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1 Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA
2 Mycobacteriology Laboratory Branch, Division of Tuberculosis Elimination, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
3 Infectious Disease Pathology Activity, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
4 Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX 77845, USA
Correspondence
Frederick D. Quinn
fquinn{at}vet.uga.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Dye et al., 1999). Although the M. bovis BCG vaccine is widely used to prevent mycobacterial meningitis in children, its efficacy against pulmonary TB is highly variable (0–80 %; World Health Organization, 1979; Rodrigues & Smith, 1990).
Understanding how M. tuberculosis bacilli adapt and survive upon encountering hostile environments inside the host is critical to producing effective vaccines and therapies. Bacteria employ multiple secondary sigma factors that sense specific stress signals and transiently coordinate expression of genes encoding functions that facilitate bacterial adaptation to those particular stresses. Sequence analysis of the M. tuberculosis strain H37Rv suggested the presence of 12 secondary sigma factor genes (sigB–sigM) and a primary sigma factor gene, sigA (Cole et al., 1998).
Studies of knockout mutations of secondary sigma factor genes in M. tuberculosis strains suggest some sigma factors play roles in virulence. While the sigD and sigF genes are apparently not required for survival in macrophage infections, a sigD mutant of H37Rv and a sigF mutant of strain CDC1551 were each attenuated for pathogenesis in BALB/c mice (Raman et al., 2004; Chen et al., 2000). The sigF mutant was subsequently shown to exhibit reduced persistence in the lungs and spleen of BALB/c mice (Geiman et al., 2004). A sigH mutant of CDC1551 survived in macrophages and persisted in the lungs and spleen of C57BL/6 mice, suggesting that SigH had no role in vivo. However, in C3H/HeJ mice, SigH contributed to tissue damage and death of the host (Kaushal et al., 2002). A sigC mutant of CDC1551 was reported to be less virulent, but just as persistent as the parental strain in DBA/2 mice (Sun et al., 2004). SigJ appeared not to be required for survival in BALB/c mice (Hu et al., 2004). SigE, however, was reported to be required for survival of H37Rv in macrophages (Manganelli et al., 2001). In addition, a sigE mutant persisted less effectively than the H37Rv parent strain in the lungs, spleen and liver of BALB/c mice, and mice infected with the mutant survived longer than those infected with the parent strain (Manganelli et al., 2004). Interestingly, while C3H/HeJ mice infected with a sigE mutant of the CDC1551 strain also survived longer than those infected with the parent strain, the mutant was not impaired for lung persistence (Ando et al., 2003). Genetic differences between the parent strains of the sigE mutants or differential susceptibilities in the murine models were posited to explain the observed disparity in persistence (Manganelli et al., 2004). Such phenotypic variability highlights the importance of examining mutants in different genetic backgrounds and the need for study in multiple animal model systems.
While murine models of TB have the advantages of low cost, availability of immunological reagents, and the choice of inbred populations with varying susceptibility to aerosol infection, guinea pigs are considered a superior model of human M. tuberculosis infection in terms of lung granuloma formation and function, subsequent necrosis, caseation and disease progression (Baldwin et al., 1998; Turner et al., 2003; McMurray et al., 1996; Orme et al., 2001; Ridley & Ridley, 1987). The classical human lung granuloma contains a caseous necrotic core with varying degrees of calcification surrounded by epithelioid macrophages and layers of T lymphocytes and fibrous proteins (Cotran et al., 1999). While these features are evident in the guinea pig TB model, the same does not hold for murine models. Murine granulomas are structured differently, containing significant numbers of B lymphocytes (Gonzalez-Juarrero et al., 2001). Mice also produce an unusually strong cellular immune response to M. tuberculosis bacilli that limits lung damage and enables sustained bacillary loads in excess of 106 per lung for many weeks (Orme et al., 2001). In addition, it may take months, if ever, for murine granulomas to exhibit central necrosis, but calcification never occurs (Rhoades et al., 1997).
