The PPE2 protein of Mycobacterium tuberculosis is responsible for the development of hyperglycemia and insulin resistance during tuberculosis

Reviewer #1 (Public review):

Summary:

In this manuscript, Bisht et al. investigate the role of PPE2, a Mycobacterium tuberculosis (Mtb) secreted virulence factor, in adipose tissue physiology during tuberculosis (TB) infection. Previous work by this group established the significance of PPE proteins in Mtb virulence and their role in modulating the innate immune response. Here, the authors present compelling evidence that PPE2 regulates host cell adipogenesis and lipolysis, thereby establishing a link to the development of insulin resistance during TB infection. These fundamental findings demonstrate, for the first time, that a bacterial virulence factor is directly involved in the profound body fat loss, or "wasting," which is a long-established clinical symptom of active TB.

Key Strengths:

The confidence in the major findings of this study is significantly strengthened by the authors' comprehensive approach. They judiciously employ multiple experimental systems, including:

(1) Purified PPE2 protein.

(2) A non-pathogenic Mycobacterium strain engineered to express PPE2.

(3) A pathogenic clinical Mtb strain (CDC1551) utilizing a targeted PPE2 deletion mutant.

(4) While the presence of Mtb in adipose tissues in human and animal models is well-documented, this study is groundbreaking in demonstrating that an Mtb virulence-associated factor actively modulates host fatty acid metabolism within the adipose tissue.

Key Weakness:

Although the manuscript provides solid evidence associating the presence of PPE2 with transcriptional changes in host fatty acid machinery within the adipose tissue, the underlying mechanistic details remain elusive. A focused, deep mechanistic follow-up study will be essential to fully appreciate the complex biological implications of the findings reported here.

Reviewer #2 (Public review):

Summary:

In the manuscript entitled "The PPE2 protein of Mycobacterium tuberculosis is responsible for the development of hyperglycemia and insulin resistance during tuberculosis" the authors identify PPE2, a secretory protein of Mycobacterium tuberculosis, as a modulator of adipose function. They show that PPE2 treatment in mice causes fat loss, immune cell infiltration into adipose, reduced gene expression of PPAR-γ, C/EBP-α, and adiponectin, and glucose intolerance. Overall, the authors link PPE2 with adipose tissue perturbation and insulin resistance following infection with M. tuberculosis. PPE2, a secretory protein of Mycobacterium tuberculosis, is a modulator of adipose function. They show that PPE2 treatment in mice causes fat loss, immune cell infiltration into adipose, reduced gene expression of PPAR-γ, C/EBP-α, and adiponectin, and glucose intolerance. Overall, the authors link PPE2 with adipose tissue perturbation and insulin resistance following infection with M. tuberculosis.

Strengths:

While it is known that M. tuberculosis persists in adipose, the mycobacterial factors contributing to adipose dysfunction are unknown. The study uses multiple mechanisms, including recombinant purified protein, non-pathogenic mycobacterium expressing PPE2, and a clinical strain of M. tuberculosis depleted of PPE2, to show that PPE2 may play an important role in causing fat loss, lipolysis, and insulin resistance following infection. The authors show that PPE2, through unknown mechanisms, decreases gene expression of proteins involved in adipogenesis. Although the mechanisms are unclear, this study advances the field as it is the first to identify a secreted factor (PPE2) from M. tuberculosis to play a role in disrupting adipose tissue.

Weaknesses:

There is a lack of completeness amongst the figures that greatly diminishes the claims and impact of the manuscript. For example, in Figures 2 and 5, the authors measure adipocyte area in H&E-stained adipose tissue to show adipose hypertrophy. However, this was not completed in Figures 3 and 4 despite the authors claiming that treatment with rPPE2 induces adipose hypertrophy. It is unclear why the adipocyte area was not measured in these figures, and having this included would support the author's claim and strengthen the manuscript. The same is true for immune cell infiltration, where the authors say there is increased immune cell infiltration following PPE2 treatment. This is based on H&E staining, but the data supporting this is limited. Although the authors measure CD3+ T cell infiltration in adipose tissue from mice infected with the clinical strain where PPE was depleted, staining was performed in only this experiment. Completing these experiments by showing data to support that PPE2 induces immune cell infiltration would greatly strengthen the manuscript.

