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Infection and Immunity, March 2004, p. 1733-1745, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1733-1745.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Attenuation of Late-Stage Disease in Mice Infected by the Mycobacterium tuberculosis Mutant Lacking the SigF Alternate Sigma Factor and Identification of SigF-Dependent Genes by Microarray Analysis

Deborah E. Geiman,1 Deepak Kaushal,1,{dagger} Chiew Ko,1 Sandeep Tyagi,1 Yukari C. Manabe,1 Benjamin G. Schroeder,2,{ddagger} Robert D. Fleischmann,2 Norman E. Morrison,1 Paul J. Converse,1 Ping Chen,1,§ and William R. Bishai1*

Center for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore,1 The Institute for Genomic Research, Rockville, Maryland2

Received 30 September 2003/ Returned for modification 16 November 2003/ Accepted 9 December 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Mycobacterium tuberculosis alternate sigma factor, SigF, is expressed during stationary growth phase and under stress conditions in vitro. To better understand the function of SigF we studied the phenotype of the M. tuberculosis {Delta}sigF mutant in vivo during mouse infection, tested the mutant as a vaccine in rabbits, and evaluated the mutant's microarray expression profile in comparison with the wild type. In mice the growth rates of the {Delta}sigF mutant and wild-type strains were nearly identical during the first 8 weeks after infection. At 8 weeks, the {Delta}sigF mutant persisted in the lung, while the wild type continued growing through 20 weeks. Histopathological analysis showed that both wild-type and mutant strains had similar degrees of interstitial and granulomatous inflammation during the first 12 weeks of infection. However, from 12 to 20 weeks the mutant strain showed smaller and fewer lesions and less inflammation in the lungs and spleen. Intradermal vaccination of rabbits with the M. tuberculosis {Delta}sigF strain, followed by aerosol challenge, resulted in fewer tubercles than did intradermal M. bovis BCG vaccination. Complete genomic microarray analysis revealed that 187 genes were relatively underexpressed in the absence of SigF in early stationary phase, 277 in late stationary phase, and only 38 genes in exponential growth phase. Numerous regulatory genes and those involved in cell envelope synthesis were down-regulated in the absence of SigF; moreover, the {Delta}sigF mutant strain lacked neutral red staining, suggesting a reduction in the expression of envelope-associated sulfolipids. Examination of 5'-untranslated sequences among the downregulated genes revealed multiple instances of a putative SigF consensus recognition sequence: GGTTTCX18GGGTAT. These results indicate that in the mouse the M. tuberculosis {Delta}sigF mutant strain persists in the lung but at lower bacterial burdens than wild type and is attenuated by histopathologic assessment. Microarray analysis has identified SigF-dependent genes and a putative SigF consensus recognition site.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Control of Mycobacterium tuberculosis infection is difficult due to the complex and long-term nature of the host-pathogen interactions in this disease. Initial infection is followed by bacterial multiplication within mononuclear phagocytes, release of intracellular organisms, and dissemination (15). The subsequent development of specific immunity often results in containment of the infection but not eradication of the organism. Therefore, reactivation of tuberculosis may occur years after initial exposure (56). The mechanisms enabling M. tuberculosis to survive during the late stages of active infection in the mouse tuberculosis model may have a role in the development of latent tuberculosis (54). Disease pathogenesis in these different stages is likely to involve various virulence factors which are differentially deployed as well as a system of genetic adaptation by the pathogen (32, 47).

In the mouse tuberculosis model, one key transition point during pathogenesis occurs at 4 to 8 weeks after infection, as host acquired cell-mediated immune responses mount (42). At this time, tubercle bacilli may employ specific adaptive mechanisms that lead to a state of contained, multibacillary pulmonary infection, which shares some features of the infection that occur in humans. While no widely accepted animal model of human latent M. tuberculosis infection is currently available (19), the late-stage multibacillary plateau of lung bacterial counts seen in the mouse has been proposed to correlate in some aspects with the arrested paucibacillary state in human latent tuberculosis infection (32). Consequently, the identification of bacterial genes required for survival in chronic murine infection may be valuable for understanding the pathogenesis of human latent M. tuberculosis infection.

Many studies have implicated sigma factors in the regulation of virulence gene expression by M. tuberculosis (11, 27, 36, 45). The M. tuberculosis sigF gene was discovered by degenerate PCR (17) and is a close homologue of sporulation sigma factors in Streptomyces coelicolor and Bacillus subtilis as well as stress response sigma factors in B. subtilis, Staphylococcus aureus, and Listeria monocytogenes (16). It is strongly induced during the stationary phase of growth and under certain stress conditions, such as nitrogen depletion, cold shock (17), and exposure to certain antibiotics (38). There was no marked change of sigF mRNA expression in M. tuberculosis H37Rv after a short 2-h exposure to a variety of stresses in culture (34), but expression was upregulated during growth within macrophages (21). Recently, M. tuberculosis sigF has been shown to be expressed during nutrient starvation, which may be a model of the nongrowing drug-resistant state that mimics the persistence of M. tuberculosis in vivo (5). The M. tuberculosis {Delta}sigF mutant strain, in which the sigF gene is deleted and replaced by a hygromycin resistance gene, has been shown to be less virulent in mice by time-to-death analysis (8).

