study-of-phagolysosome-biogenesis-in-live-macrophages

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Helmholtz Centre for Infection Research

national institute for medical research

Autophagy is the unique, regulated mechanism for the degradation of organelles. This intracellular process acts as a prosurvival pathway during cell starvation or stress and is also involved in cellular response against specific bacterial infections. Vibrio cholerae is a noninvasive intestinal pathogen that has been studied extensively as the causative agent of the human disease cholera. V. cholerae illness is produced primarily through the expression of a potent toxin (cholera toxin) within the human intestine. Besides cholera toxin, this bacterium secretes a hemolytic exotoxin termed V. cholerae cytolysin (VCC) that causes extensive vacuolation in epithelial cells. In this work, we explored the relationship between the vacuolation caused by VCC and the autophagic pathway. Treatment of cells with VCC increased the punctate distribution of LC3, a feature indicative of autophagosome formation. Moreover, VCC-induced vacuoles colocalized with LC3 in several cell lines, including human intestinal Caco-2 cells, indicating the interaction of the large vacuoles with autophagic vesicles. Electron microscopy analysis confirmed that the vacuoles caused by VCC presented hallmarks of autophagosomes. Additionally, biochemical evidence demonstrated the degradative nature of the VCC-generated vacuoles. Interestingly, autophagy inhibition resulted in decreased survival of Caco-2 cells upon VCC intoxication. Also, VCC failed to induce vacuolization in Atg5-/- cells, and the survival response of these cells against the toxin was dramatically impaired. These results demonstrate that autophagy acts as a cellular defense pathway against secreted bacterial toxins.

Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae.

Macrophages can potentially kill all mycobacteria by poorly understood mechanisms. In this study, we explore the role of NF-kappaB in the innate immune response of macrophages against Mycobacterium smegmatis, a nonpathogenic mycobacterium efficiently killed by macrophages, and Mycobacterium avium which survives within macrophages. We show that infection of macrophages with M. smegmatis induces an activation of NF-kappaB that is essential for maturation of mycobacterial phagosomes and bacterial killing. In contrast, the pathogenic M. avium partially represses NF-kappaB activation. Using microarray analysis, we identified many lysosomal enzymes and membrane-trafficking regulators, including cathepsins, LAMP-2 and Rab34, were regulated by NF-kappaB during infection. Our results argue that NF-kappaB activation increases the synthesis of membrane trafficking molecules, which may be rate limiting for regulating phagolysosome fusion during infection. The direct consequence of NF-kappaB inhibition is the impaired delivery of lysosomal enzymes to M. smegmatis phagosomes and reduced killing. Thus, the established role of NF-kappaB in the innate immune response can now be expanded to include regulation of membrane trafficking during infection.

NF-kappa B activation controls phagolysosome fusion-mediated killing of mycobacteria by macrophages.

We have shown that several lipids can modulate the macrophage innate immune response against mycobacteria and enhance their killing. Since NF-kappaB is required for mycobacterial killing, we tested the ability of lipids to activate NF-kappaB in uninfected macrophages and those infected with mycobacteria. In uninfected cells, sphingomyelin (SM), phosphatidylinositol-4-phosphate (PIP) and arachidonic acid (AA) enhanced NF-kappaB activation and the cell surface expression of CD69, a macrophage activation marker regulated by NF-kappaB. Sphingosine (Sph), sphingosine-1-phosphate (S1P), diacylglycerol (DAG), eicosapentanoic acid (EPA) and phosphatidyl choline (PC) failed to activate either NF-kappaB or CD69. Ceramide (Cer) activated CD69 expression without activating NF-kappaB. In Mycobacterium smegmatis-infected cells, NF-kappaB was transiently activated in a manner that was enhanced by SM, PIP and AA. In contrast Mycobacterium avium mostly repressed NF-kappaB activation and only SM and AA could induce its partial activation. While lipids that activate NF-kappaB in uninfected cells tend to kill mycobacteria in macrophages Sph and S1P failed to activate NF-kappaB under most conditions but nevertheless enhanced killing of M. smegmatis, M. avium and M. tuberculosis H37Rv. Our results argue that both NF-kappaB-dependent and -independent mechanisms are involved in macrophage killing of mycobacteria and that both mechanisms can be enhanced by selected lipids.

Role of lipids in killing mycobacteria by macrophages: evidence for NF-kappaB-dependent and -independent killing induced by different lipids.

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.

Porins facilitate nitric oxide-mediated killing of mycobacteria.

Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.

Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

Compartmentalization in cells of the immune system, the focus of this review, facilitates the spatiotemporal organization of cellular responses essential for specialized immune functions. In this process of compartment maintenance, Rab proteins are central regulators of protein-mediated transport and fusion of intracellular structures. It is widely believed that the intracellular concentration of proteins that regulate intracellular transport, including Rab proteins, is constitutively mantained. However, there is a growing body of evidence indicating that transcriptional rates of Rab proteins can be modified. This process is especially evident during immune activation and argues that after activation, these cells require higher levels of Rab proteins. The aim of this review is to discuss evidence showing the increasing links between Rab protein expression and intracellular transport, particularly in monocytes and macrophages. We highlight here biological processes in which the expression of Rab GTPases is selectively regulated, leading to the activation of specific intracellular routes. Further, we focus on the immune regulation of intracellular transport after cytokine activation and microbial infection, with an emphasis in mycobacterial infection.

