No abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death due to bacterial infections in mankind, and BCG, an attenuated strain of Mycobacterium bovis, is an approved vaccine. BCG sequesters in immature phagosomes of antigen presenting cells (APCs), which do not fuse with lysosomes, leading to decreased antigen processing and reduced Th1 responses. However, an Mtb derived ΔfbpA attenuated mutant underwent limited phagosome maturation, enhanced immunogenicity and was as effective as BCG in protecting mice against TB. To facilitate phagosome maturation of ΔfbpA, we disrupted an additional gene sapM, which encodes for an acid phosphatase. Compared to the wild type Mtb, the ΔfbpAΔsapM (double knock out; DKO) strain was attenuated for growth in mouse macrophages and PMA activated human THP1 macrophages. Attenuation correlated with increased oxidants in macrophages in response to DKO infection and enhanced labeling of lysosomal markers (CD63 and rab7) on DKO phagosomes. An in vitro Antigen 85B peptide presentation assay was used to determine antigen presentation to T cells by APCs infected with DKO or other mycobacterial strains. This revealed that DKO infected APCs showed the strongest ability to present Ag85B to T cells (>2500 pgs/mL in 4 hrs) as compared to APCs infected with wild type Mtb or ΔfbpA or ΔsapM strain (<1000 pgs/mL in 4 hrs), indicating that DKO strain has enhanced immunogenicity than other strains. The ability of DKO to undergo lysosomal fusion and vacuolar acidification correlated with antigen presentation since bafilomycin, that inhibits acidification in APCs, reduced antigen presentation. Finally, the DKO vaccine elicited a better Th1 response in mice after subcutaneous vaccination than either ΔfbpA or ΔsapM. Since ΔfbpA has been used in mice as a candidate vaccine and the DKO (ΔfbpAΔsapM) mutant is more immunogenic than ΔfbpA, we propose the DKO is a potential anti-tuberculosis vaccine.

Oxidation of methionine (Met) residues in proteins by reactive oxygen species and reactive nitrogen intermediates results in altered protein structures, which subsequently affect their functions. Oxidized Met (Met-O) residues are reduced to Met by the methionine sulfoxide reductase (Msr) system, which includes mainly MsrA and MsrB. MsrA and MsrB show no sequence and structural identity with each other but both reduce methionine sulfoxides. MsrA is specific to the reduction of methionine-S-sulfoxide, whereas MsrB is specific to the reduction of methionine-R-sulfoxide. Genes encoding the enzymes MsrA and MsrB exist in most living organisms including bacteria. In recent times, absence of these enzymes has been implicated in the virulence of bacterial pathogens. In particular, pathogens deficient in Msr have been reported to have reduced ability to adhere with eukaryotic cells, to survive inside hosts and to resist in vitro oxidative stress. Bacterial proteins that are susceptible to Met oxidation, in the absence of Msr, have also been identified. This review discusses the current knowledge on the role of Msr in bacterial virulence.

Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis due to M. tuberculosis (Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG 85C5 induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger T H 1 cytokines (IL-12, IL-1β, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG 85C5 also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG 85C5 infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG 85C5 induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG 85C5 markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG 85C5 induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.