In this study, we evaluated the phenotype of the M. tuberculosis {Delta}sigF mutant during mouse infection by organ CFU counts and histopathologic analysis. Finding an attenuated phenotype in this model, we also tested the efficacy of the {Delta}sigF mutant strain as a possible candidate for vaccination against M. tuberculosis using the rabbit aerosol challenge model (6, 12). Through microarray analysis, we studied the global expression of genes under the influence of SigF during the different stages of in vitro growth. Evaluation of genes underexpressed in the absence of SigF has permitted the identification of a putative consensus binding site for SigF.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial cultivation. The virulent CDC1551 (also known as CSU93 or Oshkosh) strain of M. tuberculosis (18, 60) and the Erdman strain were grown at 37°C on Löwenstein-Jensen medium or in roller bottles in 7H9-albumin-dextrose complex (7H9-ADC) broth (Difco Laboratories, Detroit, Mich.) supplemented with 0.2% glycerol and 0.05% Tween 80. The {Delta}sigF mutant strain and the complemented {Delta}sigF mutant strain were generated from CDC1551 as described previously (8). M. bovis BCG (Pasteur) was maintained under similar conditions. For animal inoculation, liquid cultures were declumped by brief bath sonication and settling and were diluted in complete 7H9 medium. Colony counts from mouse organs were performed by using Middlebrook 7H10-ADC agar plates, made selective by adding carbenicillin, polymyxin B, trimethoprim, and amphotericin B to final concentrations of 100 µg/ml, 200 U/ml, 20 µg/ml, and 10 µg/ml, respectively.

Mouse virulence assays. For mouse organ CFU assays, BALB/c mice (Harlan Sprague Dawley), 6 to 8 weeks old, were inoculated intravenously with 0.1 ml of dispersed preparations of mycobacteria. Three inocula were prepared using dispersal techniques as described previously (40) and counted on the day of infection. Plate counts showed the actual inocula to be 6.1 x 105 CFU for wild-type M. tuberculosis bacteria and 3.6 x 105 CFU for the {Delta}sigF mutant strain of M. tuberculosis. Groups of six mice were sacrificed at weeks 1, 2, 4, 8, 12, 16, and 20. The lungs and spleen were removed, and the tissues were homogenized in phosphate-buffered saline-Tween. The homogenates were transferred to plates with complete 7H10-ADC agar, and the colonies were enumerated to determine infectious burden in these organs. A similar experiment was conducted with outbred Swiss-Webster mice, in which the inoculum of the {Delta}sigF mutant strain was 3.8 x 105 CFU and that of the wild type was 3.4 x 105 CFU.

Vaccination of rabbits. Strains of {Delta}sigF mutant M. tuberculosis CDC1551, M. bovis BCG, and wild-type M. tuberculosis Erdman were grown to log phase, bead vortexed, and then allowed to settle. Supernatants were pooled, mixed with glycerol to a final concentration of 10%, and then frozen in aliquots at -70°C. The bacterial titers of the aliquots were determined by plating serial dilutions on Middlebrook 7H10 agar. Vaccine aliquots were thawed and diluted in 10% oleic acid albumin at the time of vaccination. Pathogen-free New Zealand White rabbits (2.5 kg each, female) were purchased from Covance Research Products, Inc. (Denver, Pa.). Animals were vaccinated with 5 x 106 organisms intradermally on each flank at time zero. Nine weeks later, animals were aerosol challenged with M. tuberculosis Erdman strain (a kind gift of Frank Collins) by a nose-only manifold system at the U.S. Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Frederick, Md. Each animal was exposed for 10 min to an aerosolized 10-ml inoculum of aerosol containing 106 organisms/ml diluted in 10% oleic acid albumin. Whole-body plethysmographs and impinger samples of the aerosols were obtained for each rabbit. The oleic acid-albumin solutions containing the aerosolized bacteria were cultured at various dilutions on both Löwenstein-Jensen slants and 7H10 Middlebrook agar (Fisher). For each rabbit, the number of viable bacilli inhaled (104 to 105 organisms) was calculated based on the volume of inhaled air during exposure and the number of CFU per milliliter cultured from the impinger samples (6). The animals were housed in biosafety-level-3 facilities at the George Washington University Medical Center immediately following infection. After 5 weeks, the rabbits were euthanized with intravenous pentobarbital. The lungs were removed, and the number and volume of grossly visible primary tubercles in the lungs were assessed by Lurie's tubercle count method (14). Lung specimens for histological examination were fixed in 10% formalin and paraffin embedded. All animals were maintained in accordance with protocols approved by the institutional Animal Care and Use Committee of the three institutions where the work was performed.

