Mycobacterial Pathogenicity Research Articles

Genome wide screening to discover novel toxin-antitoxin modules in Mycobacterium indicus pranii; perspective on gene acquisition during mycobacterial evolution.

Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram-negative sepsis and COVID-19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin-antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome-wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery.

Overview of Small Molecules as Fluorescent Probes of Mycobacterium tuberculosis.

Tuberculosis (TB) remains one of the leading infectious causes of death worldwide. Detecting and precisely quantifying viable Mycobacterium tuberculosis (M. tuberculosis) is crucial for comprehending mycobacterial pathogenicity; the progression and outcomes of tuberculosis; and the action, efficacy, and resistance of drugs. Fluorescent probes have emerged as indispensable tools for studying the intricate structure and dynamic interactions of M. tuberculosis with its host environment. This minireview underscores the significance of small molecules as fluorescent probes in advancing our understanding of mycobacterial biology and highlights their potential for guiding the development of novel therapeutic interventions against tuberculosis.

Computational insights into potential marine natural products as selective inhibitors of Mycobacterium tuberculosis InhA: A structure-based virtual screening study

Several factors are associated with the emergence of drug resistance mechanisms, such as impermeable cell walls, gene mutations, and drug efflux systems. Consequently, bacteria acquire resistance, leading to a decrease in drug efficacy. A new and innovative strategy is required to combat drug resistance in tuberculosis (TB) effectively. Therefore, targeting the mycolic acid biosynthesis pathway, which is involved in synthesising mycolic acids (MAs), essential structural components responsible for mycobacterial pathogenicity, has garnered interest in TB research and the concept of drug resistance. In this context, InhA, which plays a crucial role in the fatty acid synthase-II (FAS-II) system of the MA biosynthetic pathway, was selected as a druggable target for screening investigation. To identify potential lead molecules against InhA, diverse marine natural products (MNPs) were collected from the comprehensive marine natural products database (CMNPD). Virtual screening studies aided in selecting potential lead molecules that best fit within the substrate-binding pocket (SBP) of InhA, forming crucial hydrogen bond interaction with the catalytic residue Tyr158. Three MNPs, CMNPD30814, CMNPD1702, and CMNPD27355, were chosen as prospective alternative molecules due to their favorable pharmacokinetic properties and lack of toxicity according to ProTox-II predictions. Additionally, improved reactivity of the MNPs was observed in the results of density functional theory (DFT) studies. Furthermore, comparative molecular dynamics simulation (MDS), principal component (PC)-based free energy landscape (FEL) analysis, and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) were employed to show enhanced structural stability, increased H-bond potential, and high binding affinity toward the target InhA. Moreover, the hot spot residues that contributed to the high binding energy profile and anchored the stability of the complexes were revealed with their individual interaction energy. The computational insights from this study provide potential avenues to combat TB through the multifaceted mode of action of these marine lead molecules, which can be further explored in future experimental investigations.

Comparison of Proline-glutamate-proline-glutamate-polymorphic GC-rich Sequences Family Protein Wag22 (Rv1759c), PE_PGRS31 (Rv1768), PE_PGRS32 (Rv1803), and PE_PGRS33 gene (Rv1818c) in Exponential State and Under In vitro Model of Latency in Same Clinical Isolates of Mycobacterium tuberculosis: Frameshift Mutation in Extensively Drug-resistant and Totally Drug-resistant tuberculosis Bacilli

