ritcher l._lipf promoter of mtb is upregulated by acidic ph_2009

The lipF promoter of Mycobacterium tuberculosis is upregulatedspecifically by acidic pH but not by other stress conditions

Laetitia Richter1,2 and Beatrice Saviola1,*

1 Basic Medical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, 309 E.Second St. Pomona CA 91766

2 Department of Biological Sciences California State Polytechnic University Pomona 3801 West Temple Ave,Pomona CA 91768

AbstractThe lipF gene of Mycobacterium tuberculosis has been implicated in pathogenesis and its promoterhas been shown to be upregulated by acidic stress. To further define the acidic pH that upregulatesthe lipF promoter from M. tuberculosis and to establish that it is specifically upregulated by acidstress and not by other environmental stresses, promoter expression levels were measured under avariety of conditions. The conditions measured were pH, temperature, oxidative stress and hypoxicstress.

KeywordsAcidic stress; lipF; lipase; Mycobacterium tuberculosis

IntroductionThe lipF gene of Mycobacterium tuberculosis encodes an esterase and has been shown to beimportant in pathogenesis (Zhang et al., 2005; Camacho et al, 1999). A transposon insertionbetween the promoter region for lipF and the gene resulted in a bacterium significantly reducedin its ability to grow in a mouse lung (Camacho et al., 1999). The same mutant grew well innutrient broth medium in vitro and an additional transposon insertion into the gene showed itto be non-essential (Lamichane et al., 2003; Sassetti et al., 2003). We had previously identifieda 479 base pair (bp) region of DNA upstream of the lipF gene that is transcriptionallyupregulated by exposure to growth media at pH 4.5 (Saviola et al., 2003). In support of thelipF gene’s involvement in a response to acidic stress, disruption of a homologue of the genein M. smegmatis led to a decreased ability to replicate at lower pHs (O’Brien at al., 1996; Raoet al., 2001; Tran et al., 2005).

As there may be additional pH’s that upregulate the lipF promoter, we sought to define themaximum pH that can induce promoter expression. In addition, to determine if the lipFpromoter is responsive to many stresses, or if it is responsive specifically to acidic stress wesought alternative stress conditions that may result in upregulation of the lipF promoter. We

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NIH Public AccessAuthor ManuscriptMicrobiol Res. Author manuscript; available in PMC 2010 January 1.

Published in final edited form as:Microbiol Res. 2009 ; 164(2): 228–232. doi:10.1016/j.micres.2007.06.003.

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examined lipF’s inducibility due to heat shock, cold stress, oxidative stress, hypoxic stress,and alkaline stress.

Materials and MethodsStrains and Media

The Mycobacterium smegmatis strain MC2155 (ATCC) was used in all experiments.Mycobacteria were grown in Middlebrook 7H9 broth (Difco) supplemented with 10% ADC(bovine serum albumin, dextrose and NaCl), 0.025% Tween 80 (polyoxyethylenesorbitanmonooleate) and 0.2% glycerol. For exposure to acidic stress, 7H9 growth media was adjustedin pH by the addition of concentrated HCl, and then filter sterilized through a Nalgenedisposable polyethersulfone filter with a 0.45 μm pore size. For exposure to alkaline stress,7H9 growth media was adjusted in pH by the addition of concentrated NaOH, and then filtersterilized through a Nalgene disposable filter as previously described.

