Cytokine effects on maturation of the phagosomes containing Mycobacteria avium subspecies paratuberculosis in J774 cells

Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages. Intracellular survival of several species of pathogenic mycobacteria is dependent on inhibition of maturation of the phagosomes containing these pathogens into functional phagolysosomes. In activated macrophages, however, this capacity is reduced, leading to increased bacterial killing. It is the hypothesis of this study that there is increased acidification and maturation of the phagosome containing M. a. ptb in interferon gamma and lipopolysaccharide (IFN-gamma/LPS) activated macrophages. In activated macrophages colocalization of M. a. ptb with either a marker of acidic compartments (Lysotracker Red) or compartments containing a late phagosome maturation marker lysosome-associated membrane protein-1 (Lamp-1) were evaluated by laser confocal microscopy. Intracellular survival of M. a. ptb in activated macrophages was evaluated directly using differential fluorescent live/dead staining. The results of this study demonstrated increased colocalization of both Lysotracker Red and Lamp-1 with FITC labeled M. a. ptb, which correlated with decreased survival of M. a. ptb within activated macrophages.     

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.     

Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.     

On the killing of mycobacteria by macrophages

Both pathogenic and non-pathogenic mycobacteria are internalized into macrophage phagosomes. Whereas the non-pathogenic types are invariably killed by all macrophages, the pathogens generally survive and grow. Here, we addressed the survival, production of nitrogen intermediates (RNI) and intracellular trafficking of the non-pathogenic Mycobacterium smegmatis , the pathogen-like, BCG and the pathogenic M. bovis in different mouse, human and bovine macrophages. The bacteriocidal effects of RNI were restricted for all bacterial species to the early stages of infection. EM analysis showed clearly that all the mycobacteria remained within phagosomes even at late times of infection. The fraction of BCG and M. bovis found in mature phagolysosomes rarely exceeded 10% of total, irrespective of whether bacteria were growing, latent or being killed, with little correlation between the extent of phagosome maturation and the degree of killing. Theoretical modelling of our data identified two different potential sets of explanations that are consistent with our results. The model we favour is one in which a small but significant fraction of BCG is killed in an early phagosome, then maturation of a small fraction of phagosomes with both live and killed bacteria, followed by extremely rapid killing and digestion of the bacteria in phago-lysosomes.     

Porins facilitate nitric oxide-mediated killing of mycobacteria

Non-pathogenic mycobacteria such us Mycobacterium smegmatis reside in macrophages within phagosomes that fuse with late endocytic/lysosomal compartments. This sequential fusion process is required for the killing of non-pathogenic mycobacteria by macrophages. Porins are proteins that allow the influx of hydrophilic molecules across the mycobacterial outer membrane. Deletion of the porins MspA, MspC and MspD significantly increased survival of M. smegmatis in J774 macrophages. However, the mechanism underlying this observation is unknown. Internalization of wild-type M. smegmatis (SMR5) and the porin triple mutant (ML16) by macrophages was identical indicating that the viability of the porin mutant in vivo was enhanced. This was not due to effects on phagosome trafficking since fusion of phagosomes containing the mutant with late endocytic compartments was unaffected. Moreover, in ML16-infected macrophages, the generation of nitric oxide (NO) was similar to the wild type-infected cells. However, ML16 was significantly more resistant to the effects of NO in vitro compared to SMR5. Our data provide evidence that porins render mycobacteria vulnerable to killing by reactive nitrogen intermediates within phagosomes probably by facilitating uptake of NO across the mycobacterial outer membrane.     

Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium

16S rRNA sequences from Mycobacterium tuberculosis, M. avium, M. gastri, M. kansasii, M. marinum, M. chelonae, M. smegmatis, M. terrae, M. gordonae, M. scrofulaceum, M. szulgai, M. intracellulare, M. nonchromogenicum, M. xenopi, M. malmoense, M. simiae, M. flavescens, M. fortuitum, and M. paratuberculosis were determined and compared. The sequence data were used to infer a phylogenetic tree, which provided the basis for a systematic phylogenetic analysis of the genus Mycobacterium. The groups of slow- and fast-growing mycobacteria could be differentiated as distinct entities. We found that M. simiae occupies phylogenetically an intermediate position between these two groups. The phylogenetic relatedness within the slow-growing species did not reflect the Runyon classification of photochromogenic, scotchromogenic, and nonchromogenic mycobacteria. In general, the phylogenetic units identified by using rRNA sequences confirmed the validity of phenotypically defined species; an exception was M. gastri, which was indistinguishable from M. kansasii when this kind of analysis was used.     

Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates

Mycobacterium avium subsp. paratuberculosis is genetically similar to other members of the Mycobacterium avium complex (MAC), some of which are nonpathogenic and widespread in the environment. We have utilized an M. avium subsp. paratuberculosis whole-genome microarray representing over 95% of the predicted coding sequences to examine the genetic conservation among 10 M. avium subsp. paratuberculosis isolates, two isolates each of Mycobacterium avium subsp. silvaticum and Mycobacterium avium subsp. avium, and a single isolate each of both Mycobacterium intracellulare and Mycobacterium smegmatis. Genomic DNA from each isolate was competitively hybridized with DNA from M. avium subsp. paratuberculosis K10, and open reading frames (ORFs) were classified as present, divergent, or intermediate. None of the M. avium subsp. paratuberculosis isolates had ORFs classified as divergent. The two M. avium subsp. avium isolates had 210 and 135 divergent ORFs, while the two M. avium subsp. silvaticum isolates examined had 77 and 103 divergent ORFs. Similarly, 130 divergent ORFs were identified in M. intracellulare. A set of 97 ORFs were classified as divergent or intermediate in all of the nonparatuberculosis MAC isolates tested. Many of these ORFs are clustered together on the genome in regions with relatively low average GC content compared with the entire genome and contain mobile genetic elements. One of these regions of sequence divergence contained genes homologous to a mammalian cell entry (mce) operon. Our results indicate that closely related MAC mycobacteria can be distinguished from M. avium subsp. paratuberculosis by multiple clusters of divergent ORFs.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Bactericidal activity of alkaline glutaraldehyde solution against a number of atypical mycobacterial species

The mycobactericidal activity of 2% alkaline glutaraldehyde solution was determined using standardized suspensions of 10 species of atypical mycobacteria and compared with that for virulent Mycobacterium tuberculosis. Suspensions of M. avium, M. intracellulare and M. gordonae were more resistant to disinfection by the glutaraldehyde than were virulent tubercle bacilli while M. kansasii, M. scrofulaceum and M. szulgae were somewhat more susceptible. Mycobacterium marinum, M. smegmatis and M. fortuitum were highly sensitive to the disinfectant action of the alkaline glutaraldehyde solution. This variation in sensitivity shown by apparently closely related strains of mycobacteria to this disinfectant has important practical implications.     

Modulation of cellular phosphatidylinositol 3-phosphate levels in primary macrophages affects heat-killed but not viable Mycobacterium avium's transport through the phagosome maturation process

Most disease causing mycobacteria are intramacrophage pathogens which replicate within nonacidified phagosomes that can interact with the early endosomal network but fail to mature to a phagolysosome. The mycobacterial phagosome retain some proteins required for fusion with endocytic vesicles including Rab5 but lack others such as early endosomal autoantigen 1 (EEA1). As the membrane lipid phosphatidylinositol 3-phosphate (PtdIns-3-P) is required for EEA1 membrane association and phagosome maturation, it may be a potential target of pathogenic mycobacteria. To test this hypothesis, macrophage cellular levels of PtdIns-3-P were altered by retroviral introduction of the type III Phosphoinositide 3-Kinase (VPS34) and the PtdIns-3-P phosphatase myotubularin 1 (MTM1). By utilizing the PtdIns-3-P-specific probes FYVE and PX coupled to EGFP (EGFP-2-FYVE and EGFP-PX, respectively), the expression of PtdIns-3-P on the mycobacterial phagosome was addressed. All phagosomes containing viable Mycobacterium avium stained positive for EGFP-2-FYVE and EGFP-PX despite obvious differences in PtdIns-3-P concentrations in cells expressing MTM1 or VPS34. Altering PtdIns-3-P cellular concentrations did not affect trafficking of live bacilli. However, a significant increase in the transport of killed bacilli to a late endosomal/lysosomal compartment was observed in VPS34-compared to MTM1-transduced macrophages. Therefore, although overexpression of PdtIns-3-P in macrophages can facilitate phagosome maturation, its effect on phagosomes containing viable M. avium was negligible.     

