance, Ca2+-mediated signalling []. Prominent PFs in the context oneonatal sepsis are listeriolysin O romL. monocytogenes,-hemolysin/cytolysin rom GBS (Streptococcus agalactiae),-hemolysin and cytolysin A rom E. coli, and -hemolysin,-hemolysin, and the leukocidins rom S. aureus. It has

long been appreciated that PFs are especially importantduring initiation o bacterial inections through induction onecrosis and apoptosis o host epithelial and endothelial cells,which promotes microbial invasion and subverts deencemechanisms. However, PFs may also contribute to sepsis byreceptor mediated or membrane damaging mechanisms inimmune cells, which respond with the ormation o inam-matory mediators, as was shown or-hemolysin/cytolysinrom GBS[] and listeriolysin O[,] and-hemolysinand Panton-Valentine leukocidins romS. aureus[,].

5.-Hemolysin/Cytolysin from

Group B StreptococciStreptococcus agalactiae(group B streptococci, GBS) are themajor cause o sepsis and meningitis in newborn inantswithout underlying disease in the western world. In addition,they are a signicant cause o invasive inections in pregnantwoman and immuneocompromised patients [, ]. Tepore-orming toxin-hemolysin/cytolysin is one o the main

virulence actors o GBS. It has been implicated in thepathogenesis o early- [] and late-onset neonatal sepsis,although its role in both cases remains controversial. Rabbitsinected with wild-type GBS had signicantly higherbacterialblood counts than those inected with GBS mutants lack-ing the-hemolysin/cytolysin []; mortality also increaseddramatically. Similarly, in a neonatal rat model o meningitiswild-type GBS induced more neuronal damage in the cortexand the hippocampus than cytolysin-decient mutants [].Te clinical outcome score, assessed in this study by weightedchanges and motor activity, decreased prooundly uponpresence o the cytolysin. Te -hemolysin/cytolysin lyticprotein agent is thought to be encoded by the cylE geneo the cyl operon [], since expression o cylE induces -hemolytic activity in nonhemolytic E. coli. However, the exactmolecular nature o the protein component responsible orhemolysis and pore ormation remains obscure, as it hasevaded purication to homogeneity as o yet. Interestingly,-hemolysin/cytolysin is also necessary or the synthesis

o an orange carotenoid pigment [], with which it alsoassociates []. Hemolytic activity o partially puried -hemolytic activity containing the carotenoid pigment couldbe inhibited by addition o the lipid dipalmitoylphosphatidyl-choline (DPPC), the major component o suractant in thelung. Accordingly, suractant deciency may explain in partthe particular susceptibility o preterm inants to GBS sepsisand meningitis [, ]. As outlined above, breaching oepithelial barriers by GBS is the rst step in sepsis patho-genesis. Accordingly, it appears to be important that -hemolysin/cytolysin mediates not only injury o lung epithe-lial [] and o lung microvascular endothelial cells [] andinvasion o brain endothelial cells [] but also injury o

proessional phagocytes [] and neurons []. Moreover,GBS mutants lacking clyE were more readily cleared rommouse and human blood []. Interestingly, Rubens andcolleagues initially proposed that-hemolysin/cytolysin wasno longer needed or systemic disease maniestation once theepithelial barriers have been breached []. However, pro-

and anti-inammatory activity o-hemolysin/cytolysin inmacrophages was demonstrated, indicating more prooundimmunomodulatory unctions o the cytolysin [, ].One direct or indirect molecular-hemolysin/cytolysin tar-get with important implications or mononuclear phago-cyte activation is the NLPRP inammasome. Activationo the NLRP inammasome requires GBS expressing -hemolysin/cytolysin. Tis pathway is essential in a mousemodel o GBS sepsis, where deciency in NLRP or its sig-nalling partners apoptosis-associated speck-like protein andcaspase- increases lethality and bacterial dissemination [].Yet direct evidence or binding, engagement, and activationo LRs by the-hemolysin/cytolysin is not available.

