Báo cáo khoa học: Polyphosphates from Mycobacterium bovis – potent inhibitors of class III adenylate cyclases pptx

We identified mycobacterial polyphosphates with a mean chain length of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M.. In test-ing AC

inhibitors of class III adenylate cyclases

Ying Lan Guo1, Hermann Mayer2, Waldemar Vollmer3, Dorothea Dittrich4, Peter Sander4,5,

Anita Schultz1and Joachim E Schultz1

1 Pharmazeutisches Institut, Universita¨t Tu¨bingen, Germany

2 Institut fu¨r anorganische Chemie, Fakulta¨t fu¨r Chemie und Pharmazie, Universita¨t Tu¨bingen, Germany

3 Institute for Cell and Molecular Biosciences, Newcastle University, UK

4 Institut fu¨r Medizinische Mikrobiologie, Universita¨t Zu¨rich, Switzerland

5 Nationales Zentrum fu¨r Mykobakterien, Zurich, Switzerland

cAMP is a key signaling molecule in virtually all living

organisms This ubiquity is mirrored by the abundance

and diversity of the synthetic enzymes, adenylate

cyc-lases (ACs) Currently, six classes of ACs exist, which

share no identifiable sequence similarities Here we

deal with ACs grouped together in class III, which

contains by far the most AC isozymes Among these

are all cyclases from eukaryotes and the overwhelming

majority of those from bacteria [1] In eukaryotic cells,

ACs are typically pseudoheterodimeric, i.e the result

of a gene duplication [2] Both pseudomonomers

con-tribute amino acids to a single catalytic center [3,4]

Bacterial class III ACs are generally monomers that must dimerize to form two catalytic centers that are essentially identical to those of eukaryotic class III ACs [5,6] Usually, cAMP is generated intracellularly

in response to extracellular signals such as hormones, changes in ion compositions, pH or nutrients, and a variety of stress conditions Although stimulatory conditions often persist for considerable periods of time, cAMP formed in vivo is mostly short-lived This requires activated ACs to be quickly returned to

a basal activity state [7–9] In eukaryotic cells, GTP hydrolysis and dissociation of the activated

Keywords

adenylate cyclase; cAMP; Mycobacterium;

polyphosphate; stress response

Correspondence

J E Schultz, Pharmazeutisches Institut,

Universita¨t, Tu¨bingen, Auf der Morgenstelle

8, 72076 Tu¨bingen, Germany

Fax: +49 7071 295952

Tel: +49 7071 2972475

E-mail: joachim.schultz@uni-tuebingen.de

(Received 7 October 2008, revised 7

November 2008, accepted 10 December

2008)

doi:10.1111/j.1742-4658.2008.06852.x

cAMP generation in bacteria is often stimulated by sudden, but lasting, changes in extracellular conditions, whereas intracellular cAMP concen-trations quickly settle at new levels As bacteria lack G-proteins, it is unknown how bacterial adenylate cyclase (AC) activities are modulated Mycobacterium tuberculosishas 15 class III AC genes; therefore, we exam-ined whether mycobacteria contain a factor that may regulate AC activi-ties We identified mycobacterial polyphosphates with a mean chain length

of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M tuberculosis and other bacteria The identity of the inhibitor was established by phosphatase degradation, 31P-NMR, acid

or base hydrolysis, PAGE and comparisons with commercial standards, and functional substitution by several polyphosphates The data indicate that each AC dimer occupies 8–15 phosphate residues on a polyphosphate strand Other polyionic polymers such as polyglutamate, polylysine and hyaluronic acid do not affect cyclase activity Notably, the structurally unrelated class I AC Cya from Escherichia coli is unaffected Bacterial polyphosphate metabolism is generally viewed in the context of stress-related regulatory networks Thus, regulation of bacterial class III ACs by polyphosphates could be a component of the bacterial stress response

Abbreviations

AC, adenylate cyclase; poly-P, polyphosphate.