Neutral red staining. Following growth on Löwenstein-Jensen medium for 6 weeks, wild-type, knockout, and complemented strains were washed with 50% methanol at 37°C for 60 min. After decanting and draining the solvent, 5 ml of 5% NaCl in 0.5% Trizma base, brought to pH 9.5 with 2 N HCl, was added to the cells. For color development, 50 µl of 0.05% neutral red was added, and the washed cells were incubated for 60 min at 37°C. Neutral red binding was determined by the color of the cell pellet (39).

RNA isolation. Cultures of the M. tuberculosis {Delta}sigF mutant strain, wild-type M. tuberculosis, and the complemented {Delta}sigF mutant strain were grown to A600 values of 0.6, 2.2, and also for 3 days after an A600 of 2.2 was reached. Total RNA was extracted from the cultures using Trizol. Briefly, the bacterial cultures were centrifuged and the pellet washed in phosphate-buffered saline. The pellet was resuspended in 1 to 3 ml of Trizol along with 0.1-mm-diameter Zirconia/silica microbeads (BioSpec Products, Bartlesville, Okla.) and agitated on a bead beater three times at 30-s intervals. Cells were chilled on ice for 1 min following each disruption. The sample was centrifuged at 14,000 rpm (Eppendorf model 5417R centrifuge), and 200 µl of chloroform/ml of sample was added to the supernatant, followed by vortexing and centrifuging for 3 min at 4°C. Isopropanol was added to the aqueous phase, the RNA precipitated at room temperature, and the pellet washed with 80% ethanol. After air drying, the pellet was resuspended in diethyl pyrocarbonate-treated water and stored at -70°C.

Microarray probe labeling, hybridization, and analysis. Gene-specific PCR primers were designed to amplify internal fragments from a total of 4,016 open reading frames from the annotated sequences of M. tuberculosis CDC1551 and H37Rv (10, 18, 27). Individual purified PCR products were spotted in duplicate on High Contact Angle slides (Corning, Ithaca, N.Y.) using a 96-well format IAS arrayer (Intelligent Automation Systems, Cambridge, Mass.). Bacterial RNA prepared by the Trizol method was reverse transcribed and labeled with Cy3 or Cy5 (Amersham Pharmacia) using the aminoallyl labeling method (27). The slides were scanned with a Genepix (Axon Instruments, Union City, Calif.) scanner using Genepix Pro 3.0 software. Spot intensities were defined and quantified using the TIGR Spotfinder and Array Viewer software systems. For each bacterial growth point, two independent RNA preparations from wild type and mutant were prepared. For each RNA sample pair, reverse labeling was performed by switching the Cy3 and Cy5 dyes (two hybridizations for each growth point). Each amplicon was spotted in duplicate, yielding four relative hybridization values for each gene at each growth point. The ratio of wild type to mutant was determined. Any ratio greater than 1.7-fold was operationally considered a significant down-regulation in the {Delta}sigF mutant strain. In the same respect, any ratio less than 0.5-fold was considered to be up-regulated in the {Delta}sigF mutant strain. The median value was used for comparison of wild type and mutant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation and survival of the M. tuberculosis {Delta}sigF mutant strain in mouse tissues. We investigated the in vivo growth phenotype of the M. tuberculosis {Delta}sigF mutant strain in the mouse tuberculosis model, which earlier time-to-death analysis had shown to be less virulent than wild-type (8). Dispersed preparations of the {Delta}sigF mutant strain and wild-type M. tuberculosis were administered by injection of 3.6 x 105 and 6.1 x 105 CFU, respectively, into the tail veins of groups of BALB/c mice. At the indicated times, mice were euthanized and the CFU counts were determined by plating homogenates of the spleen and lung tissues. Although the initial levels of {Delta}sigF mutant were lower at week 1 than those of the wild type, in keeping with the smaller inoculum given, the in vivo growth rates of the mutant and wild-type strains were essentially identical from 1 to 8 weeks in lungs (Fig. 1). However, at 8 weeks the M. tuberculosis {Delta}sigF mutant strain persisted in the lung while the wild-type strain continued to proliferate slowly from 8 to 20 weeks (Fig. 1A). At 20 weeks, the difference in CFU between mutant and wild type was 40-fold in the lung and 43-fold in the spleen, compared to only 3- and 4-fold differences at week 1 in the lung and spleen, respectively. This level of attenuation seen in organ CFU counts is notably greater than that found with mutant tubercle bacilli lacking either sigH (27) or sigC (58) or sigE (1), which maintained CFU counts comparable to those of the wild type with reduced pathology. On the other hand, the {Delta}sigF mutant strain persists at considerably higher levels and for a longer period in immunocompetent mice than do auxotrophic mutants such as the pantothenate auxotroph (48). A similar experiment conducted with outbred Swiss-Webster mice, in which the inoculum of the {Delta}sigF mutant strain (3.8 x 105) exceeded that of the wild type (3.4 x 105) by a factor of 1.1, revealed a similar pattern of stable lung persistence by the mutant during late-stage infection (data not shown).



