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2 0 08 ---Folia Biologica et O ecologica 4: 5 - 2 4
(Acta Univ. Lodz., Folia Biol. et Oecol.)
An t o n i Ró ż a l s k i
D e p ar tm en t o f Im m u n o b io lo g y o f B acteria , Institu te o f M icro b io lo g y and Im m u n o lo g y , U n iv ersity o f Ł ó d ź, 12/16 B an a ch a S tr., 9 0 -2 3 7 Ł ó d ź . P o la n d ; e-m a il: ro za la @ b io l.u n i.lo d z.p l
LIPOPOLYSACCHARIDE (LPS, ENDOTOXIN) OF PROTEUS BACTERIA
- CHEMICAL STRUCTURE, SEROLOGICAL SPECIFICITY
AND THE ROLE IN PATHOGENICITY
A b s tra c t: Lipopolysaccharides (LPS) o f Gram -negative bacteria are com posed o f three regions: О-specific chain (OPS), the core oligosaccharide and lipid A. All three regions of Proteus LPS were studied. T he differences in the structure o f OPS serve as the basis for the serological classification o f P roteus strains. T he serological classification schem e o f these bacteria currently consists o f 76 serogroups. The structural diversity o f the core region is characteristic for Proteus sp. and distinguishes this genus from other bacteria. In this paper the results of structural, imm unochem ical and serological studies o f all three regions o f P roteus LPS, as well as a function o f LPS as endotoxin and its role in the form ation o f urinary stones, swarm ing phenom enon and bacterial growth in biofilm are reported.
Key w ords : bacterial lipopolysaccharide, endotoxin, Proteus.
1. IN T R O D U C T IO N
The genus Proteus was described in 1885 by HAUSER and originally had
two species P. mirabilis and P. vulgaris. It belongs to the Enterobacteriaceae family and currently consists of five species: P. mirabilis, P. vulgaris, P. penneri,
P. hauseri and P. myxofaciens, a well as three unnamed Proteus genomospecies
4, 5 and 6. Proteus myxofaciens is the only Proteus species without any significance in the pathogenicity of humans, it has been isolated form living
and death larvae of the gypsy moth Porthetia dispar ( J a n d a , A b b o t 2 0 06 ).
Proteus rods are widely distributed in the natural environment where they are
involved in the decomposing of the organic matter o f the animal origin, they are also present in the intestines o f humans and animals. They are opportunistic bacterial pathogens which under favorable conditions cause urinary tract infections (UTI), wound infections, meningitis in neonates or infants
and rheumatoid arthritis (MOHR O ’H a r a et al. 2000; J a n d a , A b b o t 2006). UTI caused by these bacteria usually take place in patients with urinary catheter in place or with structural and/or functional abnormalities in urinary tract, as well as after surgical intervention in the urogenital system. Proteus rods are also associated with nosocomial infections. P. mirabilis causes UTI with the highest frequency among all Proteus species. It causes complicated infections and infections in long catheterized patients. Proteus rods can cause hematogenous infections and ascending infections, however, the latter are more common for
these microorganisms ( W a r r e n 1996).
The most characteristic features of Proteus bacteria is their ability to swarm
on solid surfaces ( B e l a s 1996). Swarming results from the bacterial
transfor-mation of peritrichously flagellated short rods called swimmer cells to elongated, multinucleated nonseptated forms with increased number o f flagella termed as swarmer cells. Swimmer cells grow in liquid media, whereas swarmer cells are formed in solid media. Population of swarmer cells can migrate in coordinated way on the solid media and then disintegrate into short rods. The process of differentiation of swimmer cells to swarmer cells, their migration on solid media and disintegration to short rods, known as swarming phenomenon or
swarming growth, is cyclical ( R a t h e r 2005). Both morphologically and
phy-siologically different cells - swimmer short rods and swarmer elongated cells play an important role in pathogenesis. Highly flagellated swarmer cells are thought to be crucial in ascending UTI infections, whereas short rods containing
fimbriae are responsible for colonization of host mucosal surface ( B e l a s 1992).
Proteus rods have evolved multiple virulence factors which act in concert
but not individually. These are fimbriae, flagella, enzymes (urease, hydrolyzing
urea to C 02 and NH3; proteases degrading antibodies, tissue matrix proteins
and proteins of complement system; deaminases hydrolyzing amino acids to a-keto-acids, playing a role of siderophores binding iron) and toxins -
hemo-lysins as well as endotoxin - lipopolysaccharide (LPS) (COKER et al. 2000;
M o b l e y 1996; R ó ż a l s k i et al. 1997; R ó ż a l s k i 2002).