In this study, we employed low-dose aerosol infection of guinea pigs for screening sigma factor knockout mutants of strain H37Rv to identify sigma factors that may be important regulators of virulence. Mutants defective in production of SigC produced the most-attenuated phenotypes, exhibiting limited pathogenesis in the lungs and spleen. This work suggests that SigC has a role in granuloma formation in the lungs and in successful dissemination and establishment of infection in other organs.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid constructions.
Plasmid pPR27
sigMhyg, used to create a M. tuberculosis sigM deletion mutant (sigMhyg) marked with a hygromycin resistance gene from p16R1, hyg, was made as follows. The sigM gene+
500 bp flanking regions obtained by PCR with M. tuberculosis DNA template, Pfu DNA polymerase, dNTPs and primers sigMF1/sigMR1 was cloned into plasmid pCR2.1TOPO (Invitrogen). An internal deletion of sigM was obtained by digesting the resulting plasmid with SacII and AgeI, blunting the DNA ends with T4 DNA polymerase, and ligating with the blunted 1300 bp BspHI–SmaI hyg gene from p16R1 (Yuan et al., 1998) to form plasmid pCR2.1sigMhyg. The SpeI fragment containing the sigMhyg+500 bp flanking regions was then cloned into plasmid pPR27 (Pelicic et al., 1997) to form plasmid pPR27sigMhyg.
Plasmid pPR27sigFhyg, used in the creation of a hyg-marked sigF deletion mutant in M. tuberculosis (sigFhyg), was produced as follows. The sigF gene plus flanking regions (1140 bp upstream, 500 bp downstream), obtained by PCR from M. tuberculosis template with primers usfXF1/sigFR5, was TA-cloned into plasmid pCR2.1TOPO, creating plasmid pRKK413. The sigF gene was deleted from this plasmid by PCR using primers sigFF1/sigFR1 to form plasmid pCR2.1sigF1. The hyg gene was inserted into the PacI site of pRKK413 to create pCR2.1sigFhyg. The BamHI fragment containing sigFhyg plus flanking sequences was then ligated into the BamHI site of pPR27 to form pPR27sigFhyg.
Plasmid pPR27sigChyg, used in generating a hyg-marked disruption mutation in the SigC-coding region of M. tuberculosis (sigChyg), was constructed as follows. A DNA segment carrying sigC plus 500 bp flanking regions obtained from M. tuberculosis template DNA (by PCR with primers sigCF1 and sigCR1) was TA-cloned into vector pCR2.1TOPO to form plasmid pCR2.1sigC. A 1293 bp hygromycin resistance gene, hyg, obtained as a SmaI–BspHI fragment from plasmid p16R1, was inserted into the unique EcoNI site within sigC to form plasmid pCR2.1sigChyg. The BamHI fragment encoding the hyg-disrupted sigC gene plus 500 bp flanking regions from this plasmid was ligated into plasmid pPR27 to form pPR27sigChyg.
Plasmid pLJsigC used in the creation of an unmarked internal sigC deletion mutant of M. tuberculosis (Rv-sigC) was constructed as follows. The 1569 bp BamHI fragment containing sigC plus 500 bp flanking regions was excised from pCR2.1sigC and inserted into the BamHI site of plasmid pUC19spf' (Erickson & Gross, 1989). An internal deletion of the carboxy-terminal third of sigC was obtained by removing the sequence between the EcoNI and XhoI sites to form pUC19spfsigC. The BamHI fragment containing sigC plus 500 bp flanking sequences was excised from this plasmid and ligated into the BamHI site of plasmid pLCG2 to form plasmid pLCG2sigC. Plasmid pLCG2 is a derivative of p2NIL (Parish & Stoker, 2000) in which the PacI site was destroyed by insertion of a PmeI linker (New England Biolabs). The PacI fragment encoding the lac–sac region from pGOAL17 was then inserted into the PmeI site of pLCG2sigC to create pLJsigC.