AC–G-protein complex terminates signaling, possibly

also with the involvement of secondary modifications

such as phosphorylation [10,11] Termination of

acti-vation of bacterial class III ACs has not been

investi-gated G-proteins or G-protein-like mediators of AC

stimulation are unknown in bacteria For example, in

the cyanobacterium Anabaena, the ACs CyaB1 and

CyaB2 are stimulated by the enzymatic product cAMP

via an N-terminal tandem GAF domain [12,13]

Theoretically, this would result in perpetual

self-activa-tion until ATP is exhausted, an unlikely physiological

situation Recently, it has been shown that sodium

ions may be involved in the process of autoinactivation

of CyaB1 AC [14]

In Mycobacterium tuberculosis, 15 class III AC genes

have been identified [15,16], and for at least 10, AC

activity has been demonstrated [1,17,18] Deletion of

single AC isoforms in M tuberculosis did not result in

obvious phenotypes [17,19] The stressor conditions

employed may have been insufficient and⁄ or functional

replacement could compensate for the loss of an

indi-vidual AC isoform Therefore, two major questions

exist: how are the bacterial ACs stimulated, and how

is stimulation terminated while stimulatory conditions

continue to prevail? To address these questions, we

examined whether mycobacteria contain endogenous

factors that modulate AC activities We used a cell

homogenate from Mycobacterium bovis BCG, the live

vaccine strain against tuberculosis, to search for the

presence of such modulators We have isolated

poly-phosphate (poly-P) from M bovis BCG, which is a

well-known bacterial constituent [20,21], and

demon-strated that it is a most powerful inhibitor of class III

ACs from M tuberculosis and other bacteria In

test-ing ACs from the class IIIa, IIIb and IIIc subfamilies,

we found that all were strongly inhibited by this cell

constituent, whereas a class I AC from Escherichia coli

was not affected

Results

In an exploratory experiment, we homogenized 1 g

(wet weight) of M bovis BCG cells (grown as a settling

culture under hypoxia) with a French press, and

prepared a 100 000 g supernatant (60 min) The

super-natant strongly inhibited the activity of recombinant

Rv1625c, a membrane-bound, mammalian-like AC

from M tuberculosis [22] The virtual IC50 was 1.6 lg

of protein (data not shown) The suspended pellet

from the above centrifugation was inactive To assess

the specificity of inhibition, several controls were used:

(a) suspended cell pellets and supernatants from

Myco-bacterium smegmatis or E coli (BL-21) grown without

stress in well-oxygenated rich media had only very low inhibitory potency; (b) M bovis BCG cells were washed extensively with 50 mm Tris⁄ HCl buffer con-taining 150 mm NaCl, yet this treatment did not detach an AC inhibitor; and (c) we tested unused as well as spent medium after harvesting of BCG No inhibition (or activation) was observed This led us to believe that M bovis BCG produced a soluble intra-cellular inhibitor of Rv1625c

What is the chemical nature of the inhibitor? Boiling removed 98% of protein, but AC inhibition was unim-paired Similarly, extended digestion with trypsin did not abolish inhibition, virtually excluding a protein The inhibitory factor was not DNA or RNA This was verified by nuclease digestion and controlled by agarose gel electrophoresis After ether extraction, the inhibitor remained in the aqueous phase, excluding lipids Next,

we incubated the enriched inhibitory fraction with lyso-zyme or cellosyl, a promiscuous bacterial muramidase from Streptomyces coelicolor The results were equivo-cal After digestion with cellosyl, AC inhibition was retained, whereas incubation with lysozyme resulted in loss of inhibition An HPLC analysis failed to identify muropeptides, the expected products of lysozyme or cellosyl digestion (data not shown) Lysozyme has an isoelectric point of 11 Therefore, we hypothesized that the inhibitor might be an acidic compound such as poly-P and bind to lysozyme and not to cellosyl This would explain the contradictory results with both muraminidases When we incubated a sample with 1.5 units of acid phosphatase at pH 5.5, AC inhibition was lost Similarly, inhibition was almost completely lost upon treatment with HCl or NaOH at room temperature, indicating hydrolysis of a poly-P

