Volume LXXI, 2002, no. 1 VOJENSKÉ ZDRAVOTNICKÉ LISTY - SUPLEMENTUM 39

MICROBIAL PROTEINS: FROM STRUCTURE TO PATHOGENICITY — GROELAS AN EXAMPLE .

lAles MACELA, lLenka HERNYCHOVA, 2Jana HAVLASOVÁ, 2Jiři STULÍKlInstitute Of Nuclear and Biological Medical Defence, Purkyně Military Medical Academy,

Hradec Králové, Czech Republic

2Proteome Center for the Study Of Intracellular Parasitism of Bacteria, Purkyně Military Medical Academy,Hradec Králové, Czech Republic '

Introduction

Genomic and proteomic analysis bring to bio-medical labs qualitatively new technologies, whichenable the complex look to genes and gene-relatedproducts — proteins. In contrary to genes, whichnumber is definitive for given living system, thenumber of protein species varied substantiallyaccording the immediate living conditions of the _system studied. Moreover, most Of the proteinshave the function associated With the tertiary Struc-ture Of their mOlecule, e.g. with the space Orientationso called folding and intracellular compartmentwhere are they located. In in vitro Systems the pro-cess Of folding is inefficient and Only minority Ofsynthesized molecules have correct shape. In vivomost mOlecules mustrapidly fold to proper tertiaryconformation, otherwise unfolded and misfoldedproteins would be degraded. The promotion of pro-tein folding is most probably the chaperones andchaperonins role. These molecules create a familyof conserved proteins found in all compartments ofprokaryotic and eukaryotic cells. Moreover, in invı'vO systems synthesized and folded proteins mustoccupy the proper intracellular niche to expresstheir function. Very frequently the intracellularlocation, in connection with posttranslational modi-fication ofproteins produced, dictated the spectrumof their biological functions. This assumption isgenerally accepted and is valid for chaperonins aswell. Recent report accentuate the evaluation of .proteomic results obtained from extensive bacterialanalysis On the example Of GroEL protein With res-pect to structure-function relationship and to intra-cellular localization of individual protein specieswhat might have significant impact for the under- ’standing of pathogenicity of bacteria.

Molecular chaperones and chaperonins

. According their function and Space Orientation,chaperones can be divided into two chaperone clas-ses. Chaperones that bind and stabilize a widerange of unfolded or partially folded proteins, pre-

vented them from being degraded, are called mole-cular chaperones. The function of molecular chape-rones is to bind to all nascent polypeptide chains asthey are synthesized on ribosomes. Chaperonesexerted the protective function in cytosol, mito-

_ chondrial matrix, chloroplasts, and nucleus of thecell. Molecular chaperones are represented by theheat shock protein 70 family of proteins, whichincludes Hsp70 in the cytosol and mitochondria, Bipprotein in endoplasmic reticulum, and DnaK cha-perone in bacterial cells. The second group of cha-perones represents chaperonins. Chaperoninsdirectly facilitate the folding of proteins. The typi-cal representative of chaperonins is bacterialGroEL molecule that creates multimeric barrel-sha-ped complexes composed from 14 identical subu-nits (Fig. l). The partially folded or misfolded pro-teins enter the cavity of GroEL complex, wherethey bind to inner wall and undergo the process offolding. The exit of folded protein from GroELcavity is an ATP-dependent process assisted by co-chaperonin GroES. The identical function has theTCiP molecule in eukaryotic cells. TCiP is thus theeukaryotic homologue of bacterial GroEL molecu-le with one substantial difference —- TCiP lacks theanalogue of GroES co-chaperonin. The parametersofGroEL/GroES cavity were estimated to be 18.4 nmlong and 3.3 nm in diameter, what enables thefolding of Small- and medium-Sized proteins [forreviews See publications 1 or 2-].

