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Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice

Ryan H. Senaratne¹, Ben Sidders^1,2,3,, Patricia Sequeira^1,, Grainne Saunders², Kathleen Dunphy¹, Olivera Marjanovic¹, J. Rachel Reader⁴, Patricia Lima¹, Stephen Chan¹, Sharon Kendall³, Johnjoe McFadden² and Lee W. Riley¹

¹ School of Public Health, University of California, Berkeley, CA 94720, USA

² School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK

³ Department of Pathology and Infectious Disease, Royal Veterinary College, Royal College Street, London NW1 0TU, UK

⁴ Comparative Pathology Laboratory, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

Correspondence
Lee W. Riley
lwriley{at}berkeley.edu

Received 16 June 2007
Accepted 6 October 2007

Abbreviations: H&E, haematoxylin and eosin; p.i., post-infection; WT, wild-type.

These authors contributed equally to this work.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
It is estimated that 2 billion people worldwide are latently infected with Mycobacterium tuberculosis, the aetiologic agent of tuberculosis (Dye et al., 2002), and 2–23 % of these latently infected persons ultimately develop active disease in their lifetime (Parrish et al., 1998). We previously showed that an M. tuberculosis strain disrupted in an operon called mce1 (mce1) was more virulent in mice than its parent wild-type (WT) strain, H37Rv; the mutant was also diminished in its ability to induce a Th1-type immune response (Shimono et al., 2003). We thus suggested that mce1 genes may temper the ability of M. tuberculosis to cause overt disease and play a role in establishing latent infection in mice (Shimono et al., 2003).

The mce1 operon is a member of a family of related operons comprising mce1, 2, 3 and 4 containing homologous genes arranged similarly (Cole et al., 1998; Tekaia et al., 1999). Six of the mce1 genes (mce1A–F) encode proteins that localize to the cell wall (Chitale et al., 2001; Shimono et al., 2003; Tekaia et al., 1999). One of the mce1 proteins (Mce1A) was previously shown to confer upon a nonpathogenic Escherichia coli strain an ability to enter HeLa cells (Arruda et al., 1993; Chitale et al., 2001). The corresponding protein in the mce2 operon (Mce2A), which is 67 % similar in amino acid sequence to Mce1A, did not exhibit this activity. Santangelo et al. (2002) have shown that mce3 genes are negatively regulated by a gene (Rv1963) belonging to the tetR family. In contrast, mce1 has a negative transcriptional regulator (mce1R) belonging to the GntR family at the corresponding position (Casali et al., 2006; Cole et al., 1998). Recently we have shown that the M. tuberculosis strain disrupted in mce1R exhibits enhanced virulence in mice, indicating that the level of expression of the mce1 products has a profound effect on the clinical outcome of the infection (Uchida et al., 2007). Gioffre et al. (2005) have studied mce1, mce2 and mce3 in BALB/c mice. The above authors used two methods of infection: intratracheal and intraperitoneal routes. When mice were infected via the intratracheal route, all three mce mutants showed reduced bacterial counts; when mice were infected via the intraperitoneal route, mce1 showed 50 % increased bacterial counts while the counts decreased for mce2 and remained unchanged for mce3. The mouse survival study was carried out only up to 20 weeks (Gioffre et al., 2005). Joshi et al. (2006) studied mce4 in C57BL/6 mice using intravenous infection. They co-infected mice with mce4 and WT H37Rv and studied the infection up to 100 days. Thus these other studies have not assessed the long-term survival of mce2–4 operon mutations in mice. The long-term effects of mce mutations on mice are important to assess the suggested role of the mce operons in latency. We therefore studied the long-term survival (>60 weeks) of mice infected with mutants disrupted in mce3 and mce4 using a low-dose aerosol infection model.

	METHODS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Mycobacteria and culture conditions. WT M. tuberculosis H37Rv and its derivative strains (mce mutants) were grown in Middlebrook 7H9 broth containing 10 % ADC (Becton Dickinson), 0.2 % glycerol and 0.05 % Tween 80 (7H9-ADCT) or on Middlebrook 7H11 agar containing OADC (Becton Dickinson), 0.5 % glycerol and the antifungal agent cycloheximide (100 µg ml^–1) (Sigma-Aldrich). Bacteria were passed through a 5 µm pore filter to prepare single cell suspensions prior to mouse infection and measuring growth curves.