For final inhibitor purification, the heat-treated sam-ple was bound to DEAE–Sephacel, released with

400 mm NaCl, and fractionated by Superose 6 chro-matography Fractions were assayed for activity using recombinant Rv1625c (Fig 1A), and analyzed by elec-trophoresis [23] In active fractions, poly-Ps with a chain length of about 70 residues were stained, whereas inactive fractions did not stain (Fig 1B) A dose–response curve with the combined fractions established the high inhibitory potency (Fig 1C) This indicated that the inhibitor was poly-P

Final chemical identification was carried out by31 P-NMR spectroscopy of concentrated fractions 50–64 from the Superose 6 column (Fig 2, trace E) The intense resonance at )21.8 p.p.m is characteristic for interior phosphate residues, and the weak, broad peaks

at )6.2 and )20.2 p.p.m are indicative of terminal and penultimate phosphate groups, respectively Thus, the 31P-NMR spectrum identified the sample as linear

poly-Ps [24–26] This was confirmed by comparisons with 31P-NMR spectra of commercial polyphosphate samples (Fig 2 and Table 1) The cyclic triphosphate (trimetaphosphate) showed a singlet at )21.5 p.p.m., due to exclusively interior residues (Fig 2, trace A) The two terminal groups and the interior residue of the linear triphosphate gave two resonances at )7.1 and )21.4 p.p.m at an integrated area ratio of 2 : 1 that were split by each other into a doublet and a triplet (Fig 2, trace B) Extending the chain length to

25 phosphate residues allowed detection of the terminal groups (d =)5.8 p.p.m.), the penultimate groups (d =)20.8 p.p.m.), and the interior residues (d =)21.8 p.p.m.) The resonance of the terminal groups displayed characteristic doublets and doublets

of doublets (Fig 2, trace C) Further increases in chain length led to broadening of terminal phosphate reso-nances, which agreed with a reduced T2 for polymeric systems (Fig 2, trace D, poly-P75) Moreover, calcula-tion of the peak areas of the resonances allowed an estimate of the chain length Calculated chain lengths for poly-P25 and poly-P75 were in agreement with those given by the supplier (Table 1) The mycobacte-rial sample yielded an average chain length of 72, in agreement with the electrophoretic analysis (Fig 1B) Finally, commercial poly-Ps, which were further char-acterized by 31P-NMR and hydrolysis by acid or base, inhibited class III ACs identically to the material from

M bovis BCG With 167 nm Rv1625c in the assay,

200 nm poly-P75 inhibited enzyme activity completely Owing to the scarcity of poly-P isolated from

M bovis BCG, we routinely used biological material for initial studies and commercial poly-Ps for in-depth

45 232

40

A

B

C

45 kDa

30

120 AC activity (%)

46

10

48

0

40

0 20 40 60 80 100

0

Fraction number Fraction # P75 P45 P25 50 52 56 58 60 62 64

Poly P

46 48

100

80

60

20

40

0

Mycobacterial poly-P ( M )

Fig 1 Purification and analysis of an AC inhibitor from M bovis

BCG (A) Superose 6 gel filtration and inhibition of Rv1625c by

indi-vidual fractions Solid line, D 280 nm; stippled lines (d), inhibition

of 167 n M Rv1625c; arrows on top denote molecular mass

mark-ers (B) Electrophoretic analysis (15% polyacrylamide gel containing

6 M urea) of inhibitory fractions from (A) Note almost empty

lanes 46 and 48, which are barely inhibitory Controls of linear

poly-Ps with average chain lengths of 75 (14 lg), 45 (7 lg), and 25

(7 lg) residues are on the left The gel was stained with toluidine

blue O to detect poly-Ps [23] The wide bands indicate chain length

variations in the commercial standards and column fractions One

hundred per cent activity corresponds to 360 nmol cAMPÆmg)1Æ

min)1 (C) Dose–response curve of Rv1625c inhibition of combined

fractions from the Superose 6 fractionation in (A).

A B C D E

p.p.m.