2. L PS - G E N E R A L IN FO R M A T IO N
Lipopolysaccharide of Gram-negative bacteria are composed of three gene-tically and structurally distinct regions: О-specific chain (O-antigen, O-specific polysaccharide), the core oligosaccharide and lipid A, which anchors the LPS
molecule to the bacterial outer membrane ( R a e t z , W h i t f i e l d 2002). All three
regions o f Proteus LPS have been studied (RÓŻALSKI 2002). LPS containing all
these three regions is produced by smooth forms of bacteria. Rough strains or rough mutants o f different classes (Ra-Re mutants) synthesize LPS containing
lipid A and core region or part of the core region (RÓŻALSKI et al. 2002). Lipopolysaccharide participates in the physiological function of outer membrane of bacterial cells and is essential for its growth and survival. It is also a target for interaction with antibacterial drugs and immune mechanisms of the host. LPS, released from the bacterial surface in infected macroorganism induces a spectrum of biological activities important in the pathogenesis, particularly in
septic shock ( L u k a s i e w i c z , Ł u g o w s k i 2003).
T h e d iffe re n ce s in the structure o f O -a ntig ens serv e as the b asis for the ser olog ica l c la ssific a tio n o f Proteus strains. T he se rolog ical cla ssific atio n sc h em e
o f K A U FM A N and P e r c h in clu des 49 d ifferent P. mirabilis and P. vulgaris
O -serogro u ps and 19 se r olo g ica lly d istinct fla gellar H -an tigen s ( K a u f f m a n n
1966). T h e ch e m ica l and sero lo gica l stu die s perform ed in th e Institute o f
M ic ro b io lo g y and Im m u no log y , U niver sity o f L o d z in co lla bo ra tion w ith N. D. Z e l i n s k y Institute o f O rganic C hem istry, R u ssian A ca d em y o f S c ie n c e s in M o s co w , R u ssia, h av e led to esta b lish in g ad dition al O -sero gro u ps, w hich w ere
created to c la s sif y P. penneri and P. hauseri strains, as w ell as P. myxofaciens,
Proteus g e n o m o sp e c ie s and som e P. mirabilis and P. vulgaris strains u nclassified before ( K n i r e l et al. 1993; K n i r e l et al. 1999; R ó ż a l s k i 2004). T h e serological c la s sific a tio n sc h e m e currently c o n sists o f 76 serogro ups ( D r z e w i e c k a et al.,
2004; D r z e w i e c k a , S i d o r c z y k 2005; Z a b ł o t n i , 2006). In this re view I report
on the ch e m ic al structure o f the O -an tig ens o f Proteus b a cilli and their
sero-lo g ic a l s p ec ificity , as w e ll as on the co re and lip id A reg ion o f e n d oto xin and on se lec te d b io lo g ica l ro les o f L PS o f these bacteria.
3. O -S P E C IF IC P A R T O F L PS
Proteus O-antigens are linear or branched polysaccharides built up of
oligosaccharide repeating units. All polysaccharides with one exception of
P. vulgaris 0 5 3 contain am ino sugars either D -glucosam ine (GlcN) or
D-galactosam ine (GalN). Acidic О-specific polysaccharides (OPS) represent the 90% of Proteus O-antigens. OPSs of these bacteria are acidic due the presence of uronic acids - D-glucuronic acid (GlcA), D-galacturonic acid (GalA), L-altruronic acid (L-AltA). Some OPSs are acidic also due to the presence o f (R) or (5) lactic acid or less often malonic (Mai), pyruvic (Pyr) or succinic (Sue) acids, which are linked to the sugar residues. Hexuronic acids GlcA and GalA usually have a free carboxyl group, however, they are very often amidated with a-am ino group of am ino acids - L-alanine (L-Ala), L-lysine (L-Lys), L-serine (L-Ser) and L-threonine (L-Thr). Two OPSs of P. mirabilis 0 1 3 and P. myxofaciens 0 6 0
contain amides of GlcA and GalaA, respectively with /VE- [ ( R ) -1 -carboxyethy] 1-L-
