Petra Selchow

Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting.

The protective effect of antibodies (Abs) is generally attributed to neutralization or complement activation. Using Legionella pneumophila and Mycobacterium bovis bacillus Calmette–Guérin as a model, we discovered an additional mechanism of Ab-mediated protection effective against intracellular pathogens that normally evade lysosomal fusion. We show that Fc receptor (FcR) engagement by Abs, which can be temporally and spatially separated from bacterial infection, renders the host cell nonpermissive for bacterial replication and targets the pathogens to lysosomes. This process is strictly dependent on kinases involved in FcR signaling but not on host cell protein synthesis or protease activation. Based on these findings, we propose a mechanism whereby Abs and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation.

Relief from Zmp1-mediated arrest of phagosome maturation is associated with facilitated presentation and enhanced immunogenicity of mycobacterial antigens.

ABSTRACT Pathogenic mycobacteria escape host innate immune responses by blocking phagosome-lysosome fusion. Avoiding lysosomal delivery may also be involved in the capacity of mycobacteria to evade major histocompatibility complex (MHC) class I- or II-dependent T-cell responses. In this study, we used a genetic mutant of Mycobacterium bovis BCG that is unable to escape lysosomal transfer and show that presentation of mycobacterial antigens is affected by the site of intracellular residence. Compared to infection with wild-type BCG, infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zinc metalloprotease 1 (Zmp1) resulted in increased presentation of MHC class II-restricted antigens, as assessed by activation of mycobacterial Ag85A-specific T-cell hybridomas. The zmp1 deletion mutant was more immunogenic in vivo, as measured by delayed-type hypersensitivity (DTH), antigen-specific lymphocyte proliferation, and the frequency of antigen-specific gamma interferon (IFN-γ)-producing lymphocytes of both CD4 and CD8 subsets. In conclusion, our results suggest that phagosome maturation and lysosomal delivery of BCG facilitate mycobacterial antigen presentation and enhance immunogenicity.

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.

Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis

Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1′. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.

Intrinsic rifamycin resistance of Mycobacterium abscessus is mediated by ADP-ribosyltransferase MAB_0591.

Objectives: Rifampicin, a potent first-line TB drug of the rifamycin group, shows only little activity against the emerging pathogen Mycobacterium abscessus. Reportedly, bacterial resistance to rifampicin is associated with polymorphisms in the target gene rpoB or the presence of enzymes that modify and thereby inactivate rifampicin. The aim of this study was to investigate the role of the MAB_0591 (arrMab)-encoded rifampicin ADP-ribosyltransferase (Arr_Mab) in innate high-level rifampicin resistance in M. abscessus. Methods: Recombinant Escherichia coli and Mycobacterium tuberculosis strains expressing MAB_0591 were generated, as was an M. abscessus deletion mutant deficient for MAB_0591. MIC assays were used to study susceptibility to rifampicin and C25 carbamate-modified rifamycin derivatives. Results: Heterologous expression of MAB_0591 conferred rifampicin resistance to E. coli and M. tuberculosis. Rifamycin MIC values were consistently lower for the M. abscessus &Dgr;arrMab mutant as compared with the M. abscessus ATCC 19977 parental type strain. The rifamycin WT phenotype was restored after complementation of the M. abscessus &Dgr;arrMab mutant with arrMab. Further MIC data demonstrated that a C25 modification increases rifamycin activity in WT M. abscessus. However, MIC studies in the M. abscessus &Dgr;arrMab mutant suggest that C25 modified rifamycins are still subject to modification by Arr_Mab. Conclusions: Our findings identify Arr_Mab as the major innate rifamycin resistance determinant of M. abscessus. Our data also indicate that Arr_Mab-mediated rifamycin resistance in M. abscessus can only in part be overcome by C25 carbamate modification.

Discovery of the first potent and selective Mycobacterium tuberculosis Zmp1 inhibitor.

The Mycobacterium tuberculosis extracellular zinc metalloprotease 1 (Zmp1) has been proposed to play a key role in phagosome maturation and to enhance the survival of Mycobacterium tuberculosis in the host. Consequently, small molecule inhibitors of Zmp1 are of pivotal importance as a tool to better understand the pathogenicity of Zmp1 and as lead candidates for pharmacological intervention. Here we combined in silico structure-based inhibitor design with biochemical studies to discover and characterize the first potent competitive Zmp1 inhibitor showing a Ki of 94 nM and a high selectivity for Zmp1 with respect to human Neprilysin.

Phenylethyl Butyrate Enhances the Potency of Second-Line Drugs against Clinical Isolates of Mycobacterium tuberculosis

ABSTRACT Ethionamide (ETH) is a second-line drug for the treatment of tuberculosis. As a prodrug, ETH has to be activated by EthA. ethA is controlled by its repressor EthR. 2-Phenylethyl-butyrate (2-PEB) inhibits EthR binding, enhances expression of EthA, and thereby enhances the growth-inhibitory effects of ethionamide, isoxyl, and thiacetazone in Mycobacterium tuberculosis strains with resistance to ETH due to inhA promoter mutations but not ethA mutations.

Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.

Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Lipoprotein Glycosylation by Protein-O-Mannosyltransferase (MAB_1122c) Contributes to Low Cell Envelope Permeability and Antibiotic Resistance of Mycobacterium abscessus

Lipoproteins are important components of the mycobacterial cell envelope due to their function in cell wall homeostasis and bacterial virulence. They are post-translationally modified with lipid- and glycosyl-residues in various species and interference with acylation or glycosylation leads to reduced growth and attenuated virulence in Mycobacterium tuberculosis. Lipoproteins are also expressed in the emerging and highly drug resistant pathogen Mycobacterium abscessus which frequently affects the lungs of patients with chronic pulmonary disease or cystic fibrosis. We investigated post-translational modification, acylation and glycosylation, of heterologously expressed (M. tuberculosis LppX and Mpt83) and endogenous (SodC) lipoproteins at the molecular level in M. abscessus and identified MAB_1122c as protein O-mannosyltransferase (Pmt). Both, heterologous and endogenous lipoproteins carried a characteristic lipid anchor with palmitic acid (C16), palmitoleic acid (C16:1), oleic acid (C18), or tuberculostearic acid (C19) modifications. Multiple hexose-moieties were detected in the N-terminal region of the model lipoproteins expressed in M. abscessus. Conservation of lipoprotein glycosylation in M. tuberculosis and M. abscessus was revealed and points toward the existence of an O-glycosylation motif or other regulatory mechanisms regarding this post-translational modification. Deletion of MAB_1122c prevented glycosylation and affected susceptibility to specific antibiotics which are large or target peptidoglycan synthesis and to lysozyme. Cell envelope permeability of M. abscessus Δpmt was increased and mutant bacteria showed reduced survival inside macrophages. The results provide a link between post-translational modification of lipoproteins and the permeability of the mycobacterial cell envelope which stresses the importance of lipoproteins as components of this complex structure.