The authors state that a Student's t-test was performed to calculate the significance between two samples. However, there is no discussion of what statistical method was used when there were more than 2 groups, which occurs throughout the manuscript, such as in Figure 5, where 4 groups are analyzed. Having the appropriate statistical analysis is important for the impact of the manuscript.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "The PPE protein of Mycobacterium tuberculosis is responsible for the development of hyperglycemia and insulin resistance during tuberculosis", Bisht et al describe that PPE2 protein from Mtb is a key modulator of adipose tissue physiology that contributes to the development of insulin resistance. The authors have used 3T3-L1 preadipocyte cell lines, M. smegmatis overexpression strain, mice model, and genetically modified Mtb deletion strains to demonstrate that PPE promotes persistence in adipose tissue and regulates glucose homeostasis. Using qPCR and RNA-seq experiments, the authors demonstrate that PPE2 regulates the expression of key genes involved in adipogenesis.

Strengths:

Using purified protein, the authors show that PPE2 regulates adipose tissue physiology, and this effect was neutralised in the presence of anti-PPE2. The expression of several adipogenic markers was also reduced in 3TL-1 adipocytes treated with rPPE2 and in mice infected with M. smegmatis strains overexpressing PPE2. Using a mouse model of infection, the authors show that PPE2 contributes to enhanced mycobacterial survival within fat tissues. The authors also show infiltration of immune cells in the fat tissues of mice infected with wild-type and ppe2-complemented strains compared to the ppe2 KO strain. In order to gain a better mechanistic understanding of how PPE2 regulates adipogenesis, the authors employed an RNA-seq approach and identified 191 genes that were significantly differentially expressed in the fat tissues of mice infected with wild-type and ppe2 KO Mtb strains. The differentially expressed genes included transcripts encoding for proteins involved in chemokine/cytokine signalling, ER stress response. The expression of a few of these markers was also validated by qPCR and western blot analysis. Finally, the authors also show that PPE2 promotes lipolysis by reducing phosphodiesterase levels and activating PKA-HSL signalling. The experimental design is overall reasonable, and the methods used are reliable. Overall, the current study did provide some new information on the contribution of PPE2 in regulating adipose tissue physiology.

Weaknesses:

(1) The authors have used several methodologies to show that PPE2 regulates adipose tissue physiology and glucose homeostasis. But the exact mechanism is still not clear.

(2) Mtb encodes several PE/PPE proteins? The authors have used PPE2 for their study. Will secretory PPE2 homologs also regulate similar cellular processes?

(3) How do the authors rule out that the differences observed in the fat tissues of mice infected with wild-type and mutant strains are not associated with reduced bacterial burdens? Is it possible to include another Mtb attenuated strain as a control in mice experiments for few critical experiments?

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In this manuscript, Bisht et al. investigate the role of PPE2, a Mycobacterium tuberculosis (Mtb) secreted virulence factor, in adipose tissue physiology during tuberculosis (TB) infection. Previous work by this group established the significance of PPE proteins in Mtb virulence and their role in modulating the innate immune response. Here, the authors present compelling evidence that PPE2 regulates host cell adipogenesis and lipolysis, thereby establishing a link to the development of insulin resistance during TB infection. These fundamental findings demonstrate, for the first time, that a bacterial virulence factor is directly involved in the profound body fat loss, or "wasting," which is a long-established clinical symptom of active TB.

Key Strengths:

The confidence in the major findings of this study is significantly strengthened by the authors' comprehensive approach. They judiciously employ multiple experimental systems, including:

(1) Purified PPE2 protein.

