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Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients.

Vemula MH, Ganji R, Sivangala R, Jakkala K, Gaddam S, Penmetsa S, Banerjee S - Front Microbiol (2016)

Bottom Line: The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23).A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases.This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad Hyderabad, India.

ABSTRACT
Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

No MeSH data available.


Related in: MedlinePlus

Zmp1 is expressed and secreted by in vitro grown Mycobacterium tuberculosis H37Rv under granuloma-like conditions.(A) The culture supernatants of in vitro grown M. tuberculosis H37Rv under normal conditions and under various stress conditions, such as, acidic pH5.5, H2O2 induced oxidative stress, nutrient starvation and iron depletion, were precipitated by 10% trichloroacetic acid and the resultant precipitate was subjected to Western blot using Mouse anti-Zmp1 antibody (1:1000 dilution; Upper panel). Rabbit anti-CFP10 antibody (1:1000 dilution) against CFP10, a culture filtrate protein 10, was used as a positive control (Middle Panel) and Mouse anti-GroEL1 antibody (1:1000 dilution) against GroEL1, a cytoplasmic chaperone was used as a negative control (Lower Panel) to evaluate for cell lysis products in supernatants. The absence of band corresponding to GroEL1 in the supernatant fractions suggests the purity of culture filtrate preparations. (B) Figure represents the Western blots with the whole bacterial lysates as control experiment.
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Figure 2: Zmp1 is expressed and secreted by in vitro grown Mycobacterium tuberculosis H37Rv under granuloma-like conditions.(A) The culture supernatants of in vitro grown M. tuberculosis H37Rv under normal conditions and under various stress conditions, such as, acidic pH5.5, H2O2 induced oxidative stress, nutrient starvation and iron depletion, were precipitated by 10% trichloroacetic acid and the resultant precipitate was subjected to Western blot using Mouse anti-Zmp1 antibody (1:1000 dilution; Upper panel). Rabbit anti-CFP10 antibody (1:1000 dilution) against CFP10, a culture filtrate protein 10, was used as a positive control (Middle Panel) and Mouse anti-GroEL1 antibody (1:1000 dilution) against GroEL1, a cytoplasmic chaperone was used as a negative control (Lower Panel) to evaluate for cell lysis products in supernatants. The absence of band corresponding to GroEL1 in the supernatant fractions suggests the purity of culture filtrate preparations. (B) Figure represents the Western blots with the whole bacterial lysates as control experiment.

Mentions: The functionally active, endotoxin free, purified recombinant Zmp1 (rZmp1) was used as antigen to capture anti-Zmp1 antibodies in the sera samples of TB patients (results presented and discussed later). The presence of anti-Zmp1 antibodies in M. tuberculosis infected patients confirmed that Zmp1 was indeed expressed by M. tuberculosis during infection. Zmp1 was identified as one of the culture filtrate proteins of in vitro grown H37Rv (de Souza et al., 2011). We extended the study to check if the same holds true for granuloma-like conditions. To study the same, H37Rv was grown under different stress conditions known to simulate acellular caseous environment of TB granulomas, that is, acidic pH 5.5, H2O2 induced oxidative stress, nutrient deprivation and iron deficiency (Stallings and Glickman, 2010). Bacteria were grown to mid-log phase and then subjected to various stresses for 36 h. The mycobacterial cells were then harvested and the culture supernatants separated. Culture supernatants were then precipitated using 10% TCA and the precipitate was used to detect presence of Zmp1 protein using Western blotting with in-house generated anti-Zmp1 antibody (Figure 2A, upper panel). CFP10 and GroEL1, which were probed with their respective antibodies, were used as positive and negative controls respectively (Figure 2A, middle and lower panel). CFP10 is a known mycobacterial secretory protein (Malen et al., 2007, 2008) and hence was used as a positive control for culture supernatant preparations while GroEL1 is an intrabacterial, membrane associated protein which is not secreted out (de Souza et al., 2011; Malen et al., 2011) and is used as negative control. Absence of GroEL1 in the culture supernatants indicated absence of cell lysis products in the culture supernatant (Figure 2A, lower panel). The presence of band corresponding to Zmp1 in Western blots suggested secretion of the protein under all the tested stress conditions (Figure 2A, upper panel). The assay was a qualitative check to confirm the secretion of Zmp1, though it is possible that the levels of secreted Zmp1 in culture supernatants may vary with stress conditions. Figure 2B represents the Western blots with the whole bacterial lysates as control experiment. This suggested that Zmp1 is indeed a secreted protein, possibly secreted within granuloma of infected host.