Generation of mce mutants. The M. tuberculosis H37Rv mce mutants were constructed by the method of Parish & Stoker (2000). Deleted alleles were created by amplifying (approx.) 1 kb regions upstream and downstream of each operon. Following digestion with the appropriate restriction enzymes, these PCR products were subcloned into the p2NIL vector in tandem. Mutant selection and additional vector information have been previously described by Parish & Stoker (2000). Deletion mutations were confirmed by Southern blot hybridization.

Mouse infections. Eight-week-old C57BL/6 mice (Jackson Laboratories) were infected with the M. tuberculosis strains via inhalation by the Inhalation Exposure System (Glas-col). The inoculum doses were assessed from harvest of the right lungs of three mice (per infection) 24 h post-infection (p.i.). The dose of infection was 72–108 bacilli per lung for all infections. Lungs were homogenized and plated onto 7H11 agar, followed by enumeration of c.f.u. 21 days later. At different time points p.i., the right lung, liver and spleen from three mice were collected, homogenized in PBS-Tween (0.05 %), appropriately diluted, and plated onto 7H11 agar plates (supplemented as above). At 21 days, the bacterial load of each organ was determined by c.f.u. enumeration.

Determination of mouse morbidity. Four groups of eight to nine mice each, infected with each of the above strains, were followed until they exhibited moribund features that occur just before death. At this point, mice were anaesthetized with a mixture of ketamine HCl, xylazine and acepromazine injected subcutaneously, and then euthanized by cervical dislocation. Loss of weight accompanied by failure to groom, ruffled fur and lethargy were used to assess morbidity, in addition (in some instances) to the recommendation of the veterinary staff of the North Animal Facility of UC Berkeley. The health of the mice was monitored daily by the above veterinary staff.

Determination of survival and induction of cytokines in macrophages by mce mutants. The RAW 264.7 murine macrophage-like cell line (ATCC) was cultured and maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum (Omega Scientific) at 37 °C in a 5 % CO₂ humidified incubator. Cells were plated at 2x10⁵ cells per well in 24-well tissue culture plates. The macrophages were incubated (i.e. infected) with either 2x10⁵, 2x10⁶ or 2x10⁷ bacteria for 6 h. After 6 h of infection, macrophages were washed three times with DMEM to remove extracellular bacteria. To examine the intracellular invasion, growth and survival of bacteria, we lysed the macrophages with 1 ml PBS-0.5 % Triton X-100 and their serial dilutions were plated onto 7H11 agar. This was performed 6, 48 and 72 h p.i. c.f.u. on plates were enumerated 21 days after plating. Supernatants of the above cell cultures were used to measure TNF, IL-12, IL-6, IL-10 and monocyte chemoattractant protein 1 (MCP-1) produced by macrophages in response to infection with WT or the mce mutant strains. Lipopolysaccharide (1 µg ml^–1) (Sigma) was used as a positive control and uninfected macrophages served as negative controls for each experiment. The above cytokines and MCP-1 were measured by ELISA with the reagents purchased from eBioscience.

Histology. The mouse left lung fixed in 10 % neutral (PBS) buffer formalin was embedded in paraffin, sectioned and stained for histology with either haematoxylin and eosin (H&E) or the Ziehl–Neelsen technique. Sectioning and staining were performed by Histology Consultation Services, Everson, Washington, USA. For comparative purposes, sections were obtained from the same regions of all lungs; three sections were obtained from each lung from three mice. Sections obtained from the top and the bottom parts of the lung were stained by H&E, while the section obtained from the middle region was stained by the Ziehl–Neelsen technique. The histopathology of each lung was assessed for two to four H&E-stained sections and one to two sections stained by the Ziehl–Neelsen technique. Histopathology of 6 weeks and 15 weeks p.i. lungs was analysed. Pathological analysis was done by a veterinary pathologist from the School of Veterinary Medicine UC Davis, USA.

Statistics. Mouse survival was compared by Kaplan–Meier curves. The mean c.f.u. counts of M. tuberculosis recovered from organs of each mouse group (three per group) were compared by the Student's t-test.