A second pore-orming toxin o GBS is the CAMP actorthat has long been used or microbiological identicationo GBS, since it characteristically synergizes with secretedsphingomyelinase oS. aureusto lyse erythrocytes on bloodagar plates []. However, its role in neonatal sepsis is notclear, as it was not required or systemic inection in a mousemodel o GBS inection [].

6. Listeriolysin O fromListeria monocytogenes

Listeriamonocytogenes (L. monocytogenes) is a Gram-positivebacterium that causes early- and late-onset neonatal sep-sis and meningitis. L. monocytogenes has the capacity tobreach the intestinal barrier, thereby causing ood-bornelisteriosis, the blood-brain barrier, causing meningitis, andthe maternal-placental barrier, causing early-onset listerio-sis. Listeriolysin O (LLO), a member o the PF class ocholesterol-dependent cytolysins (CDCs), is a major viru-lence actor oL. monocytogenes with multivalent unctions[]. In the late s, Kathariou et al. and Portnoy etal. reported that L. monocytogenes mutants lacking unc-tional LLO were avirulent in mice [, ]. Furthermore,LLO mutants did not induce secretion o cytokines suchas NF-, IL-, or IFN-, when injected intravenouslyinto CBL/ mice []. Recently, a single-gene signature-

tag-based approach was used to assess the contribution oindividual amino acids o LLOs to its virulence in mice [].

Based on structural homology with other toxins such aspneumolysin rom Streptococcus pneumoniae [] and per-ringolysin rom Clostridium perfringens [], common pore-orming properties can be proposed [] (seeFigure (a)).LLO engages cholesterol as a native membrane receptorin dependence on the two amino acids threonine andleucine , oligomerises to a prepore complex o up to monomers, and orms a membrane pore in a concertedreolding step with each protomer contributing two beta-hairpins to the membrane-spanning -barrel, which origi-nates rom ve-helices in the soluble state[,]. Te


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allosteric monomer assembly and prepore reolding processwere recently shown to rely on an undecapeptide sequence(-ECGLAWEWWR-), which was originally thoughtto be soley responsible or cholesterol binding []. Teexact nature o the membrane pore remains controversial,as arciorm pores, that is, membrane pores with a seemingly

incomplete protein ring lining the aqueous membrane hole,are ofen observed in electron microscopic and atomic orcemicroscopic imaging o various CDCs [, ]. Moreover,LLO does not lose its membrane targeting properties aferincubation with cholesterol[].

Listeria are classical intracellular pathogens [] and LLOpore ormation was traditionally thought to only mediateescape oListeriarom the phagolysosome []. Tis conceptwas based on the nding that LLO was active only at acidicand not at neutral pH, which is ound in the maturingphagolysosome []. However, host actors also play animportant part in regulating the activity o LLO in thephagolysosome. LLO hijacks the reductive capacity o the-intereron-inducible lysosomal thiol reductase GIL tomaintain cysteine o LLO in its reduced thiol state [],thus greatly increasing bacterial escape rom the phagolyso-some. Intriguingly, CDCs were originally, that is, beorethe identication o cholesterol as a membrane receptor,termed sulydryl-activated[] or thiol-activated, oxygen-labile cytolysins [], as chemical reduction activated thetoxins towards hemolysis o red blood cells. Additionally,the cystic brosis transmembrane conductance regulator(CFR), which transports chloride not only across the apicalplasma membrane o epithelial cells in the lung but alsointo the phagolysosomes o macrophages, potentiates LLOoligomerisation on the phagosomal membrane and its lyticactivity and phagolysosomal escape oL. monocytogenes [].However, the role o LLO extends beyond mediating phago-somal escape. LLO reduces ormation o reactive oxygenspecies (ROS) by inhibiting the NADPH oxidase NOXin RAW . macrophages []. Tis activity seems torely on pore ormation in the phagosomal membrane andprevents degradation o bacteria inside the phagosome. Poreormation at the plasma membrane o target cells inducesthe dynamin-/F-actin-dependent but clathrin-independentuptake o L. monocytogenes into HepG cells []. Tisnding questions the traditional model o LLO pore-ormingactivity being strictly dependent on the low phagosomal pH[], whereby premature lysis o target cells by (secreted)LLO is prevented[]. Residual lytic activity and structural