Mycobacterium tuberculosis Zinc Metalloprotease-1 Elicits Tuberculosis-Specific Humoral Immune Response Independent of Mycobacterial Load in Pulmonary and Extra-Pulmonary Tuberculosis Patients.

Vemula MH, Ganji R, Sivangala R, Jakkala K, Gaddam S, Penmetsa S, Banerjee S - Front Microbiol (2016)

Zmp1 is expressed and secreted by in vitro grown Mycobacterium tuberculosis H37Rv under granuloma-like conditions.(A) The culture supernatants of in vitro grown M. tuberculosis H37Rv under normal conditions and under various stress conditions, such as, acidic pH5.5, H2O2 induced oxidative stress, nutrient starvation and iron depletion, were precipitated by 10% trichloroacetic acid and the resultant precipitate was subjected to Western blot using Mouse anti-Zmp1 antibody (1:1000 dilution; Upper panel). Rabbit anti-CFP10 antibody (1:1000 dilution) against CFP10, a culture filtrate protein 10, was used as a positive control (Middle Panel) and Mouse anti-GroEL1 antibody (1:1000 dilution) against GroEL1, a cytoplasmic chaperone was used as a negative control (Lower Panel) to evaluate for cell lysis products in supernatants. The absence of band corresponding to GroEL1 in the supernatant fractions suggests the purity of culture filtrate preparations. (B) Figure represents the Western blots with the whole bacterial lysates as control experiment.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814508&req=5

Figure 2: Zmp1 is expressed and secreted by in vitro grown Mycobacterium tuberculosis H37Rv under granuloma-like conditions.(A) The culture supernatants of in vitro grown M. tuberculosis H37Rv under normal conditions and under various stress conditions, such as, acidic pH5.5, H2O2 induced oxidative stress, nutrient starvation and iron depletion, were precipitated by 10% trichloroacetic acid and the resultant precipitate was subjected to Western blot using Mouse anti-Zmp1 antibody (1:1000 dilution; Upper panel). Rabbit anti-CFP10 antibody (1:1000 dilution) against CFP10, a culture filtrate protein 10, was used as a positive control (Middle Panel) and Mouse anti-GroEL1 antibody (1:1000 dilution) against GroEL1, a cytoplasmic chaperone was used as a negative control (Lower Panel) to evaluate for cell lysis products in supernatants. The absence of band corresponding to GroEL1 in the supernatant fractions suggests the purity of culture filtrate preparations. (B) Figure represents the Western blots with the whole bacterial lysates as control experiment.
Mentions: The functionally active, endotoxin free, purified recombinant Zmp1 (rZmp1) was used as antigen to capture anti-Zmp1 antibodies in the sera samples of TB patients (results presented and discussed later). The presence of anti-Zmp1 antibodies in M. tuberculosis infected patients confirmed that Zmp1 was indeed expressed by M. tuberculosis during infection. Zmp1 was identified as one of the culture filtrate proteins of in vitro grown H37Rv (de Souza et al., 2011). We extended the study to check if the same holds true for granuloma-like conditions. To study the same, H37Rv was grown under different stress conditions known to simulate acellular caseous environment of TB granulomas, that is, acidic pH 5.5, H2O2 induced oxidative stress, nutrient deprivation and iron deficiency (Stallings and Glickman, 2010). Bacteria were grown to mid-log phase and then subjected to various stresses for 36 h. The mycobacterial cells were then harvested and the culture supernatants separated. Culture supernatants were then precipitated using 10% TCA and the precipitate was used to detect presence of Zmp1 protein using Western blotting with in-house generated anti-Zmp1 antibody (Figure 2A, upper panel). CFP10 and GroEL1, which were probed with their respective antibodies, were used as positive and negative controls respectively (Figure 2A, middle and lower panel). CFP10 is a known mycobacterial secretory protein (Malen et al., 2007, 2008) and hence was used as a positive control for culture supernatant preparations while GroEL1 is an intrabacterial, membrane associated protein which is not secreted out (de Souza et al., 2011; Malen et al., 2011) and is used as negative control. Absence of GroEL1 in the culture supernatants indicated absence of cell lysis products in the culture supernatant (Figure 2A, lower panel). The presence of band corresponding to Zmp1 in Western blots suggested secretion of the protein under all the tested stress conditions (Figure 2A, upper panel). The assay was a qualitative check to confirm the secretion of Zmp1, though it is possible that the levels of secreted Zmp1 in culture supernatants may vary with stress conditions. Figure 2B represents the Western blots with the whole bacterial lysates as control experiment. This suggested that Zmp1 is indeed a secreted protein, possibly secreted within granuloma of infected host.

Bottom Line: The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23).A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases.This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad Hyderabad, India.

ABSTRACT
Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

No MeSH data available.


Related in: MedlinePlus