Promoter inductions with acidic mediaMycobacterium smegmatis containing either, the plasmid pFPV27 with a promoterless genefor enhanced green fluorescent protein (gfp) from the jelly fish Aequorea Victoria (Cormacket al., 1996; Valdivia et al., 1996), or plipF with a 59 bp DNA region of the lipF promoterfused to gfp, was grown overnight to an approximate optical density of 0.7 at 600 nm(OD600). M. smegmatis was centrifuged and resuspended in 7H9 culture media (middlebrook)at pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5 and neutral pH 7.0. All promoter inductions wereperformed at 37°C and were exposed for 3 hrs, and 20 hrs. All samples were vortexed with 4mm glass beads to eliminate clumping and were diluted to the same optical density at 600 nm.The samples were measured on a TD-700 Turner designs fluorometer with a 486 nm excitationfilter and a 510–700 nm emission filter. All pH points were tested in triplicate. Adjustedfluorescence units were determined to be fluorescence units/O.D. units. To further narrow themaximum pH that induces the lipF promoter, M. smegmatis bearing pFPV27, or plipF wasexposed to 7H9 media at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, and pH 7.0 for 20hours and was measured on a TD-700 Turner designs fluorometer as previously described.Fluorescence was confirmed on a Nikon AFX-DX fluorescence microscope with a 450–490nm excitation filter and a 510 nm long pass emission filter (data not shown).

Induction with other environmental conditionsM. smegmatis containing pFPV27, plipF, or pBEN with a constitutive heat shock promoterfrom M. tuberculosis fused to gfp, was grown to OD600= 0.7 in 7H9 culture media pH 7.0. Totest for temperature stress, 1 ml samples were pipetted into test tubes and exposed to atemperature of 4°C in a Kenmore refrigerator or to a temperature of 42°C in a VWR heat blockfor 3 hours. To test for oxidative stress, hydrogen peroxide was added to a final concentrationof 5 mM in 7H9 culture media pH 7.0 for 3 hours and 20 hours. This hydrogen peroxideconcentration was used as it had previously been determined to be minimally lethal to themycobacteria (Springer et al., 2001). For hypoxic stress, mycobacterial aliquots weremaintained in 5 ml culture tubes in 7H9 culture media pH 7.0 and placed into an anaerobic jarusing the GasPak system (BBL) for 3 hours and for 20 hours. As a control, M. smegmatis wasalso exposed to culture media pH 4.5 for 3 hours. All samples were measured as previouslydescribed with a TD-700 Turner Designs fluorometer. Fluorescence was confirmed on a NikonAFX-DX fluorescence microscope as previously described (data not shown).

M. smegmatis bearing pFPV27, plipF, or pBEN was grown to an OD600= 0.7 in 7H9 media.Cells were spun down and resuspended in 7H9 media pH 4.5, 7.0, pH 8.0, pH 9.0 and pH 10.0and measured after 3 hours on a TD-700 Turner Designs fluorometer as previously described.

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Fluorescence was confirmed with a Nikon AFX-DX fluorescence microscope as previouslydescribed (data not shown).

Results and DiscussionThe lipF promoter of M. tuberculosis has already been shown to be significantly upregulatedby exposure to acidic media pH 4.5 (Saviola et al., 2003). We sought to test the maximum pHthat can induce the lipF promoter of M. tuberculosis to determine if higher pH’s can alsoupregulate promoter activity. As a pH below pH 4.5 is unlikely to be encountered in vivo, wetested pHs above 4.5 (Clemens and Horwitz, 1995; Deretic and Fratti; 1999; Schaible et al.,1998, Via et al., 1997; Xu et al., 1994; MacMicking et al., 2003).

By exposing M. smegmatis bearing a plipF-gfp fusion to a number of increasingly less acidicpH’s, we have determined that the maximum pH that induces the lipF promoter after 3 hoursof exposure is pH 5.5 (fig 1a). In this same experiment, the maximum pH to induce the lipFpromoter after overnight exposure is pH 6.0 (fig 1a). Thus higher pHs may result intranscriptional upregulation of the lipF promoter but require a longer period of exposure toproduce a full effect. Neither pH 6.5 nor pH 7.0 induced the lipF promoter after 3 hours ofexposure or after overnight exposure. pH 6.0 had previously been found to induce theexpression of lipF in M. tuberculosis after 36 hours of exposure (Zhang et al., 2005).