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  		FIG. 1. Comparison of the survival of the {Delta}sigF mutant strain ({circ}) and wild-type M. tuberculosis strains (CDC1551) ({square}) in the mouse tuberculosis model. Following intravenous inoculation of mice with 3.6 x105 (5.56 log) mutant or 6.1 x 105 (5.79 log) wild-type bacteria, groups of six mice were sacrificed at the indicated times. A virulence analysis by whole organ CFU counts was done in the lung (A) and spleen (B) at each indicated time. Error bars, standard deviations.

 
Histopathological analysis of mouse tissues at the 4th week after infection showed that both wild-type and mutant strains produced comparable degrees of interstitial inflammation with small, scattered foci of organizing lesions (Fig. 2A and D, respectively). Granulomatous inflammation began to form in both strains at the 12th week. However, wild-type M. tuberculosis displayed increasing granuloma size, coalescence of the lesions, and progressive loss of lung parenchyma, while the disease in lungs of mice infected with the {Delta}sigF mutant strain showed retarded disease progression (Fig. 2B and E, respectively). Indeed, at 20 weeks, lungs of mice infected with the mutant strain (not shown) showed degrees of pneumonitis similar to those of wild-type-infected animals at 12 weeks (Fig. 2B), suggesting a delay in the development of classic tuberculosis pathology with the mutant. Acid-fast stains revealed a paucity of bacilli in lung tissues infected with the {Delta}sigF mutant strain at 20 weeks compared with the wild type (Fig. 2C and F, spleen not shown), which is in agreement with the organ CFU assay results shown in Fig. 1. At the level of gross pathology, there were readily apparent differences between tissues infected by the mutant versus wild type. The organs of animals that were infected by the mutant strain had smaller and fewer granulomas and less inflammation than those infected with wild type (e.g., spleen, Fig. 2G). Hence, the {Delta}sigF mutant strain showed a mouse phenotype of persistent, high-level organ survival but reduced histopathologic evidence of disease. In contrast, reduced pathology was observed in the face of equivalent bacterial replication in the M. tuberculosis {Delta}sigH mutant (27).



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  		FIG. 2. Microscopic histopathology of mouse lung tissues during the course of infection by wild-type M. tuberculosis (A to C) and the {Delta}sigF mutant strain (D to F). Low-power views are representative of lung specimens after formalin fixation, paraffin embedding, and azure eosin staining, corresponding to an infection duration of 4 weeks (A and D) and 12 weeks (B and E). Representative high-power views of lung sections are shown after acid-fast staining at 20 weeks of infection (C and F). Also shown are the unfixed representative murine spleens (G) at necropsy after infection for 20 weeks: no infection (left), infection with wild type (middle), or infection with the {Delta}sigF mutant strain (right). See text for details.

 
Testing the efficacy of the {Delta}sigF mutant strain as a vaccine in the rabbit model. Since it has been shown by these and previous data (8) that the M. tuberculosis {Delta}sigF mutant strain is attenuated in the mouse infection model, we tested this mutant for its efficacy as a vaccine in the rabbit model. Pathogen-free New Zealand White rabbits, six per group, were inoculated intradermally with 5 x 106 CFU of preparations of the {Delta}sigF mutant strain and BCG. Five unvaccinated rabbits, matched for age and weight, were used as controls. No skin lesions formed at the sites of intradermal vaccination with either the M. tuberculosis {Delta}sigF mutant strain or BCG. Nine weeks after inoculation, the animals were aerosol challenged with the M. tuberculosis Erdman strain, which is a relatively virulent strain for rabbits (33). At 5 weeks the rabbits were euthanized and the lungs were removed. The number and size of the visible primary tubercles were assessed by Lurie's tubercle count method (6). Although fewer tubercles formed in rabbits vaccinated with the {Delta}sigF mutant strain compared to those vaccinated with BCG (Table 1), the difference was not statistically significant. The tubercle numbers in the rabbits vaccinated with the {Delta}sigF mutant strain were 56% of the control numbers (P = 0.06 by one-tailed t test analysis for vaccine-mediated protection, justified by the expectation, based on the mouse data, and the observation, in the rabbit data, of no exacerbation of disease caused by the vaccine strain [41]), while BCG-vaccinated rabbits formed 68% of the control numbers (P = 0.11). The diameters of the tubercles in the rabbits vaccinated with the {Delta}sigF mutant strain and BCG were 76 and 70% of the unvaccinated control animals, respectively. These results suggest that intradermal vaccination of rabbits with the M. tuberculosis {Delta}sigF mutant strain may offer some degree of protection from tubercle formation in the rabbit aerosol challenge model.


