)

Indian Journal of Biochemistry & Biophysics Vol. 38, August 200 I, pp. 235-240

Biochemical effects of sparfloxacin on cell envelope of mycobacteria

Devinder Kaur, Indu Verma and G K Khuller*

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 1600 12, India

Received 23 Jun e 2000; revised 1 February 2001; accepted 29 March 2001

Sparfioxacin, a difluorinated quinolone is a potent an ti -mycobacteri al agent used in the treatment of mycobacterial infections. We have investi gated whether sparfloxac in had other, more subtle effects on mycobacteria besides its interac tion with DN A gyrase that could contribute to its therapeut ic efficacy. MycobacteriulII slIIegmatis cell s grown in media with sub­inhibitory concentrat ion of sparfioxac in were observed to have significant reducti on in the biosy nthesis of vital macromolecules, as shown by the incorporation of va ri ous radiolabelled precursors. The analysis of subcellular di stribution of phospholipids of sparfloxacin-treated cell s demonstrated an increase in the cell membrane and reducti on in the cell wal l, suggesting changes in the cell envelope architecture by sparfioxacin. Significant changes were also obscrved in other chemical constituents of the ce ll wa ll. especially in the arabinose and glucosamine contents. Mycolic ac ids, the major component of mycobacterial cell wall were reduced in the presence of Ml C.\o of sparfloxac in. There was a decrease in the limi ting fluorescence intensi ty (F rna.) of l-anilinonaphthalene 8-sulfonate CA NS) indicating alterations in the organi zation and confo rmation of mycobacterial cell surface. These results suggest that the mechan ism of ac ti on of ami-mycobacte ri al ac ti on of sparfl oxac in in volves mycobacterial cell envelope.

The global resurgence of tuberculosis and the emergence of multi-drug resistant (M DR) strains of M. Tuberculosis have emphasized an urgent need for new effective anti-mycobacterial drugs1.2. Among recently developed drugs, f1u oroquinolones represent an important class of therapeutic agents3

, Fluoro­quinolones such as ciprofloxacin and sparfloxacin have proven clinical activ ity in the treatment of patients with tuberculosis and leprosy4.5 . Of all the fluoroquinolones evaluated in experimental studies, sparfloxacin appeared to have the greatest acti vity against M. tuberculosis including the vast majority of MDR clinical isobtes6

. Regarding the mechani sm of action, fluoroquinolones are known to act by inhibit ing the topoisomerase II enzyme, DNA gyrase and topoi somerase IV (ref. 7). However, the events that result in cell death are yet poorly defincd8

, 1n E. coli , substantial alterations in the integrity of ce ll membrane by a DNA gyrase inh ibitor has also been reported which results in to cell death9

, In mycobacteria, DN A gyrase is the only targe t so far identifiedlO. Although , many physio logica l aspects of quinolone action in mycobacteria are in parallel with earlier findings in E. coli, it is still important to

Author for correspondence Phone: 747 585, Ext 282,274; Fax-744 40 1,745078: Email- Illcd in st @pg i.chd.nic. in

evaluate the effect of sparfloxacin on the mycobacterial envelope, which is unusually thick and lipophilic. The ai m of the present study was, therefore, to explore a possible relationship between anti-mycobacterial ac tivity of sparfloxacin and its effect on mycobacterial cell envelope integrity.

Materials and Methods Bac/erial strain and determillation of M IC and MIC50

Mycobacterium smegmatis MTCC6 obtained from Microbial Type Culture Collection (MTCC), Chandigarh was grown in modified Youman's medium. The MIC was determined by broth dilu tion method. Briefly, the drugs were added to liquid media to produce seri al two-fold dilutions. These tubes and a control tube containing no drug were inocul ated wi th a suspension of bacteria to yie ld a final concentrat ion of approximately 105_106 CFU/ml. Growth was monitored by measuring absorbance at 650 nm till the control cell s reached mid-exponenti al phase. The MIC was defined as the minimum concentration of drug, which resulted in no visib le growth and MICso (sub­inhibitory concentration) as the concentrat ion inhibiting growth by 50%.

