Tuberculosis caused by the intracellular pathogen Mycobacterium tuberculosis (M. tb), remains a potential threat regardless of strong efforts to alleviate its toll on humanity. Intracellular/intraphagosomal survival of this pathogen plays a critical role in the infection cycle of the pathogen, a process which majorly relies on array of virulence factors to colonize and replicate within the host macrophages (Ehrt and Schnappinger, 2009). Virulence factors which have been known to play a vital role in host pathogen interaction at the molecular level include stress responders, heat shock proteins (HSPs), foldases, and chaperones (Henderson, 2010). Studies involving immunological characterization of these effector molecules can bridge huge gaps in our understanding of M. tb biology and facilitate better therapeutic and diagnostic interventions.

Protein folding in the cell is assisted by molecular chaperones and foldases. The foldases generally include peptidyl-prolyl isomerases and protein disulfide isomerases. In addition to peptidyl prolyl isomerase activity, Ppiases have been shown to play role in diverse biological processes such as receptor signaling, apoptosis, stress response, and RNA-mediated gene expression (Sykes et al., 1993; Lu et al., 1996; Wu et al., 2000; Mark et al., 2001). Human cyclophilin A and also M. tb Ppiases have been reported to possess chaperone activity (Zhang et al., 2013; Pandey et al., 2016). Many such chaperones and HSPs have also been known to have immune modulatory role during bacterial infections. For example, mycobacterial HSP65 induces a strong cellular and humoral immune response (Peetermans et al., 1994; Friedland et al., 2008).

M. tb is known to possess two Ppiases (cyclophilins), PpiA and PpiB. M. tb PpiA is a part of the secretome and is known to interact with host proteins involved in immune defense mechanism and signal transduction (Henriksson et al., 2004; Bhaduri et al., 2014) while PpiB has been reported in membrane fraction and mannosylation enriched culture filtrate (Cole et al., 1998; Gu et al., 2003). Immunological characterization of these enzymes in terms of their possible role in modulating host immune response, virulence and intracellular survival, has not been investigated so far. PpiB, being an essential protein (Sassetti et al., 2003), makes it an important target for developing new interventions.

In the present study, we describe the immunogenic potential of M. tb cyclophilins, their involvement in eliciting host immune response, altering the host cytokine profile and promoting the intracellular survival of the pathogen, significant attributes which highlight the seminal role of these proteins in the infection process of M. tb.

Besides their biological function as helpers in protein folding, bacterial molecular chaperones have a distinctive role in virulence and stress tolerance. They are also termed as moonlighting proteins (Vanghele and Ganea, 2010). Hsp70 chaperone is present on bacterial surface and functions also as a plasminogen receptor in pathogens like M. tb, Neisseria meningitides, and Listeria monocytogenes (Knaust et al., 2007; Xolalpa et al., 2007). We investigated whether mycobacterial cyclophilins also have roles beyond protein folding to modulate the host immune response and assist in pathogenesis. Since B cells can exert influence on T cells, they are considered as important determinants in the outcome of infection with M. tb. In-silico analysis of M. tb PpiA and PpiB proteins displayed major antigenic stretches with a peak value ≥1.7. This was vindicated by the observation of significantly increased presence of antibodies to M.tb Ppiases in sera from TB patients as compared to healthy individuals. This is consistent with earlier findings that members of stress family of proteins such as Hsp70 and Hsp10 elicit strong B-cell response (Mehlert and Young, 1989; Young and Garbe, 1991). These findings clearly indicate the immunodominant nature of these cyclophilins expressed during infection and also highlight their likely diagnostic potential.

In M. tb infection cycle, macrophages act as the first line of defense and the pathogen hires a plethora of strategies to counteract the host immune response. Skewing of the balance in secretion of pro-inflammatory and anti-inflammatory cytokines results in intracellular bacterial clearance or survival, respectively (Newman et al., 1991; Byrd, 1997). Chronic inflammatory pathology is the hallmark of tuberculosis, which indicates that overproduction of pro-inflammatory cytokines lies at the heart of the infection. TNF-α is crucial for the formation and maintenance of granuloma that are main effector sites of antimicrobial activity against the pathogen (Kindler et al., 1989). Estimation of proinflammatory cytokines secreted by THP-1 cells on treatment with increased doses of rPpiA revealed that this Ppiase is a potent stimulator of TNF-α and IL-6 and thus may play an important role in host inflammatory pathology. Mycobacterial HSPs Hsp65 and chaperones have been earlier shown to act as strong immune modulators and are potent stimulators of pro-inflammatory cytokines (Peetermans et al., 1994; Naffin-Olivos et al., 2014).