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  		TABLE 1. Efficacies of M. tuberculosis {Delta}sigF mutant strain and BCG as vaccines in the rabbit aerosol-challenge model and tubercle count methoda

 
Microarray identification of SigF-regulated genes. In order to identify genes under SigF control or influence, the global expression patterns of the M. tuberculosis wild-type and {Delta}sigF mutant strains were explored at different growth stages by complete genomic microarrays (3, 27, 34, 53, 61). Total RNA was isolated from cultures of the M. tuberculosis {Delta}sigF mutant strain and wild-type CDC1551 grown to A600 values of 0.6, 2.2, and for 3 days after an A600 of 2.2 was reached (i.e., exponential phase, early stationary [S] phase, and late S-phase, respectively). The cDNA from bacterial transcripts was labeled and allowed to hybridize to a microarray containing 4,016 PCR products, each specific for a unique M. tuberculosis CDC1551 gene. By examining the relative intensities (i.e., wild type over mutant) and using an operational cutoff of >=1.7-fold, underexpression was found for 38 genes in exponential phase, 187 genes in early S-phase, and 277 genes in late S-phase (Table 2). Of those genes underexpressed in the mutant strain during the three stages of growth, nearly 50% encode hypothetical proteins or proteins of unknown function. In addition, there were 68, 22, and 58 relatively overexpressed genes at exponential, early S-, and late S-phases, respectively, of which ~40% encode hypothetical proteins (Table 2). Table 3 lists the most highly down-regulated genes in the different growth phases, and a complete list of downregulated genes can be found at the Gene Expression Omnibus (GEO) database at NCBI (www.ncbi.nlm.nih.gov/geo). Importantly, the expression profile of the complemented M. tuberculosis {Delta}sigF mutant strain (8) was virtually identical to that of the wild type at each of the three time points studied (data not shown).


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  		TABLE 2. Numbers of SigF-dependent genes during different growth stagesa

 

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  		TABLE 3. Genes showing greatest down-regulation in the M. tuberculosis {Delta}sigF mutant strain at different growth phasesa

 
Exponential-phase microarray analysis. In exponential growth phase (A600 = 0.6), 16 of the 38 underexpressed genes encode hypothetical unknown proteins (Table 2). Some genes with relatively lower expression in the mutant strain are involved in fatty acid and phospholipid metabolism, such as MT2304/Rv2244 (acpM, which codes for an acyl carrier protein) and MT0928/Rv0905 (echA6, which codes for enoyl coenzyme A (enoyl-CoA) hydratase/isomerase and in detoxification, such as MT2912/Rv2846c (efpA, which codes for efflux protein) and MT4033/Rv3914 (trxC, which codes for thioredoxin). Of note the ahpC gene (MT2503/Rv2428), which encoded alkyl hydroperoxidase, is strongly down-regulated by loss of SigF in exponential phase. The M. tuberculosis ahpC gene has been implicated in isoniazid resistance (52, 63), as well as virulence (22, 55, 62). SigF may also regulate genes involved in protein folding such as MT3527/Rv3418c (groES, which codes for 10-kDa chaperonin) and MT0265/Rv0251c (hsp, which codes for an Hsp20 homologue).

Stationary-phase microarray analysis. SigF has been shown to be more highly expressed in stationary growth phase and during stress conditions (17, 38). Therefore, it is not surprising the microarray analysis revealed that more genes were down-regulated in the {Delta}sigF mutant strain in stationary phase and under growth stress conditions than in the exponential growth phase (Tables 2 and 4). The underexpressed genes include those involved in energy metabolism (such as electron transport, fermentation, anaerobic metabolism, and polysaccharide synthesis and degradation), nucleotide synthesis, and central intermediary metabolism (including oxidoreductases, arylsulfatases, methyl transferases, acyltransferases, monooxygenase flavin adenine dinucleotide [FAD] binding protein, and a glutamine amidotransferase). This broad range of genes whose expression is influenced in part by SigF supports its role as a stress-response regulator.


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  		TABLE 4. Genes down-regulated in early stationary phase in the M. tuberculosis {Delta}sigF mutant strain, classified by gene function

 
Cell envelope genes. In stationary phase several genes involved in the biosynthesis and structure of the cell envelope were relatively down-regulated in the {Delta}sigF mutant strain, as shown by microarray analysis, as were several involved in the biosynthesis and degradation of surface polysaccharides and lipopolysaccharides (Table 4). Examples include pimB (MT0583/Rv0557), which is involved in lipoarabinomannan biosynthesis (49), and murB (MT0500/Rv0482), which is involved in cell wall formation and peptidoglycan biosynthesis (4).

Since the microarray studies showed that in the absence of sigF a number of polyketide synthase genes, including pks2 (13, 46), are underexpressed, which might affect the structure of the cell envelope in M. tuberculosis, we evaluated wild-type and mutant strains for the presence of cell envelope sulfolipids. We found the {Delta}sigF mutation conferred negative neutral red staining (Fig. 3) to the M. tuberculosis strain, suggesting reduced synthesis of cell-envelope associated sulfolipids (39).



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  		FIG. 3. The M. tuberculosis {Delta}sigF mutant produces reduced levels of cell envelope-associated sulfolipids as assessed by neutral red staining of whole bacteria. The wild-type and complemented mutant M. tuberculosis strains retained the dye, whereas the {Delta}sigF mutant strain is devoid of neutral red-retaining activity.