integrity o LLO at neutral pH [,,] are in line withLLO-mediated calcium inux into epithelial Hep- [] andHEK cells [] along with concomitant L. monocytogenesuptake. Indeed LLO seems to orm pores at neutral pHin the plasma membrane, which do not result in lysis othe target cell but rather in uptake o the pathogen [].As pneumolysin rom S. pneumoniae can replace LLO inthe uptake o L. monocytogenes into HepG cells, CDCsrom other bacterial pathogens may similarly induce cellularuptake. Te contribution o LR signalling in responseto CDC has been subject o several studies. As examples,the LLO homologues anthrolysin (Bacillus anthracis) andpneumolysin can signal via LR [, ]. On the other

hand, LLO induces an inammatory cellular response in aLR-independent ashion []. Moreover, LLO activity atthe plasma membrane induces clustering o lipid rafs[],suppressiono antigen-induced -cell activation [], inam-masome activation, and histone H dephosphorylation [],all o which might contribute to sepsis progression either at

the stage o heightened inammation or at later stages oimmune suppression.

7.-Hemolysin,-Hemolysin, and LeukocidinsfromStaphylococcus aureus

Staphylococcus aureus (S. aureus) is well recognised as asignicant cause o neonatal sepsis []. Around ten bacteriaare sufficient to colonise the umbilical cord. Afer birth, S.aureuscan colonise the upper respiratory tract in up to %o inants []. S. aureus produces a number o PFs withdistinct specicity or target cell membranes. Although mostclinical isolates produce the PFs -hemolysin, bicomponent

-hemolysins, and bicomponent leukocidins, none o these

toxins was ound to be a necessary and sufficient virulencedeterminant o neonatal sepsis. In contrast, other actorssuchas the antigenic, peptidoglycan-associated protein A [],superantigens [], and sphingomyelinase C (-hemolysin,[]) contribute to host invasion, subversion o the immunesystem, and sepsis maniestation. However, there is evidencethat pore ormation by -hemolysin (-toxin, AF) con-tributes to the pathogenesis o sepsis []. As an example,erythrocyte lysis by AF could be directly imaged [].Downregulation o AF expression in vivo clearly reduces

virulence oS. aureus [, ]. In a model using CBL/Jmice, AF activates the NLRP inammasome, thereby pro-moting necrotising pneumonia []. Moreover, monoclonalantibodies to AF are protective against staphylococcal pneu-monia[]. Indeed, a nonhemolytic variant o AF was usedto vaccinate rabbits, and antisera could be used to passivelyimmunize mice against an otherwise lethal challenge withwild-type S. aureus []. In a mouse model o mastitis,coagulase and AF proved to be the primary virulence deter-minants[]. Heat-inactivatedS. aureusand an AF mutantgreatly reduced the bacterial burdens in a mouse brainabscess model and attenuated the expression o inammatorymediators [].Inan in vivo model o corneal virulence, AFalso proved to be a decisive virulence actor [].

AF is one o the best-studied PFs to date. It was therst toxin o which the membrane structure was solved by

X-ray crystallography (seeFigure (c),[]). AF consistso amino acids and oligomerises on the plasma mem-brane o target cells to a heptamer (potentially a hexamer)prior to membrane insertion and pore ormation. It wasshown to have an important role in bacterial pathogenesis,especially by its ability to induce necrotic cell death [ ],by which it can cause vascular leakage when perused intothe lung[]. Recently the metalloprotease ADAM wasidentied as the membrane receptor o AF []. At lowtoxin concentrations, ADAM is required to mediate thecytotoxic effects o AF. Interestingly, binding o AF toADAM resultedin the upregulation o ADAM in alveolarepithelial cells and concomitant cleavage o E-cadherin. Tis


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leads to epithelial barrier disruption thereby aggravatingstaphylococcal pneumonia in mice []. Moreover, activa-tion o the NLRP-inammasome by AF might contributeto later stages o sepsis, although the molecular mechanismunderlying inammasome activation remains elusive at thisstage []. In this respect, it is interesting to note that, whereas

direct LR activation has not been demonstrated or AF,NOD-dependent sensing oS. aureus was dependent onAF [].

