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Selective enrichment of mycobacterial proteins from infected host macrophages.

Chande AG, Siddiqui Z, Midha MK, Sirohi V, Ravichandran S, Rao KV - Sci Rep (2015)

Bottom Line: Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host.This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence.The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-India.

ABSTRACT
Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

No MeSH data available.


Related in: MedlinePlus

The ER stress response of the uninfected (UI) macrophages and macrophages infected with the indicated panel of strains:(A) representative images of ER stress documented by probing an ER stress marker (Grp78) and visualized with secondary Alexa488 conjugated antibody (green). Nuclei were counterstained with Hoechst; (B) Graph represents the quantification of Grp78 from three independent experiments, with at least 100 cells scored from each field. (**pValue ≤ 0.001, calculated with paired t-tests).
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f6: The ER stress response of the uninfected (UI) macrophages and macrophages infected with the indicated panel of strains:(A) representative images of ER stress documented by probing an ER stress marker (Grp78) and visualized with secondary Alexa488 conjugated antibody (green). Nuclei were counterstained with Hoechst; (B) Graph represents the quantification of Grp78 from three independent experiments, with at least 100 cells scored from each field. (**pValue ≤ 0.001, calculated with paired t-tests).

Mentions: Given the known role of mycobacterial GroEL in both assisting protein folding, as well as in suppressing aggregation of unfolded proteins, we speculated that GroEL1 may function in the host macrophage by suppressing the cellular ER stress response and, thereby, prevent the activation of apoptosis. Several earlier studies have demonstrated that suppression of host cell apoptosis represents an important facet of mycobacterial virulence3334353637. To test this we separately infected THP-1 cells with either H37Ra, H37Rv (the respective virulent and avirulent counterparts), a GroEl1-knockout variant of H37Rv (GroEL-KO), or GroEL1-KO that had been complemented with the GroEL1 gene (Comp)38. Infection was for 24 hrs, after which cells were stained for the ER stress marker Grp78 and then visualized by confocal microscopy. Whereas H37Ra infection produced significant ER stress in the host cells this, however, was not the case in H37Rv-infected cells (Fig. 6). That is, H37Rv appeared to be capable of suppressing the ER stress response of the host cell to infection. Interestingly though, this ability to suppress ER stress was partially diminished in the GroEL1-KO bacteria, although complementation with the GroEL1 gene (Comp) then again fully restored this capacity (Fig. 6). Collectively, therefore, these results confirm that GroEL1 contributes at least partially to the mycobacterial virulence-specific suppression of ER stress in the host cells. It is highly likely here that the partial effect seen with GroEL1-KO may be due to at least some degree of functional redundancy with the other nodes of the chaperone network (Fig. 5B,C). Similarly, although GroEL1 is transiently produced in H37Ra-infected cells, its activity could likely be muted because of the absence of the other chaperones that may contribute to its function. Both of these aspects, however, remain to be experimentally verified.


Selective enrichment of mycobacterial proteins from infected host macrophages.

Chande AG, Siddiqui Z, Midha MK, Sirohi V, Ravichandran S, Rao KV - Sci Rep (2015)

The ER stress response of the uninfected (UI) macrophages and macrophages infected with the indicated panel of strains:(A) representative images of ER stress documented by probing an ER stress marker (Grp78) and visualized with secondary Alexa488 conjugated antibody (green). Nuclei were counterstained with Hoechst; (B) Graph represents the quantification of Grp78 from three independent experiments, with at least 100 cells scored from each field. (**pValue ≤ 0.001, calculated with paired t-tests).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548221&req=5

f6: The ER stress response of the uninfected (UI) macrophages and macrophages infected with the indicated panel of strains:(A) representative images of ER stress documented by probing an ER stress marker (Grp78) and visualized with secondary Alexa488 conjugated antibody (green). Nuclei were counterstained with Hoechst; (B) Graph represents the quantification of Grp78 from three independent experiments, with at least 100 cells scored from each field. (**pValue ≤ 0.001, calculated with paired t-tests).
Mentions: Given the known role of mycobacterial GroEL in both assisting protein folding, as well as in suppressing aggregation of unfolded proteins, we speculated that GroEL1 may function in the host macrophage by suppressing the cellular ER stress response and, thereby, prevent the activation of apoptosis. Several earlier studies have demonstrated that suppression of host cell apoptosis represents an important facet of mycobacterial virulence3334353637. To test this we separately infected THP-1 cells with either H37Ra, H37Rv (the respective virulent and avirulent counterparts), a GroEl1-knockout variant of H37Rv (GroEL-KO), or GroEL1-KO that had been complemented with the GroEL1 gene (Comp)38. Infection was for 24 hrs, after which cells were stained for the ER stress marker Grp78 and then visualized by confocal microscopy. Whereas H37Ra infection produced significant ER stress in the host cells this, however, was not the case in H37Rv-infected cells (Fig. 6). That is, H37Rv appeared to be capable of suppressing the ER stress response of the host cell to infection. Interestingly though, this ability to suppress ER stress was partially diminished in the GroEL1-KO bacteria, although complementation with the GroEL1 gene (Comp) then again fully restored this capacity (Fig. 6). Collectively, therefore, these results confirm that GroEL1 contributes at least partially to the mycobacterial virulence-specific suppression of ER stress in the host cells. It is highly likely here that the partial effect seen with GroEL1-KO may be due to at least some degree of functional redundancy with the other nodes of the chaperone network (Fig. 5B,C). Similarly, although GroEL1 is transiently produced in H37Ra-infected cells, its activity could likely be muted because of the absence of the other chaperones that may contribute to its function. Both of these aspects, however, remain to be experimentally verified.

Bottom Line: Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host.This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence.The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi-India.

ABSTRACT
Upon infection, Mycobacterium tuberculosis (Mtb) deploys specialized secretion machinery to deliver virulent proteins with the capacity to modulate a variety of host-cellular pathways. Studies on the identification of intra-macrophage Mtb proteins, however, are constricted by an inability to selectively enrich these virulent effectors against overwhelming protein content of the host. Here, we introduce an Mtb-selective protein labeling method based on genetic incorporation of azidonorleucine (Anl) through the expression of a mutant methionyl-tRNA synthetase. Exclusive incorporation of Anl, into native Mtb proteins, provided a click handle to pull out low abundant secretory proteins from the lysates of infected cells. Further, temporal secretome profiling, upon infection with strains of varying degree of virulence, revealed the proficiency of virulent Mtb to secrete chaperones. This ability contributed at least partially to the mycobacterial virulence-specific suppression of ER stress in the host macrophage, representing an important facet of mycobacterial virulence. The Anl labeling approach should facilitate new exciting opportunities for imaging and proteomic investigations of differently virulent Mtb isolates to understand determinants of pathogenicity.

No MeSH data available.


Related in: MedlinePlus