Effect (~fspO/f70xacin on DNA and protein synThesis M. smegma/is cells grown in the presence of MICso

of sparfloxacin and control cells were suspended in

236 INDIAN J. BIOCHEM. BIOPHYS. , VOL. 38, AUGUST 200 1

sterile Youman's medium. The cell s were then pulsed wi th e H]-thymidine and [3H]-leucine, fo r DNA and protein synthes is, respectively for di ffe rent time intervals with constant shaking at 37°C. This was followed by the addi tion of 10% trichloroacetic acid and filtration on preweighed filter papers (0.45 /lm ), which were then dried and weighed again. The radioactivity was measured in a liquid scintillation counter, and the results were calculated as the cpm incorporated per mg dry weight of cell s.

EXlractioll and identification oj lipids Lipids were ex trac ted and purified by the method

of Folch et ai I. Indi vidual phospholipid components were separated by thin layer chromatography (TLC) on silica gel H plates by using the solvent system chloroform-methanol-7N ammoni a (65:25:4, vollvoll vol). Phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidylinositol were identified with specific spray reagents. For phosphatidylino­sitolmannosides (PIMs), TLC plates were sprayed with a -naphthol reagent (containing 10.5 ml of 15% ethanolic a-naphthol, 6.5 ml of concentrated H2S04, 40.5 ml of ethanol and 4.0 ml of water) and then heated till the browni sh purple spots appeared l2. Individual phospholipids were quantitated by estimating lipid phosphorus in the spots separated on TLC by the method of Bartlett as modified by Marinetti 13.

Synthesis oj phospholip ids Cells were harvested in the mid-exponenti al phase

of growth , washed with normal saline and suspended in Kreb's Ringer buffer under sterile conditi ons. Cell s were incubated at 37°C fo r I hr under shaking conditions to obtain a homogenous suspension and [1_1 4C] sodium acetate (25 /lCiIiOO ml of medium) was added to the cell s and incubation was continued for 90 min. From each culture 10 ml portions were transferred to tubes containing 0.5 ml of I M KCN . The tubes were centrifuged at 27,000xg for IS min . The cell pellet was recovered and lipids were ex tracted. Radi oacti vity was counted in a liquid scintillation fluid containing 0.4% (wtlvo l) PPO, (2, 5-diphenyloxazole) and 0.05 % (wtlvol) POPOP [1 , 4-bis (5-phenyloxazolyl) benzene].

Biochemical characterization oj cell wall /cell membrane fra ctions

The cell wall and cell membrane were isolated by the differential centrifugation method of Kearney and

Goldmanl4. The crude cell wall fraction thus obtained was purifi ed by diges tion with 0.2% trypsi n in 0.67 M phosphate buffe r (P H 8.0) fo r 3 hr at 37°C with continuous stirring ls. The sediment that was obtained by centrifugation at 10,000xg for 30 min was washed by centrifugation once with phosphate buffer, three times with I M NaC I solution, and then twice with di stilled water. The fin al sediment was suspended in a small amount of distilled water and lyophilized. The purity of cell wall and cell membranes was determined by measuring the acti vity of ATPase, a marker of cell membrane according to the method described by Penumarti and Khul ler l6 . Total and indi vidual phospholipids were extracted and estimated in cell wall and cell membrane fract ions by the methods as described earlier l6 . Samples of the cell wall fraction were ac id hydrolyzed by the method of Takeya et ail S for the quantitati ve determination of amino sugars and reducing sugars. Mycolic ac ids of the cell wall were ex tracted by the method of Winder and Collinsl7 dried under vacuu m weighed (grav imetric method).

Structural studies Structural studies with I-ani li nonaphthalene-8-

sulfonate (ANS) were carried out to monitor the changes on cell surface of M. smegmatis grown in the presence of sub-inhibitory concentration of sparfloxacin . The assay mixture consisted of a total volume of 2 ml in 10 mM citrate phosphate buffer (P H 6.5) containing 0.7 M NaCI , 10 )J.M ANS and whole-cell protein concentration ranging from 5 to 100 )J.g as estimated by the method of Lowry et a1 18

•

The fluorescence emission was recorded in a spectrofluorimeter (SFM-25; Kontron AG Zurich Switzerl and) with the excitati on wavelength at 390 nm and the emission wavelength of 5 LO nm. The fluorescence developed was recorded and the data was plotted as the reciprocal of the fluorescence signal (arbitrary unit) versus the reciprocal of the concentration of whole cell protein. A straight line was obtained whose extrapolation with the ordinate gave the reciprocal of limiting fluorescence (Frn,. ) of ANS in the membrane.