 
SigF as a member of the M. tuberculosis regulatory gene hierarchy. Microarray analysis revealed that several regulatory genes are SigF dependent. The expression of sigF itself is down-regulated in the absence of functional SigF protein indicating that sigF expression is at least in part autoregulated. Indeed, a promoter upstream of sigF and the anti-sigma factor gene usfX preceding it has been shown by in vitro transcription to be SigF dependent (2). Moreover, the expression of alternate sigma factor gene sigC also appears to be SigF dependent (Table 4). sigC is one of the most abundantly expressed sigma factor genes (34), and a recent study has shown that M. tuberculosis sigC controls a large multifaceted regulon and is essential for lethality in the mouse model (58). Additionally, SigF appears to play a role in the expression of several other repressor/activators from the MarR, GntR, and TetR family of DNA binding regulators (Table 4). The finding of overlapping transcriptional influence on other regulators suggests that SigF participates in a hierarchical network of M. tuberculosis gene regulation—an observation previously made for M. tuberculosis SigH (27, 35, 45), SigE (1, 36), and SigC (58).

Identification of a SigF promoter recognition consensus sequence. As shown by the microarray analysis, several genes were relatively underexpressed in the {Delta}sigF mutant strain. We would expect some of these genes to be directly regulated by SigF, while others may be indirectly regulated. To search for shared promoter sequences, we used the Search Pattern function in Tuberculist (http://genolist.pasteur.fr/TubercuList/) and the B. subtilis SigB and M. tuberculosis SigF-dependent promoter sequence GTTTX17GGGTAT for the gene upstream of sigF, i.e., usfX or rsbW (MT3386, Rv3287c) recently determined by biochemical approaches (2). We identified several SigF-influenced genes which are preceded by sequences similar to that in the usfX promoter. Analysis was restricted to genes, downregulated in the microarray analysis, with a clear-cut 5'-untranslated region rather than those that are downstream members of an operon. Allowing no more than two mismatches in each hexamer with a spacer size ranging from 16 to 20 nucleotides (nt), putative promoter sequences were found in intergenic regions up to 500 bp from the start site. Tables 5 and 6 show sets of genes repressed in the mutant in stationary phase with either no more than three or a maximum of four, respectively, total mismatches in the -35 and -10 regions compared to the usfX promoter sequence. Restricting the number of mismatches to three or fewer revealed a set of 14 genes whose expression was downregulated in the {Delta}sigF mutant strain in the microarray analysis and revealed a consensus sequence of -35 GGTTTC and -10 GGGTAT (Table 5). The consensus promoter for these 14 genes has a consensus spacer size of 18 nt with the GGG motif in the -10 region being absolutely conserved. The distance from the translational start site to the 5' end of the promoter region averages approximately 200 nt. The stacking energy (measured on a scale of -3.82 kcal/mol for TA dinucleotides to -14.59 kcal/mol for GC dinucleotides; see btwisted at http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html) in the -10 element of the promoter region is a relatively low -7.55 kcal/mol/bp within the promoter unwinding region. The GC content of the consensus promoter hexamers is 50%, notably lower than the 66% found in the M. tuberculosis genome as a whole.


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  		TABLE 5. Genes down-regulated in stationary phase in the M. tuberculosis {Delta}sigF mutant strain having no more than three mismatches in the -35 and -10 consensus SigF promoter regionse

 

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  		TABLE 6. Genes having four mismatches in the -35 and -10 consensus SigF promoter regions that were found to be down-regulated in stationary phase in the M. tuberculosis {Delta}sigF mutant strain

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A previous study from our group showed that the M. tuberculosis {Delta}sigF mutant strain and its otherwise-isogenic parental wild-type strain CDC1551 have the same growth rates both in broth culture and during intracellular growth in human monocyte-derived macrophages (8). However, following intravenous infection in mice a time-to-death study showed that the {Delta}sigF mutant strain was significantly attenuated, with mouse survival times for mutant infection of up to 334 days (median time to death, 246 days) compared to 184 days (median time to death, 161 days) for wild-type-infected mice (8). In the present study, we show that the {Delta}sigF mutant strain is capable of early proliferation followed by persistence at high CFU counts in mouse lungs for at least 20 weeks. While the {Delta}sigF mutant strain persisted in lung tissues it did not achieve levels as high as those observed with wild-type infection, which exceeded 7.7 x 106 CFU when BALB/c mice were used. The histopathology of the lung and spleen confirms that there is less disease progression in the mice infected with the mutant strain. We did not assess the phenotype of the complemented {Delta}sigF mutant strain in this organ CFU count assay; however, previous studies have indicated that sigF complementation restores wild-type levels of resistance to rifampin and wild-type levels of chenodeoxycholate uptake (8). Moreover, in the present study we observed that sigF complementation reverses the loss of neutral red staining seen in the {Delta}sigF mutant strain and also restores the microarray gene expression pattern of the {Delta}sigF mutant strain to one virtually identical to that of wild type. Thus, complementation of sigF restores multiple phenotypes observed in the {Delta}sigF mutant strain back to their wild-type state, indicating that the mutant phenotypes observed are due solely to replacement of the sigF gene and not some unanticipated second site mutation. In contrast to the {Delta}sigF mutant strain, the pattern of infection by M. bovis BCG, an attenuated strain widely used as a tuberculosis vaccine, showed a pattern similar to that of so-called persistence (per) mutants (37) with initial proliferation in lungs for 2 to 3 weeks followed by gradual host clearance in BALB/c mice (data not shown).