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o O S al o o с v> o 0 Q i o чО O VO O o:T a b le 1 : (c d .) S tr a in /S e ro g ro u p C h e m ic a l s tr u c tu re o f O P S L it e ra tu re P . p e n n e ri 0 6 6 ß -L -R h a p N A c 3 N A c -( l— i 3 — > 4 )-a -D -G lc p -( 1 — » 3 )-a -L -6 d T a lp 2 A c -( 1 — > 3 )-ß -D -G lc p N A c -( 1 -» D r z e w ie c k a , S id o r c z y k 2 0 0 5 P ro te u s g e n o m o - -s p e c ie s 6 (0 6 9 ) o -D -G lc p A 3 /4 A c -( 1 -i 4 — > 6 )-ß -D -G lc p N (L -A la )3 A c -( l-> 4 )-ß -D -G lc p A -( l-» 3 )-ß -D - -G lc p N A c ó A c -( 1 — » Z y c h e t a l. 2 0 0 5 P . p e n n e ri 0 7 3 E tn /’ -i 6 -» 4 ) -R ib -o l-5 -/4 0 — > 4 )-ß -D -G lc p -( l— » 3 )-ß -D -G a lp -( 1 -» 3 )-ß -D -G a Ip N A c -( 1 -> D r z e w ie c k a , S id o r c z y k 2 0 0 5 RÓ ŻALSKI
The pyranose form is typical for most monosaccharides, except for ribose
which was found in furanose form in Proteus endotoxins (K N IR E L et al. 1997). In
the furanose form GalN in P. penneri 063 OPS is present (A r b a t sk y et al. 1998). Phosphorylation is also characteristic for Proteus OPSs - glycerol and ribitol phosphates, as well as other phosphate-linked substituents such as ethanolamine and choline were found in these O-antigens. The amino group of most amino sugars is acetylated or is substituted by acyl components such as (Ä)-hydroxybutanoyl group (/?-3H0Bu), amino acids (L/D-alanine, D-alanine dipeptide, D-aspartic acid) as well as by residue of malonic or succinic acids. Sugar residues in ca. 35% OPSs are usually non-stoichiometrically substituted with O-acetyl groups. Proteus O-antigens contain sugar constituents rarely found in bacterial products or even in nature, including deoxy sugars such as 6-deoxy-L-talose (L-6dTal), 2-amino-2,6-dideoxy-L- -glucose (L-quinovosamine, L-QuiN), 3-amino-3,6,-dideoxy-D-gIucose (Qui3N), 4-amino-4,6-dideoxy-D-glucose (Qui4N), 2-amino-2,6-dideoxy-L-galactose (L-fuco- samine, L-FucN), 3-amino-3,6-dideoxy-D-galactose (Fuc3N), 2,4-diamino-2,4,6- -trideoxy-D-galactose (FucN4N), 2-amino-2,6-dideoxy-L-mannose (L-rhamnosamine, L-RhaN) and 2,3-diamino-2,3,6-trideoxy-L-mannose (L-RhaN3N), as well as acidic amino sugars - 2-amino-2-deoxy-D-galacturonic acid (GalNA) and 5,7-diamino- -3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (pseudaminic acid, Pse) (R ó ż a lsk i et al. 2002; R ó ż a lsk i 2004). О-specific polysaccharides representing selected O-serogroups of Proteus sp. are shown in Table 1.
4. S E R O L O G IC A L S P E C IF IC IT Y O F P R O TE U S O -A N T IG E N S
The serological specificity of Proteus O-antigens was studied by use of polyclonal rabbit anti-0 sera specific to the particular serogroups. In this study the О -specific polysaccharides, their partial structures, as well as in some studies also synthetic antigens corresponding to Proteus O-antigens were used to identify the epitopes determining the O-specificity. As it was expected uronic acids and hexosamines, the characteristic compounds of Proteus OPSs, play an important role in the serological specificity of these antigens. GlcA and GalA with free carboxyl groups or amidated with amino acids, as well /V-acetylated amino-acids can be often found in the serological determinants of Proteus O-antigens. a-D -G lcA and ß-D-GlcA present as a branch of О -specific polysaccharides chain, serve as immunodominant sugars in specific epitopes in P. mirabilis 0 6
and P. vulgaris 0 4 7 , respectively (Fig. 1) (C E D Z Y N SK I et al. 1998; RÓ ŻA LSKI
et al. 2002). Hexuronic acid present in linear O-polysaccharides can also play an im portant role in the im munospecificity. It was found for OPSs of
P. vulgaris 0 2 2 and 0 3 2 in which ß-D-GlcA and a-D -G alA respectively, were
P. mirabilis 0 6
a-n-Glc/jA -l!
