Phagocytosis influences the intracellular survival of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> via the endoplasmic reticulum stress response

Background

Mycobacterium smegmatis, a rapidly growing non-tuberculosis mycobacterium, is a good model for studying the pathogenesis of tuberculosis because of its genetic similarity to Mycobacterium tuberculosis (Mtb). Macrophages remove mycobacteria during an infection. Macrophage apoptosis is a host defense mechanism against intracellular bacteria. We have reported that endoplasmic reticulum (ER) stress is an important host defense mechanism against Mtb infection.

Results

In this study, we found that M. smegmatis induced strong ER stress. M. smegmatis-induced reactive oxygen species (ROS) play a critical role in the induction of ER stress-mediated apoptosis. Pretreatment with an ROS scavenger suppressed M. smegmatis-induced ER stress. Elimination of ROS decreased the ER stress response and significantly increased the intracellular survival of M. smegmatis. Interestingly, inhibition of phagocytosis significantly decreased ROS synthesis, ER stress response induction, and cytokine production.

Conclusions

Phagocytosis of M. smegmatis induces ROS production, leading to production of proinflammatory cytokines. Phagocytosis-induced ROS is associated with the M. smegmatis-mediated ER stress response in macrophages. Therefore, phagocytosis plays a critical role in the induction of ER stress-mediated apoptosis during mycobacterial infection.

     

Production of recombinant proteins in Mycobacterium smegmatis for structural and functional studies

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over‐express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non‐pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.     

The mycobacterial DNA-binding protein 1 (MDP1) from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG influences various growth characteristics

Background  

Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene.     

Acanthamoeba polyphaga-enhanced growth of Mycobacterium smegmatis

Background

Mycobacterium smegmatis is a rapidly-growing mycobacterium causing rare opportunistic infections in human patients. It is present in soil and water environments where free-living amoeba also reside, but data regarding M. smegmatis-amoeba relationships have been contradictory from mycobacteria destruction to mycobacteria survival.

Methodology/Principal Findings

Using optic and electron microscopy and culture-based microbial enumeration we investigated the ability of M. smegmatis mc² 155, M. smegmatis ATCC 19420^T and M. smegmatis ATCC 27204 organisms to survive into Acanthamoeba polyphaga trophozoites and cysts. We observed that M. smegmatis mycobacteria penetrated and survived in A. polyphaga trophozoites over five-day co-culture resulting in amoeba lysis and the release of viable M. smegmatis mycobacteria without amoebal cyst formation. We further observed that amoeba-co-culture, and lysed amoeba and supernatant and pellet, significantly increased five-day growth of the three tested M. smegmatis strains, including a four-fold increase in intra-amoebal growth.

Conclusions/Significance

Amoebal co-culture increases the growth of M. smegmatis resulting in amoeba killing by replicating M. smegmatis mycobacteria. This amoeba-M. smegmatis co-culture system illustrates an unusual paradigm in the mycobacteria-amoeba interactions as mycobacteria have been mainly regarded as amoeba-resistant organisms. Using these model organisms, this co-culture system could be used as a simple and rapid model to probe mycobacterial factors implicated in the intracellular growth of mycobacteria.     

Improved mycobacterial protein production using a <Emphasis Type="Italic">Mycobacterium smegmatis groEL1ΔC</Emphasis>expression strain

Background  

The non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1.     

Molecular modeling and <Emphasis Type="Italic">in silico</Emphasis> characterization of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> TlyA: Possible misannotation of this tubercle bacilli-hemolysin

Background  

The TlyA protein has a controversial function as a virulence factor in Mycobacterium tuberculosis (M. tuberculosis). At present, its dual activity as hemolysin and RNA methyltransferase in M. tuberculosis has been indirectly proposed based on in vitro results. There is no evidence however for TlyA relevance in the survival of tubercle bacilli inside host cells or whether both activities are functionally linked. A thorough analysis of structure prediction for this mycobacterial protein in this study shows the need for reevaluating TlyA's function in virulence.     