Results

Anti-mycobacterial activity oJ spmjloxacin The effect of sparfloxacin against M. smegmatis

was examined by growing the cell s in the presence of sparfloxacin at concentrations ranging from 0.02-

A

-

KAUR et at.: BIOCHEMICAL "EFFECTS OF SPARFLOXACIN ON CELL ENVELOPE OF MYCOBACTERIA 237

0.2 IJ.g/ml until the mid-exponential phase. Results were expressed as the relative percent of growth as compared to that of the control, plotted against the concentration of sparfloxacin. The MIC and MICso were found to be 0.08 IJ.g/ml and 0.2 IJ.g/ml, respectively and were confirmed on the basis of colony forming units.

Measurement of DNA and protein synthesis There was 41.7% decrease in the synthesis of DNA

within 5 min in the cells grown in the presence of MICso of sparfloxacin and remained almost constant till 30 min . The effect of sub-inhibitory concentration of the drug on protein synthesis as measured by incorporation of eHJ-leucine was observed to decrease by 36% as compared to control.

Effect of sparfloxacin on lipids and phospholipids of M. smegma lis

Cells grown in the presence of sub-inhibitory concentration of sparfloxacin revealed significant quantitative alterations (Table I). There was a significant reduction in the total lipids (p<O.OS) and total phospholipids (p<O.OI). This decrease was also reflected in the content of individual phospholipids with significant decrease (p< 0.001) in the content of PIMs with no change in the levels of PE and CL.

Synthesis of lipids Since quantitative changes were observed in lipids

and total phospholipids of cells grown in the presence of sub-inhibitory concentration of sparfloxacin, precursor incorporation studies were carried out to determine the rate of phospholipid synthesis. Incorporation of radioactivity into total phospholipids was found to be significantly decreased (p<O.O 1) (Table 2). This decrease in total phospholipids was mainly due to decrease in the synthesis of PIMs (p<0.00 1) with no change in the synthesis of PE and CL.

Quantitation of mycolic acids On quantitating mycolic acids by gravimetric

method (Fig. 1), sparfloxacin grown cells were found to contain reduced content (p<O.O 1) as compared to control. [1_14C] sodium acetate was also used as precursor to study the in vivo synthesis of mycolic acids . Incorporation of radioactivity into mycolic acids of cells grown in the presence of sparfloxacin revealed net reduction (p<0 .0 1) in the synthesis of mycolic acids (Fig. 1).

Analysis of cell wall and cell membrane phospholipids

Subcellular fractions of M. smegma tis grown in the

Table I-Total lipids, phospholipids and individual phospholipids of M. smegmatis grown at MICsoof sparfloxacin.

[Values are mean ± SD of three independent batches analyzed in duplicates]

Total lipids Tota l phospholipids Individual phospholipids (mg/g dry wt. of cells) (mg/g dry wt. of cells) (mg/g dry wt. of cells)

PIMs PE CL

Control 177.9± 1.63 32.8 ± 1.96 9.82 ± 0.54 4.24 ± 0.87 7.8 1 ±0.52

SPX" 145.3±19.0* 26.07 ± 1.45** 4.66±0.23*** 4.7± 0.43 NS 8.24±0.49NS

***p<O.OO I; **p<O.O I; *p<0.05; NS , non significant; a, sparfloxacin .

Table 2-Incorporation of [1_14q sodium acetate into total lipids, phospholipids and individual phospholipids of M. smegl1latis grown in the presence of MICslJ of sparfloxacin.