Complementation plasmid pMV306sigC was created as follows. The M. tuberculosis sigC region (containing 565 bp of upstream regulatory sequence to 92 bp downstream of sigC), obtained by PCR using primers up565sigC/down92sigC, was ligated into the HindIII site of the mycobacterial integrating vector pMV306 (Kong & Kunimoto, 1995).
Isolation of M. tuberculosis mutants.
The hygromycin-marked M. tuberculosis sigma factor knockout strains (sigChyg, sigFhyg, sigMhyg) were obtained using the homologous recombination method of Pelicic et al. (1997) following electroporation of H37Rv with a plasmid derived from the mycobacterial temperature-sensitive replicating plasmid pPR27 (pPR27sigChyg, pPR27sigFhyg, pPR27sigMhyg, respectively). The unmarked sigC mutant (Rv-sigC) was obtained following electroporation of H37Rv with the suicide plasmid pLJsigC using the unmarked deletion method (Parish & Stoker, 2000). Strain sigChyg was complemented with a wild-type copy of the sigC gene by introduction of plasmid pMV306sigC, resulting in strain sigCcomp. All plasmids were sequenced to check for the absence of PCR-generated mutations.
THP-1 infections.
The human monocytic cell line THP-1 was cultured in RPMI medium supplemented with 10 % fetal bovine serum (RPMI+10 % FBS). For infection assays, 106 monocytes were seeded into each well of 24-well dishes. Differentiation into macrophages was achieved by incubation for 3 days following addition of 100 nM phorbol esters (Sigma-Aldrich). The macrophage monolayers were incubated for 24 h in medium lacking phorbol esters prior to infection. Cells were infected for 6 h with the indicated M. tuberculosis strains at an m.o.i. of approximately 10 bacilli per host cell by addition of identical volumes from bacterial stocks previously stocked at identical cell densities (OD580=0.5). Non-adherent bacilli were removed by washing the monolayers with Hank's Balanced Salts Solution (HBSS, Invitrogen). External bacilli not removed by washing were prevented from replicating by 2 h incubation of the monolayers in RPMI+10 % FBS containing 200 µg amikacin ml–1. The monolayers were again washed with HBSS and then covered with 1 ml RPMI+10 % FBS. At the indicated timepoints, THP-1 cells were lysed by the addition of Triton X-100 to 0.1 %. Viable bacilli were enumerated by serial dilution in Middlebrook 7H9 medium containing 0.05 % Tween 80 and supplemented with 10 % ADC (BD/Difco) and plating on Middlebrook 7H11 agar supplemented with 10 % OADC (BD/Difco) and 0.05 % Tween 80. Over the 7 day infection period, uninfected THP-1 monolayers progressed from 50 % to almost complete confluence. Over a comparable period, infected monolayers remained at approximately 50 % confluence although an increased number of detached cells were detected as the infections proceeded.
Animal infections.
Female outbred Hartley guinea pigs (500 g) used in this study were purchased from the Charles River Laboratories. M. tuberculosis H37Rv and mutant derivatives were grown to exponential phase in Dubos medium containing 0.05 % Tween 80 and supplemented with 10 % Middlebrook OADC (BD/Difco). Cultures were divided into 1 ml aliquots and frozen at –80 °C until used. Thawed aliquots were diluted in double-distilled sterile water to the desired inoculum concentration. Animals were infected using a Madison aerosol generation chamber (Wiegeshaus et al., 1970) calibrated to deliver approximately 30 c.f.u. M. tuberculosis bacilli into the lungs of each guinea pig. Except as otherwise indicated, four animals infected with each strain were sacrificed at each timepoint. The lower left lung lobe and distal third of the spleen from each animal were fixed in 10 % formalin for 20 h and then placed in 70 % ethanol. Tissues with the most pathology for each specimen were paraffin embedded and 4 µm tissue sections were stained with haematoxylin and eosin for histopathology and Ziehl–Neelsen stains for acid-fast bacilli (AFB).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Mutants were identified by PCR and confirmed by Southern analysis (data not shown).