Quantitative Mass Spectrometry Reveals Plasticity of Metabolic Networks in Mycobacterium smegmatis

Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.The genus Mycobacterium comprises more than 100 known species of obligate and opportunistic pathogens as well as nonpathogenic saprophytes such as Mycobacterium smegmatis, which shares many characteristics with its pathogenic relatives (1, 2). Because of its similarity in basic cellular processes and the ease and safety in handling nonpathogenic bacteria, M. smegmatis is widely used as a model system for pathogenic Mycobacterium tuberculosis. These organisms share a common framework of carbon metabolism that is complemented with additional reactions and pathways suiting their pathogenic and saprophytic lifestyles. Although the M. smegmatis genome has been sequenced, understanding the many cellular processes is limited by the fact that a large number of genes, many of which are unique to mycobacteria, have not been functionally annotated. Furthermore, genes encoding proteins that mediate specialized functions may be expressed only in response to specific environmental cues. These gaps in our current understanding of mycobacterial metabolism warrant genome-scale studies aimed at delineating the adaptive mechanisms employed under specific growth conditions (3–6).Comparative proteome profiling is a powerful tool for investigating differences in global protein abundance that occur in response to different environmental stimuli (7, 8), and it can provide new insights into the metabolic and regulatory pathways involved in adaptation to the associated stimuli (9–11). Stable isotope dimethyl labeling is a technique that allows precise quantitative mass spectrometry-based analysis of proteome-wide changes within an organism (12, 13). Here, we demonstrate that this technique can also be exploited for deep mining of mycobacterial proteomes and simultaneous comparative analysis of proteomes originating from up to three different experimental conditions. We demonstrate this comparative approach by quantitative analysis of the M. smegmatis proteome in bacterial cells grown with one of three different carbon sources representing glycolytic (glucose) or gluconeogenic (acetate and propionate) substrates. Acetate and propionate are the immediate downstream products of fatty acid β-oxidation, a committed pathway for entry of long-chain fatty acids into the central carbon-metabolizing network.Using this approach, we identified 3,067 proteins in M. smegmatis with high confidence using at least two unique peptides per protein. Among the proteins that we identified, 162 proteins showed differential abundance in cells grown with either acetate or propionate as the carbon source compared with glucose-grown cells. The majority of these proteins clustered within the functional category related to energy metabolism, providing new insights into how these carbon sources are assimilated in M. smegmatis. An interesting feature that emerges from this study is the plasticity of metabolic networks in M. smegmatis, where multiple pathways can be co-utilized for assimilation of the same metabolite. This finding, along with the recent demonstration that mycobacteria can co-catabolize multiple carbon sources (14), underscores the flexibility and metabolic potential of M. smegmatis. In the future, this approach could be used to explore the metabolic adaptations that play a role in growth of M. tuberculosis on fatty acid substrates, which are thought to be an important carbon source for the bacteria in the lungs of the infected host (5, 6, 15, 16).     

The Novel Responses of Ethambutol Against <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> mc<Superscript>2</Superscript>155 Revealed by Proteomics Analysis

Ethambutol (EMB), one of the effective anti-mycobacterial drugs, inhibits the biosynthesis of mycobacterium cell wall. To elucidate the molecular mechanism of EMB against tuberculosis (TB), Mycobacterium smegmatis mc²155 was employed as a model of mycobacterial system in this study. We compared the protein profiles on M. smegmatis mc²155 treated by EMB and untreated using fluorescence difference two-dimensional gel electrophoresis (2-D DIGE). A total of 40 differential protein spots were selected and 22 proteins were identified by HPLC-nano ESI–MS/MS analysis, including 16 over-expressed proteins and 6 under-expressed proteins. These proteins mainly affected energy metabolism, as well as synthesis and modification of macromolecules. The expressions of correspondent genes were confirmed by RT-PCR. This investigation provided some clues for searching potential drug targets.     