Abstract Background: Proline-glutamate (PE)/proline-PE (PPE) proteins play an important role in the development of mycobacterial pathogenicity by modulating the host immune system. In the present investigation, the structural changes in PE-polymorphic GC-rich sequences (PGRS) family protein Wag22 (Rv1759c), PE_PGRS31 (Rv1768), PE_PGRS32 (Rv1803), and PE_PGRS33 gene (Rv1818c) were compared and analyzed in exponential state and under in vitro model of latency in same clinical isolates of Mycobacterium tuberculosis (MTB). Methods: MTB strains were isolated from clinically and laboratory-confirmed cases of tuberculosis (TB). The TB isolates were subjected to the Xpert MTB/rifampin test and then, further susceptibility testing using proportional methods was performed on them. The isolates were characterized using both 16S–23S RNA and hsp65 genes spacer polymerase chain reaction-restriction fragment length polymorphism. Selected isolates studied at two experimental set–up at exponential phase OD 600 = 0.05 (5 cfu/mL × 106 cfu/mL) and under zero oxygen and nutrition for 26 months to selected isolates studied at two experimental setup in exponential phase OD600 = 0.05 (5 cfu/mL × 106 cfu/mL) and under zero oxygen and nutrition after 26 months. Whole-genome sequencing was performed on studied isolates and the protein structures were analyzed using a bioinformatics web server. Results: No deletion, insertion, or substation occurred in susceptible, mono-drug and multidrug resistant-TB isolates were observed at PE-PGRS family protein Wag22 (Rv1759c) and PE_PGRS31 (Rv1768) at exponential phase. Although, a large deletion (at Rv1759c; Rv1768) was observed in extensively drug-resistant (XDR) and totally drug-resistant (TDR) TB isolates at the exponential phase. All studied TDR-TB isolates had a common deletion position from amino acid 1 (methionine) to amino acid 83 (glycine) and from amino acid 725 (proline) to amino acid 914 (threonine) at PE-PGRS family protein Wag22 (Rv1759c). At PE_PGRS32 (Rv1803), deletion occurred from amino acid 1 (methionine) to amino acid 212 (glycine) in latent TDR-TB bacilli. No changes in Rv1803 were observed in other studied isolates. In contrast, 66.6% of studied isolates had either insertion, deletion, substitution, or combination of changes at PE_PGRS33 (Rv1818c). However, the majority of changes at Rv1818c occurred in drug-resistant isolates. We also documented the region of deletion and insertion at PE_PGRS33 (Rv1818c) is different in active and latent TDR-TB isolates. Conclusions: Changes in these PE-PGRS family protein was associated with drug susceptibility patterns of individual isolates. Our result showed a total frameshift mutation of protein that had a different length in comparison to the original protein. These changes might disturb the interactions between XDR and TDR-TB isolates and immune responses, which needs further investigation.

Contraction and expansion dynamics: deciphering genomic underpinnings of growth rate and pathogenicity in Mycobacterium.

Mycobacterium bacteria, encompassing both slow growth (SGM) and rapid growth mycobacteria (RGM), along with true pathogenic (TP), opportunistic pathogenic (OP), and non-pathogenic (NP) types, exhibit diverse phenotypes. Yet, the genetic underpinnings of these variations remain elusive. Here, We conducted a comprehensive comparative genomics study involving 53 Mycobacterium species to unveil the genomic drivers behind growth rate and pathogenicity disparities. Our core/pan-genome analysis highlighted 1,307 shared gene families, revealing an open pan-genome structure. A phylogenetic tree highlighted clear boundaries between SGM and RGM, as well as TP and other species. Gene family contraction emerged as the primary alteration associated with growth and pathogenicity transitions. Specifically, ABC transporters for amino acids and inorganic ions, along with quorum sensing genes, exhibited significant contractions in SGM species, potentially influencing their distinct traits. Conversely, TP strains displayed contraction in lipid and secondary metabolite biosynthesis and metabolism-related genes. Across the 53 species, we identified 26 core and 64 accessory virulence factors. Remarkably, TP and OP strains stood out for their expanded mycobactin biosynthesis and type VII secretion system gene families, pivotal for their pathogenicity. Our findings underscore the importance of gene family contraction in nucleic acids, ions, and substance metabolism for host adaptation, while emphasizing the significance of virulence gene family expansion, including type VII secretion systems and mycobactin biosynthesis, in driving mycobacterial pathogenicity.