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.     

Disruption of the actin filament network affects delivery of endocytic contents marker to phagosomes with early endosome characteristics: the case of phagosomes with pathogenic mycobacteria

Phagosomes containing live virulent mycobacteria undergo fusion with early endosomes, but they are unable to mature normally. Accordingly, they do not fuse with lysosomes. Although M. avium-containing phagosomes retain fusion and intermingling characteristics of early endosomes indefinitely, fusions with early endosomes are increasingly restricted as bacteria multiply. In addition, when endocytic tracers, such as horseradish peroxidase (HRP), are added to M. avium-infected macrophages at 1 or up to 15 days after infection, an atypical time course of acquisition of the tracer by the phagosomes is observed, i.e., a 10 to 20 min lag, instead of immediate acquisition as is typical for early endosomes (and phagosomes with early endosome characteristics). These events coincide with a marked disorganization of the actin filament network in M. avium-infected macrophages. In the present study, we have therefore addressed the following question: Do actin filaments play a role in fusion and intermingling of contents between early endosomes and immature phagosomes that undergo homotypic fusion with early endosomes? We examined the time course of acquisition of subsequently internalized endocytic marker (HRP) by early endosome-like preexisting phagosomes, i.e. 2 hour-old phagosomes with either hydrophobic latex particles, virulent or avirulent M. avium, after depolymerization of the actin filament network with cytochalasin D or after repolymerization of the actin filament network with jasplakinolide, in cases where the network had been depolymerized (macrophages infected with M. avium, at 1 or up to 7 days after infection). By direct morphological observation at the electron microscope level and by a kinetic approach, we show here that depolymerization of the actin filament network with cytochalasin D delays acquisition of HRP whereas repolymerization restores immediate acquisition of the marker. We conclude that the actin filament network is involved in fusion and intermingling of endocytic contents between early endosomes and early endosome-like phagosomes, and that disruption of this network by M. avium is the cause for the atypical acquisition of content marker by phagosomes containing these pathogenic mycobacteria.     

Genome scale portrait of cAMP-receptor protein (CRP) regulons in mycobacteria points to their role in pathogenesis

cAMP Receptor Protein (CRP)/Fumarate Nitrate Reductase Regulator (FNR) family proteins are ubiquitous regulators of cell stress in eubacteria. These proteins are commonly associated with maintenance of intracellular oxygen levels, redox-state, oxidative and nitrosative stresses, and extreme temperature conditions by regulating expression of target genes that contain regulatory cognate DNA elements. We describe the use of informatics enabled comparative genomics to identify novel genes under the control of CRP regulator in Mycobacterium tuberculosis (M.tb). An inventory of CRP regulated genes and their operon context in important mycobacterial species such as M. leprae, M. avium subsp. paratuberculosis and M. smegmatis and several common genes within this genus including the important cellular functions, mainly, cell-wall biogenesis, cAMP signaling and metabolism associated with such regulons were identified. Our results provide a possible theoretical framework for better understanding of the stress response in mycobacteria. The conservation of the CRP regulated genes in pathogenic mycobacteria, as opposed to non-pathogenic ones, highlights the importance of CRP-regulated genes in pathogenesis.     

Higher order Rab programming in phagolysosome biogenesis

Phagosomes offer kinetically and morphologically tractable organelles to dissect the control of phagolysosome biogenesis by Rab GTPases. Model phagosomes harboring latex beads undergo a coordinated Rab5-Rab7 exchange, which is akin to the process of endosomal Rab conversion, the control mechanisms of which are unknown. In the process of blocking phagosomal maturation, the intracellular pathogen Mycobacterium tuberculosis prevents Rab7 acquisition, thus, providing a naturally occurring tool to study Rab conversion. We show that M. tuberculosis inhibition of Rab7 acquisition and arrest of phagosomal maturation depends on Rab22a. Four-dimensional microscopy revealed that phagosomes harboring live mycobacteria recruited and retained increasing amounts of Rab22a. Rab22a knockdown in macrophages via siRNA enhanced the maturation of phagosomes with live mycobacteria. Conversely, overexpression of the GTP-locked mutant Rab22aQ64L prevented maturation of phagosomes containing heat-killed mycobacteria, which normally progress into phagolysosomes. Moreover, Rab22a knockdown led to Rab7 acquisition by phagosomes harboring live mycobacteria. Our findings show that Rab22a defines the critical checkpoint for Rab7 conversion on phagosomes, allowing or disallowing organellar transition into a late endosomal compartment. M. tuberculosis parasitizes this process by actively recruiting and maintaining Rab22a on its phagosome, thus, preventing Rab7 acquisition and blocking phagolysosomal biogenesis.     