Fig 2 31 P-NMR spectra of the concentrated fractions (50–64) of the Superose 6 gel filtration and of commercial poly-P standards: (A) cyclic poly-P3; (B) linear poly-P3; (C) linear poly-P25; (D) linear poly-P75; and (E) concentrated inhibitor PP1, terminal phosphate groups; PP2, penultimate groups; PPn, interior phosphate residues The concentrations of the poly-P standards were calculated to be uniformly 100 m M Pi, i.e 33.3 m M poly-P3 [(A) and (B)], 4 m M poly-P25 (C), and 1.33 m M poly-P75 (D).

characterizations We never noted disparities in results

between the different poly-P sources Rv1625

inhibi-tion by poly-P75 was instantaneous, as demonstrated

experimentally (Fig 3A) This established that

inhibi-tion was reversible [27] This was confirmed in diluinhibi-tion

experiments in which the enzyme–inhibitor complex

was diluted eight-fold immediately prior to the start of

the reaction Reaction velocities of Rv1625c were

determined in the presence of 10, 20 and 25 nm

poly-P75 as a function of the substrate Mn-ATP A

Linewe-aver–Burk plot showed that Kmvalues were unchanged

whereas Vmax decreased, indicating noncompetitive

inhibition (Fig 3B) We excluded any chelating effect

of poly-Ps on the Mn2+concentration, because even if

the poly-P concentration was calculated in terms of

molarity of orthophosphate, it did not exceed low

micromolar values, whereas Mn2+ was fixed at 2 mm;

that is, poly-P could not act as a chelating agent Next,

we investigated whether phosphates of different chain

length had differing inhibitory potencies

Orthophos-phate up to 11 mm had no effect on Rv1625c,

pyro-phosphate inhibited it with an IC50 of 210 lm, the

linear triphosphate had an IC50of 20 lm, and the

cyc-lic trimetaphosphate an IC50 of 2 mm; that is, these

compounds were poor inhibitors (Table 2) In contrast,

all IC50 concentrations of poly-Ps with a chain length

of 16 or more were in the submicromolar range (Fig 4

and Table 2) When we normalized the individual IC50

concentrations of various poly-P compounds with

chain lengths > 16 to the concentration of

orthophos-phate, i.e the poly-P IC50 concentrations in Table 2

were multiplied by the average phosphate chain length,

we obtained a mean IC50 concentration of

660 ± 62.8 nm phosphate (± SEM; range 561–

825 nm) This indicated that once a critical length of

the poly-P chain is reached, the increasing affinity

of poly-P is linearly related to the increase in the

cal-culated total phosphate concentration For poly-P75

and cyclic poly-P17, the apparent Ki values were

determined with 100 nm Rv1625c, 30 and 60 lm

Mn-ATP, and different inhibitor concentrations

Table 1 Chemical shifts and coupling constants of the purified inhibitor and the commercial poly-Ps d, chemical shift; 2 J pp , coupling constant of two adjacent phosphate groups; PP1, terminal phosphate groups; PP2, penultimate phosphate groups; PPn, interior phosphate residues; s, singlet; d, doublet; t, triplet; br, broad; br s, broad singlet; br d, broad doublet.

Sample

d (p.p.m.)

Calculated chain length (average)

100

A

B

40

60

40 n M

50 n M

20

80 n M

0 7

Time of preincubation (s)

5

25 n M

4

10 20

3

0

2

1

0 0.02

1

1/S (µ M ) –1

Fig 3 Kinetics of the inhibition of Rv1625c by poly-P75 (A) Rv1625c at 134 n M was used in the assay The concentrations of poly-P75 added at the beginning are indicated The first assay was started after 7 s of preincubation of protein and poly-P75 by addition

of the substrate ATP Assays were run for 4 min One hundred per-cent activity corresponds to 448 nmol cAMPÆmg)1Æmin)1 (B) Double reciprocal plot (Lineweaver–Burk) of substrate kinetics of Rv1625c

in the presence of three concentrations of poly-P75 as an inhibitor.