This study taken together with the earlier one by Chen et al. (8) reveals that the M. tuberculosis {Delta}sigF mutant strain achieves and persists at high colony counts in mouse tissues but is attenuated in eliciting histopathologic evidence of tissue damage and in producing lethality in mice. This general pattern of attenuation (high lung CFU counts but delayed time to death) has been referred to as the immunopathology phenotype (Imp or Pat [23]) and has been observed in three other M. tuberculosis alternate sigma factor mutants—the M. tuberculosis {Delta}sigH (27),{Delta}sigE (1), and {Delta}sigC (58) mutants. Additionally, the immunopathology pattern of attenuation was noted in M. tuberculosis lacking the whiB3 gene (57) and the RD1 region of deletion (29). Since there is evidence that RD1 contains regulatory genes (31) and that WhiB3 is a regulator of RNA polymerase (57), the immunopathology phenotype appears to result from several diverse defects in the M. tuberculosis regulatory apparatus. It is noteworthy that the expression of sigC was reduced in the M. tuberculosis {Delta}sigF mutant strain. Since both the {Delta}sigF and the {Delta}sigC mutant strains have been observed to display the immunopathology phenotype in mice, it is conceivable that these two sigma factors form a regulatory hierarchy which governs the expression of a key group of immunopathology antigens. To date no specific effector genes or proteins of the immunopathology phenotype have been identified which might account for failure of this class of M. tuberculosis mutants to elicit wild-type levels of tissue damage in the absence of a clear-cut bacterial survival defect.

Because of its ability to persist but not elicit the same degree of lethality, we tested the M. tuberculosis {Delta}sigF mutant strain as a tuberculosis vaccine using the rabbit aerosol challenge model with tubercle counting as the efficacy parameter. The rabbit is an important model for vaccine testing against tuberculosis because of the similarities between the rabbit and human forms of disease (15). After aerosol challenge with M. tuberculosis, the ability of various vaccines to prevent grossly visible primary tubercles has allowed us to differentiate the relative efficacy of candidate vaccines (6, 14). Using this model we observed fewer tubercles in the lungs of rabbits vaccinated intradermally with the {Delta}sigF mutant strain than in either rabbits vaccinated with BCG or unvaccinated controls. The protection conferred by the {Delta}sigF mutant strain approached statistical significance (P = 0.06) in this experiment using relatively small groups of outbred rabbits. Hence, in the rabbit model, the M. tuberculosis {Delta}sigF mutant strain confers some protection against tubercle formation after infection with M. tuberculosis. The M. tuberculosis {Delta}sigF mutant strain is among the first immunopathology-type of mutant to be evaluated as a live attenuated vaccine against tuberculosis. Hsu et al. (24) reported that a {Delta}RD1 M. tuberculosis mutant conferred protection similar to that observed for BCG, all strains of which lack the RD1 region. Interestingly, they also found that the loss of cytolytic activity for pneumocytes coincided with the deletion of a more specific region, namely, the deletion of the Rv3874/Rv3875 (cfp 10/esat-6 or esxB/esxA) segment of RD1. These encouraging results suggest that the immunopathology mutant class, with its unique ability to persist without aggressive pathological tissue damage, may be useful in future vaccination strategies for tuberculosis. This may be attributable to the presence of important immunogenic proteins still present in these attenuated immunopathology phenotype (Imp) mutants and/or their greater persistence in tissues (7, 27). Whether these mutants also persist after sensitization with environmental mycobacteria remains to be tested. Studies to test the M. tuberculosis {Delta}sigF mutant strain and other immunopathology mutants both as stand-alone vaccines or as boosters to previously administered BCG vaccines are under way in other animal models.

