Reversing BCG-mediated autophagy inhibition and mycobacterial survival to improve vaccine efficacy.

BACKGROUND: Autophagy is an important mechanism for promoting Mycobacterium clearance from macrophages. Pathogenic and non-pathogenic mycobacterium can activate the mTOR pathway while simultaneously inducing autophagy. M. tuberculosis and M. bovis BCG inhibit autophagy and favor intracellular bacteria survival. RESULTS: We observed that pre-infection of live or heat-killed BCG could prevent autophagy induced by pharmacological activators or M. smegmatis, a strong autophagy-inducing mycobacterium. BCG-derived lipids are responsible for autophagy inhibition. However, post-infection with BCG could not stop the autophagy initiated by M. smegmatis, which increases further autophagy induction and mycobacteria clearance. Coinfection with BCG and heat killed M. smegmatis enhanced antigen specific CD4+ T cell responses and reduced mycobacterial survival. CONCLUSION: These results suggest that autophagy-inducing M. smegmatis could be used to promote better innate and consequential adaptive immune responses, improving BCG vaccine efficacy.

Identification of Autophagy-Inhibiting Factors of Mycobacterium tuberculosis by High-Throughput Loss-of-Function Screening.

The interaction of host cells with mycobacteria is complex and can lead to multiple outcomes ranging from bacterial clearance to progressive or latent infection. Autophagy is recognized as one component of host cell responses that has an essential role in innate and adaptive immunity to intracellular bacteria. Many microbes, including Mycobacterium tuberculosis, have evolved to evade or exploit autophagy, but the precise mechanisms and virulence factors are mostly unknown. Through a loss-of-function screening of an M. tuberculosis transposon mutant library, we identified 16 genes that contribute to autophagy inhibition, six of which encoded the PE/PPE protein family. Their expression in Mycobacterium smegmatis confirmed that these PE/PPE proteins inhibit autophagy and increase intracellular bacterial persistence or replication in infected cells. These effects were associated with increased mammalian target of rapamycin (mTOR) activity and also with decreased production of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). We also confirmed that the targeted deletion of the pe/ppe genes in M. tuberculosis resulted in enhanced autophagy and improved intracellular survival rates compared to those of wild-type bacteria in the infected macrophages. Differential expression of these PE/PPE proteins was observed in response to various stress conditions, suggesting that they may confer advantages to M. tuberculosis by modulating its interactions with host cells under various conditions. Our findings demonstrated that multiple M. tuberculosis PE/PPE proteins are involved in inhibiting autophagy during infection of host phagocytes and may provide strategic targets in developing therapeutics or vaccines against tuberculosis.

Mycobacterium tuberculosis PPE51 Inhibits Autophagy by Suppressing Toll-Like Receptor 2-Dependent Signaling.

Autophagy is an ubiquitous homeostatic pathway in mammalian cells and plays a significant role in host immunity. Substantial evidence indicates that the ability of Mycobacterium tuberculosis (Mtb) to successfully evade immune responses is partially due to inhibition of autophagic pathways. Our previous screening of Mtb transposon mutants identified the PPE51 protein as an important autophagy-inhibiting effector. We found that expression of PPE51, either by infecting bacteria or by direct expression in host cells, suppressed responses to potent autophagy-inducing stimuli and interfered with bacterial phagocytosis. This phenotype was associated with reduced activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a key component of signaling pathways that stimulate autophagy. Multiple lines of evidence demonstrated that the effects of PPE51 are attributable to signal blocking by Toll-like receptor 2 (TLR2), a receptor with known involvement of activation of ERK1/2 and autophagy. Consistent with these results, mice with intact TLR2 signaling showed striking virulence attenuation for an Mtb ppe51 deletion mutant (Δ51) compared to wild-type Mtb, whereas infection of TLR2-deficient mice showed no such attenuation. Mice infected with Δ51 also displayed increased T cell responses to Mtb antigens and increased autophagy in infected lung tissues. Together, these results suggest that TLR2 activates relevant host immune functions during mycobacterial infection, which Mtb then evades through suppression of TLR2 signaling by PPE51. In addition to its previously identified function transporting substrates across the bacterial cell wall, our results demonstrate a direct role of PPE51 for evasion of both innate and adaptive immunity to Mtb. IMPORTANCE Tuberculosis is a significant global infectious disease caused by infection of the lungs with Mycobacterium tuberculosis, which resides and replicates mainly within host phagocytic cells. During coevolution with humans, Mtb has acquired various mechanisms to inhibit host cellular processes, including autophagy. Autophagy is a complex host cellular process that helps control intracellular infections by enhancing innate and adaptive immune responses. We identified the Mtb protein PPE51 as a mycobacterial effector that inhibits autophagy. We discovered TLR2 and mitogen-activated protein kinase signaling as the axis by which PPE51 mediates this effect. Autophagy regulation by PPE51, along with suppression of other TLR2-activated host cell functions, leads to increased bacterial survival in phagocytic cells and tissues of infected mice. A better understanding of how Mtb regulates autophagy and other host immune effectors could facilitate the design of new therapeutics or vaccines against tuberculosis.