(5–80 nm) Dixon diagrams gave apparent Kivalues of

14 nm for poly-P75 and 20 nm for poly-P17, i.e lower

than the enzyme concentration This indicated that a

single poly-P strand inhibited more than one AC

mole-cule Next, we determined IC50concentrations of

poly-P25 at different concentrations of Rv1625c Here, IC50

values increased linearly with the increasing protein

concentration (Fig 5) With a slope of the curve of

0.154 (R = 0.982; Fig 5), and considering that at

IC50, 50% of the dimers are bound to poly-P, this

indi-cated a molar ratio between the Rv1625c dimer and

poly-P25 of 3 : 1, close to the results obtained with

poly-Ps with different chain lengths (Table 2)

Next, we examined whether poly-Ps inhibit different

bacterial AC isoforms On the basis of systematic

differences in key amino acids and on small, strictly localized length variations, class III ACs have been divided into four subfamilies, IIIa to IIId [1] Rv1625c

is a class IIIa AC, as are all mammalian membrane-bound ACs A concatenated Rv1625c homodimer with

an identical domain sequence as the membranous mammalian ACs, (Rv1625c)2, is active [17] and inhib-ited by poly-P, just like Rv1625c (data not shown) Do poly-Ps also inhibit AC isozymes from other class III subfamilies? We examined the following ACs: cyaG from Arthrospira platensis as another class IIIa isoform containing a HAMP domain; as class IIIb ACs, myco-bacterial Rv3645, which has a HAMP domain between its membrane anchor and the catalytic domain, and CyaB1 from Anabaena sp., which has an N-terminal cAMP-binding tandem GAF domain; as a class IIIc

AC, the M tuberculosis pH sensor Rv1264 [6] In general, all class III ACs were potently inhibited by poly-P75 (Fig 6A) Although the IC50 concentrations differed slightly (11, 57, 315, 102 and 31 nm for Rv1625c, CyaG, Rv3645, CyaB1 and Rv1264, respec-tively), all were in the nanomolar range (Fig 6A) Because the catalytic centers of class III ACs appear

to be highly similar [3,5,6], it is likely that poly-Ps gen-erally inhibit class III ACs Rv1264 has been shown to

be a pH sensor that is strongly activated by pH values around 5.5 [6] This allowed testing of whether poly-Ps affected the basal and the activated states of a

clas-s IIIc AC clas-similarly Poly-P75 inhibited the baclas-sal clas-state

at pH 8 and the activated state at pH 5.5, but with markedly different efficacies The activated enzyme was fully inhibited at 400 nm, whereas the basal state was not yet fully inhibited at 30 lm (Fig 6B) The

Table 2 IC 50 values of poly-Ps for the AC Rv1625c The protein

concentration in the assays was 83 n M

Poly-P16

80

60

20

40

–9

0

10

Concentration ( M )

Fig 4 Inhibition of Rv1625c by poly-Ps of different strand lengths.

Rv1625c at 167 n M was used in the assays One hundred per cent

activity corresponds to 370 nmol cAMPÆmg)1min)1.

26

22

18

10

14

Fig 5 Poly-P25 IC50values increase with increasing AC concentra-tions Assays were carried out at different Rv1625c concentraconcentra-tions.

IC 50 values (y-axis) were plotted against the protein concentrations

at which only dimers exist [22] (regression coefficient r = 0.999).

reported structures of Rv1264 showed that a canonical

catalytic cleft of class III ACs is formed at pH 5.5,

whereas at pH 8, the catalytic amino acids are far

apart [6] The differences in the IC50 concentrations

may indicate that poly-P binds in the catalytic crevice

Another question was whether poly-P specifically inhibits class III ACs or also acts on class I ACs We expressed Cya, the class I AC from E coli, and purified

it to homogeneity by Ni2+–nitrilotriacetic acid chro-matography The specific activity of the purified protein was 27.2 nmolÆmg)1Æmin)1 with Mg-ATP as a sub-strate This class I AC was unaffected by up to 30 lm poly-P75 in the assay (Fig 6A, stippled line) Finally, the specificity of poly-P inhibition was investigated using other ionic polymers We employed anionic poly-glutamate (Mr‡ 15 000, Sigma, Munich, Germany), cationic polylysine (Mr4000–15 000, Sigma), and acidic hyaluronic acid (from Streptococcus equi, Fluka, Munich, Germany) We tested these compounds at 0.2 and 2 lm with Rv1625c None inhibited AC activity, demonstrating that the effect of poly-P was specific