3
->4)-a-L-Fuc/)N Ac-( 1 ->3)-ß-D-Glc/;NAc-( I —>
P. mirabilis 04 7
ß-n-GIcpA X
4
—»3)-ß-D-Gal/>N Ac-( I -»4)-a-D-GalpN АсЗ Ac-( 1 ->3)-ß-D-GalpN Ac-( I ->
P. vulgaris 02 2 0t-D-Qui/?3NAc2,4Ac i 3 —>2 )-ß -L -R h ap -( 1 —> 4 )-a -L -R h a/> -(l-* 4 )-/?-.D -G /< y v l-(l-» 3 )-ß -D -G lc/)N A c -( I -> P. vulgaris 0 32
->4)-ot-D-GalpA-( 1 -»2)-cx-L-Rhap-{ I ->2)-a-L-Rha/>-( 1 -M yß-D-GalpA-( l-»3)-ß-D-Glc/>NAc-<1 ->
P. mirabilis 0 2 7 ß-D-GIcpNAc
/ ET
4 6
—»3)-ß-D-Glc/>A6(L-Lys)-( 1 —>3)-a-D-GalpA6(L-Ala)-( I —>3)-ß-D-GlcpNAc-( I —»
P. mirabilis 01 4
D-AlaEtnjP 6
-»4)-a-D-Galp-( I -»4)-ß-D-GalpNAc-( 1 ->3)-a-D-Galp6Ac-( 1 ->3)-ß-D-GalpNAc-( l->
P. mirabilis 013 CH)-Gal/)A6(2.V,Ä/?-Alal.ys) 4 —>3)-a-D-Galp-( I —>3)-ß-D-Glc/jNAc-( 1 —> P. myxofaciens (060) -»6)-ß-D-Glc/)NAc-(l—>3)-ß-D-GlcpNAc-(l-»4)-ß-D-Glc/)A6(2Ä',#/f-AlaLys)-(l-^6)-a-D-Gal/)NAc-(l- P. mirabilis 0 3 <x-D-Gal/>A6(l,-Lys) a-D-Glcp i ' I 4 2
—>6)-ß-D-GalpNAc-( 1 —>4)-ß-D-GlcpA-( l->3)-ß-D-GalpNA c-( 1 —>
P. mirabilis O l 1
ß-D-GIcpNAc a-D-Glcp
4, X
2 6
—>4)-ß-D-Glc/)A-( 1 —>3)-ß-D-GalpA6(L-Thr)-( 1 ->3)-ß-D-GlcpNAc-( I -4
P. mirabilis 0 26
—>4)-a-n-Gal/>A6(l.-Lys)-( 1 ->4)-a-D-Gal/>-( I ->3)-ß-D-GalpA4Ac-( 1 ->3)-ß-D-GlcpNAc-( 1
P. mirabilis 0 28
et al. 1999; B a r t o d z i e j s k a et al. 1998). The immunodominat role of the lateral N-acetyl-D-glucosamine residue linked to the ß-D-GlcA(-L-Lys) was
noticed in the specificity of P. mirabilis 0 2 7 (VINOGRADOV et al. 1989). In
some Proteus О-specific polysaccharides unusual compounds can also play immunodominant role, however, it is not a rule. Such a role is played by a unique component /V-[(/?)-l-carboxyethyl]ethanolamine phosphate linked to
the Gal residue in P. mirabilis 0 1 4 OPS ( P e r e p e l o v et al. 1999). The
immunodominat position in OPSs of P. mirabilis 0 1 3 and P. myxofaciens 0 6 0
is occupied by AlaLys linked to the GalA and GlcA, respectively (SwiERZKO
et al. 2001; SlDORCZYK et al. 2003).
The most com mon epitopes showed for Proteus O-antigens were uronic acids substituted by amino aids. The importance o f a-D -G alA(-L-Lys) in the
specificity of P. mirabilis 0 3 ( K a c a et al. 1987), 0 2 6 (SHASHKOV et al. 1996)
and 0 2 8 ( R a d z i e j e w s k a - L e b r e c h t et al. 1995), as well as a-D-GalA(-L-Thr)
in the specificity of P. mirabilis O i l (Arbatsky et al. 2000) were found, however a-D-GalA (-L-Ser) in P. mirabilis 0 2 8 appeared to be out of importance in the specificity.
