TheMycobacterium tuberculosisComplex-Restricted Genecfp32Encodes an Expressed Protein That Is Detectable inTuberculosis Patients and Is Positively Correlated with PulmonaryInterleukin-10

Abstract

Human tuberculosis (TB) is caused by the bacillus Mycobacterium tuberculosis, a subspecies of the M. tuberculosis complex (MTC) of mycobacteria. Postgenomic dissection of the M. tuberculosis proteome is ongoing and critical to furthering our understanding of factors mediating M. tuberculosis pathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. The cfp32 gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, the cfp32 gene sequence is conserved within the MTC, as no polymorphisms were found in the tested cfp32 PCR products upon sequence analysis. Western blotting of M. tuberculosis subcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to the M. tuberculosis pathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies.The Mycobacterium tuberculosis complex (MTC) is a group of highly related pathogenic mycobacteria that include M. tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG] vaccine strain), Mycobacterium bovis subsp. caprae, and Mycobacterium microti (13). The MTC taxon is extraordinary in that its members exhibit a restricted number of fixed single-nucleotide polymorphisms between subspecies but differ from one another by the presence or absence of large chromosomal deletion loci, severity of disease, and mammalian host spectra (13,43,66

Tuberculosis Is Associated with a Down-Modulatory Lung Immune Response That Impairs Th1-Type Immunity

“…The percentages of cell types obtained by induced sputum in the current study also appeared similar to those in our previous study using BAL cells (18). In addition, we previously observed that levels of IL-10 protein in induced sputum samples of pulmonary TB patients positively correlated with the amount of CFP32, a M. tuberculosis Ag, indicating that IL-10 levels correlate with the failure of immune control M. tuberculosis (37). In the current study, using lung cells from induced sputum we confirmed and further extended our prior results (18,37); TB cases expressed significantly higher levels of mediators associated with Th1-like immunity (IFN-␥, IL-12p35, IL-12p40, and NEMO) compared with patients with OLD or with clinically well HCW.…”

“…In addition, we previously observed that levels of IL-10 protein in induced sputum samples of pulmonary TB patients positively correlated with the amount of CFP32, a M. tuberculosis Ag, indicating that IL-10 levels correlate with the failure of immune control M. tuberculosis (37). In the current study, using lung cells from induced sputum we confirmed and further extended our prior results (18,37); TB cases expressed significantly higher levels of mediators associated with Th1-like immunity (IFN-␥, IL-12p35, IL-12p40, and NEMO) compared with patients with OLD or with clinically well HCW. Our observations are supported by other studies that measured IFN-␥, TNF-␣, or IL-12 (41,43) and by microarray evaluation of BAL cells from TB patients (8,44).…”

“…Protein cytokines were quantified from supernatants collected from an in vitro challenge study and from induced sputum supernatants using commercial ELISA kits (Immunotech-Coulter) (18,37). The limit of detection for IL-10 and IFN-␥ are 5 pg/ml and 0.08 UI/ml, respectively.…”

Rapid detection of Mycobacterium tuberculosis complex in cattle and lechwe (Kobus leche kafuensis) at the slaughter house

“…M. bovis was identified from the results of the multiplex-PCR, which targets three genetic regions that are cfp32, RD9 and RD12. 6,[22][23][24] In summary, our study clearly provides evidence that the LAMP assay is a method that allows direct detection of MTC from processed clinical/abattoir specimens. This means that the technique can also be applied in the field of veterinary medicine for rapid confirmation of suspected cases.…”

O ressurgimento da tuberculose e o impacto do estudo da imunopatogenia pulmonar

“…Recently published studies conducted by our group at the University of Cornell show that one of these proteins, known as CFP32, is expressed only by members of the Mtb complex and is not expressed by other species of mycobacteria. In vitro and ex vivo studies have shown that this protein is able to induce considerable production of IL-10, which, as previously mentioned, facilitates the progress of the infection (7) . The presence of this gene in the mycobacteria has advanced the development of new diagnostic methods as well as aiding the identification of subspecies within the genus, with implications for studies that focus on molecular epidemiology (8) .…”