Extraction and preliminary characterizaion of a mycobacteriolytic preparation (stazyme) from aStaphylococcus strain: mycobactericidal activity and its use in rapid extraction of mycobacterial DNA

The clear culture filtrate from 1-L culture of a laboratory contaminant ofStaphylococcus (coagulase^– strain, designated Clavelis) was filtered, concentrated, dialyzed, and the proteins were precipitated. The precipitate was washed, concentrated, and aliquoted (about 4 mg of total proteins/ml, designated as Stazyme). The crude preparation was subjected to gel filtration on Sephadex G-75, and the fractions were screened for their lytic ability againstMycobacterium smegmatis. Native proteins in the stazyme were electrophoretically separated, electroeluted, and their lytic activity againstM. smegmatis was compared with parallel controls (partially purified proteins extracted from the same quantity of the uninoculated bacterial growth medium). Only stazyme preparations caused significant growth inhibition ofM. smegmatis, M. chelonae, M. xenopi, M. tuberculosis, andM. kansasii. Stazyme essentially possessed a lytic activity measured with purifiedM. smegmatis andMicrococcus lysodeikticus cell walls and showed high bactericidal activity againstM. smegmatis, M. chelonae, andM. tuberculosis. It was also able to rapidly lyse intactM. smegmatis organisms, permitting significant yield of mycobacterial DNA.     

Phenotypic heterogeneity in mycobacterial stringent response

Background  

A common survival strategy of microorganisms subjected to stress involves the generation of phenotypic heterogeneity in the isogenic microbial population enabling a subset of the population to survive under stress. In a recent study, a mycobacterial population of M. smegmatis was shown to develop phenotypic heterogeneity under nutrient depletion. The observed heterogeneity is in the form of a bimodal distribution of the expression levels of the Green Fluorescent Protein (GFP) as reporter with the gfp fused to the promoter of the rel gene. The stringent response pathway is initiated in the subpopulation with high rel activity.     

Decaprenylphosphoryl-β-D-ribose 2'-epimerase, the target of benzothiazinones and dinitrobenzamides, is an essential enzyme in Mycobacterium smegmatis

Background

The unique cell wall of bacteria of the suborder Corynebacterineae is essential for the growth and survival of significant human pathogens including Mycobacterium tuberculosis and Mycobacterium leprae. Drug resistance in mycobacteria is an increasingly common development, making identification of new antimicrobials a priority. Recent studies have revealed potent anti-mycobacterial compounds, the benzothiazinones and dinitrobenzamides, active against DprE1, a subunit of decaprenylphosphoribose 2′ epimerase which forms decaprenylphosphoryl arabinose, the arabinose donor for mycobacterial cell wall biosynthesis. Despite the exploitation of Mycobacterium smegmatis in the identification of DprE1 as the target of these new antimicrobials and its use in the exploration of mechanisms of resistance, the essentiality of DprE1 in this species has never been examined. Indeed, direct experimental evidence of the essentiality of DprE1 has not been obtained in any species of mycobacterium.

Methodology/Principal Findings

In this study we constructed a conditional gene knockout strain targeting the ortholog of dprE1 in M. smegmatis, MSMEG_6382. Disruption of the chromosomal copy of MSMEG_6382 was only possible in the presence of a plasmid-encoded copy of MSMEG_6382. Curing of this “rescue” plasmid from the bacterial population resulted in a cessation of growth, demonstrating gene essentiality.

Conclusions/Significance

This study provides the first direct experimental evidence for the essentiality of DprE1 in mycobacteria. The essentiality of DprE1 in M. smegmatis, combined with its conservation in all sequenced mycobacterial genomes, suggests that decaprenylphosphoryl arabinose synthesis is essential in all mycobacteria. Our findings indicate a lack of redundancy in decaprenylphosphoryl arabinose synthesis in M. smegmatis, despite the relatively large coding capacity of this species, and suggest that no alternative arabinose donors for cell wall biosynthesis exist. Overall, this study further validates DprE1 as a promising target for new anti-mycobacterial drugs.     