Polyclonal anti-0 sera contain antibodies of different types of specificity. Usually, the major fraction of antibodies recognizes the main epitope which defines the group specificity, whereas the minor fractions can bind other epitopes in O-antigen or in core region of LPS. О-specific antibodies may cross react with LPS of strains belonging to the same species or genus but classified into other serogroups, as well as even with LPS of taxonomically different bacteria. Indeed, the characteristic feature of Proteus O-antisera is cross reactivity with heterologous lipopolysaccharides of the same genus and
less often with LPS from other bacterial genera (R Ó Ż A L SK I et al. 2002). Due
to the com mon a-L -FucN A c-(l->3)-a/ß-D -G lcN A c disaccharide such cross reactivity was observed in antigen - antibodies systems of LPS and hetero-logous antisera of P. vulgaris 0 8 , 01 2 , 0 3 9 and in P. mirabilis 0 6 (Fig. 2) ( R ó ż a ls k i 2004). The marked serological relationship showed LPS of
P. mirabilis 0 7 and 0 4 9 , containing D-Qui4N N-acetylated with malonic and
succinic acid, respectively (K o n d a k o v a et al. 2004). Close serological related-ness was also shown between P. vulgaris 0 2 2 and 0 3 2 , due to the presence of sim ilar trisaccharides fragments a-L -R h a-(l—»4)-ß-G lcA -(l—»3)-ß-D-GlcNAc
and a-L -R h a-(l—»4)-ß-GalA -(l—»3)-ß-D-GlcNAc, respectively (T O U K A C H et al.
1999; B a r t o d z ie js k a et al. 1998). Comparison of the O-antigen structures of P. vulgaris 0 1 7 and P. vulgaris 04 5 revealed the presence of similar trisaccharide epitopes a-D-GlcpN Ac-( 1 - > 2)-ß-D-Fuc3N[/?3HOBuJ-( 1 —->6)-a-D- Glc and ß-D-Glc/?NAc-(l —>2)-ß-D-Fuc3NAc-(l—>6)-ot-D-GlcNAc, respectively, which may account for the serological relatedness of these strains (To rzew s-
Fig 2: Cross-reacting epitopes in OPSs from: P. vulgaris 0 8 , 0 1 2 , 0 3 9 and P. m irabilis 0 6 ;
P. m irabilis 0 7 and 0 4 9 ; P. vulgaris 0 2 2 and 0 3 2 ; P. vulgaris 0 1 7 and 0 4 5 (for literature see
text and T able 1). E pitopes are shown in bold type
P. vulgaris 0 8 a-D-Galp I i 3 ->3)-ß-D-Glc/>A-( I —>4)-a-L-Fiic/»NAc-(l-»3)-a-D-Glc/>NAc-(l—> P. vulgaris 0 12 a-D-Glc/K 1 —>6)-a-D-GalpNAc4Ac 1 i 3 —>6)-ß-D-Glc/>-( 1 —>4)-oc-L-l'uc/;NAc-(l—>3)-ß-D-Gky)NAc-( 1 -*3)-Gro-1 -/>-( 0 -> P. vulgaris 0 39 ->8)-ß-Pse/35Ac7Ac-(2-»3)-a-L-FucpNAc-(l->3)-a-D-GlcpNAc-(l-> P. mirabilis 0 6 a-D-Glc/)A-1 i 3 ->4)-a-I.-Fuc/)NAc-(l-»3)-ß-D-Gl(yNAc41-> P. mirabis 0 7 ß-D-Quip4NMal 1 i 6 ->2)-ß-D-Gal/>-( 1 ->4)-ß-D-Glc/>-( 1 —>3)-ß-D-Glc/)N Ac-( 1 -> P. mirabilis 04 9 a-D-Qui/>4NSuc 1 I 4
—» 2 )-a-D -G al/)A -( 1 —> 3 )-a-L -R h ap -( 1 —>4)-ot-D-Glc/>-( 1 —> 2 )-a-L -R h ap -( 1 —» 3 )-ß -D -G lcp N A c-( 1 —> P. vulgaris 0 22 a-D-Qui/)3NAc2,4Ac 2 I 3 —>2)-ß-L-Rha/)-( I —>4)-a-L-Rha/>-(l—>4)-ß-D-Glc/jA-(l-»3)-ß-D-Glc/>NAc-(l—» P. vulgaris 0 32
-»4)-a-D-Gal/)A-( 1 ->2)-a-L-Rha/j-( I ->2)-a-L-R!ia/>-( I-*4)-ß-D-C al/jA-0 ->3)-ß-D-GlcpNAc-(l -»
P. vulgaris 0 17
—»2)-ß-D-Fuc/>3N(3HOBu)4Ac-(l—»6)-<x-D-Glcp3Ac-(l->4)-ß-D-Glc/)A-(l—>3)-a-D-GlcpNAc-(l—»
P. vulgaris 0 45
Proteus O-antigens show also similarity with OPSs from bacteria of other
genera. This fact can be reflected in the cross reactivity of Proteus O- -antisera with heterologous LPS. For example, such cross reactivity was found betw een anti-A mirabilis 0 1 3 serum, as well anti-P. myxofaciens serum and LPS from Providencia alcalifaciens 0 1 4 and 0 2 3 . Serological studies revealed an important role of AlaLys for the specificity of these
O-antigens ( K o c h a r o v a et al. 2003; T o r z e w s k a et al. 2004a). The common
compound D-Qui4N carrying N-linked N-acetyl-aspartic acid is responsible for antigenic relationship of O-antigens of P. mirabilis 0 3 8 , as well as P.