Folding of proteins and something more

Generally, the protection and folding of proteinsis only a basic but not exclusive function of chape-rones. Another fimction, which is related tO thecommon property Of chaperones to interact withproteins, is a selective transport of proteins among

- individual cellular compartments. The chaperonesof the Hsp70 family within eukaryotic cells excel inthis respect. The transportation of pre-proteins

~ through the mitochondrial membrane is facilitatedby their binding to mitochondrial Hsp70, which

40 VOJENSKÉ ZDRAVOTNICKÉ LISTY - SUPLEMENTUM Volume LXXI, 2002, no. ._ı

Fig. 1: Horizontal (A) and vertical (B) view on the model ofFrancisella tularensis strain SCHU GroEL molecule. Model was createdusing the Deep View Swiss-d Viewer — Swiss-Model program version 36. 0002.

limit the dependency of the transport on the membra-ne potential of mitochondria [3]. The chaperone--mediated autophagy is another example of selectivetransport associated with the function of chaperones.In the frame of this process, the cytosolic proteins aretransported to lysosomes to be degraded. The chape-rones ofHsp70 type are associated With the lysosomalmembrane from the cytosolic site and together withother molecular chaperones (Hsp90, Hsp40, Hop, Hip,and BAG-1) create a molecular complex that facilitatethe transport across the lysosomal membrane [4].

Molecules of HSp60 family have apparentlybroader spectrum of functions, which originatedfrom several forms of HSp60 molecule and theirsubcellular and extracellular locatiOns recognizeduntil now. Based on original studies of GroEL inbacteria and yeast, Hsp60s were thought to residein cytoplasm and/or stroma oforganelles. This notionwas further supported by the fact that no member ofGroEL family possesses the leader sequence or othermotifs, which. predict the molecule to be located inthe cell membrane or to be secreted. However, anumber of recent studies documented an extracyto-plasmic location for chaperonins in bacteria [5, 6].Membrane-associated localization of bacterial cha-peronins were reported in Salmonella typhimurium[7], Mycobacterium leprae [8], and several othermicrobes [9, 10]. Moreover, under the in vitro con-ditions L. pneumophila Hsp60 molecules are asso-ciated predominantly with the bacterial cell wallmainly and transported across the bacterial mem-brane and accumulated inside the vesicles of euka-ryotic host cells where the Legionella pneumophilaresides [11]. Proteome analyses ofFrancisella tula-rensz’s LVS whole cell lysates revealed the domi-

nant expression of several Hsp60 molecule speciesknown as CH6OFRATU under the accession num-ber P94798 (see Fig. 2). Western blot analyses ofouter membrane proteins extracted from Francisel-la tularensis LVS microbes with subsequent immu—nodetection with tularemia patient sera and combi-ned with identification of proteins using massspectrometry revealed the CH6OFRATU moleculesamong the integral membrane proteins (Fig. 3).

As the existence of multiple protein species of'HSp60 molecule differing in pl and molecularmass, and according the localization inside and out-side the bacterial cell seems to be proved from pro-teome studies, the functional profile of individualprotein. species should be determined. The mem-brane-associated molecular form will have probablydifferent structure and different biological func-tion than cytosolic or secreted ones. The main bio-logical function accomplished by cytosolic form ofchaperonins was mentioned above. The recognizedfunctional profile of membrane-associated stressproteins covers the adherence of bacteria to struc-tural components ofeukaryotic cell membranes andthe promotion of bacterial invasiveness, whichaccompanies the process of adhered bacteria inter—nalization. This can be demonstrated by the fact thatvarious stresses augmented the adherence of bacteria

' to cultured cells. The implication ofGroEL moleculesat the process of cell adherence was demonstratedfor microbes Haemophilus ducreyi [12, 13], Heli-cobacterpylori [14], and Clostridium a’ififzcile [15].

Among the ligands for surface-exposed Hsp(s)recognized till now are sulfoglycolipids for 70-kDa--related heat Shock protein of Haemophilus influen-zae [16], membrane sulfated glycolipids for Hsp70

Volume LXXI, 2002, no.1 vOJENSKÉ ZDRAVOTNICKÉ LISTY - SUPLEMENTUM 41

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Fig. 2: Identification OfFrancisella tularensis Cpn60 molecule. (A) Two—dimensional gel electrophoretogram with identified spot con-taining Cpn60 molecules. (B) Mass spectrum obtained by MALDI TOF analysis ofSpot marked in (A)

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ofHelicobacterpylori [17], galactosylceramide for62 kDa Hsp ofBorrelia burgdorferi [18], and com-ponents of intestinal mucus for membrane associa-ted 66-kDa stress proteins of Salmonella typhimu-rium [19]. However, none of them was explicitlyproved to be the ligand for Hsp60 family members.The protein receptors with signaling function, asare Toll-like receptors or CD14, engaged in theinduction of innate immune response rather thanstructural components of surface membranes or

extracellular matrix seems to have the capacity tobind effectively 60 kDa family stress proteins ofbacterial as well as eukaryotic origin [20, 21].