Comparative genome analysis of mycobacteria focusing on tRNA and non-coding RNA

BackgroundThe Mycobacterium genus encompasses at least 192 named species, many of which cause severe diseases such as tuberculosis. Non-tuberculosis mycobacteria (NTM) can also infect humans and animals. Some are of emerging concern because they show high resistance to commonly used antibiotics while others are used and evaluated in bioremediation or included in anticancer vaccines.ResultsWe provide the genome sequences for 114 mycobacterial type strains and together with 130 available mycobacterial genomes we generated a phylogenetic tree based on 387 core genes and supported by average nucleotide identity (ANI) data. The 244 genome sequences cover most of the species constituting the Mycobacterium genus. The genome sizes ranged from 3.2 to 8.1 Mb with an average of 5.7 Mb, and we identified 14 new plasmids. Moreover, mycobacterial genomes consisted of phage-like sequences ranging between 0 and 4.64% dependent on mycobacteria while the number of IS elements varied between 1 and 290. Our data also revealed that, depending on the mycobacteria, the number of tRNA and non-coding (nc) RNA genes differ and that their positions on the chromosome varied. We identified a conserved core set of 12 ncRNAs, 43 tRNAs and 18 aminoacyl-tRNA synthetases among mycobacteria.ConclusionsPhages, IS elements, tRNA and ncRNAs appear to have contributed to the evolution of the Mycobacterium genus where several tRNA and ncRNA genes have been horizontally transferred. On the basis of our phylogenetic analysis, we identified several isolates of unnamed species as new mycobacterial species or strains of known mycobacteria. The predicted number of coding sequences correlates with genome size while the number of tRNA, rRNA and ncRNA genes does not. Together these findings expand our insight into the evolution of the Mycobacterium genus and as such they establish a platform to understand mycobacterial pathogenicity, their evolution, antibiotic resistance/tolerance as well as the function and evolution of ncRNA among mycobacteria.

Mycobacterium tuberculosis PPE7 Enhances Intracellular Survival of Mycobacterium smegmatis and Manipulates Host Cell Cytokine Secretion Through Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase Signaling.

The PE/PPE family proteins of Mycobacterium tuberculosis have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of M. tuberculosis possesses a lineage-specific PPE gene (PPE7), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing M. tuberculosis PPE7 in Mycobacterium smegmatis. We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in M. smegmatis could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1β, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.

A multi-omics investigation into the mechanisms of hyper-virulence in Mycobacterium tuberculosis

ABSTRACT Clinical manifestations of tuberculosis range from asymptomatic infection to a life-threatening disease such as tuberculous meningitis (TBM). Recent studies showed that the spectrum of disease severity could be related to genetic diversity among clinical strains of Mycobacterium tuberculosis (Mtb). Certain strains are reported to preferentially invade the central nervous system, thus earning the label “hypervirulent strains”.However, specific genetic mutations that accounted for enhanced mycobacterial virulence are still unknown. We previously identified a set of 17 mutations in a hypervirulent Mtb strain that was from TBM patient and exhibited significantly better intracellular survivability. These mutations were also commonly shared by a cluster of globally circulating hyper-virulent strains. Here, we aimed to validate the impact of these hypervirulent-specific mutations on the dysregulation of gene networks associated with virulence in Mtb via multi-omic analysis. We surveyed transcriptomic and proteomic differences between the hyper-virulent and low-virulent strains using RNA-sequencing and label-free quantitative LC-MS/MS approach, respectively. We identified 25 genes consistently differentially expressed between the strains at both transcript and protein level, regardless the strains were growing in a nutrient-rich or a physiologically relevant multi-stress condition (acidic pH, limited nutrients, nitrosative stress, and hypoxia). Based on integrated genomic-transcriptomic and proteomic comparisons, the hypervirulent-specific mutations in FadE5 (g. 295,746 C >T), Rv0178 (p. asp150glu), higB (p. asp30glu), and pip (IS6110-insertion) were linked to deregulated expression of the respective genes and their functionally downstream regulons. The result validated the connections between mutations, gene expression, and mycobacterial pathogenicity, and identified new possible virulence-associated pathways in Mtb.

MIR337-3p Enhances Mycobacterial Pathogenicity Involving TLR4/MYD88 and STAT3 Signals, Impairing VDR Antimicrobial Response and Fast-Acting Immunity.