Mycobacterium tuberculosis PE_PGRS20 and PE_PGRS47 Proteins Inhibit Autophagy by Interaction with Rab1A.

Autophagy is a fundamental cellular process that has important roles in innate and adaptive immunity against a broad range of microbes. Many pathogenic microbes have evolved mechanisms to evade or exploit autophagy. It has been previously demonstrated that induction of autophagy can suppress the intracellular survival of mycobacteria, and several PE_PGRS family proteins of Mycobacterium tuberculosis have been proposed to act as inhibitors of autophagy to promote mycobacterial survival. However, the mechanisms by which these effectors inhibit autophagy have not been defined. Here, we report detailed studies of M. tuberculosis deletion mutants of two genes, pe_pgrs20 and pe_pgrs47, that we previously reported as having a role in preventing autophagy of infected host cells. These mutants resulted in increased autophagy and reduced intracellular survival of M. tuberculosis in macrophages. This phenotype was accompanied by increased cytokine production and antigen presentation by infected cells. We further demonstrated that autophagy inhibition by PE_PGRS20 and PE_PGRS47 resulted from canonical autophagy rather than autophagy flux inhibition. Using macrophages transfected to express PE_PGRS20 or PE_PGRS47, we showed that these proteins inhibited autophagy initiation directly by interacting with Ras-related protein Rab1A. Silencing of Rab1A in mammalian cells rescued the survival defects of the pe_pgrs20 and pe_pgrs47 deletion mutant strains and reduced cytokine secretion. To our knowledge, this is the first study to identify mycobacterial effectors that directly interact with host proteins responsible for autophagy initiation. IMPORTANCE Tuberculosis is a significant global infectious disease caused by infection of the lungs with Mycobacterium tuberculosis, which then resides and replicates mainly within host phagocytic cells. Autophagy is a complex host cellular process that helps control intracellular infections and enhance innate and adaptive immune responses. During coevolution with humans, M. tuberculosis has acquired various mechanisms to inhibit host cellular processes, including autophagy. We identified two related M. tuberculosis proteins, PE_PGRS20 and PE_PGRS47, as the first reported examples of specific mycobacterial effectors interfering with the initiation stage of autophagy. Autophagy regulation by these PE_PGRS proteins leads to increased bacterial survival in phagocytic cells and increased autophagic degradation of mycobacterial antigens to stimulate adaptive immune responses. A better understanding of how M. tuberculosis regulates autophagy in host cells could facilitate the design of new and more effective therapeutics or vaccines against tuberculosis.

Targeting Autophagy as a Strategy for Developing New Vaccines and Host-Directed Therapeutics Against Mycobacteria.

Mycobacterial disease is an immense burden worldwide. This disease group includes tuberculosis, leprosy (Hansen's disease), Buruli Ulcer, and non-tuberculous mycobacterial (NTM) disease. The burden of NTM disease, both pulmonary and ulcerative, is drastically escalating globally, especially in developed countries such as America and Australia. Mycobacteria's ability to inhibit or evade the host immune system has contributed significantly to its continued prevalence. Pre-clinical studies have highlighted promising candidates that enhance endogenous pathways and/or limit destructive host responses. Autophagy is a cell-autonomous host defense mechanism by which intracytoplasmic cargos can be delivered and then destroyed in lysosomes. Previous studies have reported that autophagy-activating agents, small molecules, and autophagy-activating vaccines may be beneficial in restricting intracellular mycobacterial infection, even with multidrug-resistant strains. This review will examine how mycobacteria evade autophagy and discusses how autophagy could be exploited to design novel TB treatment strategies, such as host-directed therapeutics and vaccines, against Mycobacterium tuberculosis and NTMs.

Use of Soluble Extracellular Regions of MmpL (SERoM) as Vaccines for Tuberculosis.

The current vaccine for tuberculosis (TB) is a live attenuated strain of Mycobacterium bovis (BCG) and while effective at reducing the potential for disseminated TB in young children its disease protection rates in adults is highly variable while it confers little protection against latent TB. With these limitations a new vaccine is desperately needed. We investigated the efficacy of three members of the mycobacterial membrane protein Large (MmpL) family as potential subunit vaccines for TB. MmpLs are large, multifunctional integral membrane proteins, and as such are recalcitrant to purification. Here, we describe a strategy of producing synthetic antigens comprised of the soluble, extracellular regions of MmpL (SERoM)-1, MmpL8 and MmpL10 (SERoM-8 and 10 respectively) as potential vaccine candidates. SERoM-1 and SERoM-8 were determined to be highly immunogenic by IFN-γ ELISpot assays, with 0.1% of all splenocytes from SERoM-1 vaccinated mice producing IFN-γ when re-stimulated with MmpL1. A combined SERoM-1, -8 and -10 vaccine demonstrated significant protection against M. tuberculosis challenge in a murine model of TB, resulting in approximately 10-fold reduction in bacterial numbers following challenge in both the lungs and spleens compared to adjuvant only vaccinated mice. These protective effects were comparable to that achieved with BCG.

Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method.

Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3'→5' exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.