Discussion

cAMP in bacteria is discussed in numerous publica-tions as a second messenger involved in regulatory pathways However, reliable studies in which intracel-lular cAMP concentrations were determined and regu-lation of cAMP biosynthesis in vivo was examined are rare This is due to its low intracellular concentrations and secretion of up to 95% of total cAMP into the medium, which causes unusual experimental difficulties and ambiguities [28–35] Generally, conditions that stimulate cAMP formation in bacteria, e.g changes in

pH, ion or nutrient concentrations, are related to stress conditions [28,36] In this study, we could not remedy the lack of knowledge of bacterial cAMP metabolism, and the physiological relevance of bacte-rial class III AC inhibition by poly-P merits further experiments

At the outset, we asked whether M bovis BCG con-tains endogenous factors that regulate AC activities The isolated AC inhibitor was unequivocally identified

by chemical means (31P-NMR, acid and base hydro-lysis, SDS⁄ PAGE), biochemical means (phosphatase degradation), and full functional substitution by com-mercial poly-P In addition, other bacterial constitu-ents were excluded, such as DNA, RNA, proteins and peptides, and peptidoglycans of the cell wall Although poly-P is a metabolic staple that is present in cells from all the kingdoms of life – bacteria, fungi, plants and animals – it has never been studied in conjunction with regulation of ACs [20,37] Mycobacteria produce poly-Ps under a variety of stress conditions, and pos-sess two poly-P kinases [21,38] Bacterial poly-Ps vary

in size and solubility, and intracellular concentrations

of poly-P fluctuate considerably (2–15 ngÆmg)1protein) [20,37,39,40] Depending on the organism, growth and

100

A

B

80

40

60

20

0

Poly-P75 ( M )

10 10 10 10

80

pH 5.5

40

60

20

–8

0

10 Poly-P75 ( M )

10 10 10

Fig 6 Inhibition of bacterial ACs from class III and class I by

poly-P75 (A) The following amounts of protein were used:

Rv1264(1–397), 660 n M (d); CyaG(370–672), an N-terminal truncated

version with only HAMP and catalytic domains, 640 n M (s);

CyaB1(1–859), 22 n M ( ); Rv3645(1–549), 153 lg of total membrane

proteins (h); Cya(1–446) from E coli, 876 n M ( , stippled line) (B)

Inhibition of basal and activated states of 660 n M Rv1264 by

poly-P75 The protein concentrations used were based on the linear

sec-tions of the protein dependency of the respective AC reacsec-tions This

ensured that dimerization of respective monomers was complete.

physiological conditions, poly-P may amount to up to

20% of bacterial dry weight [40,41]

For poly-P in bacteria, several physiological

functions in many locations have been proposed, e.g

poly-P accumulation in stress sensing [37,39,42–46]

Furthermore, poly-P is discussed as a primordial

pre-cursor of ATP, a flexible scaffold for the assembly of

macromolecules, a cellular phosphate store, a buffer

system in pH regulation, being involved in chelation of

cations such as Ca2+, Mn2+ and Mg2+ [20,37,39,47]

Our data suggest a new potential role for poly-P as an

inhibitor of bacterial class III ACs Because cAMP as

a bacterial alarmone is a global signaling molecule, the

reported actions of poly-P on bacterial class III ACs

may affect several cellular functions simultaneously It

was most interesting to note that the class I AC from

E coli was not inhibited by poly-P This may indicate

that the sequence dissimilarities between different

clas-ses of ACs reflect functional differences The presence

in some bacteria of ACs from different classes would

then enable different modes and levels of regulations

Actually, it may be useful for certain bacteria to

con-tain AC isoforms from different classes (such as

Pseu-domonas aeroginosa, which has AC isoforms from

classes I, II and III), because this would broaden the

modes of cellular regulation and pathogenicity [48]