Active form of vitamin D (VitD) enhances human innate immunity against Mycobacterium tuberculosis (Mtb) infection. Our previous studies showed that MIR337-3p was highly expressed in lymphocytes of tuberculosis (TB) patients. Here, we identified the mechanism of MIR337-3p in the regulation of fast-acting anti-TB immunity by inhibiting VitD-dependent antimicrobial response pathways. While high-level MIR337-3p expression was induced by mycobacterial infection in cellular models and mice, TB patients exhibited significantly increased MIR337-3p in CD14+ monocytes/macrophages, innate-like Vγ2+ T cells, and CD8+ lymphocytes containing natural killer (NK)/innate lymphoid cells. MIR337-3p promoted the mycobacterial entry/infection and replication/growth in host target cells: macrophages and lung epithelial cells. Such MIR337-3p-enhanced pathogenicity coincided with the MIR337-3p depression of VitD-dependent antimicrobial response of cytochrome P450, family 27, subfamily b, polypeptide 1 (CYP27B1)/Beta-defensin 4 (DEFB4A)/ cathelicidin antimicrobial peptide CAMP pathways. Surprisingly, single MIR337-3p species could specifically target both the Toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) 3′-untranslated regions (UTRs) to depress the TLR4/MYD88 and STAT3 signals and impair either of the two signals inhibiting the VitD-dependent antimicrobial pathways in macrophages. Concurrently, human peripheral blood mononuclear cells (PBMCs) expressing high-level MIR337-3p exhibited a reduced ability of innate cell populations to mount fast-acting cellular immunity against intracellular mycobacterial infection. Furthermore, a higher expression of Mir337-3p after mycobacterial infection of mice coincided with much greater colony-forming unit (CFU) counts in lungs and even the death of infected animals, whereas Mir337-3p inhibitor treatment of infected mice reduced Mir337-3p levels and reversed Mir337-3p-mediated increases in CFU counts. Thus, TB-driven single MIR337-3p species could specifically target/impair both TLR4/MYD88 and STAT3 activation signals, inhibiting VitD-dependent antimicrobial response and fast-acting anti-TB immunity, leading to enhanced pathogenicity.

A fresh look at mycobacterial pathogenicity with the zebrafish host model.

The zebrafish has earned its place among animal models to study tuberculosis and other infections caused by pathogenic mycobacteria. This model host is especially useful to study the role of granulomas, the inflammatory lesions characteristic of mycobacterial disease. The optically transparent zebrafish larvae provide a window on the initial stages of granuloma development in the context of innate immunity. Application of fluorescent dyes and transgenic markers enabled real‐time visualization of how innate immune mechanisms, such as autophagy and inflammasomes, are activated in infected macrophages and how propagating calcium signals drive communication between macrophages during granuloma formation. A combination of imaging, genetic, and chemical approaches has revealed that the interplay between macrophages and mycobacteria is the main driver of tissue dissemination and granuloma development, while neutrophils have a protective function in early granulomas. Different chemokine signaling axes, conserved between humans and zebrafish, have been shown to recruit macrophages permissive to mycobacterial growth, control their microbicidal capacity, drive their spreading and aggregation, and mediate granuloma vascularization. Finally, zebrafish larvae are now exploited to explore cell death processes, emerging as crucial factors in granuloma expansion. In this review, we discuss recent advances in the understanding of mycobacterial pathogenesis contributed by zebrafish models.

Flow cytometry method for absolute counting and single-cell phenotyping of mycobacteria

Detection and accurate quantitation of viable Mycobacterium tuberculosis is fundamental to understanding mycobacterial pathogenicity, tuberculosis (TB) disease progression and outcomes; TB transmission; drug action, efficacy and drug resistance. Despite this importance, methods for determining numbers of viable bacilli are limited in accuracy and precision owing to inherent characteristics of mycobacterial cell biology—including the tendency to clump, and “differential” culturability—and technical challenges consequent on handling an infectious pathogen under biosafe conditions. We developed an absolute counting method for mycobacteria in liquid cultures using a bench-top flow cytometer, and the low-cost fluorescent dyes Calcein-AM (CA) and SYBR-gold (SG). During exponential growth CA + cell counts are highly correlated with CFU counts and can be used as a real-time alternative to simplify the accurate standardisation of inocula for experiments. In contrast to CFU counting, this method can detect and enumerate cell aggregates in samples, which we show are a potential source of variance and bias when using established methods. We show that CFUs comprise a sub-population of intact, metabolically active mycobacterial cells in liquid cultures, with CFU-proportion varying by growth conditions. A pharmacodynamic application of the flow cytometry method, exploring kinetics of fluorescent probe defined subpopulations compared to CFU is demonstrated. Flow cytometry derived Mycobacterium bovis bacillus Calmette-Guérin (BCG) time-kill curves differ for rifampicin and kanamycin versus isoniazid and ethambutol, as do the relative dynamics of discrete morphologically-distinct subpopulations of bacilli revealed by this high-throughput single-cell technique.