[Values are mean ± SD of three independent batches analyzed in duplicates]

Control

SPX

Total lipids (cpmx \04/g dry wt. of cells)

33.0 ± 1.40

35.0 ± 3.74 NS

**p<O.O I; *p<O.05; NS, non significant.

Total phospholipids (Cpmx \04/g dry wt. of cells)

16.8 ± 0.55

14.8 ± 0.73*

Individual phospholipids (cpmx 104/g dry wt. of cells)

PIMs PE CL

7.7±0. 18

4.5 ± 0.63"

3.5 ± 0.32

3.8 ± 0.28NS

5.36 ± 0.26

5.86 ± 0.30 NS

238 INDIAN J. B10CHEM. BIOPHYS., VOL. 38, AUGUST 200 1

SOD

Q)

!:' -5 Q)

~" 400 400 U C. C1l

"U E en en ::J-:-

:c~ E 0 300 I/) ~ .- ,

t: "C Q) ..... .- Utn t: ... -~ on 0 ' 0 u ~ ..... ~ 200 200 -)(

u 0 E fO r:::: c. t) .2 u --(5 C1l

L-() 0 >. tOO t OO C. :E L-

0 U r::::

Con Spx Spx Con

Fi g. I- Mycol ic acid cOllle lll and incorporati on of r l _l~C] sod iu m acetate into total mycolic ac ids oj M. slIIegllla/is grow n in the presence of MICso of sparnoxac in. I_ Mycoli c acid content; III Mycolic ac id sy nt hesis. Va lues arc mea n ± SD of th ree independent batchesl.

Tab le 3-Effect of sparfi oxaci n on the distribu tion of phospholipids in ce ll wa ll and cell membrane of M. smegllla/is

I Values arc mean ± SD of three indcpende nt batches analyzed in dupli catesl

Cell fraction

Cell wall Control

Total phospholi pids (mg/g dry wt. or ce ll frac tion)

39.7 ± 1.35

PIMs

16.3 ± 0.6 1

Individual phospholipids (mg/g dry wt. of cell frac tion)

PE

14.4 ± 0.99

CL

6.5 ± 0.29

SPX 17.4± 1.89*** 7. 1 ± 0.26*** 3.5 ± 0. 14*** 5.4 ± 0.43 *

Cell membrane

Control 33 .4 ± 1.37 17.83± 0.38 5.9± 0.29 8.7 ± 037

SPX 44.4 ± 1.78*** 27.33 ± 0.49*** 11 .56 ± 0.30*** 1.6 ± 0.29***

***p<O.OO I ; *17<0.05

presence of sub-inhibitory concentrations of sparfloxac in were isolated by di ffe renti a l centri­fu gation as described in M ateria ls and Methods. The compari son of AT Pase activity in the two fracti ons showed the me mbrane to have much hi gher activity

( 1.30 j.lmo lesPi li berated/mg prote in/30 min ) as

co mpared to cell wa ll (0 . 10 j.lmo les Pi libe rated/mg protein/30 min). Thi s confirms the purity of these frac tions. Further, to ta l y ie ld o f purifi ed ce ll wall was found to be approximate ly 400 mg/g dry we ight of cells. Phospho lipid profiles o f the subcellular

components VIZ. cell wall and cell membrane were investigated (T ab le 3). A sig ni ficant decrease (p<0.00 I) in the ce ll wa ll phospho lipids was observed in ce ll s grown in the presence o f M ICso of sparfloxac in . Thi s reduction in to ta l phospho lipids was manifested in all the indi vidual phospho lipids i.e. PIMs, PE and C L. On the contrary, there was a significant increase (p <O.OOI ) in the phosphol ipids of ce ll membrane as compared to contro l, whi ch was mainly due to the accumulatio n of PIM s and PE (T able 3).

A

KAUR el al.: BIOCHEMI CAL EFFECTS OF SPARFLOXACIN ON CELL ENVELOPE OF MYCOBACTERI A 239

Tablc 4--Biochemical composition of cc ll wa ll of M. slIIeglllalis grown in the presence of MI Cso of sparnoxac in

I Va lues are mean ± SO of three indcpcndcnt batches analyzcd in du plicatcs l

Arabinose Galac tosc Glucosaminc Mycoli c acid (mg/g dry wt of cell wall )

COlll ro l 35.7 ± 5.2 38. 1 ±7.7 11.7 ± 1.0 9 11 ±33 .0

SPX 56.7 ± 3.98* 34.7 ± 2.99NS 14.8 ± 0.5** 7 18± 14.3** *

***1'<0.00 I; **1'<0.0 I; *1'<0.05; NS, non signi ficant.

Biochemical composition of cell wall Representati ve constituents of cell wall viz

arabinose, galac tose, glucosamine and mycolic ac ids were analyzed (Table 4). Similar to whole cells, cell wall of M. smegmatis grow n in the presence of MICso of sparfloxac in was fo und to have less amount of mycolic acids (p<0.001) as compared to cell wall of control cell s. However, there was an apparent increase in the content of arabinose (p<0.05) and glucosamine (p<O.O 1) with no change in the content of galactose.