Next to AF,S. aureusexpresses the bicomponent cyto-toxins leukocidins (Luk) and the-hemolysins (Hlg). Bicom-ponent implies that class S toxins (LukS-PV, LukE, HlgA,HlgC, and LukS) have to associate with class F toxins (LukF,LukD, LukF-PV, and HlgB) in a : stoichiometric ratio toorm a unctional oligomer beore insertion into the mem-brane (see Figure (c)). Pathogenic S. aureus can produceseveral different bicomponent toxin pores, among the mostprominent are LukE/LukD [], Panton-Valentine leuko-cidin LukS-PV/LukF-PV, -hemolysins LukF/HlgA [],HlgA/HlgB, HlgB/HlgC, and the M/F-PV-like leukocidins,all o which might be expressed at different stages duringsepsis. Te Panton-Valentine leukocidin (PVL), which wasrst isolated rom uruncles in [], is probably themostwidely studied member. In a mouse model, secreted PVL pro-motes tissue invasion and causes necrotizing pneumonia, viamechanisms including upregulation o protein A and otheradhesins[]. PVL is an important actor in the early stageso skin inection, as shown in a rabbit skin inection model[]. Association opvl and spa (protein A) genes seemsto be an important virulence determinant in methicillin-resistant S. aureus (MRSA). Moreover, PVL was reported todirectly bind to LR and induce inammation in the mouselung []. LukE/LukD promotes systemic bacterial growth invivo by specically targeting neutrophils []. Interestingly,

via engagement o their native receptor CCR (CC-moti-chemokine-receptor type ), LukE/LukD toxin pores clearantigen-presenting cells (macrophages and dendritic cells)and S. aureus-specic CCR-positive T/T cells [],thus greatly contributing to the spread oS. aureus in the host.

AF, Hlg, and the leukocidins belong to the class o-pore-orming toxins []. Despite their moderate sequenceidentity (around % or the pairwise alignment with AF),they share a common structural old (see Figure (c)). Testructures o the soluble, monomeric LukF (Figure (c),top, []), LukF-PV (Figure (c), middle, []), LukS-PV(Figure (c), bottom, []), and the octameric, membrane

complex o Hlg (LukF/HlgA, Figure (c), []) are available.A common molecular mechanism could be proposed inwhich a prestem, triple stranded -sheet in the solublemonomer, reolds to a double, stranded, membrane-inserted-sheet (Figures (c) and(c), lef), resulting in a hexadeca-or tetradecastranded-barrel that penetrates the membrane.

Te target membrane specicities oS. aureusPFs hinttowards their role in neonatal sepsis. Whereas AF has abroad specicity and might thus be involved at the initialstages o sepsis maniestation, where the lung epitheliumneeds to be breached, bicomponent leukocidins and Hlgsmainly attack polymorphonuclear neutrophils, macrophagesand lymphocytes [,]. Bearing in mind that PVL does

not attack lymphocytes and Hlg can be hemolytic, at leastin vitro, their restricted but highly directed mode o attackpredisposes the bicomponent leukocidins to be importantactors or the subversion o the immune system once theinitial barrier has been breached.