alcalifaciens 0 4 and 0 3 3 (K O C H A R O V A et al. 2004; T O R Z E W SK A et al. 2004b). Strong structural and serological similarity was also found between
P. vulgaris 0 2 1 , P. mirabilis 0 4 8 and Hafnia alvei 744 and PCM 1194 ( B a r t o d z i e j s k a et al. 2000).
P. mirabilis strains classified into OXK serogroup (0 3 ) cross-react with
sera directed to Orientia tsutsugamushi. Similar cross reactivity was shown between P. vulgaris 0 X 1 9 (0 1 ), as well as P. vulgaris 0 X 2 (0 2 ) and antibodies from patients with rickettsial infections. Strains belonging to these serogroups are comm only used in unspecific W eil-Felix test for sérodia-gnostics of rickettiosis. Structural and immunochemical studies revealed that the common epitopes which are recognized by these antibodies reside in
Proteus OPSs, however, their exact structures remain unknown (R Ó Ż AL SK I et al. 1997).
5. C O R E R E G IO N
The core region of Proteus lipopolysaccharides was studied in rough
mutants or in smooth forms classified into different serogroups ( R a d z i e j e w s k a -
- L e b r e c h t et al. 1989; V i n o g r a d o v et al. 1994; V i n o g r a d o v et al. 2002). The structural diversity of the core is characteristic for Proteus sp. and makes
it different from E. coli and Salmonella ( H o l s t 1999; V i n o g r a d o v et al.
2002). The core region of Proteus strains is com posed of two parts - inner part, comm on for several number of strains and second, outer part, which is characterized by a structural variability from strain to strain. The common part is not identical in all Proteus strains and is subdivided to three forms known as glycoforms I—III (Fig. 3). The outer part of core region (outer core) contains
oligosaccharide characteristic for particular Proteus strains (Table 2) (V IN O G
Fig. 3: Chem ical structure of the inner core region o f Proteus LPS (for chem ical structure of the outer core see Table 2). (Vi n o g r a d o v et al. 2002, Ró ż a l s k i 2004)
G lycoform I
EtN-/> ß-Glc ß-Ara4N
Outer core
16 14 |8
->4 )-a -G alA -( 1 ->3)-cc-LDHep-( 1 ->3)-a-LD H ep-( 1 -» 5)-a-K d o -(2->
17 14
ß-Gal A-( 1 ->7)-a-LDHep a-K do
G lycoform II
D D H e p EtN-P ß-Glc ß-Ara4N
Outer core ->4 )-a-G alA -( 1 ->3)-a-LDHep-( 1 -» 3)-a-L D H ep -(l-> 5 )-a -K d o -(2 ->
17 14
ß-G alA -(l-»7)-a-L D H ep a-K do
G lycoform III
ß -G a lA -(l—>7)-a-DDHep EtN-/3 ß-Glc ß-Ara4N
1з 1б I4 Is
->4)-a-G alA -( 1 ->3)-a-LDHep-( 1 —>3)-a-LDHep-( 1 ->5)-a-K d o-(2 ->
| 7 14
LDHep a-K do
Outer core
T ab le 2: T he outer core oligosaccharides o f selected P roteus strains (Vi n o g r a d o v et al. 2002)
P roteus s tra in s O u te r c ore s tru c tu re
P. mirabilis 0 3 ; R110 a-D D-H ep-( 1 -> 6)-a -G lc N
P. m irabilis 0 6 ß-Qui4NAlaA la-(l -» 3)-a-G al-( 1 - 4 6)-a-G lc N A c-( 1 -> 4)-a-G alN
P. m irabilis 0 2 7 ß-G lc-( 1 5)-(/S)-GaloN Ac-( l->4,6)-cx-GalN
P. mirabilis 0 2 8 , P. penneri 42
ß-G alA L ys4PEtn-(l—»3)-ß-G lcN A c-(l-» 6)-G lcN G ly
P. m irabilis 0 5 7 ß -Q u i4N A la A la -(l-» 3 )-a-G a lN A c -(l-^6 )-a -G lc -(l-» 4)-a -G alN
P. vulgar is 0 2 , 0 4 , 0 8 ,
0 X 1 9; P. penneri 2, 11, 17, 19, 107
T ab le 2: (cont.)