Our results implicate M. tb PpiA as a stimulator of pro-inflammatory cytokines thereby pointing to its vital role in the inflammatory pathology of tuberculosis (Figure 7). Conversely, assessment of pro-inflammatory cytokines secreted by THP-1 cells upon treatment with increasing concentrations of rPpiB revealed that the protein is an inhibitor of TNF-α and IL-6 along with stimulating secretion of IL-10. That M. tb PpiB is able to subvert the innate immune response points to its role in aiding the establishment of infection, which fits well with previous findings that PpiB is essential for the survival of the pathogen (Sassetti et al., 2003).

The kinetics of production along with balance between pro-inflammatory and anti-inflammatory cytokines released by macrophages upon exposure to mycobacterial antigens regulate the T cell responses (Dheenadhayalan et al., 2006; Kim et al., 2011). Production of pro-inflammatory cytokines is known to contribute to the host response against mycobacteria (Ladel et al., 1997a; Tsao et al., 1999; Sasindran and Torrelles, 2011). Our apparently paradoxical observation that PpiA increases intracellular survival despite the pro-inflammatory role suggests an alternative role of PpiA mediated TNF-α and IL-6. Since PpiA is known to be expressed late in the infection cycle (Pathakumari et al., 2015), the pro-inflammatory cytokines induced by PpiA is likely associated with immunopathological responses in tuberculosis leading to necrosis and cachexy that aid in disease progression. Production of TNF-α has earlier been shown to be directly related to virulence correlating with intracellular viability (Newman et al., 1991; Engele et al., 2002) and is also known to promote growth of virulent M. tb in monocytes (Byrd, 1997). Thus, induction of PpiA mediated TNF-α by macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism employed by M. tb. This pro-inflammatory response induced by high bacterial load is also known to induce apoptosis that is associated with mycobacterial survival (Santucci et al., 2000). Earlier studies have also shown that mycobacterial components increase the production of pro-inflammatory cytokine TNF-α in macrophages/monocytes and this elevated level of TNF-α is regarded as reason for mycobacterial persistence and virulence within human macrophages (Dheenadhayalan et al., 2006; Kim et al., 2011). IL-6, although important for control of tuberculosis in mice model (Ladel et al., 1997b), in humans is considered as a correlate of disease progression because of its role in inflammation and pathology (Tsao et al., 1999; Ilonidis et al., 2006). Conversely, PpiB employs the classical approach to aid mycobacterial growth by production of inhibitory cytokine IL-10 (Figure 7). The different strategies employed by the same classes of protein is possibly a function of the difference in their expression kinetics: PpiB being expressed early during infection employs IL-10 to dampen the inflammatory host response, whereas PpiA, expressed late in infection (Pathakumari et al., 2015) aids in immunopathology. IL-10 secreted by host cell after mycobacterial infection is also known to increases the intracellular bacterial survivability by blocking phagosomal maturation (O'Leary et al., 2011). Although Ppiases are constitutively expressed for their action as a peptidyl prolyl isomerase to catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides, the convergent evolution has adapted the mycobacterial proteins for moonlighting functions. Thus, different immunological response could be based upon the different expression patterns at different stages of infection cycle. Although currently there are no gene expression data to claim this but an earlier study wherein PpiA was shown to induce IFN-γ in LTBI as compared to that of PTB suggested enhanced expression of this protein, late in infection cycle (Pathakumari et al., 2015). Moreover PpiA is also known to be upregulated in intraphagosomal niche during infections (Mattow et al., 2006). On the contrary PpiB being essential for the in vitro growth of M. tb, suggests its expression early during infection.

Intracellular infectious agents that have co-evolved in long standing association with the host have acquired mechanisms to persist within the host cell. The persistence of pathogenic mycobacteria within the hostile environment of host macrophages is in part due to the bacterial ability to adapt to the stress conditions encountered. Our data suggest that M. tb Ppiases might play a role in the intracellular survival by subverting the host cell defenses, such as oxidative stress as well as by immunomodulation. M. tb Hip1 has been shown to modulate macrophage responses through proteolysis of GroEL2 (Naffin-Olivos et al., 2014) and this augers well with our earlier report of chaperone activity of M.tb Ppiases (Pandey et al., 2016), though the exact mechanism remains to be elucidated. In conclusion, our study demonstrates immunomodulatory potential of M. tb Ppiases and in the process unveils previously unknown functions of peptidyl-prolyl isomerases. The novel immunological features attributed herein to PpiB, an essential protein of M. tb, highlights the importance of this protein/enzyme as an important target for the development of more efficacious therapeutic interventions against TB.

This study was carried out in accordance with the recommendations of Institutional Bioethics Committee, Bhagwan Mahavir Medical Research Centre, Hyderabad, India with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Bioethics Committee.

NE and SH conceptualized and designed the research; SP, DT, and MK performed research; SP, DT, AK, JS, SH, and NE carried out data analysis; GS recruited and classified human subjects; SP, DT, JS, SH, and NE wrote the manuscript.

This work was partially funded by a Centre of Excellence research grant (BT/PR12817/COE/34/23/2015) to SH and NE from the Department of Biotechnology, Ministry of Science and Technology (DBT), Government of India.