In addition to the function in bacterial adherenceto eukaryotic cells, the surface-associated Hsp60 cha-peronin of Legionella pneumophila was demonstra-ted to be a bacterial invasin, which promotes invasive-ness of two virulent Legionella strains and in in vitroartificial system predetermine the shape ofendosomesin which the particles expressed GroEL resided [22].

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Protein Structure, pathogenicity Of infectionand autoimmune diseases

The consequence of bacterial stress proteininteraction with cell surface receptors after subse-quent signal transduction is a functional activationof cell. The proliferation cell response [20], as wellas pro-inflammatory cytokine production [23],enzyme secretion (metalloproteinase) [24] and phe-notype transformation (expression of adhesionmolecules) [25] was reported as a cell reaction tothe bacterial heat shock 60 kDa proteins. All activi-ties mentioned above are prerequisite for the properfunctioning of innate immune mechanism that createsfirst barrier against invaded microbes. Macropha-ges, once infected, produce pro-inflammatory cyto-kines, attract the lymphocyte subpopulation to thesite of infection, and present peptides to correspon-ding T lymphocyte subsets. The epitopes of bacte—rial Hsp60 can be presented by the MHC class Imolecules or by non-polymorphic class lb molecu-le Qa—l. The activated CD8+ cytotoxic T lympho-cytes recognized the bacterial peptide derived fromHsp60 in the context of self-MHC molecule andcross-recognize also a homologous peptide derivedfrom mammalian Hsp60 [26, 27]. It seems likelythat the surface expression or secretion of heatshock proteins is common immediate response ofboth interacting organisms and can be consideredas a “danger” signal for defence systems. Due tothe homology of this phylogenetically conservedproteins there are the identical epitopes on peptidesderived from prokaryotic and eukaryotic Hsps [28].Cross-recognition of self-Hsps peptides expressedon stressed cells, based on the stress induced byinfection and activation of relevant lymphocyteclones by prokaryotic molecules, is an unwantedtribute of effective anti-bacterial immune responseof organism, which might constitute the linkbetween infection and autoimmunity. The complexeffect of both chlamydial and human Hsp60 duringthe atherogenesis was recognized recently what canserve as an example and can help to understand theassociation of infections with pathophysiology ofsever human illnesses [29].

Moreover, when we consider the role of Hsp60molecules for the pathogenesis of infections andtheir consequences, we should still take in mind thechaperonin function. The example of chaperone’ssubstantial role in the pathogenicity of infection istheir involvement in the formation of beta-sheetaggregate of prion protein (PrPSc), which is theinfectious form derived by conformational conver-sion from PrPC, largely alpha-helical isoform ofprion protein. The efficacy to bind and to convertthe PrPC into protease-resistant form PrPSc was

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demonstrated for eukaryotic chaperonin and bacterialGroEL molecules as well [30, 31]. In spite of the arti-ficial system, this data documented the broad range offunctional activities of bacterial GroEL molecules.

Conclusion

The members of Hsp60 chaperonin family aremultifunctional molecules, which are over-expressedin prokaryote as well as eukaryote cells in responseto stress conditions. Bacterial GroEL molecules haveprobably the function associated with the preciselydefined structural forms (protein species) and theirlocalization inside or outside the bacterial body.Until now, we have a limited knowledge about thenumber of protein species, which are coded by gra-ESL gene and we rather assess the spectrum of acti-vities associated with this gene products. On theother hand we anticipate the deep impact of bacte-rial chaperonine molecules to the pathogenesis ofhuman illnesses. From these reasons, the care shouldbe paid to detailed structure-function relationshipstudies of individual candidate protein species. In thisrespect, the proteome study of individual subcellu-lar compartments, which can contain the microbialcomponents, can bring decisive knowledge to theunderstanding of pathogenicity of infections.

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Acknowledgement: The presented study was fully supported byMinistry OfEducation, Youth and Sport, grant No. LNOOA 033.

Correspondence: Aleš MacelaInstitute of Nuclear and Biological MedicalDefencePurkyně Military Medical AcademyTřebešská 1575500 01 Hradec KrálovéCzech Republic

Received 16. 9. 2002