(2) A non-pathogenic Mycobacterium strain engineered to express PPE2.

(3) A pathogenic clinical Mtb strain (CDC1551) utilizing a targeted PPE2 deletion mutant.

(4) While the presence of Mtb in adipose tissues in human and animal models is well-documented, this study is groundbreaking in demonstrating that an Mtb virulence-associated factor actively modulates host fatty acid metabolism within the adipose tissue.

We thank the reviewer for his appreciation that in this work we demonstrated for the first time that an Mtb virulent factor is directly linked to TB-associated wasting.

Weakness:

Although the manuscript provides solid evidence associating the presence of PPE2 with transcriptional changes in host fatty acid machinery within the adipose tissue, the underlying mechanistic details remain elusive. A focused, deep mechanistic follow-up study will be essential to fully appreciate the complex biological implications of the findings reported here.

We agree with the reviewer that a deep-focused, mechanistic follow-up study is necessary to further elucidate the complex biological implications of PPE2 actions. However, we believe that we have uncovered at least one of the possible mechanisms by which PPE2 increases lipolysis and circulating free fatty acids during infection by targeting cAMP-PKA-HSL pathway (Figure 7). In future studies we will aim to dissect out the mechanisms by which PPE2 triggers hyperglycaemia and insulin resistance.

Reviewer #2 (Public review):

Summary:

In the manuscript entitled "The PPE2 protein of Mycobacterium tuberculosis is respon,sible for the development of hyperglycemia and insulin resistance during tuberculosis" the authors identify PPE2, a secretory protein of Mycobacterium tuberculosis, as a modulator of adipose function. They show that PPE2 treatment in mice causes fat loss, immune cell infiltration into adipose, reduced gene expression of PPAR-γ, C/EBP-α, and adiponectin, and glucose intolerance. Overall, the authors link PPE2 with adipose tissue perturbation and insulin resistance following infection with M. tuberculosis. PPE2, a secretory protein of Mycobacterium tuberculosis, is a modulator of adipose function. They show that PPE2 treatment in mice causes fat loss, immune cell infiltration into adipose, reduced gene expression of PPAR-γ, C/EBP-α, and adiponectin, and glucose intolerance. Overall, the authors link PPE2 with adipose tissue perturbation and insulin resistance following infection with M. tuberculosis.

Strengths:

While it is known that M. tuberculosis persists in adipose, the mycobacterial factors contributing to adipose dysfunction are unknown. The study uses multiple mechanisms, including recombinant purified protein, non-pathogenic mycobacterium expressing PPE2, and a clinical strain of M. tuberculosis depleted of PPE2, to show that PPE2 may play an important role in causing fat loss, lipolysis, and insulin resistance following infection. The authors show that PPE2, through unknown mechanisms, decreases gene expression of proteins involved in adipogenesis. Although the mechanisms are unclear, this study advances the field as it is the first to identify a secreted factor (PPE2) from M. tuberculosis to play a role in disrupting adipose tissue.

We thank the reviewer for his appreciation of our findings presented in the manuscript.

Weaknesses:

(1) There is a lack of completeness amongst the figures that greatly diminishes the claims and impact of the manuscript. For example, in Figures 2 and 5, the authors measure adipocyte area in H&E-stained adipose tissue to show adipose hypertrophy. However, this was not completed in Figures 3 and 4 despite the authors claiming that treatment with rPPE2 induces adipose hypertrophy. It is unclear why the adipocyte area was not measured in these figures, and having this included would support the author's claim and strengthen the manuscript. The same is true for immune cell infiltration, where the authors say there is increased immune cell infiltration following PPE2 treatment. This is based on H&E staining, but the data supporting this is limited. Although the authors measure CD3+ T cell infiltration in adipose tissue from mice infected with the clinical strain where PPE was depleted, staining was performed in only this experiment. Completing these experiments by showing data to support that PPE2 induces immune cell infiltration would greatly strengthen the manuscript.