Mycobacterial fatty acid catabolism is repressed by FdmR to sustain lipogenesis and virulence

Host-derived fatty acids are an important carbon source for pathogenic mycobacteria during infection. How mycobacterial cells regulate the catabolism of fatty acids to serve the pathogenicity, however, remains unknown. Here, we identified a TetR-family transcriptional factor, FdmR, as the key regulator of fatty acid catabolism in the pathogen Mycobacterium marinum by combining use of transcriptomics, chromatin immunoprecipitation followed by sequencing, dynamic 13C-based flux analysis, metabolomics, and lipidomics. An M. marinum mutant deficient in FdmR was severely attenuated in zebrafish larvae and adult zebrafish. The mutant showed defective growth but high substrate consumption on fatty acids. FdmR was identified as a long-chain acyl-coenzyme A (acyl-CoA)-responsive repressor of genes involved in fatty acid degradation and modification. We demonstrated that FdmR functions as a valve to direct the flux of exogenously derived fatty acids away from β-oxidation toward lipid biosynthesis, thereby avoiding the overactive catabolism and accumulation of biologically toxic intermediates. Moreover, we found that FdmR suppresses degradation of long-chain acyl-CoAs endogenously synthesized through the type I fatty acid synthase. By modulating the supply of long-chain acyl-CoAs for lipogenesis, FdmR controls the abundance and chain length of virulence-associated lipids and mycolates and plays an important role in the impermeability of the cell envelope. These results reveal that despite the fact that host-derived fatty acids are used as an important carbon source, overactive catabolism of fatty acids is detrimental to mycobacterial cell growth and pathogenicity. This study thus presents FdmR as a potentially attractive target for chemotherapy.

De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp. paratuberculosis

Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1β, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.

Effect of peptidoglycan amidase MSMEG_6281 on fatty acid metabolism in Mycobacterium smegmatis

Mycobacterium smegmatis MSMEG_6281, a peptidoglycan (PG) amidase, is essential in maintaining cell wall integrity. To address the potential roles during the MSMEG_6281-mediated biological process, we compared proteomes from wild-type M.smegmatis and MSMEG_6281 gene knockout strain (M.sm-ΔM_6281) using LC-MS/MS analysis. Peptide analysis revealed that 851 proteins were differentially produced with at least 1.2-fold changes, including some proteins involved in fatty acid metabolism such as acyl-CoA synthase, acyl-CoA dehydrogenase, MCE-family proteins, ATP-binding cassette (ABC) transporters, and MmpL4. Some proteins related to fatty acid degradation were enriched through protein-protein interaction analysis. Therefore, proteomic data showed that a lack of MSMEG_6281 affected fatty acid metabolism. Mycobacteria can produce diverse lipid molecules ranging from single fatty acids to highly complex mycolic acids, and mycobacterial surface-exposed lipids may impact biofilm formation. In this study, we also assessed the effects of MSMEG_6281 on biofilm phenotype using semi-quantitative and morphology analysis methods. These results found that M.sm-ΔM_6281 exhibited a delayed biofilm phenotype compared to that of the wild-type M.smegmatis, and the changes were recovered when PG amidase was rescued in a ΔM_6281::Rv3717 strain. Our results demonstrated that MSMEG_6281 impacts fatty acid metabolism and further interferes with biofilm formation. These results provide a clue to study the effects of PG amidase on mycobacterial pathogenicity.