	RESULTS

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Generation of mce mutants

We generated three mutants by in-frame deletion of the mce3, mce4 and both mce3 and mce4 operons in M. tuberculosis H37Rv (mce3, mce4 and mce3/4, respectively). The mutations were designed so that eight genes (yrbE3A–mce3F) within mce3 (Tekaia et al., 1999) and eight genes and the first 250 bp of the ninth gene (yrbE4A to the first 250 bp of Rv3493c) within mce4 (Tekaia et al., 1999) would be deleted (Figs 1 and 2). Deletions of the genes were confirmed by Southern blot analysis (Figs 1 and 2) and PCR (data not shown). The resulting mutants, mce3, mce4 and mce3/4, displayed in vitro growth characteristics similar to those of the WT parent strain (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Southern blot analysis of the mce3 operon deletion. (a) Genomic organization of the region surrounding the mce3 operon in the WT (M. tuberculosis H37Rv). Black arrows depict the deleted genes [yrbE3A–mce3F: TubercuList coordinates (http://genolist.pasteur.fr/TubercuList/) 2207697–2216573] of the mce3 operon. Two black boxes (labelled 5' probe and 3' probe) indicate regions of DNA probes used in Southern blot analysis. (b) Genomic organization of the region surrounding the mce3 deletion in mce3 and mce3/4. White arrows in (a) and (b) depict the genes neighbouring the region of the deletion: mce3R (5' end) and Rv1972 (3' end). Genomic DNA was digested with PvuI for Southern blot analysis. (c) Southern blot analysis of genomic DNA from WT (lane 2), mce3 (lane 3) and mce3/4 (lane 4). DIG-labelled molecular mass standards II (lane 1) and VII (lane 5) are from Roche Diagnostics. Arrows indicate the molecular mass standards in kbp.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Southern blot analysis of the mce4 operon deletion. (a) Genomic organization of the region surrounding the mce4 operon in the WT (M. tuberculosis H37Rv). Black arrows depict the completely or partially deleted genes [first 250 bp of Rv3493c to yrbE4A: TubercuList (http://genolist.pasteur.fr/TubercuList/) coordinates 3911426–3920856] of the mce4 operon. Two black boxes (labelled 5' probe and 3' probe) indicate the regions of DNA probes used in Southern blot analysis. (b) Genomic organization of the region surrounding the mce4 deletion in mce4 and mce3/4. The black arrow depicts the partially deleted Rv3493c (479 bp of the 3' end). In (a) and (b), white arrows depict the genes neighbouring the region of deletion: Rv3492c (5' end) and Rv3502c (3' end). Genomic DNA was digested with PvuII for Southern blot analysis. (c) Southern blot analysis of genomic DNA from WT (lane 2), mce3/4 (lane 3) and mce4 (lane 5). DIG-labelled molecular mass standards II (lane 1) and VII (lanes 4 and 6) are from Roche Diagnostics. Arrows indicate the molecular mass standards in kbp.

Bacterial burden and survival of mice

Similar bacterial burdens were detected in organs from mice infected with mce3 or the WT at all time points (Fig. 3). However, at 15 weeks p.i., recovery of mce4 and mce3/4 c.f.u. from mouse lungs was significantly less than that of the WT (P <0.05). All other c.f.u. recoveries (from all three organs) at 15 weeks p.i. from mouse organs infected with mce mutants were not significantly different compared to c.f.u. recoveries from mouse organs infected with the WT (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Bacterial burdens of mouse organs infected with the mce mutants. Recovery of c.f.u. from the right lung at different time points p.i. (a), and recovery of c.f.u. at 105 days p.i. from the right lung [re-representation of (a) at 105 p.i.] (b), spleen (c) and liver (d) for WT, mce3, mce4 or mce3/4 from C57BL/6 mice after aerosol infection (n=3 mice per group per time point).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Survival of mice infected with mce mutants. Survival of C57BL/6 mice after aerosol infection with WT and mce mutants; n=8 for WT, n=8 for mce3, n=9 for mce4 and n=9 for mce3/4-infected groups.

View larger version (67K): [in this window] [in a new window]   Fig. 5. Histology of pulmonary lesions in infected mice. Sections of C57BL/6 mouse lungs magnified x20 (a) and x200 (b) stained with H&E. Bars, 300 µm (a) and 30 µm (b). Mouse lungs from all groups were harvested at 15 weeks p.i. All the mice were infected as in Fig. 3. The photographs were taken by a veterinary pathologist so as to represent each group. mce4- and mce3/4-infected mice had pulmonary lesions that were nodular and more contained, involving a smaller area of pulmonary parenchyma, than those of mce3- and WT-infected mice, where the lesions were more diffuse and involved a larger percentage of the parenchyma.