Given the importance of SigF in mycobacterial responses to stress and its increased expression in stationary phase, the finding that the overwhelming majority of underexpressed genes are detected in stationary phase is not surprising and further supports earlier studies of this sigma factor (8, 16, 17, 38). In this study we also identified a potentially significant in vitro phenotype of the M. tuberculosis {Delta}sigF mutant strain, namely, its inability to retain the neutral red stain which earlier studies have found to correlate with a reduction in the expression of envelope-associated mycobacterial sulfolipids (Fig. 3) (39). Chen et al. (8) noted that the {Delta}sigF mutant strain was hypersusceptible to rifampin and rifapentine and that its envelope showed permeability differences to radiolabeled chenodeoxycholate. That cell envelope gene expression is affected by the {Delta}sigF mutation may be further evidence that SigF influences the expression of genes involved in the structure and function of the mycobacterial cell wall and its complex network of lipids and polysaccharides, including virulence-related sulfolipids (20). The cell envelope of M. tuberculosis has long been identified as a defense against chemical injury, dehydration, and certain antibiotics (26, 30, 59). This is due in part to the low permeability of the unique sacculus and to small hydrophilic molecules. The relative underexpression of certain membrane proteins, lipoproteins, surface polysaccharides, lipopolysaccharides, and murein sacculus could affect the permeability of the cell wall and alter the antigenic profile of the {Delta}sigF mutant during infection. The {Delta}sigF mutant strain also shows reduced expression of several key regulators some of which (TetR, GntR, and MarR) govern the expression of efflux pumps in other bacteria (9, 50, 51) which could play a role in the novel immunopathology phenotype observed in the host-pathogen interaction of the {Delta}sigF mutant strain in mice. Loss of SigF was also found to reduce the expression of the sigma factor SigC. Taken together these observations suggest that SigF governs several subordinate regulons.

Sigma factors are associated with RNA polymerase and direct the recognition of DNA promoter sequences. Identification of promoter sequences based on functional or structural homology to sigma factors in other bacteria assumes a conservation not only of the sigma factor structural genes but in DNA recognition sites as well. Such selection pressure in noncoding promoter-containing sequences may not, however, be as intense as that in coding regions for the sigma factor genes themselves. The availability of a biochemically identified promoter sequence in the adjacent anti-SigF gene, usfX or rsbW, which is itself regulated by SigF (2), combined with our microarray results, allowed us to use pattern search analysis to seek additional genes likely to be regulated directly by this sigma factor. Indeed, we found a number of genes regulated by SigF in stationary phase that contain potential promoter sequences related to that found for the control of the usfX-sigF operon, and this permitted us to derive a putative SigF consensus recognition sequence, GGTTTCX18GGGTAT. This putative M. tuberculosis SigF promoter consensus sequence is remarkably similar to that for the B. subtilis stationary-phase and stress response sigma factor, SigB, which has GTTT in the -35 hexamer and GGGWAW (W = T or A) in the -10 region (43, 44). The conservation of the putative M. tuberculosis SigF -35 hexamer ranges from 43 to 93%, suggesting that certain residues may be more critical for recognition than others. In the regulon set reported here, the GGG motif is 100% conserved in the -10 region hexamer, while the remaining residues are 57, 64, and 71% conserved. For the -10 and -35 hexamers, no putative promoter sequence was less than 67% conserved, possibly indicating that the presence of at least four of the residues in each hexamer—at an appropriate distance from the other hexamer—is sufficient to permit SigF recognition. In addition, the variability of the promoter sequences within the same organism suggests that extrapolation of conservation from organisms with different GC content may not be a strong approach to promoter identification. However, as a prelude to direct biochemical assessment, homology-based searches remain a prudent and efficient search method for identifying putative members of a regulon.

In summary, we have shown that the {Delta}sigF mutant strain belongs to the newly appreciated class of mutants displaying defective immunopathology during mouse infection and reduced lethality in time-to-death assays, but with high-level bacterial persistence in mouse lung. The high degree of survival in tissues with reduced lethality suggests that this class of mutant may be valuable as live-attenuated vaccines for tuberculosis. Indeed, the {Delta}sigF mutant strain appears to confer somewhat stronger immunity than BCG against virulent M. tuberculosis challenge in the rabbit model. If this finding is confirmed in the mouse and possibly guinea pig models, it may suggest that alterations in the SigF regulon result in the presentation of a superior set of immunogens than BCG with potentially more long-lived protection. The microarray analysis reported here represents an initial characterization of that regulon.


   ACKNOWLEDGMENTS
 
This work was supported by grants from the NIH (AI36973, AI37856, and AI43846), the National Vaccine Program Office, and the Sequella Global Tuberculosis Foundation.

The technical assistance of Rafael Ruiz and editorial help by Naomi Gauchet are gratefully acknowledged. We are grateful to M. Louise M. Pitt of USAMRIID and Bernard Zook of George Washington University for their generous assistance with rabbit experiments.


   FOOTNOTES
 
* Corresponding author. Mailing address: Center for Tuberculosis Research, Johns Hopkins University School of Medicine, 1503 E. Jefferson St., Room 112, Baltimore, MD 21231-1001. Phone: (410) 955-3507. Fax: (410) 614-8173. E-mail: wbishai{at}jhsph.edu. Back

Editor: W. A. Petri, Jr.

{dagger} Present address: Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, TN 38105. Back

{ddagger} Present address: Applied Biosystems, Foster City, CA 94404. Back

§ Present address: Sequella, Inc., Rockville, Md. Back


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, March 2004, p. 1733-1745, Vol. 72, No. 3
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.3.1733-1745.2004
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