Mycobacterium marinum infection drives foam cell differentiation in zebrafish infection models

Host lipid metabolism is an important target for subversion by pathogenic mycobacteria such as Mycobacterium tuberculosis. The appearance of foam cells within the granuloma are well-characterised effects of chronic tuberculosis. The zebrafish-Mycobacterium marinum infection model recapitulates many aspects of human-M. tuberculosis infection and is used as a model to investigate the structural components of the mycobacterial granuloma. Here, we demonstrate that the zebrafish-M. marinum granuloma contains foam cells and that the transdifferentiation of macrophages into foam cells is driven by the mycobacterial ESX1 pathogenicity locus. This report demonstrates conservation of an important aspect of mycobacterial infection across species.

Blood antimicrobial activity varies against different Mycobacterium spp.

In vitro analysis of mycobacterial pathogenicity or host susceptibility has traditionally relied on the infection of macrophages, the target cell of mycobacteria, despite difficulties reproducing their antimycobacterial activity. We have employed alternative models, namely whole blood and leukocytes in plasma, from QuantiFERON negative individuals, and performed infections with the pathogenic M. tuberculosis, the less pathogenic M. avium, M. kansasii and M. chelonae and the occasionally pathogenic M. gordonae and M. bovis. The anticoagulant used in blood extraction, heparin or EDTA, had a major influence in the outcome of the infection. Thus, while in the heparinized models a similar number of bacteria were enumerated in the inoculum and after seven days, in the presence of EDTA a killing effect was observed, despite the inhibitory effect of EDTA on cellular functions like the production of cytokines or reactive oxygen species (ROS). A special case was the rapidly growing mycobacteria M. chelonae, that multiplied in heparinized models but was eliminated in models with EDTA. We verified that EDTA is not responsible for the bactericidal effect, but acts as a bacteriostatic agent. Further work will determine whether blood derived models are a better alternative to the classical macrophage.

Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model

Tuberculosis remains one of the major threats to public health worldwide. Given the prevalence of multi drug resistance (MDR) in Mycobacterium tuberculosis strains, there is a strong need to develop new anti-mycobacterial drugs with modes of action distinct from classical antibiotics. Inhibitors of mycobacterial virulence might target new molecular processes and may represent a potential new therapeutic alternative. In this study, we used a Dictyostelium discoideum host model to assess virulence of Mycobacterium marinum and to identify compounds inhibiting mycobacterial virulence. Among 9995 chemical compounds, we selected 12 inhibitors of mycobacterial virulence that do not inhibit mycobacterial growth in synthetic medium. Further analyses revealed that 8 of them perturbed functions requiring an intact mycobacterial cell wall such as sliding motility, bacterial aggregation or cell wall permeability. Chemical analogs of two compounds were analyzed. Chemical modifications altered concomitantly their effect on sliding motility and on mycobacterial virulence, suggesting that the alteration of the mycobacterial cell wall caused the loss of virulence. We characterized further one of the selected compounds and found that it inhibited the ability of mycobacteria to replicate in infected cells. Together these results identify new antimycobacterial compounds that represent new tools to unravel the molecular mechanisms controlling mycobacterial pathogenicity. The isolation of compounds with anti-virulence activity is the first step towards developing new antibacterial treatments.

Diverse effects of mycobacterial proline-proline-glutamic acid proteins upon interaction with host macrophages.

The proline-proline-glutamic acid (PPE) family proteins are abundant only in pathogenic Mycobacteria, but their general functions are far from unveiled. To investigate their roles in how Mycobacterium tuberculosis (Mtb) resists killing by the host, 25 PPE recombinant Mycobacterium smegmatis strains that overexpress Mtb PPE proteins were constructed. During phagocytosis, a similar amount of intracellular bacteria was observed at 2 h post-infection (hpi) for 24 PPE recombinants, while a 50% reduction of entrance was observed for the PPE29 recombinant. In addition, we found that 20 ppe genes significantly influenced the survival of mycobacteria within macrophage cells. Mycobacterial survival was promoted by overexpression of 18 of these genes and inhibited by the other two. Highest survival was observed for the PPE27 recombinant. We also measured the levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 secreted by macrophages. The overall effects varied among the different PPE recombinants. Moreover, we also found that various PPE recombinants exhibited increased resistance against oxidative, acidic and sodium dodecyl sulfate stresses that could be encountered in vivo. Together, our results indicate that PPE proteins play distinct roles in mycobacterial survival in macrophages. The findings described here broaden our understanding of mycobacterial pathogenicity.