8.-Hemolysin and Cytolysin A fromEscherichia coli

Pathogenic Escherichia coli (E. coli) cause around % oinvasive neonatal sepsis [], and antibiotic resistance isan emerging threat in this context []. Generally, E. colican persist in the intestine as a normal constituent o theintestinal microbiota. However, extraintestinal pathogenicE.coli(ExPECs) are the most common gram-negative bacterialspecies isolated rom neonates with bacterial inections, andneonatal mortality rom gram-negative sepsis remains high[]. Urinary tract inections o pregnant women can leadto aspiration o ExPECs during partition with subsequentuncontrolledgrowth in the lung o the newborn andpotentialprogression to a systemic inection. Pathogenic E. coliofensecrete the pore-orming toxin-hemolysin (HlyA, CylA), a kDa member o the RX class o toxins [], which isusually associated with strains causing uropathogenic inec-tions []. Deletiono HlyA in E. coli greatly reduced mortal-ity and cytokine production as compared to the isogenic wildtype bacteria in an intravenous inection model[]. HlyAurthermore induced hemorrhagic bleeding o bladder tissueand exoliation o urothelium when pathogenic bacteria wereadministered into the urethra[]. HlyA is encoded by atleast % o all ExPEC clinical isolates []. Around %o meningitis- and sepsis-associatedE. colibelong to the Kserotype [, ]. Te E. coli K strain RS expressed

HlyA in a zebrash model o systemic inection [] andthe hlyA gene was present in more than % oE. coliromthe genital tracts o pregnant women []. As a member othe repeats in toxin (RX) amily o hemolysins, the hlyAgene is part o the chromosomal hlyCABD operon, whichalso encodes a type ABC transporter or secretion o thePF. Te amino acid toxin repeats,which are locatedin the C-terminal portion o the protein,are composed o the sequenceGGXGCDXUX (with U being a large hydrophobic residue).

Tese repeats are responsible or Ca2+ binding, which is aprerequisite or membrane association and pore ormationby the N-terminal, hydrophobic, and acylated domain oHlyA []. Interestingly, other pore-orming proteins, such

as the human cytotoxic lymphocyte encoded perorin, alsorequire binding o Ca2+ or membrane association and poreormation []. HlyA oligomerises on the plasma membraneo target cells, where it accumulates in cholesterol-richmicrodomains [, ]. However, rather than cholesterolbeing a direct, lipid membrane receptor as in the case othe CDCs listeriolysin or pneumolysin, cholesterol seems tocontribute to the physicochemical environment necessaryor HlyA membrane engagement. Te exact nature o thepore ormation mechanism is under considerable debate. Itseems now accepted that HlyA orms membrane pores as anoligomer, at least in articial membrane mimics[],albeit possibly heterogeneous in size []. In this respect,


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it is intriguing that the PX receptor and pannexin wereound to mediate HlyA-dependent pore ormation [,].Sublytic doses o HlyA initiate degradation o paxillin andproteolytic cascades inside epithelial cells and macrophages,thus attenuating the inammatory host response and pro-moting epithelial exoliation []. Similarly, HlyA inhibits

epithelial cytokine production potentially promoting epithe-lial invasion oE. coli[].Another PF oE. coli is ClyA (also termed hemolysin-

E or SheA), which is expressed by various pathogenicand nonpathogenic E. coli including K strains, that arealso ound in clinical isolates o neonatal meningitis [],bacteremia [, ], and neonatal sepsis []. However,three neonatal meningitis K strains isolated by Ludwigand colleagues harboured deletion mutations at the clyAgene locus []. Nevertheless, a synergistic enhancemento extraintestinal inection was recently reported betweennonpathogenicE. coliK and pathogenic ExPEC strains ina mouse model o septicaemia [], which hints towardsa potential involvement o ClyA. ClyA is a kDa proteinbelonging to the class o-PFs. Te mechanism o poreormation is well understood, as crystal structures or thesoluble [] and membrane state [] are available (seeFigure (b)). Upon association with the membrane, insertiono the so-called -tongue induces a series o substantialstructural rearrangements in the now membrane-anchoredmonomer, resulting in a perpendicularposition to membranewith the amphiphatic helix a now lying along its surace.Afer oligomerisation to a dodecamer, helices a becomeinserted into the membrane orming a A hollow cylinderwith a A aperture protruding through the membrane.