P roteus s tra in s O u te r c ore s tru c tu re
P. vulgaris 0 2 5 ß -K d o -(2 -> 3 b
ß-GlcNAc-( 1 —>2)-ß-Gal-( 1 -»6 )-a -G lcN
P. penn er i 7, 14, 21 a-G lc-( 1 -> 4)-a-G alN Ac-( 1 -» 2)-a-D D -H e p-( 1 -> 6)-ot-GlcNGIy
P. penneri 16, 18 a-FucNH b-( 1 -> 4)-a -G al-( 1 ~>6)-ß-Glc-( 1 -> 3)-a-GalN6/>-Etn
6. L IP ID A
Lipid A is a biological domain of endotoxin. Structurally it is a conservative region o f LPS and in Proteus contains glucosamine disaccharide substituted with phosphate residues and fatty acids. Proteus lipid A differs form lipid A of
E. coli and Salmonella in the presence of 4-amino-4-deoxy-arabino-L-arabinose,
which quantitatively substitutes the ester-linked phosphate residue of glucosamine
backbone (Fig. 4) (SID O R C Z Y K et al. 1983).
7. B IO L O G IC A L R O L E O F P R O T E U S LPS
Proteus LPS as an endotoxin and as a cell surface antigen is associated
with broad spectrum of biological activities, and with interactions with bac-terial or eukaryotic cells. О-specific polysaccharide chain is exposed outside bacteria, and with capsular polysaccharides are involved in glycocalyx for-mation. Glycocalyx forms bacterial capsule, and via lectins or cations binds bacterial cells and makes possible the adherence of bacterial populations to each other and to the epithelial cells of macroorganisms or artificial surface
e.g. urological catheters (C OST E R T O N et al. 1978). These properties of
glyco-calyx enable bacteria to grow in the form of biofilm on a solid surface. Bacterial biofilm is defined as matrix-enclosed bacterial population adherent to the surfaces. Bacteria enclosed in glycocalyx capsule are protected against the action of antibodies and other antibacterial substances, as well as immune
mechanisms ( D o n l a n , C o s t e r t o n 2002). They also differ in expression of
particular virulence factors and metabolic activity, in comparison to bacteria
growing in liquid media ( L e w i s 2005).
As it was mentioned above, Proteus bacteria under appropriate conditions undergo a differentiation of vegetative rod shape short cells into longer forms called swarmer cells. The population of swarmer cells is then able to migrate
on solid surfaces termed swarming ( B e l a s 1996; R a t h e r 2005). It was shown
that surface polysaccharides are important for this migration. The migration of swarmer cells on solid media is facilitated by cell surface polysaccharides due
to the reduction of cells friction ( S t h a l et al. 1983; G y g i et al. 1995). Most
likely О-specific polysaccharide is also important for the swarming phenomenon
since the R e mutant strain P. mirabilis R45 producing LPS having only the
lipid A and Kdo region was unable to swarm. The R a mutant strain P. mirabilis
R110 containing lipid A and the complete core region expressed only a limited ability for migration on the solid medium, whereas most o f s-forms of
P. mirabilis, P. vulgaris and P. penneri can swarm vigorously ( B a b i c k a 2001;
K wiL 2003).