Because the catalytic folds of a bacterial class IIIc

and a mammalian class IIIa AC are superimposable

[3,6], it is reasonable to assume that mammalian ACs

will be inhibited by poly-P as well In preliminary

experiments using membranes prepared from a rat

brain homogenate, we found that brain AC activity

was inhibited by poly-P with an IC50 of 10 lm (data

not shown) As far as the mechanism of inhibition is

concerned, the data obtained with Rv1264 indicate

binding in the catalytic fold Possibly, poly-P bridges

and occludes the substrate-determining lysine (Lys296

in Rv1625c) and the arginine (Arg376), which stabilizes

the transition state Therefore, poly-P may be helpful

in attempts to crystallize and characterize other

bacte-rial ACs

To the best of our knowledge, poly-P metabolism

has never been studied in conjunction with cAMP

metabolism The concentration of poly-P in stressed

bacteria appears to be higher than is needed for AC

inhibition Under the hypoxic growth conditions of

M bovis BCG used in this study, the concentration of

poly-P would probably silence all class III ACs

Several possibilities exist to explain this fact One is

that at a low level of stress conditions, such as modest

oxygen deprivation or nutrient depletion, cAMP

formation is elicited as an initial response Poly-P

bio-synthesis is then initiated, and this turns off cAMP

production of class III ACs Another possibility is that the availability of poly-P is locally restricted The neg-atively charged polyanionic compound may bind to positively charged carrier molecules or it may be neu-tralized by inorganic cations The availability of poly-P for termination of class III activation would then be tied to competition for different intracellular binding sites Finally, the possibility exists that poly-P accumu-lation is controlled locally, such that ACs are partially maintained in an inhibited, inactive state Release from inhibition could occur by poly-P degradation by tightly regulated phosphatases The latter would thus attain regulatory significance

Experimental procedures

Mycobacterial strain and growth

M bovis BCG 1721, a streptomycin-resistant derivative of BCG Pasteur, carrying a non-restrictive rpsL mutation (K42R) [49], was grown as a settling culture in tissue culture flasks in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose, catalase (Difco, Heidelberg, Germany) and Tween-80 (0.05%) [50] Flasks were shaken once daily by hand; that is, cells were grown under hypoxic stress E coli was grown in LB medium, and M smegmatis

in LB + 0.05% Tween-80 under constant shaking for oxy-genation (210 r.p.m shaking speed) Bacteria were harvested

at an attenuance (D600 nm) of 0.5–0.7 by centrifugation (10 min, 4400 g at 4C) and stored at)80 C until use

Expression and purification of Rv1625c

Rv1625c(1–443) in pQE60 was expressed in E coli BL-21(DE3)(pRep4) and purified to homogeneity using 0.6% n-dodecyl-b-d-maltoside for solubilization as previously described [17] The purified protein could be stored at )80 C without loss of activity for at least 6 months The catalytic domain Rv1625c(204–443) and other bacterial ACs used were expressed and purified to homogeneity as previously reported [12,17,22,51,52]

AC assay

AC activity was measured for 10 min in a volume of

100 lL at 30C [53] The reactions contained 22% glycerol,

50 mm Tris⁄ HCl (pH 7.5), 2 mm MnCl2 or 2 mm MgCl2, the indicated concentrations of ATP with 25 kBq of [32P]ATP[aP] and 2 mm cAMP with 150 Bq of [2,8-3H]cAMP to monitor yield during product isolation For determination of kinetic constants, ATP was varied from 14 lm to 100 lm, with constant 2 mm Mn2+ The reaction was started by addition of enzyme ATP conver-sion was limited to < 10%, to guarantee linearity ATP

was separated from product cAMP by sequential

chroma-tography [53]