Structural studies Alterations in the cell envelope and the effect of

altered lipid composition on membrane structure was studied by using a tluorescent probe, ANS, which is known to bind both intact cells and cell membrane. Limiting fluorescence (FIll"x) va lues of ANS bound were calculated from double reciprocal plot of relati ve fluorescence intensity versus protein concentration. On incubating ANS with intact cell s, a significant decrease in FIll"x (54 ± 4.64) was seen in the cell s grown in the presence of MICso of sparfloxacin as compared to control (303 ± 43.2).

Statistical analysis The results of the present study were analyzed by applying student's ' t' tes t.

Discussion The major anti-bacteri al effects of tluoro­

quinolones are considered to be medi ated vi a binding to DNA gyrase or topoisomerase IV. However, the biochemi ca l events associated with anti -myco­bacterial acti vity of tluoroquinolones are still poorly understood. I n the present study, a wide range of biochemical effects of sparfl oxacin have been elucidated in M. smegmatis, which is a fast growing analogue of M. tuberculosis l9•

M. srnegmatis cell s grown in the presence of sub­inhibitory concentration of spartl oxacin showed a signifi cant inhibi tion (41.7%) of DNA sy nthesis within 5 min . In E.coli, quinolones are known to inhibit DNA synthesis rapidly, if gy rase is the target

d I I . f' . h 20 ? I 0 an s ow y t topOisomerase IS t e target '-. ur observations are consistent with these reports indicating interac ti on of spartloxac in with DNA gyrase in mycobacteri a. Earlier, Drilica el al. lo also reported a decrease in DN A sy nthesis in M. smegmatis cell s treated with cipro fl oxacin .

Besides interacti on of tluoroquinolones with the enzy mes of DN A synthesis, there are contl ic ting reports suggesting their effec t on bacteri al envelope22.23 . Due to unusuall y thick and lipid-rich mycobacterial enve lope, it is important to in vestigate the effect of flu oroquinolones on thi s component. In the present study, analysis of total lipids and total phospholipids revealed a significant decrease in these components of whole cell s and cell wall of M. smegmatis grown in the presence of MICso of spartloxac in which is in accordance with the findings of Verma et ap4 . Mycobacteri al cell envelope is also characteri zed by the presence of exceptionally hi gh molecular weight a -aklyl, ~-hydroxy acids, the mycoli c acids2s

. A signi ficant reduction in mycolic ac id content in cell s grown in the presence of sparfloxacin further confirmed the cell envelope alte .. ations. In addition. the analysis of other major constituents of mycobacteri al cell wall viz. arabinose, galactose and glucosamine also revealed signi ficant changes. The data presented here is consistent with the concept that the synthes is of entire cell wall core is dependent on the proper formati on of mycolyl arabinogalactan complex . Cell wall biosy nthesis is a dynamic process in which change in one component may lead to quantitati ve alterations in others.

In addition to cell wall changes, an increase in the phospholipid content of cell membrane was also

240 INDI AN J. BIOCHEM. BIOPHYS., VOL. 38, AUGUST 200 1

observed in the presence of sparfl oxacin . The vari ous biochemi cal alterations in cell wall and cell membrane suggested a changed organi zation of cell envelope. The fluorescent probe ANS has been widely used to study the structural changes induced by a large variety of compounds in natural and art ific ial membranes26 due to its bi nding to both lipids and proteins. In the present study, a significant reduction in the limiting flu orescence intensity of ANS further confirmed the surface alterati ons in the presence of sparfloxac in , whi ch could be attributed to changed lipid composition of the ce ll envelope.