NapM,a new nucleoid‐associated protein,broadly regulates gene expression and affects mycobacterial resistance to anti‐tuberculosis drugs

Nucleoid‐associated proteins (NAPs) play important roles in the global organization of bacterial chromosomes. However, potential NAPs and their functions are barely characterized in mycobacteria. In this study, NapM, an alkaline protein, functions as a new NAP. NapM is conserved in all of the sequenced mycobacterial genomes, and can recognize DNA in a length‐dependent but sequence‐independent manner. It prefers AT‐rich DNA and binds to the major groove. NapM possesses a clear DNA‐bridging function, and can protect DNA from DNase I digestion. NapM globally regulates the expression of more than 150 genes and the resistance of Mycobacterium smegmatis to two anti‐tuberculosis drugs, namely, rifampicin and ethambutol. An ABC transporter operon was found to be specifically responsible for the napM‐dependent ethambutol resistance of M. smegmatis. NapM also presents a similar regulation of anti‐tuberculosis drug resistance in M. tuberculosis. These results suggest that NapM is a new member of the mycobacterial NAP family. Our findings expand the range of identified NAPs and improve the understanding on the relationship between NAPs with antibiotic resistance in mycobacteria.     

Functional characterization of a small heat shock protein from <Emphasis Type="Italic">Mycobacterium leprae</Emphasis>

Background  

Small heat shock proteins are ubiquitous family of stress proteins, having a role in virulence and survival of the pathogen. M. leprae, the causative agent of leprosy is an uncultivable organism in defined media, hence the biology and function of proteins were examined by cloning M. leprae genes in heterologous hosts. The study on sHsp18 was carried out as the knowledge about the functions of this major immunodominant antigen of M. leprae is scanty.     

The studies of ParA and ParB dynamics reveal asymmetry of chromosome segregation in mycobacteria

Active segregation of bacterial chromosomes usually involves the action of ParB proteins, which bind in proximity of chromosomal origin (oriC) regions forming nucleoprotein complexes – segrosomes. Newly duplicated segrosomes are moved either uni‐ or bidirectionally by the action of ATPases – ParA proteins. In Mycobacterium smegmatis the oriC region is located in an off‐centred position and newly replicated segrosomes are segregated towards cell poles. The elimination of M. smegmatis ParA and/or ParB leads to chromosome segregation defects. Here, we took advantage of microfluidic time‐lapse fluorescent microscopy to address the question of ParA and ParB dynamics in M. smegmatis and M. tuberculosis cells. Our results reveal that ParB complexes are segregated in an asymmetrical manner. The rapid movement of segrosomes is dependent on ParA that is transiently associated with the new pole. Remarkably in M. tuberculosis, the movement of the ParB complex is much slower than in M. smegmatis, but segregation as in M. smegmatis lasts approximately 10% of the cell cycle, which suggests a correlation between segregation dynamics and the growth rate. On the basis of our results, we propose a model for the asymmetric action of segregation machinery that reflects unequal division and growth of mycobacterial cells.     

Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria

Background  

Mycobacteriophage Ms6 integrates into Mycobacterium smegmatis and M. bovis BCG chromosome at the 3' end of tRNA^ala genes. Homologous recombination occurs between the phage attP core and the attB site located in the T-loop. Integration-proficient vectors derived from Ms6 are useful genetic tools, but their insertion sites in the BCG chromosome remain poorly defined. The primary objective of this study was to identify Ms6 target genes in M. smegmatis and BCG. We then aimed to modify the attP site in Ms6-derived vectors, to switch integration to other tRNA^ala loci. This provided the basis for the development of recombinant M. bovis BCG strains expressing several reporter genes inserted into different tRNA^ala genes.