Purification and analysis of poly-Ps from

M bovis BCG

Cells were suspended in 50 mm Tris⁄ HCl (pH 7.5) and

broken with a French press The homogenate was

centri-fuged (100 000 g for 1 h), and the supernatant was heated

at 95C for 30 min Coagulated protein was removed

(100 000 g for 1 h) The supernatant was centrifuged

through Sephadex G50 spin columns to remove small

con-taminants; the inhibitory capacity was in the eluate Next,

DEAE–Sephacel was added, and the material bound to

the anion exchanger The matrix was poured into a

col-umn, washed, and eluted with 400 mm NaCl The eluate

was applied to a Superose 6 column (30· 1 cm), and

frac-tions were examined by electrophoresis and for inhibitory

activity (Fig 1) Electrophoresis of poly-Ps was carried out

as previously described [23] A 15% acrylamide⁄

bisacryla-mide gel with 6 m urea was prepared with TBE buffer

(89 mm Tris⁄ borate, 2 mm EDTA, pH 8.3) The gel was

prerun at 200 V for 60 min Poly-Ps with average chain

lengths of 25, 45 and 75 residues were used as markers

(Sigma) Probes that contained 25% of sample buffer

(50% sucrose, 0.125% bromophenol blue and 450 mm

Tris⁄ borate at pH 8.3, 13.5 mm EDTA) were loaded and

electrophoresed for 25 min Gels were stained with 0.05%

toluidine blue O in 25% methanol and 5% glycerol for

20 min Destaining was performed with the same solvent,

lacking the dye The concentration of poly-P in

mycobac-terial preparations were assessed using standard curves

with poly-P75

31P-NMR measurement

The 31P-NMR spectra were obtained on Bruker

Avance 400 and Bruker Avance 500 spectrometers

operat-ing at 161.98 and 202.45 MHz, respectively The spectra

were recorded by applying 30 pulses with a repetition time

of 1 s at 21C, and referenced against external 85%

H3PO4 Multipuls decoupling sequences were applied to

remove any proton phosphorus interactions.31P peak areas

were obtained by fitting the spectra to a set of Lorenzian

line shapes using the Bruker topspin 2.0 software package

The pH of all samples was adjusted to 7.5

The fractions of Superose 6 that showed 95% inhibitory

activity against Rv1625c in 50 mm Tris⁄ HCl and 100 mm

NaCl were concentrated with an Amicon Ultra-4 5K

cen-trifugal filter device, and subsequently diluted with D2O to

reduce noise (50% final D2O in the water) A total volume

of 0.5 mL was transferred into a Norell 507-HP sample

tube; 50 mm linear poly-P3, poly-P25 and poly-P75, and

cyclic poly-P3 with sodium cations in 50% D2O, were used

as standards

Muropeptide determination by HPLC

The concentrated Superose 6 fractions (see above) were analyzed for the presence of muropeptides according to Glauner [54] Briefly, the sample was incubated with cello-syl or lysozyme at pH 4.8 at 37C overnight, deactivated (100C, 10 min), and centrifuged (14 000 g, 8 min) Eighty microliters of the supernatant was mixed with 80 lL of sodium borate (0.5 m, pH 9.0) and 1–2 mg of sodium boro-hydride The excess of borohydride was destroyed after incubation at room temperature for 30 min Separation of muropeptides occurred on a 250· 4.6 mm 3 lm Hypersil ODS column at 55C using a 135 min gradient from buffer A (50 mm sodium phosphate, pH 4.3) to buffer B (75 mm sodium phosphate, pH 4.9, 15% methanol) at a flow rate of 0.5 mLÆmin)1 Detection of muropeptides occurred at 205 nm We confirmed that cellosyl degraded mycobacterial cell wall material in respective controls with mycobacterial homogenates

Acknowledgements

This work was supported by the Deutsche Forschungs-gemeinschaft (SFB 766) P Sander is in part supported

by the Swiss National Science Foundation (contract: 3100A0_120326) and the European Union (LSHP-CT-2006-037217)

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