Immune regulation of Rab proteins expression and intracellular transport.

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.

Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.

Immunoglobulins drive terminal maturation of splenic dendritic cells.

Human β-defensins are host defense peptides performing antimicrobial as well as immunomodulatory functions. The present study investigated whether treatment of Escherichia coli with human β-defensin 2 could generate extracellular molecules of relevance for immune regulation. Mass spectrometry analysis of bacterial supernatants detected the accumulation of purine nucleosides triggered by β-defensin 2 treatment. Other cationic antimicrobial peptides tested presented variable outcomes with regard to extracellular adenosine accumulation; human β-defensin 2 was the most efficient at inducing this response. Structural and biochemical evidence indicated that a mechanism other than plain lysis was involved in the observed phenomenon. By use of isotope ((13)C) labeling, extracellular adenosine was found to be derived from preexistent RNA, and a direct interaction between the peptide and bacterial nucleic acid was documented for the first time for β-defensin 2. Taken together, the data suggest that defensin activity on a bacterial target may alter local levels of adenosine, a well-known immunomodulator influencing inflammatory processes.

Human β-defensin 2 induces extracellular accumulation of adenosine in Escherichia coli.

Within a cell, the regulation of organelle positioning is considered to be critical in spatio-temporal responses. The position of late endocytic organelles (named here lysosomes for simplicity) is tightly controlled and has a functional impact on processes like endocytosis, phagocytosis and autophagocytosis. The cytoplasmic distribution profile of lysosomes can be easily determined in cells where the cytoplasm/nuclear ratio in a cross-section area is high. However, determining lysosomal position in cells with lower cytoplasm/nuclear ratio, such as macrophages is more challenging. Here, we describe a method that can be efficiently and accurately used to determine the position of organelles in macrophages using confocal microscopy in two-dimensional (2D) images. Using this approach in macrophages, we confirmed previous observations in epithelial cells that both changes in cytoplasmic pH and the levels of active Rab34 induced a re-distribution of lysosomes to the cell centre or periphery. Noteworthy is that this Rab34-dependent re-distribution of lysosomes did not significantly affect the spatial distribution profile of phagolysosomes in the cytoplasm. We conclude that although Rab34 regulates both lysosomal positioning and lysosome to phagosome fusion, the latter effect is not due to the regulation of the cytoplasmic accessibility of lysosomes to phagosomes by Rab34.

Spatial distribution of phagolysosomes is independent of the regulation of lysosome position by Rab34.

Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor.

Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

Rab GTPases are at the central node of the machinery that regulates trafficking of organelles, including phagosomes. Thanks to the unique combination of high quality phagosome purification with highly sensitive proteomic studies, the network of Rab proteins that are dynamically associated with phagosomes during the process of maturation of this organelle is relatively well known. Whereas the phagosomal functions of many of the Rab proteins associated with phagosomes are characterized, the role(s) of most of these trafficking regulators remains to be identified. In some cases, even when the function in the context of phagosome biology is described, phagosomal Rab proteins seem to have similar roles. This review summarizes the current knowledge about the identity and function of phagosomal Rab GTPases, with a particular emphasis on new evidence that clarify these seemingly overlapping Rab functions during phagosome maturation.

Functional role(s) of phagosomal Rab GTPases.

Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. IFN-γ stimulated Rab20 association with early phagosomes in macrophages. Using single phagosome imaging in live cells, we found that Rab20 induced an early delay in phagosome maturation and extended the time Rab5a and phosphatidylinositol 3-phosphate (PI3P) remains associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogated the phagosome maturation delay induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5 guanine nucleotide exchange factor Rabex-5 and that Rab20 knockdown impaired the IFN-γ-dependent Rabex-5/Rab5a recruitment into phagosomes. Altogether, we uncover here Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages.

Article	Year
Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 17267617	2007
NF-kappa B activation controls phagolysosome fusion-mediated killing of mycobacteria by macrophages. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 18684956	2008
Role of lipids in killing mycobacteria by macrophages: evidence for NF-kappaB-dependent and -independent killing induced by different lipids. Cellular microbiology\| PubMed ID: 19016780	2009
Porins facilitate nitric oxide-mediated killing of mycobacteria. Microbes and infection / Institut Pasteur\| PubMed ID: 19460455	2009
Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells. Cellular microbiology\| PubMed ID: 21658173	2011
Immune regulation of Rab proteins expression and intracellular transport. Journal of leukocyte biology\| PubMed ID: 22496357	2012
Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23197834	2012
Immunoglobulins drive terminal maturation of splenic dendritic cells. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23345431	2013
Human β-defensin 2 induces extracellular accumulation of adenosine in Escherichia coli. Antimicrobial agents and chemotherapy\| PubMed ID: 23817371	2013
Spatial distribution of phagolysosomes is independent of the regulation of lysosome position by Rab34. The international journal of biochemistry & cell biology\| PubMed ID: 23871933	2013
Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis. BMC microbiology\| PubMed ID: 24007602	2013
Functional role(s) of phagosomal Rab GTPases. Small GTPases\| PubMed ID: 24088602	2013
Identification of an immune regulated phagosomal Rab cascade in macrophages. Journal of cell science\| PubMed ID: 24569883	2014

Maximiliano Gabriel Gutierrez

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