Thus, the results of present study suggest that in mycobacteria, inhi bition of DNA replication in sparfloxacin-treated cell s leads to a cascade of physiologica l events culminating in cell envelope changes, whi ch may lead to lethal events in the cell s.

Acknowledgement

This work was fi nanced by a grant from the Indian Council of Medi cal Research (lCMR), New Delhi , India. D. Kaur is thankful to the PGIM ER (Chand igarh ) fo r provid ing a fe ll owshi p.

References

Rav igli one M. Snidcr D & Kochi A ( 1995) l AMA 273, 220-226

2 Bloch A B. Simonc P M, McCray E & Castro K G ( 1996) lAMA 275,487-489

3 Loun is N, Ji B, Tru ffol-Pcrnol A & Grosscl J ( 1997) AIl/il1licrob Agellls Chemo/her 4 1, 607 -6 10

4 Kcnnedy N, Fox R, Kisyombc G M, Saruni A 0, Uiso L 0 , Ramsay A R. Ngowi F I & Gill csp ie S H ( 1993) Alii Rev Res Vis 148. 1547- 155 1

5 Chan G P, Garcia- Ignacio B Y, Chavez V E, Livelo J B, Jimenez C L, Parrill a M L & Franzblau S G ( 1994) All/illlicrob Agen/s Chelllo/her 38,6 1-65

6 Lalandc V, Truffot-Pernot C. Moul in A P. Grosscl J & Ji B ( 1993) An/illl icwb Agel1ls Chelllother 37,407-4 13

7 Drli ca K & Zhao X ( 1997) Microbiol Mol BioI Rev 61. 377-392

8 Eva N G T, Trucksis M & Hooper D C (1996) Alltilllicrob Agell/s Chelllother 40, 188 1- 1888

9 Dougherty T J & Saukkoncn J ] ( 1985) Alltilll icrob Agellts Chelllo/h er 28,200-206

10 Drlica K. Xu C, Wa ng J Y, Burgcr R M & Ma li k M ( 1996) Allt illl icrob Agell ts Chell10ther 7, 1594-1 599

I I Folch J. Lccs M & Stanley G H S ( 1957) 1 BioI Chelll 226. 497-509

12 Khullcr G K & Brcn nan P J ( 1972) Alii Rei' Respir Dis 106. 892-896

13 MarinClli G V ( 1962) 1 Lipid Res 3, 1-20 14 Kearney E B & Gold man D S ( 1970) Bio('hil1l Biophys Acta

197, 197-205 15 Takcya K. Hisatsunc K & Inoue Y ( 1963) .1 Bacterial 85, 24-

30 16 Pc numart i N & Khullcr G K ( 1983) Expcrielllia 39, 882-883 17 Windcr F G & Coll ins P B ( 1970) 1 Cell Microbiol 63. 41-48 18 Lowry 0 H, Roscborough N J. Farr A L & Randa ll R J

( 195 1) 1 Bioi Chelll 193,265-275 19 Tak ifT H E, Cimino M, Musso M C, Weisbrod T, Martinez

R, Delgado M B, Salazar L, Bloom B R & Jacobs W R Jr ( 1996) Proc Nml Awd Sci USA 93, 362-366

20 Snydcr M & Drli ca K ( 1979) 1 Mol Bioi 13 1. 287-302 2 1 Khodursky A B, Zcchiedrich E L & Cozzarc lli N R ( 1995)

Pwc Na tl I\ C(l/ Sci USA 92, I 180 1- 11 805 22 DalLofl A ( 1985) in: The ill/illellce of ollti-bio/ics 011 the host

parasite relatioll ship II. (Adam H. Hahn W & Opfcrkuch W, eds) Springcr- Vcrlag Bcrli n and Heidclb.:rg, pp 16-27

23 Lewin C S, Howard N A & Smilh J T ( 199 1) 1 Med MicroiJiol 34. 19-22

24 Vcrma I. Rohi lla A & Khull cr G K ( 1999) Lellers ill I\pplied Microbiol29, 113- 11 7

25 Brenn an P J & Nikaiclo H ( 1995) AIIIIII Rev Biochelll 64. 26-63

26 Slavik J ( 1982) lJiuchim Biuphys Acta 694, 1-25