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). Initial differences at the t=0 timepoint may reflect differences in either self-adherence or adherence of the different strains to host cells following introduction into the cell culture medium as it lacks the anti-clumping non-ionic detergent Tween 80. However, each of the sigma factor knockout strains achieved similar titres by day 6 post-infection, suggesting that none are impaired in intracellular replication.
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Histopathological analyses of the lungs and spleen were performed. In general for each strain, the granulomas in the lung and spleen at early timepoints (5–6 weeks post-infection) appeared to be larger (some having necrotic centres), while fewer granulomas with reduced pathology were observed at 20 weeks post-infection. The sigC mutants produced the least pathology of all of the strains examined for granuloma formation, necrosis and mineralization (Figs 3, 4 and 5, Table 2). While medium and large granulomas were detected in the lungs of animals at both early and late times following infection with strains carrying a wild-type copy of sigC (H37Rv, sigMhyg, sigFhyg, sigCcomp), only a single small granuloma was detected in an animal 6 weeks post-infection with one of the sigC mutants (sigChyg). The granulomas from the sigFhyg-infected animals appeared less-defined in the lung compared to the spleen at early and late timepoints (Figs 3, 4 and 5, Table 2). Granulomas with detectable AFB were observed in the spleen of animals at 5–6 weeks following infection with H37Rv, sigMhyg, sigFhyg, but not with either sigC knockout mutant or, interestingly, the complemented strain (sigCcomp, Table 2). At 20 weeks post-infection, granulomas with detectable AFB were observed in the spleen of animals infected with either H37Rv or sigFhyg, suggesting that these granulomas were induced by the presence of M. tuberculosis bacilli (Table 2). While a granuloma was detected in the spleen from one animal infected with Rv-sigC at 20 weeks post-infection, no associated AFB were found. Therefore, we can not formally conclude that this granuloma was induced by mycobacteria.
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, Figs 3, 4 and 5). While a granuloma was detected in the lung of an animal infected with the complemented sigC mutant strain (sigCcomp), the absence of necrosis was unexpected. This may have been due to aberrant expression of a wild-type copy of sigC integrated at the L5 mycobacteriophage attachment site on the chromosome. Calcification of the necrotic centres was evident in lung granulomas of animals infected with either H37Rv or sigMhyg at 20 weeks post-infection (Table 2, Fig. 5). The lack of necrosis in granulomas from any animal infected with either sigC knockout (sigChyg or Rv-sigC) suggests that SigC regulates factors important for host immune modulation. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The ability of all the mutants to replicate inside THP-1 macrophages suggests that none of these sigma factors exclusively regulate genes that are critical for survival in macrophages. The ability of the sigC and sigF mutants of strain H37Rv to replicate in macrophages is consistent with reports of replication in macrophages by sigC and sigF mutants derived from M. tuberculosis strain CDC1551 (Sun et al., 2004; Chen et al., 2000).
The goal of this work was to use the guinea pig model as a screen for M. tuberculosis sigma factor mutants to identify those that mediate adaptive survival and pathogenesis upon entering the host environment. Although different murine strains have been used to model the relevance of specific sigma factors to human TB disease, this is believed to be the first reported use of the guinea pig model to examine the contributions of sigma factors to M. tuberculosis virulence. To mimic the typical route of human infection, the animals were infected via low-dose aerosols that deliver approximately 30 c.f.u. of bacilli to the lungs. Reflecting the outbred nature of guinea pigs, variability in tissue pathology was evident among replicate animals infected with the same bacterial strain (Table 2). However, general differences in granuloma size and composition were detectable among the sigma factor mutants. A sigM mutant was the least attenuated. It resulted in necrotic granulomas in the lungs and spleen much like those observed in H37Rv-infected animals. In addition, as with H37Rv-infected animals, calcification of lung granulomas was evident at 20 weeks post-infection. Thus, it would seem that SigM is not essential for virulence in this host.