Mycobacterium tuberculosis mammalian cell entry operon (mce) homologs in Mycobacterium other than tuberculosis (MOTT)

The cloned mammalian cell entry gene mce1a from Mycobacterium tuberculosis confers to non-pathogenic Escherichia coli the ability to invade and survive inside macrophages and HeLa cells. The aim of this work was to search for and characterize homologs of the four M. tuberculosis mammalian cell entry operons (mce1, mce2, mce3 and mce4) in mycobacteria other than tuberculosis (MOTT). The dot-blot and polymerase chain reaction (PCR) experiments performed on 24 clinical isolates representing 20 different mycobacterial species indicated that the mce operons were widely distributed throughout the genus Mycobacterium. BLAST search results showed the presence of mce1, mce2 and mce4 homologs in Mycobacterium bovis, Mycobacterium avium and Mycobacterium smegmatis. A homologous region for the mce3 operon was also found in M. avium and M. smegmatis. DNA and protein alignments were done to compare the M. tuberculosis mce operons and the deduced M. bovis, M. avium, and M. smegmatis homologs. The deduced proteins of M. bovis mce1, mce2 and mce4 operons had 99.6-100% homology with the respective M. tuberculosis mce proteins (MTmce). The similarity between M. avium mce proteins and the individual M. tuberculosis homologs ranged from 56.2 to 85.5%. The alignment results between M. smegmatis mce proteins and the respective MTmce proteins ranged from 58.5% to 68.5%. Primer sets were designed from the M. tuberculosis mce4a gene for amplification of 379-bp fragments. Amplification was successful in 14 strains representing 11 different mycobacterial species. The PCR fragments were sequenced from 10 strains representing eight species. Alignment of the sequenced PCR products showed that mce4a homologs are highly conserved in the genus Mycobacterium. In conclusions, the four mce operons in different mycobacterial species are generally organized in the same manner. The phylogenetic tree comparing the different mce operons showed that the mce1 operon was closely related to the mce2 operon and mce3 diverged from the other operons. The wide distribution of the mce operons in pathogenic and non-pathogenic mycobacteria implicates that the presence of these putative virulence genes is not an indicator for the pathogenicity of the bacilli. Instead, the pathogenicity of these factors might be determined by their expression.     

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

Aims:  To facilitate efficient identification of commonly encountered mycobacteria species ( Mycobacterium tuberculosis , Mycobacterium avium , Mycobacterium intracellulare , Mycobacterium fortuitum complex , Mycobacterium chelonae/abscessus , Mycobacterium kansasii , Mycobacterium gordonae ) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated.
Methods and Results:  Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40  M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88·9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1·1 with an average turn around time of less than 3 days.
Conclusions:  A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting.
Significance and Impact of the Study:  With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.     

Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes

Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 μm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome–lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.     

Detection and identification of mycobacteria by amplification of mycobacterial DNA

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.     

Impact of Mycobacterium Avium Infection on Macrophage-Liposome Interaction

Abstract

Mycobacterium avium (M. avium) can survive within macrophages, and alters various functions of its cellular host. Liposomes and other particulate drug carriers have been investigated as a means for enhancing drug delivery to the intracellular site of infection via the endocytic pathway. We have investigated the impact of M. avium infection on the endocytosis, retention, and intracellular disposition of liposomes, as these are essential macrophage functions that may determine the activity of liposome-encapsulated antibiotics. J774, a macrophage-like cell line, was infected with M. avium at various multiplicities. Infected cells accumulated and retained liposomes in a manner similar to uninfected cells, regardless of the time after infection. Moreover, the intracellular trafficking and disposition of liposomes was unaltered in J774 cells that contained live or heat-killed M. avium, as evidenced by the vesicular pH which liposomes encountered within cells. These results suggest that specific essential cellular functions involved in processing particulate drug carriers remain intact in macrophages infected with M. avium.