As per the suggestion of the esteemed reviewer, in the revised manuscript we will attempt to analyse adipocyte area in both Figures 3 and 4. In the original manuscript, immune cell infiltration analyses (H&E staining and CD3+ staining) was restricted to only M. tuberculosis-mouse infection model, which best reflects the human tuberculosis pathology. In other experiments involving infection with M. smegmatis expressing PPE2, immune cell infiltration studies will be carried out.

(2) The authors state that a Student's t-test was performed to calculate the significance between two samples. However, there is no discussion of what statistical method was used when there were more than 2 groups, which occurs throughout the manuscript, such as in Figure 5, where 4 groups are analyzed. Having the appropriate statistical analysis is important for the impact of the manuscript.

We agree with the reviewer that we missed to include ANOVA in the statistical analyses. We will include one-way ANOVA analysis where more than two groups are present and mention the statistical methods in the figure legends as well in the text of the revised manuscript.

Reviewer #3 (Public review):

Summary:

In this manuscript titled "The PPE protein of Mycobacterium tuberculosis is responsible for the development of hyperglycemia and insulin resistance during tuberculosis", Bisht et al describe that PPE2 protein from Mtb is a key modulator of adipose tissue physiology that contributes to the development of insulin resistance. The authors have used 3T3-L1 preadipocyte cell lines, M. smegmatis overexpression strain, mice model, and genetically modified Mtb deletion strains to demonstrate that PPE promotes persistence in adipose tissue and regulates glucose homeostasis. Using qPCR and RNA-seq experiments, the authors demonstrate that PPE2 regulates the expression of key genes involved in adipogenesis.

Strengths:

Using purified protein, the authors show that PPE2 regulates adipose tissue physiology, and this effect was neutralised in the presence of anti-PPE2. The expression of several adipogenic markers was also reduced in 3TL-1 adipocytes treated with rPPE2 and in mice infected with M. smegmatis strains overexpressing PPE2. Using a mouse model of infection, the authors show that PPE2 contributes to enhanced mycobacterial survival within fat tissues. The authors also show infiltration of immune cells in the fat tissues of mice infected with wild-type and ppe2-complemented strains compared to the ppe2 KO strain. In order to gain a better mechanistic understanding of how PPE2 regulates adipogenesis, the authors employed an RNA-seq approach and identified 191 genes that were significantly differentially expressed in the fat tissues of mice infected with wild-type and ppe2 KO Mtb strains. The differentially expressed genes included transcripts encoding for proteins involved in chemokine/cytokine signalling, ER stress response. The expression of a few of these markers was also validated by qPCR and western blot analysis. Finally, the authors also show that PPE2 promotes lipolysis by reducing phosphodiesterase levels and activating PKA-HSL signalling. The experimental design is overall reasonable, and the methods used are reliable. Overall, the current study did provide some new information on the contribution of PPE2 in regulating adipose tissue physiology.

We thank the reviewer for encouraging comments about the manuscript.

Weaknesses:

(1) The authors have used several methodologies to show that PPE2 regulates adipose tissue physiology and glucose homeostasis. But the exact mechanism is still not clear.

We have clearly demonstrated that PPE2 inhibit PPAR-γ and C/EBP-α expression to block adipogenic differentiation. Further, we demonstrated a possible mechanism by which PPE2 trigger lipolysis via activation of the ER stress and cAMP/PKA/HSL pathway which is responsible for increasing free fatty acids in circulation (Figure 7) as confirmed by our observation that PPE2KO (ppe2 knock-out) Mtb infected mice had lower NEFA as compared to the those infected with wild-type Mtb (Figure 7F). Crucially, we showed that this mechanism is clinically relevant since NEFA levels in the sera of TB patients were higher as compared to the healthy controls (Figure 7G) confirming presence of dyslipidemia in TB patients which is an established risk factor for insulin resistance (Karpe et al., 2011; Bhattacharya et al., 2007), As increased free fatty acids have been shown to be linked to development of insulin resistance in several studies, this mechanism links PPE2 with the regulation of glucose homeostasis.