As there may be a pH intermediary between pH 6.0 and 6.5 that can induce the lipF promoter,we sought to further define acid regulation by measuring gfp expression after 20 hrs of exposureof M. smegmatis bearing pFPV27, or plipF at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5,and pH 7.0. It was determined that induction of the lipF promoter occurred with exposure toacidic media at pH 6.0, 6.1, 6.2, 6.3, and to a lesser extent pH 6.4. There was no detectibleupregulation after exposure to acidic media at pH 6.5 or 7.0 (fig 1b). Thus the maximum pHthat can induce the lipF promoter is pH 6.4 after 20 hours of induction (fig 1b).

To verify that acidic stress uniquely induces the lipF promoter we investigated a number ofstress conditions including temperature stress, oxidative stress, and hypoxic stress. None ofthe conditions examined resulted in upregulation of the lipF promoter other than exposure toacidic culture media pH 4.5 (fig 2a). All conditions examined resulted in significant andmeasurable activity of the heat shock promoter induced production of gfp.

To investigate upregulation of the lipF promoter due to exposure to alkaline stress, gfpexpression was measured under a range of pH levels. All promoter inductions were performedat 37°C and were exposed for 3 hrs. The lipF promoter was upregulated by acidic stress at pH4.5, but not by alkaline stress conditions at pH 8.0, 9.0, or 10.0.

The lipF promoter appears to be upregulated specifically by acid stress. Other stresses thatcould be encountered in vivo, such as oxidative stress, temperature stress, and low oxygentension did not appear to upregulate this promoter. Likewise, upregulation of the promoter doesnot appear to be a consequence of a general departure from neutral pH but as a result of exposureto acidic stress, as exposure to acidic pH but not alkaline pH induces the lipF promoter.

The lipF promoter is transcriptionlly upregulated quickly by exposure to low external pH, butmore slowly upregulated by moderately acidic pH. External acidity could damage surfaceexposed cell wall components that may be lipid or protein. Low pH (pH 4.5) may inducedamage rapidly, while moderate acidity (pH 6.0) may induce damage more slowly.Mycobacterial surface damage may trigger transcriptional upregulation of the lipF promoterresulting in rapid upregulation at low pH and slow upregulation at moderate pH. Alternatively,transcriptional upregulation may be triggered by a decrease in cytosolic pH. At low externalpH the cytosol may become acidic rapidly, while with moderate external acidity the cytosolic

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pH may decrease slowly. It has been shown that pH decreases in the cytosol of mycobacteriawhen they are exposed to external acidic pH, despite an active mechanism by the bacteria tomaintain a neutral pH (Rao et al., 2001). Thus, decreasing cytosolic pH may also be anintracellular signal to increase transcription of plipF.

LipF has been shown to function as an esterase (Zhang et. al., 2005). This protein may act tomodify the mycobacterial cell wall to render the microorganism more resistant to acidic stress.Altered lipid composition within the cell wall may result in a mycobacterium that is lesssusceptible to acidic damage or may be less penetrable to external acidity. Alternatively, LipFmay function to cleave lipids which could provide metabolic energy to withstand acidic stress.This metabolic energy may be essential to export protons from the mycobacterial cytosol tothe external environment, thereby raising the internal pH. Thus specific induction of lipF mayprime mycobacteria to be more resistant to acidic stress and be more likely to survive invivo.

AcknowledgmentsThis work was funded by a National Institutes of Health grant 5R03AI054794-02, an American Lung AssociationGrant, a California Lung Association grant, and a Potts Memorial Foundation grant.

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Fig. 1.Effect of varying pHs on the lipF promoter. M. smegmatis bearing pFPV27 or plipF wasexposed to growth media at a. pH 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 for 3 hours or 20 hours (o/n),or at b. pH 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or 7.0 for 20 hours (o/n). AFU is adjusted fluorescenceunits.

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Fig. 2.Effect of environmental stresses on the lipF promoter. M. smegmatis bearing pFPV27, plipF,or pBEN was exposed separately to a. neutral pH, pH 4.5, low temperature, heat shock,oxidative stress for 3 hours or 20 hours, or to hypoxic conditions for 3 hours or 20 hours, b. orto pHs ranging from pH 7.0 to pH 10.0 for 3 hours.

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ritcher l._lipf promoter of mtb is upregulated by acidic ph_2009

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