A sigF mutant resulted in diffuse guinea pig lung granulomas lacking necrosis, but surprisingly normal-appearing granulomas with necrotic centres in the spleen. The lung phenotype is generally consistent with a murine study of a sigF mutant of CDC1551 which reported reduced lung granulomatous inflammation relative to the parent strain between 12 and 20 weeks post-infection (Geiman et al., 2004). The presence of necrotic guinea pig spleen granulomas containing AFB suggests that differences in the internal environments of these organs affect the survival of SigF-deficient bacilli. Geiman et al. (2004) reported a reduction in envelope-associated sulfolipids in a sigF mutant. Reduced amounts of this or other SigF-regulated products may result in heightened susceptibility to oxidizing agents or surfactants found exclusively in the lungs of guinea pigs and mice.
Studies of the three M. tuberculosis sigma factor mutants in guinea pigs suggest that SigC is more important for pathogenesis and survival within granulomas than either SigF or SigM. SigC-deficient bacilli resulted in fewer and smaller lung and spleen granulomas. In addition, necrotic centres were absent from granulomas in the lung of animals infected with a sigC mutant. This suggests that SigC is an important regulator of virulence. This view is consistent with observations that a sigC mutant of a recent clinical M. tuberculosis strain (CDC1551) produced fewer granulomas in DBA2 mice (Sun et al., 2004). However, a difference in persistence of sigC mutants between these models may exist. In this study, the lack of lung granulomas or detectable AFB suggests that sigC mutants derived from strain H37Rv do not persist to 20 weeks post-infection. This contrasts with the report that CDC1551 sigC mutant bacilli maintained elevated lung titres for a comparable period (Sun et al., 2004). This difference in persistence may be due to inherent differences between H37Rv and CDC1551. Alternatively, physiological differences between these animal models might also explain the divergent survival. The ability of guinea pigs to produce granulomas with necrotic cores may result in effective killing of internalized M. tuberculosis compared to murine hosts, thereby minimizing dissemination.
The differences in guinea pig lung and spleen granulomas induced by sigC or sigF mutants suggest different mechanisms of resisting host defences. A sigF mutant maintains the ability to produce necrotic granulomas in the spleen, but produces ill-defined lung granulomas. In contrast, sigC mutants fail to cause the production of necrotic granulomas in either organ. Different promoter consensuses have been proposed for SigC and SigF, suggesting that each controls a unique regulon (Sun et al., 2004; Geiman et al., 2004). Given that each mutant produces limited lung pathology, it is possible that some genes may have promoters for both sigma factors. Expression of sigC was reported to be down-regulated in a sigF mutant in early stationary phase growth (Geiman et al., 2004). The granuloma phenotypes are consistent with such a hierarchical regulation and suggest that SigC is more likely than SigF to be a direct regulator of genes that enable the bacilli to successfully adapt to critical host defences. Future efforts will seek to define the factors directly regulated by SigC, as they may serve as targets for vaccines or anti-mycobacterial therapies.
These studies support the hypothesis that SigC, and to a lesser extent SigF, is responsible for mediating adaptive survival of M. tuberculosis upon entering the host environment. It remains unclear what host signals activate expression or activity of these sigma factors. Further studies examining virulence of strains grown to stationary phase or taken from the surface pellicle of cells cultured under non-shaking conditions may result in bacilli expressing various stress response factors prior to entering the host. Bacilli pre-armed as such prior to infection would be expected to be more resistant upon initial encounter with host cells and may be more adept at redirecting the immune response away from a cell-mediated killing of this intracellular pathogen.
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ACKNOWLEDGEMENTS |
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We thank Pei-Hsiu Huang, Jason Ho, Nicole Latorre, Christine Gilmore and Sara Smiley for assistance in construction of vectors used in creating sigma factor mutants. We thank Lauren Jodi Pitkow for performing Southern blots confirming PCR analyses of the mutants. We are grateful to Susan Phalen and Christine McFarland for their assistance with the animal infections and necropsies. We thank Jeanine Bartlett for preparation of histopathological material. We thank Thomas Shinick and Jack Crawford for helpful discussions and critical reading of this paper.
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REFERENCES |
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Received 14 October 2005;
revised 16 February 2006;
accepted 22 February 2006.
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