A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.     

The interaction of mycobacterial protein Rv2966c with host chromatin is mediated through non-CpG methylation and histone H3/H4 binding

To effectively modulate the gene expression within an infected mammalian cell, the pathogen Mycobacterium tuberculosis would need to bring about epigenetic modifications at appropriate genomic loci. Working on this hypothesis, we show in this study that the mycobacterial protein Rv2966c is a 5-methylcytosine-specific DNA methyltransferase that is secreted out from the mycobacterium and gets localized to the nucleus in addition to the cytoplasm inside the host cell. Importantly, Rv2966c binds to specific DNA sequences, methylates cytosines predominantly in a non-CpG context and its methylation activity is positively influenced by phosphorylation. Interestingly, like the mammalian DNA methyltransferase, DNMT3L, Rv2966c can also interact with histone proteins. Ours is the first study that identifies a protein from a pathogenic bacteria with potential to influence host DNA methylation in a non-canonical manner providing the pathogen with a novel mechanism to alter the host epigenetic machinery. This contention is supported by repression of host genes upon M. tuberculosis infection correlated with Rv2966c binding and non-CpG methylation.     

Mycobacterium tuberculosis induces an atypical cell death mode to escape from infected macrophages

Background

Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds ∼20 per macrophage but the precise nature of this demise has not been defined.

Methodology/Principal Findings

We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.

Conclusions/Significance

Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis.     

Porins Increase Copper Susceptibility of Mycobacterium tuberculosis

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 μM copper by 40% and completely suppressed growth at 15 μM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.     

Survival of Mycobacterium tuberculosis and Mycobacterium bovis BCG in lysosomes in vivo

Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro, was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis.     

Keratinocyte Growth Factor Administration Attenuates Murine Pulmonary Mycobacterium tuberculosis Infection through Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF)-dependent Macrophage Activation and Phagolysosome Fusion

Augmentation of innate immune defenses is an appealing adjunctive strategy for treatment of pulmonary Mycobacterium tuberculosis infections, especially those caused by drug-resistant strains. The effect of intranasal administration of keratinocyte growth factor (KGF), an epithelial mitogen and differentiation factor, on M. tuberculosis infection in mice was tested in prophylaxis, treatment, and rescue scenarios. Infection of C57BL6 mice with M. tuberculosis resulted in inoculum size-dependent weight loss and mortality. A single dose of KGF given 1 day prior to infection with 10⁵ M. tuberculosis bacilli prevented weight loss and enhanced pulmonary mycobacterial clearance (compared with saline-pretreated mice) for up to 28 days. Similar effects were seen when KGF was delivered intranasally every third day for 15 days, but weight loss and bacillary growth resumed when KGF was withdrawn. For mice with a well established M. tuberculosis infection, KGF given every 3 days beginning on day 15 postinoculation was associated with reversal of weight loss and an increase in M. tuberculosis clearance. In in vitro co-culture experiments, M. tuberculosis-infected macrophages exposed to conditioned medium from KGF-treated alveolar type II cell (MLE-15) monolayers exhibited enhanced GM-CSF-dependent killing through mechanisms that included promotion of phagolysosome fusion and induction of nitric oxide. Alveolar macrophages from KGF-treated mice also exhibited enhanced GM-CSF-dependent phagolysosomal fusion. These results provide evidence that administration of KGF promotes M. tuberculosis clearance through GM-CSF-dependent mechanisms and enhances host defense against M. tuberculosis infection.     

Guinea pig neutrophil–macrophage interactions during infection with Mycobacterium tuberculosis

We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent Mycobacterium tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1β and TNF-α mRNA expression was significantly upregulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8-treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1β mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil–macrophage interactions may contribute to host defense against M. tuberculosis infection.     

Is Mycobacterium tuberculosis stressed out? A critical assessment of the genetic evidence

Mycobacterium tuberculosis is an obligate human intracellular pathogen which remains a major killer worldwide. A remarkable feature of M. tuberculosis infection is the ability of the pathogen to persist within the host for decades despite an impressive onslaught of stresses. In this review we seek to outline the host-inflicted stresses experienced by M. tuberculosis, the bacterial strategies used to withstand these stresses, and how this information should guide our efforts to combat this global pathogen.     

Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel_Mtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel_Mtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel_Mtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel_Mtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel_Mtb point mutants that were either synthetase dead (Rel_Mtb^H344Y) or hydrolase dead (Rel_Mtb^H80A). M. tuberculosis strains expressing the synthetase-dead Rel_Mtb^H344Y mutant did not persist in mice, demonstrating that the Rel_Mtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel_Mtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel_Mtb mutant, Rel_Mtb^H80A, that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel_Mtb^H80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel_Mtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.     

Human mesenchymal stem cell based intracellular dormancy model of Mycobacterium tuberculosis

Understanding the biology of the tuberculosis pathogen during dormant asymptomatic infection, called latent tuberculosis is crucial to decipher a resilient therapeutic strategy for the disease. Recent discoveries exhibiting presence of pathogen’s DNA and bacilli in mesenchymal stem cells (MSCs) of human and mouse despite completion of antitubercular therapy, indicates that these specific cells could be one of the niches for dormant Mycobacterium tuberculosis in humans. To determine if in vitro infection of human MSCs could recapitulate the in vivo characteristics of dormant M. tuberculosis, we examined survival, phenotype, and drug susceptibility of the pathogen in MSCs. When a very low multiplicity of infection (1:1) was used, M. tuberculosis could survive in human bone marrow derived MSCs for more than 22 days without any growth. At this low level of infection, the pathogen did not cause any noticeable host cell death. During the later phase of infection, MSC-residing M. tuberculosis exhibited increased expression of HspX (a 16-kDa alpha-crystallin homolog) with a concurrent increase in tolerance to the frontline antitubercular drugs Rifampin and isoniazid. These results present a human MSC-based intracelllular model of M. tuberculosis infection to dissect the mechanisms through which the pathogen acquires and maintains dormancy in the host.