9. Synopsis and Medical OutlookNeonatal sepsis is a syndrome caused by systemic inam-mation and dened by clinical criteria such as tachycardia,respiratory distress, temperature instability, and unusuallyhigh amount o immature immune cells in the blood.Pathogenic bacteria that are aspirated by the etus in thebirth canal during parturition can cause neonatal sepsis itheir inection is not controlled locally by the innate deencemechanisms o the respiratory and alveolar epithelia. oxins,either proteinaceous or o other molecular nature, are impor-tantactors contributing to neonatal sepsis. Whileendotoxinssuch as the lipopolysaccharide (LPS) rom Gram-negativebacteria contribute to the sepsis phenotype by activating

monocytes and macrophages via oll-like receptor binding,pore-orming proteinaceous exotoxins act by permeabilisingtarget membranes o host cells. Whereas the membranetargeting effects o PFs, that is, engagement o membrane,oligomerisation, and pore ormation, are well dened, theirsecondary downstream effects are maniold, owing to the actthat dened ionic and molecular gradients across cellularmembranes modulate a diverse set o signalling cascades.Unregulated cell death and its consequences are importantin neonatal sepsis []. Te PFs described above can causedirect necrotic cell death, which in the case oE. coli, GBS,and S. aureus contributes to overcoming the epithelial andendothelial barriers in the lung. LLO oL. monocytogenesis

critical or cell invasion and cell-to-cell spread o this intra-cellular pathogen and thus also contributes to breaching theepithelial barriers o the host. Necrotic cell death can releaseproinammatory cytokines rom leukocytes thus contribut-ing to the inammatory storm during sepsis. Interestingly,several PFs discussed above can induce the inammasome:

AF[], -hemolysin/cytolysin [], LLO [,], HlyA[], and leukocidins [, ]. Inammasome activationultimately may contribute to hyperinammation in newborninants similar to what has been shown in GBS and E. colisepsisin mice [, ]. PFs canalso elicitand alter apoptosiso host immune cells, that is, AF via caspase- [,],leukocidins via activation o caspases and [], LLO

via release o cytochrome C rom mitochondria [], -hemolysin/cytolysin independently o caspase activation [,],HlyA[],and ClyA [], thus potentially contributingto theapparent immunodeciencyo the patient that is char-acteristic during stages o sepsis []. Despite the ormationo hydrophilic channels by all PFs, the ways in which theyinduce the inammasome are varied, hinting towards theact that unctions o PFs at cellular membranes are moresubtle than might be expected rom their general modeo action. In this respect, effects o sublytic concentrationso PFs have recently been explored. For instance, sublyticdoses o LLO induce mitochondrial network disorganisationwith transient alteration o the metabolic state o the targetcell, thus weakening the cell or L. monocytogenes entrywithout destroying it []. Moreover, targeting o organs byPFs might also contribute to the septic phenotype. GBS-hemolysin/cytolysin, or example, had marked effects oncardiomyocyte contractility and viability [].

Due to their role in neonatal sepsis andbacterial inectionin general, PFs present attractive therapeutic targets. Incases where membrane receptors have been dened, specicinhibitors, akin to viral entry inhibitors, might preventmembrane binding and pore ormation. Monoclonal anti-bodies against PFs that prevent membrane binding and/orreolding to the pore state could be a way o neutralising thetoxin, at least in the blood stream. Moreover, vaccines basedon PFs may be used or immunizing women and therebyprotecting newborn inants through placental transer ospecic immunoglobulins, given the act that pneumolysinromS. pneumoniaeis considered a vaccine candidate [].Indeed, novel vaccines based on AF are currently developed[]. As PFs elicit specic cellular responses, it might,however, also be promising to designing therapeutics based

on the pathways that the toxins induce in the target cell, ashas been proposed or pMAPK and-hemolysin/cytolysinrom GBS []. In any case, it is exactly these cellularresponses towards PFs that need to be investigated in thecontext o neonatal sepsisin theuture to improve therapeuticstrategies.

Acknowledgment

Tis work was supported by a Grant rom the GermanBundesministerium ur Bildung und Forschung (BMBF EO ).


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