(2) Mtb encodes several PE/PPE proteins? The authors have used PPE2 for their study. Will secretory PPE2 homologs also regulate similar cellular processes?

It is known that Mtb encodes several PE/PPE family proteins and some of these have been implicated to play a role in host–pathogen interactions (Mukhopadhyay and Balaji, 2011; Dahiya et al., 2025). However, so far only PPE2 is shown to be present in the circulation (Bisht et al., 2023) which is the main reason we chose it for this study. Presence of PPE2 homologues in the circulation is not known so far.

(3) How do the authors rule out that the differences observed in the fat tissues of mice infected with wild-type and mutant strains are not associated with reduced bacterial burdens? Is it possible to include another Mtb attenuated strain as a control in mice experiments for few critical experiments?

We agree with the reviewer that the differences in bacterial burden can influence host tissue responses. Precisely for this reason, we did not rely on just one infection model alone. We used a multi-pronged approach to de-couple the effects of PPE2 from the effects of bacterial load, like;

(1) In vitro Model using recombinantly purified PPE2 protein (rPPE2) (Figure 1): In cultured 3T3-L1 adipocytes, purified rPPE2 protein directly inhibited adipogenesis by downregulating important factors like PPAR-g,C/EBP-α and Fatty acid synthase (which play a critical role in triglyceride metabolism) demonstrating a direct effect of PPE2 in the complete absence of infection.

(2) Recombinant Protein Injection (Figure 3): By injecting recombinantly purified PPE2 protein (rPPE2) into mice, we observed similar metabolic perturbations (fat loss, impaired glucose tolerance) in the complete absence of any bacteria, demonstrating that PPE2 can drive these phenotypes independent of bacterial burden. Further study of rescuing of PPE2 action in rPPE2-immunized mice strongly confirm the specific role of PPE2 in establishing hyperglycaemia and insulin resistance (Figure 4).

While the Mtb aerosol model can be questioned for bacterial load effects, it provides crucial in vivo validation that PPE2 function is relevant in the context of mycobacterial infection.

References

Bhattacharya S, Dey D, Roy SS. Molecular mechanism of insulin resistance. J Biosci. 2007 Mar;32(2):405-13. doi: 10.1007/s12038-007-0038-8. PMID: 17435330.

Bisht MK, Pal R, Dahiya P, Naz S, Sanyal P, Nandicoori VK, Ghosh S, Mukhopadhyay S. The PPE2 protein of Mycobacterium tuberculosis is secreted during infection and facilitates mycobacterial survival inside the host. Tuberculosis (Edinb). 2023 Dec;143:102421. doi: 10.1016/j.tube.2023.102421. Epub 2023 Oct 12. PMID: 37879126.

Dahiya P, Bisht MK, Mukhopadhyay S. Role of PE family of proteins in mycobacterial virulence: Potential on anti-TB vaccine and drug design. Int Rev Immunol. 2025; 44(4):213-228. doi: 10.1080/08830185.2025.2455161. Epub 2025 Jan 31. PMID: 39889764.

Karpe F, Dickmann JR, Frayn KN. Fatty acids, obesity, and insulin resistance: time for a reevaluation. Diabetes. 2011 Oct;60(10):2441-9. doi: 10.2337/db11-0425. PMID: 21948998; PMCID: PMC3178283.

Mukhopadhyay S, Balaji KN. The PE and PPE proteins of Mycobacterium tuberculosis. Tuberculosis (Edinb). 2011 Sep;91(5):441-7. doi: 10.1016/j.tube.2011.04.004. Epub 2011 May 6. PMID: 21527209.