The acid ic Proteus O -antigens may play an important role in stones formation
w ithin the urinary tract. T he crystalliza tion o f am m on iu m m a gn esiu m phosphate and ca lciu m p hosp hate is initiated by the activity o f urease, w h ich c le a v e s urea to am m o nia and carbon d iox id e , resulting in the rise o f the pH. In such alkaline co n d itio n the crysta lliz atio n pro ce ss occurs in ten siv ely (MOBLEY e t al. 1996; R ó ż a l s k i et al. 1997). T o r z e w s k a et al. ( 2 0 0 3 ) sh o w e d urease as the major factor in v o lv ed in sto n es form ation, h o w e v er she a lso fou nd that the e ffe c t o f this en zy m e m ay be m o d ifie d by bacterial О -sp e c if ic p oly sacch ar id es. It w as
n oticed that the sugar co m p o sitio n o f Proteus LPS m ay either enh ance or
structure and ability to bind cations. OPS o f P. vulgaris 12 has bound mag-nesium and calcium ions weakly, but increased the crystallization process, whereas OPSs o f P. vulgaris 0 4 7 which are able to bind large amounts of these ions inhibited the process of crystallization (for OPS structure see Table 1), Most likely, the Mg2+ and Ca2+ weakly bound to the polysaccharides, and could be then easily released from the bacterial surface. This phenomenon causes local supersaturation of the solution and leads to the increase in crys-tallization and stone formation.
LPS of S-forms of Gram-negative bacteria contributes to their resistance against bactericidal action of the serum. One of the possible explanations for the bactericidal effect is the action of membrane attack complex (MAC) of activated complement. MAC is extremely hydrophobic and forms pores in bacterial membranes, which lead to the bacterial lyses and death. In S-forms of bacteria hydrophobic MAC cannot pass the hydrophilic barrier of a long chain of О -specific polysaccharides to gain their outer and inner membranes ( K a c a , U j a z d a 1998; M i e l n i k et al. 2004). Biological studies of bactericidal activity o f sera against Proteus strains confirmed this hypothesis. It was shown that P. mirabilis R-mutants synthesizing the LPS molecule without O-specific polysaccharide are sensitive to the serum action, whereas most o f P. mirabilis S-forms, as well as around 50% of S-forms of P. vulgaris and P. penneri are
resistant ( B a b i c k a 2001; F u d a l a 2003; K w i l 2003).
The biological role of the core region is not clear. It is immunogenic which was shown by use o f R-mutants. The antibodies against the core region particularly directed to the heptose or Kdo subregions cross react with LPS of different bacteria, and can be used as endotoxin neutralizing antibodies because of a close similarity of these parts of LPS in different groups of bacteria ( P o l l a c k 1999; Di P a d o v a et al. 1999). The presence o f 4-amino-4-deoxy- -arabino-L-arabinose in the core region, as well in lipid A (see below) led to
the resistance of Proteus bacteria to polymyxin (B O L L et al. 1994).
LPS is known as endotoxin - the most important virulence factor of Gram-negative bacteria. The mechanism of biological action of endotoxin is comm on to most of bacteria. It shows pathophysiological effects when released from bacterial cells to blood-vascular system. The centre of biological activity of endotoxin is lipid A, which in the blood is bound by LPB (li-popolysaccharide binding protein). Complexes of LPS-LPB are recognized by receptors on different eukaryotic cells (monocytes/macrophages, lympho-cytes, endothelial cells of blood vessels) such as CD 14, as well as TLR2 and TLR4 (tool like receptors 2 and 4) resulting activation of transmembrane signal and induction of cells to produce biologically active mediators - TNF (tumor necrotizing factor), interleukins (IL-1, IL-6, IL-8), oxygen free radicals (OJ, H20 2, NO) and others. These mediators depending o f their concentration in macroorganisms elicit beneficial or most often detrimental effects e.g.
DIC (dis sem in ated intravascular co agu lation ) and ARDS (acu te respiratory
distress syn dr om e), resu lting in m ultiple organ sy stem failure (MOSF) and
shock syn d rom e ( L u k a s ie w i c z , Ł u g o w s k i 2 0 03 ).
8. SU M M A R Y
Bacteria from the genus Proteus synthesized lipopolysaccharides which are built according to the common scheme characteristic for Enterobacteriaceae family, however, markedly different in detail structure and biological activity, when compared with other representatives of this family. It serves as the basis of the serological classification of these bacteria. Long term structural and immunochemical studies led us to show the molecular basis of this classification. Antigenically, Proteus is heterogenous because of structural differences in the О-specific part of LPS. Until now 76 serogroups have been describe for this genus. Acidic О-specific polysaccharide and structural diversity of the core region are the characteristic features of Proteus LPS. The acidic character of LPS o f these bacilli has most likely very important biological consequence in the formation of bladder or kidneys stones during urinary tract infections. LPS as a endotoxin is also an important virulence factor playing an important pathophysiological role, particularly during sepsis. The future studies will most probably show the exact role of Proteus LPS on the particular stages of infections.
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