	DISCUSSION

TOP INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The longer survival times of mice infected with mce4 suggest that the attenuation of mce4 is greater than that of mce3. This is also indicated by the differences in c.f.u. recovery at 15 weeks p.i. from mouse lungs (Fig. 3) and the corresponding histopathology (Fig. 5). At this time point, the above two parameters did not indicate any attenuation of mce3 compared to WT in mice. However, the survival times of mice infected with mce3 were significantly longer than those of the mice infected with WT. There was also a small but significant difference between the survival times of mce3/4- versus mce4-infected mice. Therefore, it is possible that the greater attenuation of the double mce3/4 mutant is due to the combined effect of mutations in both mce3 and mce4 operons. However, the mouse survival, lung pathology and bacterial burden data suggest that the attenuation of mce4 in mice is much more prominent than that of mce3. Nevertheless, all of the above observations indicate that mce3 and mce4 operon mutants behave differently from the mce1 operon mutant in mice, suggesting that, despite similarity in gene sequences and arrangement, the functions of the mce3 and mce4 operons are distinct from that of the mce1 operon.

The decreased lung bacterial burden in mice infected with mce4 or mce3/4 mutants compared to that in mice infected with WT and mce3 at 15 weeks p.i. may be because either (1) the disruption of mce4 causes bacteria to replicate more slowly in host cells or (2) the mce4 operon mutant is killed more rapidly when the bacteria first encounter the host adaptive immune response. Since mce4 can replicate similarly to other strains in RAW macrophages (data not shown), it is unlikely that mce4 is more susceptible to the antibacterial activity inside the host cell. mce4-infected mouse lung has less extensive granulomatous pneumonia (i.e. fewer immune cells). One explanation is that the mce4 operon-related products attract proinflammatory cells to the lung. However, as observed in RAW cells, the absence of the mce4 operon did not have any effect on the ability of M. tuberculosis to induce or suppress TNF-, IL-6, IL-10, IL-12 and MCP-1 in ex vivo-infected macrophages. Thus the diminished proinflammatory cell response in lungs of mice infected with mce4 and mce3/4 and their reduced c.f.u. counts may be due to a decreased replicative ability of these strains under the host adaptive immune response.

Recently, Kumar et al. (2003) detected the expression of mce1, 3 and 4 operons in tubercle material collected from infected animals (guinea pigs and rabbits). These observations support our findings that mce operons other than mce1 are expressed during the disease state. In addition, Ahmad et al. (2004) demonstrated the expression of several mce3 genes during natural infection of humans infected with M. tuberculosis.

Our results support the findings of a recent study by Gioffre et al. (2005) that reported that the mce3 operon mutant was attenuated in mice. They did not examine mce4. Their survival study, however, was carried out only up to 20 weeks. Hence the long-term effect of their mce3 cannot be compared with our results. However, in that study, attenuation was observed only when mice were infected via the intratracheal route, and not when the intraperitoneal route was used. They suggested that the route of infection may make a difference in infection outcome. This study used a more physiologically relevant aerosol route of infection. The route of infection (aerosol vs intravenous) with another mutant (mce1R) in mice, however, did not make any difference to clinical outcome or survival (Uchida et al., 2007). Additionally, Joshi et al. (2006) studied the mice infected with mce mutants up to 100 days only, while we studied the survival of mice infected with mce3 and mce4 for >60 weeks. As with the study of Gioffre et al. (2005), we were unable to complement the mce2 and 3 operon mutants due to the large size of the deleted region.

In conclusion, the studies described in this paper on mce3 and mce4 operons suggest that despite their similarity with the mce1 operon in gene organization and sequences, the mce3 and mce4 operon-encoded proteins have a role markedly distinct from mce1 operon-encoded products.

	ACKNOWLEDGEMENTS

 
We thank Sally Cantrell, Nicola Casali and Lisa Morici for helpful discussion. This project was supported in part by the Ellison Medical Foundation and NIH R21AI063350. G. S. was supported by European Union grant ‘TB Vaccine Cluster’ Contract QLK2-CT-1999-01093.

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Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S. & other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef][Medline]

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